Osmoprotectant- Mediated Abiotic Stress Tolerance in Plants

Mohammad Anwar Hossain Vinay Kumar · David J. Burritt Masayuki Fujita · Pirjo S. A. Mäkelä Editors

Osmoprotectant- Mediated Abiotic Stress Tolerance in Plants Recent Advances and Future Perspectives Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants Mohammad Anwar Hossain Vinay Kumar • David J. Burritt Masayuki Fujita • Pirjo S. A. Mäkelä Editors

Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants Recent Advances and Future Perspectives Editors Mohammad Anwar Hossain Vinay Kumar Department of Genetics and Plant Breeding Department of Biotechnology Bangladesh Agricultural University Modern College Mymensingh, Bangladesh Pune, Maharashtra, India

David J. Burritt Masayuki Fujita Department of Botany Laboratory of Plant Stress Responses University of Otago Kagawa University Dunedin, Otago, New Zealand Kagawa, Kagawa, Japan

Pirjo S. A. Mäkelä Department of Agricultural Sciences University of Helsinki Helsinki, Finland

ISBN 978-3-030-27422-1 ISBN 978-3-030-27423-8 (eBook) https://doi.org/10.1007/978-3-030-27423-8

© Springer Nature Switzerland AG 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Preface

In nature, plants are constantly challenged by various abiotic and biotic stresses that can restrict their growth, development, and yields. In the course of their evolution, plants have evolved a variety of sophisticated and efficient mechanisms to sense, respond to, and adapt to changes in the surrounding environment. A common defensive mechanism activated by plants in response to abiotic stress is the production and accumulation of compatible solutes (also called osmolytes). These include amino acids (mainly proline), amines (such as glycinebetaine and polyamines), and sugars (such as trehalose and sugar alcohols), all of which are readily soluble in water and nontoxic at high concentrations. The metabolic pathways involved in the biosynthesis and catabolism of compatible solutes and the mechanisms that regulate their cellular concentrations and compartmentalization are well characterized in many important plant species. Numerous studies have provided evidence that enhanced accumulation of compatible solutes in plants correlates with increased resistance to abiotic stresses. New insights into the mechanisms associated with osmolyte accumulation in transgenic plants and the responses of plants to exogenous application of osmolyte will further enhance our understanding of the mechanisms by which compatible solutes help to protect plants from damage due to abiotic stress and the potential roles compatible solutes could play in improving plant growth and development under optimal conditions. Although there has been significant progress made in understanding the multiple roles of compatible solute in abiotic stress tolerance, many aspects associated with compatible solute-mediated abiotic stress responses and stress tolerance still require more research. As well as providing basic up-to-date information on the biosynthesis, compartmentalization, and transport of compatible solute in plants, this book will also give insights into the direct or indirect involvement of these key compatible solutes in many important metabolic processes and physiological functions, including their antioxidant and signaling functions, and roles in modulating plant growth, development, and abiotic stress tolerance. In this book, Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants: Recent Advances and Future Perspectives, we present a collection of 15 chapters written by leading experts engaged with compatible solute-induced abiotic stress

v vi Preface tolerance in plants. The main objective of this volume is to promote the important roles of these compatible solutes in plant biology, by providing an integrated and comprehensive mix of basic and advanced information for students, scholars, and scientists interested in, or already engaged in, research involving osmoprotectant. Finally, this book will be a valuable resource for future environmental stress-related research and can be considered as a textbook for graduate students and as a reference book for frontline researchers working on the relationships between osmoprotectant and abiotic stress responses and tolerance in plants.

Mymensingh, Bangladesh Mohammad Anwar Hossain Pune, India Vinay Kumar Dunedin, New Zealand David J. Burritt Kagawa, Japan Masayuki Fujita Helsinki, Finland Pirjo S. A. Mäkelä Contents

Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics, In Silico Genome Mapping, and Biotechnology �� 1 Éderson Akio Kido, José Ribamar Costa Ferreira-Neto, Manassés Daniel da Silva, Vanessa Emanuelle Pereira Santos, Jorge Luís Bandeira da Silva Filho, and Ana Maria Benko-Iseppon Proline Metabolism and Its Functions in Development and Stress Tolerance �� 41 Maurizio Trovato, Giuseppe Forlani, Santiago Signorelli, and Dietmar Funck Regulation of Proline Accumulation and Its Molecular and Physiological Functions in Stress Defence �� 73 Giuseppe Forlani, Maurizio Trovato, Dietmar Funck, and Santiago Signorelli Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms �� 99 Mohamed Zouari, Ameni Ben Hassena, Lina Trabelsi, Bechir Ben Rouina, Raphaël Decou, and Pascal Labrousse Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant Growth and Development �� 123 Elisa M. Valenzuela-Soto and Ciria G. Figueroa-Soto Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants: Possible Mechanisms �� 141 Tianpeng Zhang and Xinghong Yang Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses �� 153 Pirjo S. A. Mäkelä, Kari Jokinen, and Kristiina Himanen

vii viii Contents

Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth, Development, and (A)biotic Stress Tolerance �� 175 Le Cong Huyen Bao Tran Phan and Patrick Van Dijck Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants Under Stress �� 201 Suriyan Cha-um, Vandna Rai, and Teruhiro Takabe Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress Tolerance in Plants �� 225 Zsófia Bánfalvi The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression �� 241 Merve Kahraman, Gulcin Sevim, and Melike Bor Seed Osmolyte Priming and Abiotic Stress Tolerance �� 257 Danny Ginzburg and Joshua D. Klein Relationship Between Polyamines and Osmoprotectants in the Response to Salinity of the Legume-Rhizobia Symbiosis �� 269 Miguel López-Gómez, Javier Hidalgo-Castellanos, Agustín J. Marín-Peña, and J. Antonio Herrera-Cervera Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants �� 287 Susana de Sousa Araújo, André Luis Wendt dos Santos, and Ana Sofia Duque Fructan Metabolism in Plant Growth and Development and Stress Tolerance �� 319 Alejandro del Pozo, Ana María Méndez-Espinoza, and Alejandra Yáñez

Index �� 335 Editors Biography

Mohammad Anwar Hossain is a Professor in the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. He received his B.Sc. in Agriculture and M.S. in Genetics and Plant Breeding from Bangladesh Agricultural University, Bangladesh. He also received an M.S. in Agriculture from Kagawa University, Japan, in 2008, and a Ph.D. in Abiotic Stress Physiology and Molecular Biology from Ehime University, Japan, in 2011, through Monbukagakusho Scholarship. As a JSPS Postdoctoral Researcher, he has worked on isolating low-phosphorus stress- tolerant genes from rice at the University of Tokyo, Japan, during the period of 2015–2017. His current research interests include the isolation and characterization of abiotic stress-responsive genes and proteins, physiological and molecular mechanisms of abiotic stress response and tolerance with special reference to oxidative stress, antioxidants and methylglyoxal metabolism and signaling, generation of stress-tolerant and nutrient-efficient plants through breeding and biotechnology, and cross-stress tolerance in plants. He has over 50 peer-reviewed publications and has edited 9 books, including this one, published by CRC press, Springer, and Elsevier.

ix x Editors Biography

Vinay Kumar is an Associate Professor in the Department of Biotechnology, Modern College of Arts, Science and Commerce, Ganeshkhind, Pune, India, and a Visiting Faculty at the Department of Environmental Sciences, Savitribai Phule Pune University, Pune, India. He obtained his Ph.D. in Biotechnology from Savitribai Phule Pune University (formerly University of Pune) in 2009. For his Ph.D., he worked on metabolic engineering of rice for improved salinity tolerance. He has published 40 peer-reviewed research/review articles and edited 4 books, including this one, published by Springer and Wiley. He is a Recipient of Young Scientist Award of Science and Engineering Board, Government of India. His current research interests include elucidating molecular mechanisms underlying salinity stress responses and tolerance in plants.

David J. Burritt is an Associate Professor in the Department of Botany, University of Otago, Dunedin, New Zealand. He received his B.Sc. and M.Sc. (hons) in Botany and his Ph.D. in Plant Biotechnology from the University of Canterbury, Christchurch, New Zealand. His research interests include oxidative stress and redox biology, plant-based foods and bioactive molecules, plant breeding and biotechnology, cryo- preservation of germplasm, and the stress biology of plants, animals, and algae. He has over 100 peer- reviewed publications and has edited 4 books for Springer and 3 for Elsevier.

Masayuki Fujita is a Professor in the Department of Plant Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan. He received his B.Sc. in Chemistry from Shizuoka University, Shizuoka, and his M.Agr. and Ph.D. in Plant Biochemistry from Nagoya University, Nagoya, Japan. His research interests include physiological, biochemical, and molecular biological responses based on secondary metabolism in plants under biotic (pathogenic fungal infection) and abiotic (salinity, drought, extreme temperatures, and heavy metals) stresses, phytoalexin, cytochrome P-450, glutathione S-transferase, phytochelatin, and redox reaction and antioxidants. He has over 150 peer-reviewed publications and has edited 10 books including this one. Editors Biography xi

Pirjo S. A. Mäkelä is a Professor in the Department of Agricultural Sciences, University of Helsinki, Finland. She received her M.Sc. and Ph.D. in Crop Science from the University of Helsinki, Finland. Her research interests include physiological, biochemical, and agronomi- cal responses of plants to abiotic stresses, such as water deficit and salinity, as well as ways to minimize the effects of abiotic stresses on yield formation and quality of yield. She is also interested in active learning in higher education. She has over 70 peer-reviewed publications and has edited 3 books including this one. Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics, In Silico Genome Mapping, and Biotechnology

Éderson Akio Kido, José Ribamar Costa Ferreira-Neto, Manassés Daniel da Silva, Vanessa Emanuelle Pereira Santos, Jorge Luís Bandeira da Silva Filho, and Ana Maria Benko-Iseppon

1 Introduction

In nowadays, agriculture faces multiple challenges, including the adoption of sustainable methods to provide food for a growing urban population, in addition to the increase of bioenergy needs. Moreover, plants are subject to a variety of stresses, leading to damages that can negatively influence vegetative and reproductive development and compromise their yields, causing economic losses. In the last decades, the modernization of methods and tools enforced in agriculture has developed simultaneously with civilization. Scientific advances applied in traditional plant breeding have increased genetic gains of cultivated plants improving their yields and their resistance/tolerance to the environmental stresses around the world. Environmental stresses are classified into biotic stresses, those caused by organisms such as bacteria, fungi, viruses, nematodes, insects, or higher eukaryotes (e.g., weed and herbivores), or abiotic stresses, caused by nonliving organisms, including physical or chemicals stressors, such as high or low temperatures, drought or floods, and salinity, among others. These stressors can act alone or often combined, such as droughts and high temperatures. Plants under stress need to adapt in order to survive. They respond to the environment by modifying the expression of their genes to best suit the stressful situation and minimize the damages. The dynamics of the genes global expression determine the plant response to the stress-derived stimulus. Briefly, the stress stimulus is recognized by the receptors in plant cell membranes, and a generated signal is transmitted and amplified in a cascade that culmi- nates in the activation of specific genes. Those gene expressions will constitute the plant response to the stress. During the signaling process, enzymes and receptors are activated or deactivated through phosphorylation and dephosphorylation by

É. A. Kido (*) · J. R. C. Ferreira-Neto · M. D. da Silva · V. E. P. Santos J. L. B. da Silva Filho · A. M. Benko-Iseppon Department of Genetics, Federal University of Pernambuco, Recife, PE, Brazil

protein kinases and phosphatases (Ardito et al. 2017). Finally, activated transcription factors (TFs) inside the nucleus will recognize specific cis-regulatory elements in the promoters of the genes that will be expressed, helping them to modulate their expression. Besides the TFs, gene expressions might be influenced by CoRegs (co- regulator proteins; Burdo et al. 2014). These proteins, unlike TFs, do not interact directly with the DNAs but interfere with gene regulation by protein-protein interactions, even interacting with TFs (Chevalier et al. 2009). They also restrict or release DNA access, behaving as histone modifiers (Fang et al.2014 ) or chromatin remodelers (Han et al. 2016). Therefore, gene expression resulting from complex interactions acts on metabolic pathways or other processes such as RNA interference (RNAi, Saurabh et al. 2014), to respond to the triggering plant stress stimulus. Over time, plant evolving under unfavorable growth conditions presented molecular, biochemical, and physiological adjustments. Some of these alterations were induced by new alleles associated with isoforms neofunctionalizations, increasing plant variability. Such context resulted in a range of combined strategies, which plants access to minimize damages caused by environmental stresses. In general, plants under abiotic stresses rely on genes from three broad categories (Hossain et al. 2016): (a) Genes transcribing regulatory proteins, such as kinases and TFs, which are widely reported in plant responses. (b) Genes related to water channel proteins and ion transporters. (c) Genes linked to the protection of essential membranes and proteins such as chaperones, heat shock proteins, and osmoprotective osmolytes. This chapter regards genes related to osmoprotectants reported in plant transcriptomic studies, their regulation under abiotic stress, their genomes mapping, potential pathways, and, finally, their experiences as transgenes in order to improve plant breeding.

2 Osmoprotectant Definition, Classification, and Roles

Some inorganic ions in ideal concentrations contribute to the biochemical functions, but in high amounts, they disrupt protein functions. Diversely, osmoprotectants are small, electrically neutral, and highly soluble organic compounds with low toxicity. They can accumulate high amounts in the cells, balancing the intracellular with the external environment when in an unfavorable osmotic condition. Due to their high solubility and little interference in the cellular metabolic pathways, they are also known as compatible solutes. The DEOP database (Dragon Explorer of Osmoprotection Pathways; Bougouffa et al. 2014) is an online resource on osmoprotectants and its associated pathways (http://www.cbrc.kaust.edu.sa/deop/). According to the DEOP web index, osmoprotectants are classified into three distinct classes: (i) those containing quaternary ammonium compounds (QACs) and deriva- Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 3

a Polyamines b Aminoacids c Carbohydrate

1. Putrescine 1. Proline 1. Threalose 2. Spermidine 2. Fructan 3. Spermine

d Betaines e Sugar alcohol

1. Glycine betaine 1. Inositol (and its phosphorylated derivatives)

Fig. 1 Main classes of plant osmoprotectants and some representatives compounds tives, e.g., polyamines and betaines (such as glycine betaine); (ii) those containing amino acids and derivatives, e.g., proline and ectoine; and (iii) those containing sugars and derivatives, e.g., oligosaccharides (sucrose, trehalose, raffinose, stachy- ose, verbascose), fructan [fructose polymers; oligosaccharides or polysaccharides (>10 units)], and sugar alcohols (polyols: glycerol, inositol, arabitol, maltitol, sorbitol, mannitol, and D-ononitol). Further, based on the DEOP data, which involved scientific manuscripts published until 2014, covering more than 1160 organisms (including microorganisms, plants, and animals), a total of 135 osmoprotectant compounds were identified (Bougouffa et al. 2014). The major classes of plant osmoprotectants with representatives described in this chapter, whose expressions of their related genes have been reported, are shown in Fig. 1. Essentially, plants face two situations when under an abiotic stress, such as salt stress (Singh et al. 2015): (a) an osmotic stress due to the higher Na+ concentration in the rhizosphere, which decreases plant water potential, and (b) a nutritional imbalance caused by ionic stress, in which the higher concentration of Na+ and Cl− limits the availability and assimilation of essential nutrients. Thus, in plants under hypertonic conditions resulting from high NaCl, a flow of water occurs from the inside to the outside of the cell. This situation increases the concentration of the cellular constituents. A high concentration of ions can disrupt proteins, shifting the balance to their unfolded forms. In this case, protective osmolytes accumulated on the surfaces of proteins help to stabilize their structures, ten- sioning them back to their native structure. Therefore, these osmolytes are recognized as osmoprotectants, due to this protective role against osmotic and saline stresses. In plants under abiotic stress causing denaturation of macromolecular (proteins and membranes), osmoprotectants such as proline can improve protein stability by binding to hydrogen bonds without affecting the other functions (Slama et al. 2015). Further, trehalose, another osmoprotectant, can stabilize macromolecules, such as the bilayer structure of membranes, by binding to hydrogen bonds in the polar 4 É. A. Kido et al. groups of membranes and proteins, preserving their integrities (Pereira et al. 2004). However, this characteristic varies depending on the osmoprotectant considered. Additionally, some osmoprotectants present chaperone-like activities in order to keep both protein structures and functions. Those compatible osmolytes are also named chemical or molecular chaperones (Slama et al. 2015). Besides, in plants exposed to salinity and drought, osmoprotectants can accumulate in the cells, helping to maintain cellular turgor and driving the gradient for water uptake to sustain cell volume by osmotic adjustment. In this regulation, the cell tends to compartmentalize ions in the vacuoles; at the same time, it begins to synthesize and accumulate osmoprotectants in the cytoplasm, such as proline, to maintain the osmotic balance between these compartments (Gagneul et al. 2007). Nevertheless, when a cell undergoes osmotic stress, its redox potential is dis- turbed, and generated an excessive induction and accumulation of reactive oxygen species (ROS). ROS are by-products of the oxygen metabolism linked to electron transport (Bae et al. 2011). These reactive species [superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH)] are important in cell signaling once in adequate amounts but in excessive volume cause peroxidation of lipid, oxidation of proteins, and damage of nucleic acids. Further, they can inactivate antioxidative enzymes and even culminate in cells and plant deaths. Therefore, ROS regulation is crucial to avoid cytotoxicity and oxidative damage. Some of the compatible solutes can protect plants from oxidative damages by directly scavenging for ROS or protecting the enzymes from the antioxidative process (Slama et al. 2015). Other osmoprotectants functions in plant responses to abiotic stresses, especially concerning the drought and salinity tolerance, are found in the review reported by Singh et al. (2015).

3 The Basic of Plant Transcriptomic Studies

A transcriptomic study is an excellent option to look in the potential of osmoprotectants, notably their related genes, in a plant responding to an abiotic stimulus. In general, these studies allow the preview of the gene expression profile of an organism, organ, tissue, or cell, after applying a given stimulus. These global patterns are usually contrasted by comparing the stressed or treated profile with those corresponding to the negative control without the stimulus. Several methodologies (e.g., Northern blot, EST, SAGE and derivatives, microarray, RNA-Seq) can be used to generate libraries expressing these profiles (Kido et al. 2016). Some aspects will be briefly commented below. Transcriptomic libraries when properly generated and sequenced, using deep- sequencing (NGS, next-generation sequencing) technologies, provide millions of reads. After data quality inspections and the removal of adapters and low-quality bases, these reads allow (a) tag annotation, as those tags (26 pb) generated by the Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 5

SuperSAGE technique (Matsumura et al. 2012), or (b) transcriptome assembly (RNA-Seq data), using de novo strategy or based on a reference genome, generating the final assembled transcripts or unigenes (Wang et al. 2009). In both cases, tags or transcripts/unigenes must be adequately annotated, considering similarity levels with previously annotated sequences, using BLAST alignments. In this context, molecular targets involving osmoprotectant-related genes could be identified and selected considering: (a) The tag or unigene annotation. (b) The tag or unigene regulation (induced or repressed), considering their frequencies in two circumstantial libraries (stress treatment versus control). (c) The expression modulated by the tag or unigene; the fold change (FC) value representing the ratio of the normalized frequencies, usually tpm (tags per million), or FPKM (fragments per kilobase of transcript per million mapped reads) considering two comparing libraries. Furthermore, after appropriated statistical analysis (p-values) comparing the normalized frequencies of the tag or unigene based on the contrasted libraries, attention should be given to those identified as differentially expressed gene (DEGs). In this process, the p-values are corrected in order to minimize the type I error (Li et al. 2012), using the FDR (false discovery rate) method or similar. That correction diminishes false-positive episodes of differential expressions since the probability of false positives increases due to the high number of tests performed. Another advantage of a genomic-scale approach is the fact that many plant genomes and transcriptomes data, including those from crops under experimentally controlled stress, are deposited in public databases. The GenBank at NCBI (the National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/), the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/index-e.html), or the European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena) are constantly receiving biomolecules sequences from current projects. Therefore, these substantial databases provide bioinformatic tools, online analysis, and downloads of datas- ets allowing reliable annotations of DEGs, tags, or unigenes, in addition to other analysis. Diversely, although transcriptomic studies address the global expression of genes, the identification of those related to osmoprotective osmolytes is not a simple task. In transcriptomic studies, despite the detection of many genes comprising several functional metabolic categories, only a few tags or unigenes have already been more detailed. In general, the most observed are those genes related to DEGs showing relevant modulation based on the in silico analysis. Thus, if osmoprotectants are not the primary focus, then few osmoprotectant-related genes are unveiled. Essentially tags or unigenes that are strongly expressed after an applied stimulus are the mostly noted. Usually, the same set of genes is reported to be associated with plant abiotic stress profile. A compilation of some known osmoprotectants and their potential related pathways are shown in Table 1. 6 É. A. Kido et al.

Table 1 Some pathways associated with osmoprotectant biosynthesis in plants Pathway Related compound/class Choline biosynthesis Amine and polyamine/choline Ectoine biosynthesis Amine and polyamine/ectoine Fructan biosynthesis Sugar/fructan Glycine betaine biosynthesis Amine and polyamine/glycine betaine L-glutamate biosynthesis Amino acid/L-glutamate L-proline biosynthesis Amino acid/L-proline Mannitol biosynthesis Polyol/sugar alcohol Myo-inositol biosynthesis Myo-inositols Putrescine biosynthesis Amine and polyamine/putrescine Sorbitol biosynthesis Polyol/sugar alcohol Spermine biosynthesis Amines and polyamines/spermine Sucrose biosynthesis Sugar/sucrose Trehalose biosynthesis Sugar/trehalose

4 Osmoprotectant-Related Genes and Associated Pathways

Despite the importance of osmoprotectants in plants and the scientific advances over the years, a database compiling most of the generated information was not available until 2014, when Bougouffa et al. (2014) performing intensive data mining (more than 900,000 scientific articles) compiled 141 osmoprotectant compounds from 1160 organisms (microorganisms, plants, and animals). The authors connected osmoprotectant with potential pathways (biosynthesis or degradation) affecting these osmolytes (834), including reactions (1883), genes (3529), and proteins (4899). Concerning those compounds, only 34 remained not correlated with the identified pathways or reactions. This unique initiative resulted in the DEOP web- site (http://www.cbrc.kaust.edu.sa/deop/index.php), which is a database dedicated exclusively to osmoprotectants and their possible associated pathways. Based on the site’s background information, the focus of the authors was to study the potential of microorganisms accumulating osmoprotectants to become cell fac- tories. Another concern was the potential transference of such functional capability into other organisms through synthetic biology. Besides those already mentioned features, the available information provides perspectives covering microorganisms- plant interactions, with both organisms acting together against adverse conditions in the rhizosphere and soil environment. Such information can greatly assist studies of functional, comparative, and evolutionary genomics aspects involving osmoprotective genes. The searches performed on DEOP relational tables scrutinize pathways derived from the KEGG (Kyoto Encyclopedia of Genes and Genomes; https://www.genome. jp/kegg/) and MetaCyc databases (Metabolic Pathway Database, https://metacyc. org/). The MetaCyc database is a cured bank containing more than 2570 pathways of almost 3000 organisms from the various domains of life (Caspi et al. 2018). A compound can be associated with pathways representing osmoprotectant Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 7

Fig. 2 Venn diagram based on numbers of entries related to osmoprotectant-associated pathways [biosynthesis (final or intermediate product)/ degradation] prospected from DEOP database (http://www.cbrc. kaust.edu.sa/deop/index. php)

biosynthesis (related to the final or intermediate product; also, reversible or not), osmoprotectant degradation, and other osmoregulation. Furthermore, since it is possible to download data from the DEOP site, the entries related to the biosynthesis pathways of osmoprotective compounds as final products (December 2018) total- ized 120, while those addressing intermediate products were 205, and those covering degradation were 140 (Fig. 2). Some of the 120 identified entries are listed in Table 1, and the described pathways typically associate sugars and its derivatives (e.g., sugar alcohol), amines and polyamines, and amino acids, highlighting these compounds as osmoprotectants. According to the DEOP relational tables associated with the applied research, plant species presenting data associated with the pathways presented in Table 1 are listed in Table 2. The identified pathways and plants comprised mostly the biosynthesis of proline (14 plant species), sucrose (9), trehalose (8), and putrescine (8). In turn, plants concentrating studies focusing on those pathways (Table 1) comprised Nicotiana tabacum (9), Arabidopsis thaliana (7), Oryza sativa (7), and Triticum aestivum (5) (Table 2). Moreover, the set of reactions predicted in the pathways listed in Table 1, as well as the others available in the DEOP database, allow the identification of genes and enzymes associated with a specific osmoprotectant compound; that association is supported by the MetaCyc database, a comprehensive source of diagrams showing the enzymes involved in such reactions. Therefore, genes encoding the related enzymes identified above are good candidates to be noted in a transcriptomic study, as well as their expression after a signalized stress. Once some gene candidates are identified as DEGs, its expression still needs to be validated by a second method. Usually, the RT-qPCR (real-time reverse transcription-polymerase chain reaction) technique is performed; after all, it is considered a reference method in such cases (Provenzano and Mocellin 2007). After the validation process is done, the reliable candidates become promising to be applied as functional molecular markers to 8 É. A. Kido et al.

Table 2 Plant species presenting osmoprotectants data and associated pathwaysa based on the DEOP database (http://www.cbrc.kaust.edu.sa/deop/index.php) Pathwaysa Plant species 1 2 3 4 5 6 7 8 9 10 11 12 13 Subtotal Arabidopsis thaliana x x x x x x x 7 Avena sativa x 1 Brassica napus x 1 Glycine max x x x 3 Helianthus tuberosus x 1 Hordeum vulgare x x x x 4 Lycopersicon esculentum x 1 Malus x domestica x x 2 Nicotiana tabacum x x x x x x x x x 9 Oryza sativa x x x x x x x 7 Phaseolus vulgaris x x 2 Pisum sativum x 1 Populus sp. x 1 Solanum tuberosum x x 2 Spinacia oleracea x x x 3 Triticum aestivum x x x x x 5 Vigna aconitifolia x 1 Zea mays x x x 3 Subtotal 1 1 4 2 1 14 2 1 8 2 1 9 8 54 Biosynthesis pathwaysa: (1) choline; (2) ectoine; (3) fructan; (4) glycine betaine; (5) L-glutamate; (6) L-proline; (7) mannitol; (8) myo-inositol; (9) putrescine; (10) sorbitol; (11) spermine; (12) sucrose; (13) trehalose assist selection steps in plant breeding programs or to be evaluated in transgenic assays, helping breeders to develop new cultivars or varieties.

5 Expression of Osmoprotectant-Related Genes

Plants under abiotic stresses presented osmoprotectant-related genes modulating their expressions after the stress stimulus. Regarding salinity stress, at least 15 scientific articles covering 2015 until the beginning of 2019 presented osmoprotectant- related genes analyzed in 12 plant species. The investigated plant species comprised classic model plants (e.g., A. thaliana, M. truncatula, N. tabacum), important cultivated worldwide crops (e.g., G. max, O. sativa, S. bicolor), and other lesser-known plants (e.g., Bacopa monnieri, Chenopodium album) (Table 3). The experimental assays described in those articles were quite diverse, covering plants at different growth stages that were submitted to the NaCl salt, which molari- ties comprised from 75 to 400 mM, and the time of exposure ranging from less than 1 hour to days or even weeks. Some of the studies also looked at the influence of Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 9 ) ) ) ) ) ) ) ) ) ) ) ) 2015 2019 ) ) ) ) ) ) ) 2017 2017 2016 ) 2016a 2017 2018 2018 2018 2018 2016 2017 (continued) 2015 2018 2016 2016 2016 2016 2017 ) 2015 Reference Alavilli et al. ( Alavilli Wang et al. ( Wang Wang et al. ( Wang Jiang et al. ( Kissoudis et al. ( Ren et al. ( Zhai et al. ( Wei et al. ( Wei Wei et al. ( Wei Gong et al. ( Gong et al. ( Gong et al. ( Gong et al. ( Li et al. ( Rajaeian et al. ( Rajaeian et al. ( Ibragimova et al. Ibragimova ( Jung et al. ( Sun et al. ( Sun et al. ( de Freitas et al. ( Reg. UR UR UR UR UR UR UR DR UR DR UR DR UR UR UR UR UR UR DR UR UR Target genes Target P5CS1, P5CS2 TPS11 ADC1, ODC2 BADH GSTU4 P5CS MIPS1 P5CS2 P5CS1, P5CS3 ADC2, ODC1 ADC1 SAMDC1-5, SPDS2-6 SPDS1, SPDS4 P5CS2 P5CS, PDH BADH P5CS1 RS5 BADH P5CS OAT -inositol Osmoprotectant Proline Trehalose Putrescine/polyamine Glycine betaine Glycerol, hydroquinone Proline Myo Proline Proline Putrescine/polyamine Putrescine/polyamine Polyamine Putrescine/polyamine Proline Proline Glycine betaine Proline Raffinose Glycine betaine Proline Putrescine/polyamine Stress treatment NaCl 300 mM (6 h) NaCl 300 mM (0, 6, 12 h) NaCl 300 mM (0, 24, 48 h) NaCl 100 and 250 mM (1 h, 24 h, 15 days) NaCl 150 and 300 mM (30 days) NaCl 50 mM (3 weeks) NaCl 200 mM (2, 4, 6, 12, 24, 45 h) NaCl 250 mM (2, 4, 6, 9, 12 h) NaCl 250 mM (2, 4, 6, 9, 12 h) NaCl 200 mM (0.5, 1, 3, 6 h) NaCl 200 mM (0.5, 1, 3, 6 h) NaCl 200 mM (0.5, 1, 3, 6 h) NaCl 200 mM (0.5, 1, 3, 6 h) NaCl 250 mM (12, 18, 24 h) NaCl 200 mM (3 weeks) NaCl 200 mM (3 weeks) NaCl 200 and 300 mM (60 days) NaCl 400 mM (2, 4, 6 h) NaCl 100 and 200 mM (48 h) NaCl 100 and 200 mM (48 h) NaCl 75 mM (0, 14 days) Published scientific reports presenting regulation of osmoprotectant-related genes in plants under salt stress Published scientific reports presenting regulation

Plant species Arabidopsis Arabidopsis/ Gossypium Arabidopsis/ hirsutum Chenopodium album/tobacco Chenopodium quinoa Glycine max Glycine max Ipomea batatas Lilium regale Lilium regale Malus hupehensis Malus hupehensis Malus hupehensis Malus hupehensis Medicago truncatula Medicago Nicotiana rustica Nicotiana rustica Nicotiana tabacum/Arabidopsis Oryza sativa Raphanus sativus Raphanus sativus Sorghum bicolor Sorghum Table 3 Table 10 É. A. Kido et al. - ) ) ) ) ) 1- ) (pro 2019 2019 2019 2019 2019 2016 inositol- myo- ( PDH/ProDH MIPS Reference de Freitas et al. ( de Freitas et al. ( de Freitas et al. ( de Freitas et al. ( de Freitas et al. ( Alavilli et al. ( Alavilli (trehalose 6-phosphate TPS Reg. DR UR UR DR UR UR Target genes Target P5CS2 ProDH OAT P5CS2 P5CS1, ProDH P5CS1, P5CS2 (glycerol and hydroquinone), hydroquinone), and (glycerol (spermidine synthase), 1-pyrroline-5-carboxylate synthase), 1-pyrroline-5-carboxylate Δ SPDS GSTU ( P5CS Osmoprotectant Proline Proline Putrescine/polyamine Proline Proline Proline (ornithine decarboxylase), (ornithine (betaine aldehyde dehydrogenase), dehydrogenase), aldehyde (betaine ODC (S-adenosylmethionine decarboxylase), BADH SAMDC -aminotransferase), δ Stress treatment NaCl 75 mM (0, 14 days) NaCl 75 mM (0, 14 days) NaCl 75 mM + proline,30 mM (0.5–14 days) NaCl 75 mM + proline,30 mM (0.5–14 days) NaCl 75 mM + proline,30 mM (0.5–14 days) NaCl 300 mM (6 h) (arginine decarboxylase), (arginine (ornithine- (raffinose synthase), ADC RS OAT (continued)

Plant species Sorghum bicolor Sorghum Sorghum bicolor Sorghum Sorghum bicolor Sorghum Sorghum bicolor Sorghum Sorghum bicolor Sorghum Arabidopsis Table 3 Table line dehydrogenase), line dehydrogenase), synthase) phosphate synthase), phosphate Reg. (gene regulation), regulation), (gene Reg. Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 11 other factors besides NaCl, such as PEG, phytohormones, proline, and ethanolamine, among others, independently or combined with the salt. In that set of reports, the highlighted osmoprotectant compounds comprised the amino acid proline, the glycine betaine, the polyamines (putrescine and spermidine), the sugars (trehalose, oligosaccharides), and sugar derivatives (e.g., myo-inositol). Also, a total of 12 different osmoprotectant-related genes were investigated about their regulation after salt-stress exposure. Concerning drought stress, covering the period 2015–2019, at least 14 scientific articles also reported osmoprotectant-related genes modulating their expression after dehydration stress treatment. The experimental assays described in that researches showed different stress application methods, involving natural drought conditions (Yang et al. 2015c), drought simulated by root dehydration (0–72 h; Singh et al. 2015), suppression of irrigation (Rickes et al. 2019; Dastogeer et al. 2018), withholding water assay (Chen et al. 2016), addition of polyethylene glycol (30% PEG 6000 solution; Yadav et al. 2018), and even dehydrated fruits (grape berries) (Conde et al. 2018) (Table 4). These studies embraced 15 plant species, including the reference plants and crops already mentioned, and other lesser-known plants, such as Stipa purpurea (Yang et al. 2015c) and Ziziphus nummularia (Yadav et al. 2018]. Also, this set of reports encompassed 21 genes related to osmoprotectants, such as the amino acid proline, the glycine betaine, the polyamines (putrescine and spermidine), the sugars (sucrose, raffinose, and trehalose), and polyols (sorbitol and mannitol). Some of the investigated genes are presented in Table 4, taking into account their expression after drought or salt stress.

5.1 Amino Acid Proline

In respect to proline as an osmoprotectant, and considering plants responding to salt stress, eight scientific manuscripts presented expression results of only two genes. One of them encoded P5CS (Δ1-pyrroline-5-carboxylate synthase) and the other PDH (proline dehydrogenase). Only the first gene takes part in the proline biosynthesis pathway. The P5CS (EC 2.7.2.11/1.2.1.41) reduces glutamic acid to γ-glutamic semialdehyde (GSA), and GSA is converted spontaneously by P5CR (Δ1-pyrroline-5-carboxylate reductase) into Δ1-pyrroline-5-carboxylate (P5C). Finally, P5C is converted to proline by P5CR (Szabados and Savoure 2010). The other gene codifies the enzyme PDH (EC 1.5.5.2) that catalyzes proline degradation after plant dehydration. In general, except for some P5CS isoforms and depending on the analyzed tissue, the upregulation of transcripts of both genes are observed in roots after the salt application (Table 3). Still, considering proline, now taking into account plants responding to drought stress, the same genes were investigated (Table 4). About nine articles presented P5CS expression showing upregulation after drought stress, and only one research (Dastogeer et al. 2018) investigated the PDH expression, also noting the upregulation of the transcript. Interestingly, Dastogeer et al. (2018) pointed fungal endophytes 12 É. A. Kido et al. ) ) ) ) ) ) ) ) ) ) ) ) ) 2018 ) 2018 ) ) 2019 2019 2019 2019 2018 2018 2018 2018 2016 2016 2015b 2017 2016 2016 Chen et al. ( Chen et al. ( Rickes et al. ( Rickes Rickes et al. ( Rickes Rickes et al. ( Rickes Rickes et al. ( Rickes Jung et al. ( Dastogeer et al. ( Dastogeer et al. Antoniou et al. ( Gong et al. ( Gong et al. ( Wei et al. ( Wei Wei et al. ( Wei Yang et al. ( Yang Vaishnav and Vaishnav Choudhary ( Vaishnav and Vaishnav Choudhary ( Reference UR UR UR UR UR DR UR UR UR DR UR ns UR UR UR UR Reg. ADC, CPA, ODC ADC, CPA, BADH, CMO, SAMDC BADH, S6PDH SIP1 P5CS SOT1 GolS2 PDH1 P5CR, P5CS ODC2 ADC1, ADC2, ODC1 P5CS2 P5CS1, P5CS3 P5CS GolS P5CS Target genes Target Putrescine/ polyamine Glycine betaine Polyol Raffinose Proline Polyol RFO Proline Proline Putrescine/ polyamine Putrescine/ polyamine Proline Proline Proline Raffinose Proline Osmoprotectant 0,50 Mpa] 0,50 Mpa] − − seedlings seedlings 0,50 Mpa) − Withholding water (0, 5, 8 days) + re-watering (0, 5, 8 days) + re-watering water Withholding (after 1, 4 days). NaHS application in S. oleracea Withholding water (0, 5, 8 days) + re-watering (0, 5, 8 days) + re-watering water Withholding (after 1, 4 days). NaHS application in S. oleracea Irrigation suppressed (0, 4, 7, 9 days) Irrigation Irrigation suppressed (0, 4, 7, 9 days) Irrigation Irrigation suppressed (0, 4, 7, 9 days) Irrigation Drought (irrigation suppressed; 0, 4, 7, 9 days) Drought (irrigation Drought (6 days withhold water) Withholding water (8 days) water Withholding Withholding water (9 days) water Withholding NaCl 200 mM (0, 1, 3, 6, 12, 24 h) NaCl 200 mM (0, 1, 3, 6, 12, 24 h) Withholding water (7 days) water Withholding Withholding water (7 days) water Withholding PEG 6000 10% (2 days); osmotic potential ( PEG 6000 [osmotic potential: (10 days) PEG 6000 [osmotic potential: (10 days) Stress treatment Published scientific reports presenting osmoprotectant-related gene regulation in plants under water-deficit stress water-deficit in plants under Published scientific reports presenting osmoprotectant-related gene regulation

Spinacia oleracea Spinacia oleracea Prunus persica Prunus persica Prunus persica Prunus persica Oryza sativa Nicotiana benthamiana Medicago Medicago truncatula Malus hupehensis Malus hupehensis Lilium regale Lilium regale Jatropha curcas Jatropha Glycine max Glycine max Plant species Table 4 Table Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 13 - - ) ) ) ) ) ) ) ) ) ) ) (galacti 2018 2018 2018 2017 2017 2018 2018 (sorbitol- 2018 2016 2015c 2015c GolS S6PDH (spermidine syn Vaishnav and Vaishnav Choudhary ( Yadav et al. ( Yadav Yadav et al. ( Yadav Conde et al. ( Conde et al. ( Conde et al. ( Ebeed et al. ( Ebeed et al. ( Yang et al. ( Yang Yang et al. ( Yang Chen et al. ( SPDS (sucrose-phosphate synthase), UR UR UR UR n.s. UR UR UR UR UR UR SPS 1-pyrroline-5-carboxylate synthase), 1-pyrroline-5-carboxylate Δ ( (sorbitol transporter), (ornithine decarboxylase), SOT P5CS ODC (deoxyhypusine synthase), (deoxyhypusine DHS (raffinose synthase), P5CS P5CS GolS GolS1 MTD PLT1, SDH PLT1, DHS, SAMDC, SPDS ADC, ODC BADH P5CS FBPase, SPS1, TPS FBPase, SIP (sorbitol dehydrogenase), (sorbitol dehydrogenase), (raffinose) SDH Proline Proline Raffinose Raffinose Polyol/ mannitol Polyol Polyamine Putrescine/ polyamine Glycine betaine Proline Sucrose RFO 1-pyrroline-5-carboxylate reductase), 1-pyrroline-5-carboxylate (choline monooxygenase), Δ ( (N-carbamoylputrescine amidohydrolase), amidohydrolase), (N-carbamoylputrescine CMO (sterol C-4 methyl oxidase), (sterol C-4 methyl P5CR 0,50 CPA − SMO (polyol transporter), PLT (trehalose 6-phosphate synthase), TPS (arginine decarboxylase), (arginine seedlings (mannitol dehydrogenase), (mannitol dehydrogenase), ADC (betaine aldehyde dehydrogenase), dehydrogenase), (betaine aldehyde MTD PEG 6000 [with osmotic potential of Mpa] (10 days) PEG 6000 30% (6, 12, 24, 48, 72 h) PEG 6000 30% (6, 12, 24, 48, 72 h) Fruit dehydrated (5, 11 days) Fruit dehydrated Fruit dehydrated (5, 11 days) Fruit dehydrated Fruit dehydrated (5, 11 days) Fruit dehydrated Withholding water (>11 days); exogenous (>11 days); exogenous water Withholding polyamine application Withholding water (>11 days); exogenous (>11 days); exogenous water Withholding polyamine application Naturals drought conditions Naturals drought conditions Withholding water (0, 5, 8 days) + re-watering (0, 5, 8 days) + re-watering water Withholding (after 1, 4 days). NaHS application in S. oleracea BADH -inositol 3-alpha-D-galactosyltransferase), -inositol 3-alpha-D-galactosyltransferase), (S-adenosylmethionine decarboxylase, myo (fructose-1,6-bisphosphatase), SAMDC (proline dehydrogenase), (proline dehydrogenase), Glycine max Ziziphus nummularia Ziziphus nummularia Vitis vinífera Vitis Vitis vinífera Vitis Vitis vinífera Vitis Triticum aestivum Triticum Triticum aestivum Triticum Stipa purpurea Stipa purpurea Spinacia oleracea Reg. (gene regulation), (gene regulation), Reg. thase), 6-phosphate dehydrogenase), 6-phosphate dehydrogenase), PDH nol synthase/ FBPase 14 É. A. Kido et al. and inoculated virus conferring drought tolerance to Nicotiana benthamiana plants through osmolyte modulation and expression of host drought-responsive genes.

5.2 Glycine Betaine

Regarding glycine betaine (GB) and plants responding to the salt stress, the single gene investigated in the researched period encoded BADH (betaine aldehyde dehydrogenase; EC 1.2.1.8), which presented upregulation in most of the manuscripts (Table 3), and only one BADH downregulated (Sun et al. 2016). Besides this DR regulation, the analyzed radish transcriptome (Raphanus sativus L.) in response to salt stress (0, 100, and 200 mM NaCl for 48 h) presented 29 induced DEGs associated with osmoprotectants (threshold of |log2Ratio| ≥ 1 with FDR ≤ 0.001 and p-value ≤ 0.05), including 9 P5CS candidates and 5 (induced) of 7 trehalose-related ones (Sun et al. 2016). Concerning the drought stress, besides the BADH gene, another induced gene evaluated in GB biosynthesis pathway was CMO (choline monooxygenase; Chen et al. 2016]. In higher plants, choline is converted by CMO (EC 1.14.15.7) into betaine aldehyde, which is then catalyzed by BADH into GB (Chen et al. 2016; Takabe et al. 2006).

5.3 Polyamines

Concerning the osmoprotectant polyamines (PAs), which have some functions similar to plant growth regulators, five investigated genes covered this issue in plants responding to salt stress: ADC (arginine decarboxylase), ODC (ornithine decarboxylase), OAT (ornithine-δ-aminotransferase), SAMDC (S-adenosylmethionine decarboxylase), and SPDS (spermidine synthase) (Table 3). The ADC, ODC, and OAT genes are directly involved with the putrescine biosynthesis pathway, while SAMDC and SPDS are involved with spermidine pathway. The osmoprotectant putrescine (Put) can be synthesized directly from ornithine by ODC (EC 4.1.1.17) or indirectly, through a series of intermediates following arginine decarboxylation by ADC (EC 4.1.1.19). Most of the ADC and ODC transcripts are induced in responses to salt stresses (Table 3). Upregulation is also observed in OAT transcript, target only analyzed by de Freitas et al. (2019) in their study of S. bicolor after the stress of 75 mM NaCl, 14 days after the salt application. In respect to drought stress, several manuscripts (Chen et al. 2016; Ebeed et al. 2017; Gong et al. 2018) relate the upregulation of ADC and ODC transcripts. The osmoprotectant spermidine (Spd) is synthesized from Put by successive additions of aminopropyl groups catalyzed by SPDS (EC 2.5.1.16). In the other hand, the aminopropyl is provided by decarboxylated S-adenosylmethionine, a metabolite synthesized by SAMDC (EC 4.1.1.50). The SAMDC enzyme is also Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 15 implicated in cysteine and methionine metabolism, as well as the arginine and proline metabolism (https://www.genome.jp/dbget-bin/www_bget?ec:4.1.1.50). Based on the genome-wide study reported by Gong et al. (2018) in apple (Malus hupehensis Rehd.), the MhSAMDC1 and MhSPDS1 genes were induced not only by salt but also by other treatments (alkaline, abscisic acid, cold, and dehydration), suggesting that these genes have relevant roles in plant stress responses. About drought stress, also SAMDC and SPDS presented upregulation after stress application (Chen et al. 2016; Ebeed et al. 2017). However, concerning Chen et al. (2016), the enhancement of plant drought tolerance with effects on polyamines and soluble sugar contents also derived from NaHS application before the stress exposure. The CPA (N-carbamoylputrescine amidohydrolase) and DHS (deoxyhypusine synthase) genes were also investigated concerning responses to drought stress. Both genes are induced in plants under such stress (Chen et al. 2016; Ebeed et al. 2017). The CPA (EC 3.5.1.53) is implicated in Put generation from N-carbamoylputrescine (https://www.genome.jp/dbget-bin/www_bget?ec:3.5.1.53), while DHS (EC 2.5.1.46) participates in Spe degradation using it as a substrate. Wang et al. (2003) report that suppression of DHS delays premature leaf senescence induced by drought stress in A. thaliana among other pleiotropic effects.

5.4 Carbohydrates

Furthermore, in plants responding to salt stress, osmoprotectant carbohydrates (sugars) are the oligosaccharide raffinose and the complex sugar trehalose. Both were represented by the RS (raffinose synthase) and TPS (trehalose-6-phosphate synthase) genes, respectively. These genes participate directly in the raffinose and trehalose biosynthesis pathways, and both are induced after the applied salt stress (Jung et al. 2017; Wang et al. 2016a). In relation to plants responding to drought stress, the genes investigated, TPS and SIP (also a raffinose synthase; Rickes et al. 2019), presented upregulation after the applied stress (Chen et al. 2016). The RS enzyme (EC 2.4.1.82), as predicted in the galactose metabolism (https:// www.genome.jp/kegg-bin/show_pathway?ath00052+AT1G55740), converts galactinol into raffinose. In turn, the TPS enzyme (EC 2.4.1.15) participates in the trehalose biosynthesis in plants, generating T6P (trehalose-6-phosphate) from glucose-6-phosphate and UDP-glucose, with the subsequent dephosphorylation of T6P to trehalose by TPP (trehalose-6-phosphate phosphatase) (Cabid and Leloir 1958). Besides RS (SIP) gene, the GolS (galactinol synthase/myo-inositol 3-alpha-D- galactosyltransferase) is another induced gene observed during dehydration events and associated with the galactose metabolism. In the biosynthesis of raffinose family oligosaccharides (RFOs), the enzyme galactinol synthase (EC 2.4.1.123) catalyzes the first step converting UDP-galactose andmyo -inositol to galactinol, and this will be further converted to raffinose by the RS (SIP) enzyme. Overexpression of AtGolS2 in transformed Arabidopsis plants showing reduced leaves transpiration 16 É. A. Kido et al. presented increased endogenous galactinol and raffinose (Taji et al. 2002). The overexpression of GolS transcripts was observed in several manuscripts (Table 9.4). Additionally, another two genes associated to sugar biosynthesis were induced in plants responding to drought, but in this case, plant tolerance was improved by NaHS that was applied to Spinacia oleracea seedlings as pretreatment (Chen et al. 2016). The investigated genes were SPS (sucrose-phosphate synthase) and FBPase (fructose-1,6-bisphosphatase). According to the KEGG database, in the starch and the sucrose metabolism, the substrate UDP-glucose is converted by SPS (EC 2.4.1.14) into sucrose-6-P which is converted by SPP1 (sucrose-phosphatase 1) to sucrose. In turn, FBPase enzyme (EC 3.1.3.11) converts the substrate D-fructose-1,6- bisphosphate into D-fructose 6-P, which is a compound involved with many pathways, including galactose, and also starch and sucrose metabolisms. Based on the results, the NaHS pretreatment improved plant tolerance, modulating the expression levels of genes associated with sugar biosynthesis, and also polyamines, as mentioned before. Sugars, such as sucrose and trehalose, replace water molecules on the surfaces of proteins allowing them to preserve their conformations and, therefore, to restore their functions after rehydration (Hoekstra et al. 2001).

5.5 Sugar Alcohols

When talking about the osmoprotectant sugar alcohol myo-inositol, the represented gene is MIPS (myo-inositol-1-phosphate synthase), in plants responding to salt stress. The induced gene, after the salt-stress exposition, participates directly in the myo-inositol biosynthesis pathway (Zhai et al. 2015). The MIPS enzyme (EC: 5.5.1.4) catalyzes the conversion of D-glucose-6-phosphate to 1 L-myo-inositol-1- phosphate. The conversion is rate limiting in the biosynthesis of all inositol- containing compounds. Myo-inositol plays an essential role as a structural basis for generating second messengers useful in signal transduction (Gillaspy 2011). Also, inositol serves as a crucial component of the structural lipid phosphatidylinositol (PI) and its various phosphates, the phosphatidylinositol phosphate (PIP) lipids. Considering plants responding to drought stress, MIPS gene was not investigated in the set of researched articles. About polyols (polyhydric alcohol), including sugar alcohols such as sorbitol and mannitol, genes associated with these pathways were not presented in the set of manuscripts covering plants responding to salt stress. Nevertheless, Conde et al. (2018) reported a study covering dehydrated grape berries. In that study, most of the genes were associated with sorbitol, and they encoded SDH (sorbitol dehydrogenase, EC 1.1.99.21), S6PDH (sorbitol-6-phosphate dehydrogenase; EC 1.1.1.140), and two polyol transporters, SOT (sorbitol transporter) and PLT (polyol transporter). Based on gene expression results, all of them presented upregulation after the applied stress (Table 4). Another investigated gene encoded MTD (mannitol dehydrogenase, EC 1.1.1.255) and it was also induced (Conde et al. 2018). The MTD enzyme converts D-mannitol into D-mannose, a compound implicated in several Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 17 pathways, including galactose, fructose, and mannose metabolisms (https://www. genome.jp/dbget-bin/www_bget?cpd:C00159). In the polyol pathway, the unused glucose is reduced by aldose reductase to sorbitol, which is subsequently oxidized to fructose by SDH. After that, fructose can be phosphorylated by fructokinase and subsequently metabolized via dihydroxyac- etone phosphate or glyceraldehyde to D-glyceraldehyde 3-phosphate, which is a substrate in the glycolysis process. In turn, S6PDH acting on D-sorbitol 6-phosphate generates D-fructose 6-phosphate (fructose and mannose metabolism), a compound implicated in many KEGG pathways (https://www.genome.jp/dbget-bin/get_ linkdb?-t+pathway+cpd:C05345). The interrelation between sorbitol and sucrose supply due to its gene expression is observed in transgenic apple altered with S6PDH cDNA (Kanamaru et al. 2004).

6 In Silico Genome Mapping of Osmoprotectant-Related Genes

The osmoprotectants and their genes related to proline (P5CS1; P5CR1), trehalose (TPS1; TPPB, trehalose-phosphatase), glycine betaine (BADH1; CMO), cysteine (SAT1, serine acetyltransferase; OASTL1, O-acetyl serine (thiol) lyase), and myo- inositol (MIPS1) were in silico mapped on genomes of six plant species, including model plants (P. patens, A. thaliana), monocots (S. bicolor and O. sativa), and dicots (G. max, P. vulgaris). The mapping allowed a comparative analysis among them. The investigated genes were chosen based on a previous study with transcripts identified using 26 bp SuperSAGE unitags expressed in soybean roots after air dehydration in time intervals ranging from 0 up to 150 min (Kido et al. 2013). Apart from P5CS1 which is absent in moss P. patens (bryophyte), the most basal species analyzed, the remaining genes were identified in virtual chromosomes of all six plant species (Table 5). The analysis brought up that 33 loci were identified in P. patens (Table 5) on 21 of 27 chromosomes total (Fig. 3A), while A. thaliana, which is a compact angiosperm genome with five chromosomes, due to the loss of DNA by unequal homologous recombination (Devos et al. 2002), mapped 43 loci (Table 5 and Fig. 3B). This information points to the relevance of osmoprotectants in cellular homeostasis maintenance through plant evolution. Mosses and flowering plants evolution diverge in more than 400 million years (MYA, Nishiyama et al. 2003). In turn, considering the two legumes (Fabaceae family), soybean (Glyxine max) presented 112 loci (Table 5, Fig. 3D), while the common bean (Phaseolus vulgaris) presented 58 loci (Table 5. Figure 3C). It is worth mention that G. max (2n = 40) has almost the double of chromosomes of P. vulgaris (2n = 22), as a result of two genome duplications events, at approximately 59 and 13 million years ago (Schmutz et al. 2010). Therefore, soybean is a highly duplicated genome with nearly 75% of the genes present in multiple copies (Schmutz et al. 2010). 18 É. A. Kido et al.

Table 5 Loci numbers of genes associated with osmoprotectants∗ biosynthesis in six plant genome species Genome P5CS1 P5CR1 BADH1 CMO TPS1 TPPB INPS1 OASTL SAT1 Loci Physcomitrella 0 1 11 1 4 7 2 3 4 33 patens Arabidopsis 2 1 13 1 10 1 3 8 4 43 thaliana Glycine max 7 2 48 1 20 2 4 18 10 112 Phaseolus 4 1 13 3 12 9 2 8 6 58 vulgaris Sorghum bicolor 2 1 11 1 7 13 2 11 1 49 Oryza sativa 2 1 16 1 11 13 2 12 6 64 ∗Osmoprotectants [gene(s)]: proline∗ [P5CS1 (delta(1)-pyrroline-5-carboxylate synthetase), P5CR1 (delta(1)-pyrroline-5-carboxylate reductase)], Glycine betaine∗ [BADH1 (betaine aldehyde dehydrogenase), CMO (choline monooxygenase)], Myo-inositol∗ [INPS1 (myo-inositol 1-phosphate synthase)], Trehalose∗ [TPS1 (trehalose-6-phosphate synthase), TPPB (trehalose- phosphatase)], Cysteine∗ [SAT (serine acetyltransferase), OASTL (O-acetyl-serine(thiol)lyase)]

The other two analyzed genomes, representing grasses (Poaceae family), presented 49 loci (Sorghum bicolor; subfamily Panicoidae; Table 5 and Fig. 3E) and 64 loci (O. sativa; subfamily Oryzoidae; Table 5 and Fig. 3F). Some synteny and collinearity comparing the two genomes showed 1 block involving the chromosomes 1 of sorghum and 10 of rice (same gene order for P5CS1, SAT1, and TPS1) and other 2 blocks (chromosomes 8 of sorghum and 12 of rice and chromosomes 9 of sorghum and 5 of rice). Also, another difference is observed involving the gene SAT1 presenting only one copy in sorghum, while it shows six copies in rice (Table 5). Concerning P5CR1 and CMO genes, most of the species had only one locus, with P5CR1 duplicated in soybean, and CMO with three loci in P. vulgaris (Table 5). The consequence of these two extra copies needs further investigation. On the other hand, BADH1, TPS1, and OASTL mapped at multiple loci (Table 5), probably reflecting events of duplications, which is one of the sources of new gene generation. Once a duplicate segment is subjected to lower selection pressure in subsequent mutations, it may lead to new functions (Sankoff 2001). Based on this assumption, polyploid species, such as soybean and modern sugarcane (Garcia et al. 2006), which is highly polyploid and aneuploid, as a result of interspecific crosses within the Saccharum complex, are valuable sources of genes/alleles with potential to increase plant fitness in response to biotic and abiotic stresses.

7 Osmoprotectant-Related Genes as Transgenes

Regarding the scientific manuscripts addressing osmoprotectant-related genes, they acquired biotechnological relevance for the agriculture area, making them attractive targets to be manipulated also taking into account their participation in plant stress responses (Tables 3 and 4). The impact of this relevance is revealed by data mining Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 19

27 26

25 a 1 b SA

24 TPS1 T1 1 TPS1 1 TPS1 SA TPS1 BADH1 2 BADH1 T1 BADH1 TPS1 23 BADH SA BADH 5 1 T1

22 BADH1 OASTL 3 OASTL BADH1

TPPB T1 TPPB BADH1 MIPS1 SA 21 TPS1 MIPS1 P5CR1 TPPB 4 MIPS1 TPPB 20 TPS1 BADH1 BADH1 SAT1 TPPB BADH1 MIPS1 TPS1 5 BADH1 19 BADH1 SAT1 BADH1 BADH1 BADH1

CMO TPS1 6 18 BADH1 OASTL TPS1 TPS1 4 MIPS1 TPS1 OASTL BADH1 17OASTL1 7 BADH 2

TPS1 S AT1 P5CS 16 BADH1 8 OASTL 1

OASTL OASTL BADH1 OASTL SA 15 TPPB 9 OASTL OASTL P5CR1 BADH1 TPPB P5CS1 T1

TPS1 BADH1 BADH1 10 14 CMO

3 TPPB 11

20 L

OASTL O

T A 1 11 TPS1 T1 TPS1

OASTL S P5CR1 CMO TPS1 1 19 S BADH1 OASTL T SA

1 c d L

BADH1 T1

P5CS1 BADH1 2 BADH1 OASTL SA TPPB BADH1 TPS1 T1 BADH1 TPPB P5CR1 BADH1 OASTL TPPB SA OASTL S P5CS1 AT P5CS1 BADH1T1 MIPS1 -- P5CS1A 1 OASTL S 10 BADH1 OASTL 3 CMO BADH1 P5CS1 TPS1 2 TPS1 TPS1 P5CS1S AT1 AT1 P5CR1 S MIPS1 BADH1 OASTL P5CS1 MIPS1 TPS1 BADH1 17 4 BADH1 TPPB BADH1 TPS1 BADH1 CMO TPS1 TPS1 BADH1 BADH1 TPS1 9 BADH1OASTL BADH1 BADH1 TPS1 BADH1 SAT1 TPS1 16 TPPB 5 TPPB 3 BADH1 SAT1 TPS1 BADH1 MIPS1BADH1 SAT1 CMO TPS1 P5CS1 BADH1 BADH1 TPS1 TPPB TPPB BADH1TPS1 TPS1 BADH1 BADH1BADH1 15 TPPB S BADH1 6 AT1 BADH1 BADH1 BADH1 TPS1 BADH1 SA BADH1T1 8 P5CS1 BADH1 BADH1 BADH1 TPS1 BADH1 7 4 14 OASTL T1 BADH1 A BADH1 P5CS1 P5CS1S TPS1 BADH1 BADH1TPS1 BADH1 OASTL TPPB BADH1 BADH1 MIPS1 BADH1 BADH1TPS1 S OASTL BADH1 8 TPS1 BADH1A 13 T1 BADH1 BADH1 BADH1 TPS1 BADH1

O OASTL

SA OASTL TPS1

5 A OASTL T1 9

S T1 BADH1MIPS1

12 T 7 TPS1 SA TPS1

L OASTL 10

6 OASTL OASTL

OASTL 11

BADH1

10 BADH1 OASTL 12 OASTL OASTL e CMO f 1 1

BADH1 TPS1 TPPB OASTLTPPB SA OASTL TPS1 OASTL MIPS1 T1 1 BADH1 BADH1 P5CS1TPS1 AT TPPB 11 S TPS1 TPPB OASTL P5CS1 P5CR1 9 OASTL BADH1 T1 SA OASTL 2 TPPB 2 TPPB OASTL 10 BADH1 MIPS1 BADH1 TPPB TPS1 TPPB TPPB BADH1 TPS1 TPPB BADH1 TPS1 TPPB TPPB TPPB T1 TPPB SA 8 SAT1 MIPS1 OASTL BADH1 TPS1 TPS1 TPS1 9 TPPB TPPB TPPB 3 BADH1 3 TPS1 OASTL TPPB BADH1 BADH1 BADH1 TPS1 TPS1 TPPB OASTL BADH1 OASTL P5CS1 BADH1 P5CR1 7 TPS1 8 OASTL

BADH1 BADH1 4 TPPB BADH1 TPPB

TPPB 4 BADH1 BADH1 TPPB BADH1 TPPB OASTL BADH1

7 P5CS1 TPS1 TPS1 OASTL OASTL TPPB

6 1 BADH1 BADH1 CMO H 5

D OASTL

5 SA

OASTL A OASTL OASTL

B 6 T1

Fig. 3 In silico mapping of loci covering osmoprotectant-related genes in six plant genomes (A, Physcomitrella patens; B, Arabidopsis thaliana; C, Phaseolus vulgaris; D, Glycine max; E, Sorghum bicolor; F, Oryza sativa). Syntenic relationships showed by color lines: red [proline: P5CS1 (Δ1-pyrroline-5-carboxylate synthetase); P5CR1 (Δ1-pyrroline-5-carboxylate reductase)]; purple [glycine betaine: BADH1 (betaine aldehyde dehydrogenase); CMO (choline monooxygenase)]; orange (myo-inositol: MIPS1 (myo-inositol 1-phosphate synthase)); green [trehalose: TPS1 (trehalose-6-phosphate synthase); TPPB (trehalose phosphatase)]; blue [cysteine: SAT (serine acetyltransferase); OASTL (O-acetyl-serine(thiol)lyase)] 20 É. A. Kido et al. in the NCBI/PubMed (https://www.ncbi.nlm.nih.gov/pubmed/), with free access to the MEDLINE, a database presenting citations and manuscript summaries in life science. Searching by keywords (“osmoprotectant name AND plant stress AND transgenic”), and considering the osmoprotectants presented in Fig. 1 (proline, glycine betaine, polyamines, sugars, and sugar alcohols), looking for scientific manuscripts published in the last decade, excluding reviews and genes not directly involved in osmoprotectant biosynthesis pathways, the results were very inspiring (Fig. 4).

7.1 Amino Acid Proline

In light of amino acids as osmoprotectants and taking into consideration transgenic (or syngenic) plants, proline is the most studied amino acid. Several scientific manuscripts attest its participation in plant stress responses. Curiously, when associated with transgenic plants, the text mining results showed that most of the reports do not address genes from the proline biosynthesis pathway (data not showed), but other genes. Some of these genes, whose biotechnological potentials were investigated, encode the protease inhibitor chymotrypsin (Tiwari et al. 2015), transcription factors (NAC, Liu et al. 2017b), and even structural proteins, such as dehydrin, DHN-5 (Saibi et al. 2015). In those reports, proline was analyzed and associated as a positive reference of osmoprotectant acting under stressful conditions. Thus, the increase in proline content after plant post-transformation followed by stress application was a positive sign of the transgene impact. Besides those cases, the applied data mining identified five manuscripts covering genes encoding key enzymes of the proline biosynthesis in plants, basically P5CS and P5CR (Fig. 4 and Table 6). Transgenic plants of Panicum virgatum, G. max, S. bicolor, and A. thaliana, superexpressing P5CS or P5CR after stress stimulus, have been reported in abiotic stress responses to salt, heat, and drought (Table 6). In transgenic plants of P. virgatum, the physiological impacts of the P5CS gene are associated with higher ROS scavenging levels (Guan et al. 2018). However, regarding P5CR, its physiological impact still needs to be clarified.

7.2 Glycine Betaine

As for betaines, the referential osmoprotectant is glycine betaine (GB). Furthermore, genes codifying the enzymes choline oxidase (CO, EC 1.1.3.17) and betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) were the most studied in transgenic plants reports (Fig. 4 and Table 7). Nevertheless, based on the glycine, serine, and threonine metabolisms, the osmoprotectant betaine is synthesized involving two conversions: (1) choline into betaine aldehyde and (2) betaine aldehyde into betaine. In higher plants, CMO (choline Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 21

Putrescine Spermidine Spermine

NM: 11 NM: 12 NM: 13 TS: Arginine decarboxylase TS: S-adenosylmethionine TS: S-adenosylmethionine decar-boxylase decar-boxylase TP: Arabidopsis thaliana TP: Arabidopsis thaliana and TP: Arabidopsis thaliana and Sola-num lycopersicum Sola-num lycopersicum TM: Agrobacterium-mediated TM: Agrobacterium-mediated TM: Agrobacterium-mediated AS: Drought and dehydration AS: Drought and salt AS: Drought and salt BS: F. oxysporum, Al. solani, B. BS: F. oxysporum, A.solani, BS: F. oxysporum, A.solani cinerea, F. oxysporum, and B. cinerea, F. oxysporum, P. syringae P. syringae, and M. sexta

ProlineProl Threalose Fructan

NM: 5 NM: 6 NM: 3 TS: P5CS and P5CR TS: Trehalose-6-phosphate TS: Fructan synthase 6-fructosyltransferase TP: Arabidopsis thaliana, TP: Arabidopsis thaliana and TP: Triticosecale wittmack Panicum virgatum, Glycine max, Oriza sativa and Ni-cotiana tabacum and Sorghum bi-color TM: Agrobacterium-mediated TM: Agrobacterium-mediated TM: Agrobacterium-mediated AS: Salt, heat, and drought AS: Salt, drought, and chilling AS: Cold, salt, and drought BS: Not avaiable BS: Botrytis cinérea and BS: Not avaiable P. syringae

Inositol Glycine and phosphorylated derivatives betaine

NM: 22 NM: 22 TS: L-myo-inositol-1-phosphate TS: Choline oxidase and synthase betaine aldehyde dehydrogenase TP: Arabidopsis thaliana and TP: Arabidopsis thaliana and Nico-tiana tabacum Sola-num lycopersicum TM: Agrobacterium-mediated TM: Agrobacterium-mediated AS: Drought and salt AS: Drought and salt BS: Not avaiable BS: Not avaiable

Polyamine Aminoacid

Carbohydrate Sugar alcohol Betaine

Fig. 4 Data mining results covering the most representative osmoprotectants in plants by compound class (colored boxes), based on manuscripts total number (NM), target genes (TS), transgenic method (TM), transformed plant species (TP), and the analyzed stress: abiotic (AS) or biotic (BS) 22 É. A. Kido et al.

Table 6 Plants genetically modified (GM) with transgenes associated to the osmoprotectant proline Stress Transgene donor species GM plant Reg. Tol. treatment Reference Arabidopsis thaliana|P5CR Glycine max UR > Heat and De Ronde et al. Drought (2004) Arabidopsis thaliana|P5CS Arabidopsis UR < Heat Lv et al. (2011) thaliana Phaseolus vulgaris|P5CS1 & Arabidopsis UR > Salt Chen et al. P5CS2 thaliana (2013) Puccinellia Panicum UR > Salt Guan et al. chinampoensis|P5CS virgatuma (2018) Vigna aconitifolia|P5CS Sorghum bicolora UR > Salt Reddy et al. (2015) Reg. (transgene regulation), UR (upregulated), DR (downregulated), Tol. [increasing (>) or decreasing (<) plant tolerance], aAgrobacterium-mediated transformation, transgenes: P5CS (delta1-pyrroline-5-carboxylate synthase), P5CR (delta-pyrroline-5-carboxylate reductase)

Table 7 Plants genetically modified (GM) with transgenes associated to the osmoprotectant glycine betaine Stress Transgene donor species GM plant Reg. Tol. treatment Reference Arthrobacter globiformis|CO Solanum tuberosum UR > Drought Cheng et al. (2013) Spilanthes oleracea|BADH Ipomoea batatas UR > Salt, oxidative Fan et al. and cold (2012) Arthrobacter globiformis|CO Solanum – > Drought and Goel et al. lycopersicum Salt (2011) Aphanothece Nicotiana tabacum UR > Drought He et al. halophytica|2-MGMT (2011) Aphanothece Nicotiana tabacum UR > Drought He et al. halophytica|GSMT (2011) Arthrobacter globiformis|CO Oryza sativaa – > Drought Kathuria et al. (2009) Bacterial|CO Populus alba × UR > Drought, cold, Ke et al. Prosopis and salt (2016) glandulosaa Methanohalophilus Arabidopsis UR > Drought and Lai et al. portucalensis|GSMT thalianaa salt (2014) Methanohalophilus Arabidopsis UR > Drought and Lai et al. portucalensis|SDMT thalianaa salt (2014) Arthrobacter globiformis|CO Solanum UR > Heat Li et al. lycopersicum (2011b) Arthrobacter globiformis| CO Solanum – > Low- Li et al. lycopersicum phosphate salt (2011b) stress (continued) Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 23

Table 7 (continued) Stress Transgene donor species GM plant Reg. Tol. treatment Reference Oryza sativa|CMO Nicotiana tabacum UR > Salt Luo et al. (2012) Arabidopsis Arabidopsis UR < Salt Missihoun thaliana|BADH10A8 thaliana et al. (2014) Arabidopsis Arabidopsis UR < Salt Missihoun thaliana|BADH10A9 thaliana et al. (2014) Aphanothece Gossypium UR > Salt Song et al. halophytica|2-MGMT hirsutuma (2018) Aphanothece Gossypium UR > Salt Song et al. halophytica|GSMT hirsutuma (2018) Oryza sativa|BADH Oryza sativa (MT) C < Drought, cold, Tang et al. and salt (2014) Bacterial|CO Brassica chinensis UR > Salt and heat Wang et al. (2010) Suaeda liaotungensis|BADH Solanum – > Salt Wang et al. lycopersicum (2013) Suaeda corniculata|BADH Arabidopsis UR > Drought and Wang et al. thaliana salt (2016) Arthrobacter globiformis|CO Solanum UR > Salt Wei et al. lycopersicum (2017) Salicornia europaea|CMO Nicotiana tabacuma – > Salt Wu et al. (2010) Bambusa vulgaris|CMO Bambusa vulgaris C > Salt Yamada et al. (MT) (2015) Sesuvium Arabidopsis UR > Drought and Yang et al. portulacastrum|ALDH10 thaliana osmotic (2015a) Bacterial|CO water lilya UR > Cold Yu et al. (2018) Arthrobacter globiformis|CO Solanum tuberosum UR > Drought Cheng et al. (2013) GM (genetically modified plant), Reg. (transgene regulation), UR (upregulated), DR (downregulated), C (constant), Tol. [increasing (>) or decreasing (<) plant tolerance], aagrobacterium- mediated transformation, transgenes: GSMT (glycine sarcosine N-methyltransferase), SDMT (sarcosine dimethylglycine N-methyltransferase), CO (choline oxidase), BADH (betaine aldehyde dehydrogenase), ALDH (aldehyde dehydrogenase), CMO (choline monooxygenase), 2-MGMT (dimethylglycine methyltransferase) monooxygenase, EC 1.14.15.7) carries out the first step and BADH the second one (Chen and Murata 2002; Takabe et al. 2006). In animals and many bacteria, the first step is accomplished by membrane-bound choline dehydrogenase (EC 1.1.99.1) or soluble CO, while BADH enzyme achieves the second step. However, in some halo- phile bacteria, a three-step series of methylation reactions from glycine to betaine are catalyzed by methyltransferases [GSMT (glycine sarcosine methyltransferase, EC:2.1.1.156) and SDMT (sarcosine/dimethylglycine N-methyltransferase, EC:2.1.1.157) (Nyyssola et al. 2000). 24 É. A. Kido et al.

The biotechnological potential of genes encoding some of the mentioned enzymes as transgenes has been investigated (Table 7). Transgenic studies with CO and BADH genes, according to our data mining, corresponded to eight and six reports, respectively (Table 7). The CO gene as transgene was investigated in Solanum lycopersicum plants subjected to abiotic stresses, such as drought and salt (Goel et al. 2011). An explanation of the physiological impact verified in transgenic poplar plants overexpressing the CO transgene showed plants with higher efficiency of the photosystem II activity (Ke et al. 2016; Table 7). These plants presented lower levels of ion leakage and ROS under cold stress when compared to their respective controls (Ke et al. 2016). Concerning the BADH gene, Fan et al. (2012, Table 7) working with Ipomoea batatas transgenic lines demonstrated that BADH transgene increased the BADH activity and consecutive GB accumulation, besides the already known transgenic events altering A. thaliana plants after drought or osmotic stress (Yang et al. 2015a). The authors showed that transgenic plants under a variety of environmental stresses (salt, oxidative, and cold) have a boost in their protection against cell damage through maintenance of cell membrane integrity; stronger photosynthetic activity; reduced ROS production; and increased activity of free radical scavenging enzymes.

7.3 Polyamines

Transgenic plants and polyamines encompass 36 scientific manuscripts addressing the osmoprotectants putrescine (Put, 11), spermidine (Spd, 12), and spermine (Spm, 13) (Fig. 4). The identified articles revealed that in response to several stresses, including biotic and abiotic stresses (Fig. 4), there is a profound impact of these compounds on plant physiology, mostly in the acclimatization processes. As previously mentioned, as in Put, the ADC is the referential gene to be observed (Table 8). Most of the transgenic plants transformed with the ADC gene involved Agrobacterium-mediated system and A. thaliana. The effects of the ADC transgene were investigated in plants responding to abiotic stresses, such as drought (Alcázar et al. 2010) and salt (Alet et al. 2011; Table 8), but also under biotic stresses (Fig. 4; Hazarika and Rajam 2011; Brauc et al. 2012; Kim et al. 2013). Physiological reactions resulting from the ADC gene transformation in Lotus tenuis under drought (Espasandin et al. 2014; Table 8) grant biochemical and morphological responses correlated with Put levels. The authors mentioned above provide evidence that Put modulating ABA biosynthesis at the transcriptional level controls the ABA content in response to drought. Concerning the osmoprotectants Spd and Spm, both compounds are generated from Put by successive additions of aminopropyl groups provided by decarboxylated S-adenosylmethionine, a metabolite synthesized by the enzyme S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50). The aminopropyl additions to Put are catalyzed by the spermidine (EC 2.5.1.16) and spermine (EC 2.5.1.22) synthases. Based on this sharing of metabolic pathways, several reports Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 25

Table 8 Plants genetically modified (GM) with transgenes associated to the osmoprotectants putrescine, spermidine, and spermine Stress Transgene donor species GM plant Reg. Tol. treatment Reference Arabidopsis Arabidopsis UR > Drought Alcázar et al. thaliana|ADC2 thalianaa (2010) Avena sativa|ADC Leptotes tenuis UR > Drought Espasandin et al. (2014) Avena sativa|ADC Arabidopsis UR > Drought Alet et al. (2011)a thaliana (FRE) Avena sativa|ADC Arabidopsis UR C Salt Alet et al. (2011)b thalianaa Datura Arabidopsis C C$ Drought Peremarti et al. stramonium|SAMDC thaliana (2009) Medicago sativa|SAMS1 Nicotiana tabacum UR > Cold (FRE) Guo et al. (2014) Pine|GLU Populus tremula; UR > Drought Molina-Rueda and Plumeria alba Kirby (2015) Poncirus trifoliata|ADC Arabidopsis UR > Osmotic, Wang et al. (2011) thaliana drought, cold Arabidopsis Arabidopsis C > Drought Alcázar et al. thaliana|ADC thaliana (2010) Avena sativa|ADC Leptotes tenuis C > Drought Espasandin et al. (2014) Citrus sinensis|PAO Citrus sinensis DR > Salt Wang and Liu (2016) Datura Arabidopsis UR C$ Drought Peremarti et al. stramonium|SAMDC thaliana (2009) Hordeum vulgare|Hb1 Hordeum vulgare UR > Drought Montilla-Bascón et al. (2017) Malus domestica|SPDS Panorpa communis UR > Heavy metal Wen et al. (2010) Medicago sativa|SAMS1 Nicotiana tabacum C > Cold Guo et al. (2014) Nicotiana tabacum|SPDS Nicotiana tabacum DR < Salt and Choubey and (MP) drought Rajam (2018) Saccharomyces Solanum UR > Heat Cheng et al. cerevisiae|SAMDC lycopersicum (2009) Yeast|SAMDC Solanum UR > Cold Goyal et al. (2016) lycopersicum Arabidopsis Arabidopsis C > Drought Alcázar et al. thaliana|ADC2 thalianaa (2010) Arabidopsis Arabidopsis UR > Heat Sagor et al. (2013) thaliana|SPMS thalianaa Avena sativa|ADC Arabidopsis UR > Salt Alet et al. (2011)b thalianaa Avena sativa|ADC Leptotes tenuis C > Drought Espasandin et al. (2014) Citrus sinensis|PAO Citrus sinensis DR > Salt Wang and Liu (2016) (continued) 26 É. A. Kido et al.

Table 8 (continued) Stress Transgene donor species GM plant Reg. Tol. treatment Reference Datura Arabidopsis UR C$ Drought Peremarti et al. stramonium|SAMDC thaliana (2009) Human|SAMDC Lycopersicon UR > Salt, drought, Hazarika and esculentuma cold Rajam (2011) Leymus Arabidopsis UR > Cold and salt Liu et al. (2017b) chinensis|SAMDC thaliana Medicago sativa|SAMS1 Nicotiana tabacum C > Cold Guo et al. (2014) Saccharomyces Solanum UR > Heat Cheng et al. cerevisiae|SAMDC lycopersicum (2009) Saccharomyces Gossypium UR > Drought Momtaz et al. cerevisiae|SAMDC barbadenseb (2010) Yeast|SAMDC Solanum UR > Cold Goyal et al. (2016) lycopersicum Reg. (transgene regulation), UR (upregulated), C (constant), C$ (faster plant recovery), Tol. [increasing (>) or decreasing (<) plant tolerance], aagrobacterium-mediated transformation, bbioballistic method, transgenes: putrescine [ADC (arginine decarboxylase), GLU (glutamine synthetase), SAMS1 (S-adenosylmethionine synthetase 1), SAMDC (S-adenosylmethionine decarboxylase)], spermidine [ADC (arginine decarboxylase), SPDS (spermidine synthase), PAO (polyamine oxidase), SAMDC, SAMS1], spermine [ADC (arginine decarboxylase), SPMS (spermine synthase), PAO (polyamine oxidase), SAMDC, SAMS1)] discussed both compounds reporting the same gene (SAMDC; Tables 8). The SAMDC/SAMS1 transgene overexpression in transgenic Nicotiana tabacum plants promoted polyamine synthesis, which in turn improved the antioxidant protection against H2O2-induced damages, resulting in transgenic plants with enhanced tolerance to freezing and chilling stresses (Guo et al. 2014). Focusing on Spd and Spm, the transgenic plants of A. thaliana (Liu et al. 2017a) and S. lycopersicum (Cheng et al. 2009) stood out (Fig. 4 and Table 8). In most of those events, the transgenes were investigated in plants responding to abiotic stresses, such as drought (Peremarti et al. 2009) and salinity (Wang and Liu 2016), and also to biotic stresses [Fusarium oxysporum (Hazarika and Rajam 2011), Botrytis cinerea, Alternaria solani (Hazarika and Rajam 2011), Pseudomonas syringae, and the insect Manduca sexta (Nambeesan et al. 2012)]. Transgenic plants also included Citrus sinensis (Wang and Liu 2016) and Gossypium barbadense (Momtaz et al. 2010).

7.4 Carbohydrates

The association of carbohydrates with osmoprotective functions and transgenic events highlighted trehalose and fructans in the data mining results (Fig. 4). From the six identified events involving trehalose (Table 9), the reference gene encoded Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 27

Table 9 Genetically modified (GM) plants involving genes related to the osmoprotectant trehalose and fructan Stress Transgene donor species GM plant Reg. Tol. treatment Reference Brevibacterium helvolum|MTS, Oryza sativaa – > Drought Joo et al. MTH, and MTSH (2014) Gossypium hirsutum|T6PS Arabidopsis UR < Cold Wang et al. thalianaa (2016a) Manihot esculenta|T6PS1 Nicotiana UR > Drought Han et al. tabacuma (2016) Oryza sativa|T6PS Oryza sativaa UR > Salt, PEG, and Li et al. cold (2011a) Psathyrostachys Nicotiana UR > Drought, cold, He et al. huashanica|6-SFT tabacuma and salt (2015) Secale cereale|1-SST Triticosecale UR > Cold Diedhiou et al. Wittmackb (2012) Triticum aestivum|6-SFT Triticosecale UR > Cold Diedhiou et al. Wittmackb (2012) Triticum Nicotiana UR > Cold and salt Bie et al. aestivum|6-SFT|1-SST|1-FFT tabacuma (2012) GM (genetically modified), Reg. (transgene regulation), UR (upregulated), Tol. [increasing (>) or decreasing (<) plant tolerance], aAgrobacterium-mediated transformation, bbioballistic method; transgenes: trehalose [T6PS (trehalose-6-phosphate synthase), MTS (maltooligosyltrehalose synthase), MTH (maltooligosyltrehalose trehalohydrolase), MTSH (maltooligosyltrehalose synthase fused with maltooligosyltrehalose trehalohydrolase)], fructan [6-SFT (fructan-6- fructosyltransferase), 1-SST (sucrose: sucrose 1-fructosytransferase), FFT (fructan: fructan 1-fructosyltransferase)]

T6PS (TPS, EC 2.4.1.15). As mentioned before, the trehalose biosynthesis in plants involves TPS and TPP (Cabid and Leloir 1958). Additionally, transgenic plants included A. thaliana, N. tabacum, and O. sativa, while the studied stresses covered abiotic (drought, cold, and salt; Table 9) and biotic stresses (Botrytis cinerea, Pseudomonas syringae; Wang et al. 2018; Fig. 4). Considering abiotic stress, the transgenic rice plants overexpressing TPS1 transgene showed enhanced drought, cold, and salt tolerance by increasing trehalose and proline levels (Li et al. 2011a). The authors speculate that TPS1 participates in abiotic stress pathways indirectly through alteration of the expression of some stress- related genes encoding ELIP (early light-inducible protein), HSP70 (heat shock protein), RAB16C (responsive to ABA), CRP (cold-regulated protein), DHN6 (dehydrin), LEA14A (late embryogenesis abundant protein), and WSI18 (water stress-inducible protein). In turn, the data mining results for fructans and transgenic plants identified six events (three manuscripts; Table 9). The main transgene with biotechnological potential encoded the enzyme sucrose:fructan 6-fructosyltransferase (6-SFT; EC 2.4.1.10), which is involved in grass fructan biosynthesis catalyzing the initiation and extension of 2,6-linked fructans (Wei et al. 2001). Some transformed plants 28 É. A. Kido et al. comprised triticale and N. tabacum, while the analyzed abiotic stresses included cold, salt, and drought (Table 9). In N. tabacum transgenic plants overexpressing the 6-SFT transgene, the abiotic tolerance was associated with fructans and proline accumulations, together with malondialdehyde (MDA) level reduction (He et al. 2015), which is recognized as oxidative stress biomarker.

7.5 Sugar Alcohols

Furthermore, transformed plants in association with sugar alcohol, myo-inositol, and its phosphorylated derivatives are the most addressed (Fig. 4). The data mining results identified several manuscripts (22), most of them reporting the biotechnological potential of the MIPS/INPS transgenes. As mentioned before, the MIPS enzyme catalyzes the step that is the rate limiting in the biosynthesis of all inositol- containing compounds. Other investigated transgenes (Table 11.10) are IMPase (l-myo-inositol monophosphatase); 5PTase/ INPP5E (inositol polyphosphate 5-phosphatase); MIOX (myo-inositol oxygenase); IMT (myo-inositol methyltransferase); IPK (inositol polyphosphate kinase); IP6K (inositol polyphosphate 6-/3-kinase); and PIS (phosphatidylinositol synthase). Also, the transgenes encoding D-ononitol epimerase (OEP) and phospholipase C2 (PLC2, EC 3.1.4.3) have been investigated (Table 10). The PLC2 converts phosphatidylinositol to inositol 1-phosphate which is then converted into myo-inositol (https://www.genome.jp/dbget-bin/www_bget?3.1.4.3). In the halophyte Mesembryanthemum crystallinum (common ice plant) the OEP1 gene is associated with osmoprotectant accumulation, including methylated inositols, D-ononitol, and D-pinitol (Ishitani et al. 1996). The authors also highlighted differences in glycophytic and halophytic regulation of the Inps1 gene expression, since A. thaliana did not show upregulation of Inps1 or increased amounts of Ins when salt stressed. In general, the impact of these transgenes has been analyzed (Fig. 4 and Table 10) especially in A. thaliana (Kaur et al. 2013) and N. tabacum (Chatterjee and Majumder 2010) and mainly for drought (Tan et al. 2013) and salinity (Kusuda et al. 2015). Genetically modifiedOryza sativa plants overexpressing MIPS present induced Ins metabolism in association with the activation of basal metabolisms, including glycolysis, tricarboxylic acid cycle, and the pentose-phosphate pathway (PPP), one of the major antioxidant cellular defense systems (Kusuda et al. 2015).

8 Conclusion and Future Perspectives

Due to the involvement of osmoprotectants in positive plant responses to various environmental stresses that affect crops and often cause economic damage, the study of genes related to these compounds and their effects is relevant for plant breeding programs and crop production. Since traditional plant breeding requires Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 29

Table 10 Genetically modified plants involving genes related to the osmoprotectant myo-inositol Stress Transgene donor species GM plant Reg. Tol. treatment Reference Arabidopsis thaliana|ADC2 Arabidopsis thalianaa UR > Drought Alcázar et al. (2010) Arabidopsis thaliana|MIOX Arabidopsis thalianaa UR > Cold, salt, Lisko et al. (2013) heat, pyrene Arabidopsis thaliana|IP6K Lycopersicon – > Drought, Zhang et al. (2009) esculentuma cold, and oxidative Brassica napus|PLC2 Canolaa – > Cold Nokhrina et al. (2014) Brassica napus|PLC2 Canolaa UR > Drought Georges et al. (2009) Cicer arietinum|IMPase Arabidopsis thalianaa UR > Salt, PEG Saxena et al. (2013) or paraquat, cold Cicer arietinum|MIPS Arabidopsis thalianaa – > Salt and Kaur et al. (2013) osmotic Glycine max|OEP Arabidopsis thalianaa – > Drought and Ahn et al. (2018) salt Glycine max|SAL1 Arabidopsis thalianaa DR > Salt Ku et al. (2013) Glycine max|IMT Arabidopsis thalianaa – > Salt and Ahn et al. (2011) osmotic Human|5-ptases Lycopersicon DR > Light Alimohammadi esculentuma et al. (2015) Ipomoea batatas|MIPS Ipomoea batatasa UR > Drought, Zhai et al. (2015) salt Medicago falcata|MIPS Nicotiana tabacuma UR > Cold, salt, Tan et al. (2013 drought Mesembryanthemum Nicotiana tabacuma UR > Salt Patra et al. (2010) crystallinum|IMT Oryza sativa|MIOX Oryza sativaa – > PEG and Duan et al. (2012) mannitol Oryza sativa|IMPase Nicotiana tabacuma UR > Cold Zhang et al. (2017) Oryza sativa|MIPS Oryza sativaa UR > Salt Kusuda et al. (2015) Populus euphratica|MIPS Populus euphraticaa UR > Salt and Cu Zhang et al. (2018) Porteresia coarctata|MIPS Nicotiana tabacuma – > Salt Chatterjee et al. (2010) Porteresia coarctata|MIPS Nicotiana tabacuma UR > Salt Patra et al. (2010) Spartina alterniflora|MIPS Arabidopsis thalianaa – > Salt Joshi et al. (2013) Thellungiella Brassica napusa – > Salt, Zhu et al. (2009) halophila|IPK osmotic, and oxidative Zea mays|PIS Zea maysa UR > Drought Liu et al. (2013) Zea mays|PIS Nicotiana tabacuma UR > Drought Zhai et al. (2012) Reg. (transgene regulation), UR (upregulated), DR (downregulated), Tol. [increasing (>) or decreasing (<) plant tolerance], aAgrobacterium-mediated transformation, transgenes: OEP (D-ononitol epimerase), MIPS (l-myo-inositol-1-phosphate synthase), IMPase (l-myo-inositol monophosphatase), 5PTase (inositol polyphosphate 5-phosphatase), PLC2 (phospholipase C2), MIOX (myo-inositol oxygenase), PIS (phosphatidylinositol synthase), IMT (myo-inositol methyltransferase), IPK (inositol polyphosphate kinase), and IP6K (inositol polyphosphate 6−/3-kinase) 30 É. A. Kido et al. specialized human resources for parental selection, progeny generation, and selection of promising accessions, usually from assays implemented at different production areas and throughout several years, obtaining elite materials has become time-consuming and expensive. In these circumstances, the use of new techniques involving genomics/bioinformatics, transgenic application, and marker-assisted selection (MAS) can minimize the time required to release elite cultivars and the costs involving plant breeding activities. Thus, to identify osmoprotectant-related genes and their associated biosynthesis pathways, to know their transcriptional dynamics, to recognize possible genetic bottlenecks in cultivated crops, to clarify how osmoprotectants can minimize stress damages, as well as to evaluate their biotechnological potential as transgenes should be goals to be persecuted. Consequently, plant transcriptomics provides a fertile field for investigations, associating gene expression profiles according to the evaluated plant stress response. In conformity, technical analysis trying to validate the in silico profiles, with the posterior genetically improvement of the plant, could help to explore the productive potential of cultivars/varieties, frequently not cultivated in ideal conditions. Further, some great news is expected to emerge from the biotechnological research area, by the increasing number of biomolecule sequences deposited in public databases, cheaper computing resources, and more specialized and robust databases, allied with efficient, fast, and friendly bioinformatic tools. The biotechnological approaches involving osmoprotectant-related genes open new perspectives for the analysis and incorporation of these genes into cultivated plant species, improving the advances in plant breeding and crop production.

Acknowledgments The authors acknowledge the Brazilian institutions FINEP (Financiadora de Estudos e Projetos), FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco), and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support and fellowships (CNPq 311894/2017-8). The authors also thank Ms. Suzana de Aragão Britto Kido for the English language revision.

References

Ahn CH, Hossain MA, Lee E, Kanth BK, Park PB (2018) Increased salt and drought tolerance by D-pinitol production in transgenic Arabidopsis thaliana. Biochem Biophys Res Commun 504:315–320. https://doi.org/10.1016/j.bbrc.2018.08.183 Ahn CH, Park U, Park PB (2011) Increased salt and drought tolerance by D-pinitol production in transgenic Arabidopsis thaliana. Biochem Biophys Res Commun 415:669–674. https://doi. org/10.1016/j.bbrc.2011.10.134 Alavilli H, Awasthi JP, Rout GR, Sahoo L, Lee B, Panda SK (2016) Overexpression of a barley aquaporin gene, HvPIP2;5 confers salt and osmotic stress tolerance in yeast and plants. Front Plant Sci 7:1–12. https://doi.org/10.1016/j.ijepes.2015.09.006 Alcázar R, Planas J, Saxena T, Zarza X, Bortolotti C, Cuevas J, Bitrián M, Tiburcio AF, Altabella T (2010) Putrescine accumulation confers drought tolerance in transgenic Arabidopsis plants over-expressing the homologous Arginine decarboxylase 2 gene. Plant Physiol Biochem 48:547–552. https://doi.org/10.1016/j.plaphy.2010.02.002 Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 31

Alet AI, Sanchez DH, Cuevas JC, del Valle S, Altabella T, Tiburcio AF, Marco F, Ferrando A, Espasandín FD, González ME, Carrasco P, Ruiz OA (2011) Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress. Plant Signal Behav 6:278–286. https://doi.org/10.4161/psb.6.2.14702 Alimohammadi M, Lahiani MH, Khodakovskaya MV (2015) Genetic reduction of inositol tri-

phosphate (InsP3) increases tolerance of tomato plants to oxidative stress. Planta 242:123–135. https://doi.org/10.1007/s00425-015-2289-1 Antoniou C, Fragkoudi I, Martinou A, Stavrinides MC, Fotopoulos V (2018) Spatial response of Medicago truncatula plants to drought and spider mite attack. Plant Physiol Biochem 130:658– 662. https://doi.org/10.1016/j.plaphy.2018.08.018 Ardito F, Giuliani M, Perrone D, Troiano G, Lo Muzio L (2017) The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review). Int J Mol Med 40:271– 280. https://doi.org/10.3892/ijmm.2017.3036 Bae YS, Oh H, Rhee SG, Do Yoo Y (2011) Regulation of reactive oxygen species generation in cell signaling. Mol Cells 32:491–509. https://doi.org/10.1007/s10059-011-0276-3 Bie X, She M, Li J, Ye X, Lin Z, Zhang S, Gao X, Wang K, Du L (2012) Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco. Plant Cell Rep 31:2229–2238. https://doi. org/10.1007/s00299-012-1332-y Bougouffa S, Radovanovic A, Essack M, Bajic VB (2014) DEOP: a database on osmoprotectants and associated pathways. Database 2014 Brauc S, De Vooght E, Claeys M, Geuns JMC, Höfte M, Angenon G (2012) Overexpression of arginase in Arabidopsis thaliana influences defence responses againstBotrytis cinerea. Plant Biol 14:39–45. https://doi.org/10.1111/j.1438-8677.2011.00520.x Burdo B, Gray J, Goetting-Minesky MP, Wittler B, Hunt M, Li T, Velliquette D, Thomas J, Gentzel I, Dos Santos BM, Mejía-Guerra MK, Connolly LN, Qaisi D, Li W, Casas MI, Doseff AI, Grotewold E (2014) The Maize TFome - Development of a transcription factor open read- ing frame collection for functional genomics. Plant J 80:356–366. https://doi.org/10.1111/ tpj.12623 Cabid E, Leloir LF (1958) The biosynthesis of trehalose phosphate. J Biol Chem 231(1):259–275 Caspi R, Billington R, Fulcher CA, Keseler IM, Kothari A, Krummenacker M, Latendresse M, Midford PE, Ong Q, Ong WK, Paley S, Subhraveti P, Karp PD (2018) The MetaCyc database of metabolic pathways and enzymes. Nucleic Acids Res 46:D633–D639. https://doi. org/10.1093/nar/gkx935 Chatterjee J, Majumder AL (2010) Salt-induced abnormalities on root tip mitotic cells of Allium cepa: prevention by inositol pretreatment. Protoplasma 245:165–172. https://doi.org/10.1007/ s00709-010-0170-4 Chen E, Zhang X, Yang Z, Wang X, Yang Z, Zhang C, Wu Z, Kong D, Liu Z, Zhao G, Butt HI, Zhang X, Li F (2017) Genome-wide analysis of the HD-ZIP IV transcription factor family in Gossypium arboreum and GaHDG11 involved in osmotic tolerance in transgenic Arabidopsis. Mol Gen Genomics 292:593–609. https://doi.org/10.1007/s00438-017-1293-5 Chen J, Shang Y-T, Wang W-H, Chen X-Y, He E-M, Zheng H-L, Shangguan Z, He E-M (2016) Hydrogen sulfide-mediated polyamines and sugar changes are involved in hydrogen sulfide- induced drought tolerance in Spinacia oleracea seedlings. Front Plant Sci 7:1–1. https://doi. org/10.3389/fpls.2016.01173 Chen JB, Yang JW, Zhang ZY, Feng XF, Wang SM (2013) Two P5CS genes from common bean exhibiting different tolerance to salt stress in transgenic Arabidopsis. J Genet 92:461–469. https://doi.org/10.1007/s12041-013-0292-5 Chen THH, Murata N (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol 5:250–257. https://doi. org/10.1016/S1369-5266(02)00255-8 Cheng L, Zou Y, Ding S, Zhang J, Yu X, Cao J, Lu G (2009) Polyamine accumulation in transgenic tomato enhances the tolerance to high temperature stress. J Integr Plant Biol 51:489–499. https://doi.org/10.1111/j.1744-7909.2009.00816.x 32 É. A. Kido et al.

Cheng YJ, Deng XP, Kwak SS, Chen W, Eneji AE (2013) Enhanced tolerance of transgenic potato plants expressing choline oxidase in chloroplasts against water stress. Bot Stud 54:1–9. https:// doi.org/10.1186/1999-3110-54-30 Choubey A, Rajam MV (2018) RNAi-mediated silencing of spermidine synthase gene results in reduced reproductive potential in tobacco. Physiol Mol Biol Plants 24:1069–1081. https://doi. org/10.1007/s12298-018-0572-x Conde A, Soares F, Breia R, Gerós H (2018) Postharvest dehydration induces variable changes in the primary metabolism of grape berries. Food Res Int 105:261–270. https://doi.org/10.1016/j. foodres.2017.11.052 Chevalier D, Morris ER, Walker JC (2009) 14-3-3 and FHA Domains Mediate Phosphoprotein Interactions. Annual Review of Plant Biology 60(1):67–91 Dastogeer KMG, Li H, Sivasithamparam K, Jones MGK, Wylie SJ (2018) Fungal endophytes and a virus confer drought tolerance to Nicotiana benthamiana plants through modulating osmolytes, antioxidant enzymes and expression of host drought responsive genes. Environ Exp Bot 149:95–108. https://doi.org/10.1016/j.envexpbot.2018.02.009 de Freitas PAF, de Carvalho HH, Costa JH, Miranda RS, KDDC S, de Oliveira FDB, Coelho DG, Prisco JT, Gomes-Filho E (2019) Salt acclimation in sorghum plants by exogenous proline: physiological and biochemical changes and regulation of proline metabolism. Plant Cell Rep 38:403. https://doi.org/10.1007/s00299-019-02382-5 de Ronde JA, Cress WA, Krüger GHJ, Strasser RJ, Van Staden J (2004) Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress. J Plant Physiol 161:1211–1224. https://doi.org/10.1016/j.jplph.2004.01.014 Devos KM, Brown JKM, Bennetzen JL (2002) Genome Size Reduction through Illegiti mate Recombination Counteracts Genome Expansion in Arabidopsis. Genome Research 12(7):1075–1079 Diedhiou C, Gaudet D, Liang Y, Sun J, Lu ZX, Eudes F, Laroche A (2012) Carbohydrate profiling in seeds and seedlings of transgenic triticale modified in the expression of sucrose: sucrose- 1-fructosyltransferase (1-SST) and sucrose: fructan-6-fructosyltransferase (6-SFT). J Biosci Bioeng 114:371–378. https://doi.org/10.1016/j.jbiosc.2012.05.008 Duan J, Liu P, Li Z, Zhang M, Ali J, Zhang H, Li J, Xiong H (2012) OsMIOX, a myo-inositol oxygenase gene, improves drought tolerance through scavenging of reactive oxygen species in rice (Oryza sativa L.). Plant Sci 196:143–151. https://doi.org/10.1016/j.plantsci.2012.08.003 Ebeed HT, Hassan NM, Aljarani AM (2017) Exogenous applications of Polyamines modulate drought responses in wheat through osmolytes accumulation, increasing free polyamine levels and regulation of polyamine biosynthetic genes. Plant Physiol Biochem 118:438–448. https:// doi.org/10.1016/j.plaphy.2017.07.014 Espasandin FD, Maiale SJ, Calzadilla P, Ruiz OA, Sansberro PA (2014) Transcriptional regulation of 9-cis-epoxycarotenoid dioxygenase (NCED) gene by putrescine accumulation positively modulates ABA synthesis and drought tolerance in Lotus tenuis plants. Plant Physiol Biochem 76:29–35. https://doi.org/10.1016/j.plaphy.2013.12.018 Fan W, Zhang M, Zhang H, Zhang P (2012) Improved tolerance to various abiotic stresses in transgenic sweet potato (Ipomoea batatas) expressing spinach betaine aldehyde dehydrogenase. PLoS One 7:e37344. https://doi.org/10.1371/journal.pone.0037344 Fang H, Liu X, Thorn G, Duan J, Tian L (2014) Expression analysis of histone acetyltransfer- ases in rice under drought stress. Biochemical and Biophysical Research Communications 443(2):400–405 Gagneul D, Ainouche A, Duhaze C, Lugan R, Larher FR, Bouchereau A (2007) A reassessment of the function of the so-called compatible solutes in the halophytic Plumbaginaceae Limonium latifolium. Plant Physiol 144:1598–1611. https://doi.org/10.1104/pp.107.099820 Garcia AAF, Kido EA, Meza AN, Souza HMB, Pinto LR, Pastina MM, Leite CS, Da Silva JAG, Ulian EC, Figueira A, Souza AP (2006) Development of an integrated genetic map of a sugarcane (Saccharum spp.) commercial cross, based on a maximum-likelihood approach for estimation of linkage and linkage phases. Theor Appl Genet 112:298–314. https://doi.org/10.1007/ s00122-005-0129-6 Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 33

Georges F, Das S, Ray H, Bock C, Nokhrina K, Kolla VA, Keller W (2009) Over-expression of Brassica napus phosphatidylinositol-phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation. Plant Cell Environ 32:1664–1681.https://doi. org/10.1111/j.1365-3040.2009.02027.x Gillaspy GE (2011) The cellular language of myo-inositol signaling. New Phytol 192:823–839. https://doi.org/10.1111/j.1469-8137.2011.03939.x Goel D, Singh AK, Yadav V, Babbar SB, Murata N, Bansal KC (2011) Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses. J Plant Physiol 168:1286–1294. https://doi.org/10.1016/j.jplph.2011.01.010 Gong X, Dou F, Cheng X, Zhou J, Zou Y, Ma F (2018) Genome-wide identification of genes involved in polyamine biosynthesis and the role of exogenous polyamines in Malus hupehensis Rehd. under alkaline stress. Gene 669:52–62. https://doi.org/10.1016/j.gene.2018.05.077 Goyal RK, Fatima T, Topuz M, Bernadec A, Sicher R, Handa AK, Mattoo AK (2016) Pathogenesis- related protein 1b1 (PR1b1) is a major tomato fruit protein responsive to chilling temperature and upregulated in high polyamine transgenic genotypes. Front Plant Sci 7:901. https://doi. org/10.3389/fpls.2016.00901 Guan C, Huang YH, Cui X, Liu SJ, Zhou YZ, Zhang YW (2018) Overexpression of gene encoding the key enzyme involved in proline-biosynthesis (PuP5CS) to improve salt tolerance in switchgrass (Panicum virgatum L.). Plant Cell Rep 37:1187–1199. https://doi.org/10.1007/ s00299-018-2304-7

Guo Z, Tan J, Zhuo C, Wang C, Xiang B, Wang Z (2014) Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced S-adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up-regulating polyamine oxidation. Plant Biotechnol J 12:601–612. https://doi.org/10.1111/pbi.12166 Han B, Fu L, Zhang D, He X, Chen Q, Peng M, Zhang J (2016) Interspecies and intraspecies analysis of trehalose contents and the biosynthesis pathway gene family reveals crucial roles of trehalose in osmotic-stress tolerance in cassava. Int J Mol Sci 17:1–18. https://doi.org/10.3390/ ijms17071077 Hazarika P, Rajam MV (2011) Biotic and abiotic stress tolerance in transgenic tomatoes by constitutive expression of S-adenosylmethionine decarboxylase gene. Physiol Mol Biol Plants 17:115–128. https://doi.org/10.1007/s12298-011-0053-y He X, Chen Z, Wang J, Li W, Zhao J, Wu J, Wang Z, Chen X (2015) A sucrose: fructan-6- fructosyltransferase (6-SFT) gene from Psathyrostachys huashanica confers abiotic stress tolerance in tobacco. Gene 570:239–247. https://doi.org/10.1016/j.gene.2015.06.023 He Y, He C, Li L, Liu Z, Yang A, Zhang J (2011) Heterologous expression of ApGSMT2 and ApDMT2 genes from Aphanothece halophytica enhanced drought tolerance in transgenic tobacco. Mol Biol Rep 38:657–666. https://doi.org/10.1007/s11033-010-0152-9 Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends in Plant Science 6(9):431–438 Hossain MA, Wani SH, Bhattacharjee S, Burritt DJ, Tran L-SP (2016) Drought stress tolerance in plants, Molecular and genetic perspectives, vol 2, 1st edn. Springer, Berlin. https://doi. org/10.1007/978-3-319-32423-4 Ibragimova SM, Trifonova EA, Filipenko EA, Shymny VK (2015) Evaluation of salt tolerance of transgenic tobacco plants bearing the P5CS1 gene of Arabidopsis thaliana. Russ J Genet 51:1181–1188. https://doi.org/10.1134/S1022795415120078 Ishitani M, Majumder AL, Bornhouser A, Michalowski CB, Jensen RG, Bohnert HJ (1996) Coordinate transcriptional induction of myo-inositol metabolism during environmental stress. Plant J 9:537–548. https://doi.org/10.1046/j.1365313X.1996.09040537.x Jiang Y, Zhu S, Yuan J, Chen G, Lu G (2016) A betaine aldehyde dehydrogenase gene in quinoa (Chenopodium quinoa): structure, phylogeny, and expression pattern. Genes Geno 38:1013– 1020. https://doi.org/10.1007/s13258-016-0445-z 34 É. A. Kido et al.

Johnson SM, Cummins I, Lim FL, Slabas AR, Knight MR (2015) Transcriptomic analysis comparing stay-green and senescent Sorghum bicolor lines identifies a role for proline biosynthesis in the stay-green trait. J Exp Bot 66:7061–7073. https://doi.org/10.1093/jxb/erv405 Joo J, Choi HJ, Lee YH, Lee S, Lee CH, Kim CH, Cheong JJ, Do Choi Y, Song SI (2014) Over- expression of BvMTSH, a fusion gene for maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase, enhances drought tolerance in transgenic rice. BMB Rep 47:27–32. https://doi.org/10.5483/BMBRep.2014.47.1.064 Joshi R, Ramanarao MV, Baisakh N (2013) Arabidopsis plants constitutively overexpressing a myo-inositol 1-phosphate synthase gene (SaINO1) from the halophyte smooth cordgrass exhibits enhanced level of tolerance to salt stress. Plant Physiol Biochem 65:61–66. https://doi. org/10.1016/j.plaphy.2013.01.009 Jung H, Chung PJ, Park SH, Redillas MCFR, Kim YS, Suh JW, Kim JK (2017) Overexpression of OsERF48 causes regulation of OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance. Plant Biotechnol J 15:1295–1308. https://doi.org/10.1111/ pbi.12716 Kanamaru N, Ito Y, Komori S, Saito M, Kato H, Takahashi S, Omura M, Soejima J, Shiratake K, Yamada K, Yamaki S (2004) Transgenic apple transformed by sorbitol-6-phosphate dehydrogenase cDNA. Plant Science 167(1):55–61 Kathuria H, Giri J, Nataraja KN, Murata N, Udayakumar M, Tyagi AK (2009) Glycinebetaine- induced water-stress tolerance in codA-expressing transgenic indica rice is associated with up-regulation of several stress responsive genes. Plant Biotechnol J 7:512–526. https://doi. org/10.1111/j.1467-7652.2009.00420.x Kaur H, Verma P, Petla BP, Rao V, Saxena SC, Majee M (2013) Ectopic expression of the ABA-inducible dehydration-responsive chickpea L-myo-inositol 1-phosphate synthase 2 (CaMIPS2) in Arabidopsis enhances tolerance to salinity and dehydration stress. Planta 237:321–335. https://doi.org/10.1007/s00425-012-1781-0 Ke Q, Wang Z, Ji CY, Jeong JC, Lee HS, Li H, Xu B, Deng X, Kwak SS (2016) Transgenic poplar expressing codA exhibits enhanced growth and abiotic stress tolerance. Plant Physiol Biochem 100:75–84. https://doi.org/10.1016/j.plaphy.2016.01.004 Kido EA, Rc J, Neto F, Silva RLO, Belarmino LC, Neto JPB, Soares-cavalcanti NM, Pandolfi V, Silva MD, Nepomuceno AL, Benko-iseppon AM (2013) Expression dynamics and genome distribution of osmoprotectants in soybean: identifying important components to face abiotic stress. BMC Bioinformatics 14 (Suppl 1):S7 Kido EA, Ferreira-Neto JRC, Pandolfi V, Souza AMS, Benko-Iseppon AM (2016) Drought stress tolerance in plants: insights from transcriptomic studies. In: Drought stress tolerance in plants, Molecular and genetic perspectives, vol 2. Springer, pp 153–186. https://doi. org/10.1007/978-3-319-32423-4 Kim SH, Kim SH, Yoo SJ, Min KH, Nam SH, Cho BH, Yang KY (2013) Putrescine regulating by stress-responsive MAPK cascade contributes to bacterial pathogen defense in Arabidopsis. Biochem Biophys Res Commun 437:502–508. https://doi.org/10.1016/j.bbrc.2013.06.080 Kissoudis C, Kalloniati C, Flemetakis E, Madesis P, Labrou NE, Tsaftaris A, Nianiou-Obeidat I (2015) Stress-inducible GmGSTU4 shapes transgenic tobacco plants metabolome towards increased salinity tolerance. Acta Physiol Plant 37:1–11. https://doi.org/10.1007/ s11738-015-1852-5 Ku Y-S, Koo NS-C, Li FW-Y, Li M-W, Wang H, Tsai S-N, Sun F, Lim BL, Ko W-H, Lim BL, Lam H-M (2013) GmSAL1 hydrolyzes inositol-1,4,5-trisphosphate and regulates stomatal closure in detached leaves and ion compartmentalization in plant cells. PLoS One 8:e78181. https:// doi.org/10.1371/journal.pone.0078181 Kusuda H, Koga W, Kusano M, Oikawa A, Saito K, Hirai MY, Yoshida KT (2015) Ectopic expression of myo-inositol 3-phosphate synthase induces a wide range of metabolic changes and confers salt tolerance in rice. Plant Sci 232:49–56. https://doi.org/10.1016/j.plantsci.2014.12.009 Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 35

Lai SJ, Lai MC, Lee RJ, Chen YH, Yen HE (2014) Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress. Plant Mol Biol 85:429–441. https://doi.org/10.1007/s11103-014-0195-8 Li D, Zhang T, Wang M, Liu Y, Brestic M, Chen THH, Yang X (2019a) Genetic engineering of the biosynthesis of glycine betaine modulates phosphate homeostasis by regulating phosphate acquisition in tomato. Front Plant Sci 9:1–13. https://doi.org/10.3389/fpls.2018.01995 Li H, Mo YL, Cui Q, Yang XZ, Guo YL, Wei CH, Yang J, Zhang Y, Ma JX, Zhang X (2019b) Transcriptomic and physiological analyses reveal drought adaptation strategies in drought- tolerant and -susceptible watermelon genotypes. Plant Sci 278:32–43. https://doi.org/10.1016/j. plantsci.2018.10.016 Li HW, Zang BS, Deng XW, Wang XP (2011a) Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta 234:1007–1018. https:// doi.org/10.1007/s00425-011-1458-0 Li S, Li F, Wang J, Zhang W, Meng Q, Chen THH, Murata N, Yang X (2011b) Glycinebetaine enhances the tolerance of tomato plants to high temperature during germination of seeds and growth of seedlings. Plant Cell Environ 34:1931–1943. https://doi. org/10.1111/j.1365-3040.2011.02389.x Li J, Witten DM, Johnstone IM, Tibshirani R (2012) Normalization, testing, and false discovery rate estimation for RNA-sequencing data. Biostatistics 13:523–538. https://doi.org/10.1093/ biostatistics/kxr031 Li L, Li L, Wang X, Zhu P, Wu H, Qi S (2017) Plant growth-promoting endophyte Piriformospora indica alleviates salinity stress in Medicago truncatula. Plant Physiol Biochem 119:211–223. https://doi.org/10.1016/j.plaphy.2017.08.029 Lisko KA, Torres R, Harris RS, Belisle M, Martha M (2013) Elevating vitamin C content via overexpression of myo-inositol oxygenase and L-gulono-1,4-lactone oxidase in Arabidopsis leads to enhanced biomass and tolerance to abiotic stresses. In Vitro Cell Dev Biol Plant 49(6):643–655. https://doi.org/10.1007/s11627-013-9568-y Liu X, Zhai S, Zhao Y, Sun B, Liu C, Yang A, Zhang J (2013) Overexpression of the phosphatidylinositol synthase gene (ZmPIS) conferring drought stress tolerance by altering membrane lipid composition and increasing ABA synthesis in maize. Plant, Cell & Environment 36(5):1037–1055 Liu C, Zhang X, Zhang K, An H, Hu K, Wen J, Shen J, Ma C, Yi B, Tu J, Fu T (2015) Comparative analysis of the Brassica napus root and leaf transcript profiling in response to drought stress. Int J Mol Sci 16:18752–18777. https://doi.org/10.3390/ijms160818752 Liu S, Hao H, Lu X, Zhao X, Wang Y, Zhang Y, Xie Z, Wang R (2017a) Transcriptome profiling of genes involved in induced systemic salt tolerance conferred by Bacillus amyloliquefaciens FZB42 in Arabidopsis thaliana. Sci Rep 7:1–13. https://doi.org/10.1038/s41598-017-11308-8 Liu Z, Liu P, Qi D, Peng X, Liu G (2017b) Enhancement of cold and salt tolerance of Arabidopsis by transgenic expression of the S-adenosylmethionine decarboxylase gene from Leymus chinensis. J Plant Physiol 211:90–99. https://doi.org/10.1016/j.jplph.2016.12.014 Liu ZM, Yue MM, Yang DY, Zhu SB, Ma NN, Meng QW (2017c) Over-expression of SlJA2 decreased heat tolerance of transgenic tobacco plants via salicylic acid pathway. Plant Cell Rep 36(4):529–542. https://doi.org/10.1007/s00299-017-2100-9 Luo D, Niu X, Yu J, Yan J, Gou X, Lu BR, Liu Y (2012) Rice choline monooxygenase (OsCMO) protein functions in enhancing glycine betaine biosynthesis in transgenic tobacco but does not accumulate in rice (Oryza sativa L. ssp. japonica). Plant Cell Rep 31:1625–1635. https://doi. org/10.1007/s00299-012-1276-2 Lv W-T, Lin B, Zhang M, Hua X-J (2011) Proline accumulation is inhibitory to Arabidopsis seedlings during heat stress. Plant Physiol 156:1921–1933. https://doi.org/10.1104/pp.111.175810 Lyu JI, Park JH, Kim J-K, Bae C-H, Jeong W-J, Min SR, Liu JR (2018) Enhanced tolerance to heat stress in transgenic tomato seeds and seedlings overexpressing a trehalose-6-phosphate synthase/phosphatase fusion gene. Plant Biotechnol Rep 12:399–408. https://doi.org/10.1007/ s11816-018-0505-8 36 É. A. Kido et al.

Matsumura H, Urasaki N, Yoshida K, Krüger DH, Kahl G, Terauchi R (2012) RNA Abundance Analysis. Life Sci 883:230. https://doi.org/10.1007/978-1-61779-839-9 Missihoun TD, Willèe E, Guegan JP, Berardocco S, Shafiq MR, Bouchereau A, Bartels D (2014) Overexpression of ALDH10A8 and ALDH10A9 genes provides insight into their role in glycine betaine synthesis and affects primary metabolism in Arabidopsis thaliana. Plant Cell Physiol 56:1798–1807. https://doi.org/10.1093/pcp/pcv105 Molina-Rueda JJ, Kirby EG (2015) Transgenic poplar expressing the pine GS1a show alterations in nitrogen homeostasis during drought. Plant Physiol Biochem 94:181–190. https://doi. org/10.1016/j.plaphy.2015.06.009 Momtaz OA, Hussein EM, Fahmy EM, Ahmed SE (2010) Expression of S-adenosyl methionine decarboxylase gene for polyamine accumulation in Egyptian cotton Giza 88 and Giza 90. GM Crops 1:257–266. https://doi.org/10.4161/gmcr.1.4.13779 Montilla-Bascón G, Rubiales D, Hebelstrup KH, Mandon J, Harren FJM, Montilla-Bascón G, Cristescu SM, Mur LAJ, Prats E (2017) Reduced nitric oxide levels during drought stress promote drought tolerance in barley and is associated with elevated polyamine biosynthesis. Sci Rep 7:1–15. https://doi.org/10.1038/s41598-017-13458-1 Moschen S, Di Rienzo JA, Higgins J, Tohge T, Watanabe M, González S, Rivarola M, García- García F, Dopazo J, Hopp HE, Hoefgen R, Fernie AR, Paniego N, Fernández P, Heinz RA (2017) Integration of transcriptomic and metabolic data reveals hub transcription factors involved in drought stress response in sunflower (Helianthus annuus L.). Plant Mol Biol 94:549–564. https://doi.org/10.1007/s11103-017-0625-5 Nambeesan S, AbuQamar S, Laluk K, Mattoo AK, Mickelbart MV, Ferruzzi MG, Mengiste T, Handa AK (2012) Polyamines attenuate ethylene-mediated defense responses to abrogate resistance to Botrytis cinerea in tomato. Plant Physiol 158:1034–1045. https://doi.org/10.1104/ pp.111.188698 Niu GL, Gou W, Han XL, Qin C, Zhang LX, Abomohra AEF, Ashraf M (2018) Cloning and functional analysis of phosphoethanolamine methyltransferase promoter from maize (Zea mays L.). Int J Mol Sci 19:13. https://doi.org/10.3390/ijms19010191 Nishiyama T, Fujita T, Shin-i T, Seki M, Nishide H, Uchiyama I (2003) Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: Implication for land plant evolution. Proceedings of the National Academy of Sciences 100(13):8007–8012 Nokhrina K, Ray H, Bock C, Georges F (2014) Metabolomic shifts in Brassica napus lines with enhanced BnPLC2 expression impact their response to low temperature stress and plant pathogens. GM Crops Food 5:120–131. https://doi.org/10.4161/gmcr.28942 Nyyssölä A, Kerovuo J, Kaukinen P, von Weymarn N, Reinikainen T (2000) Extreme Halophiles Synthesize Betaine from Glycine by Methylation. Journal of Biological Chemistry 275(29):22196–22201 Patra B, Ray S, Richter A, Majumder AL (2010) Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol. Protoplasma 245:143–152. https://doi.org/10.1007/ s00709-010-0163-3 Pereira CS, Lins RD, Chandrasekhar I, Freitas LCG, Hu PH (2004) Interaction of the disaccharide trehalose with a phospholipid bilayer: a molecular dynamics study. Biophys J 86(4):2273– 2285. https://doi.org/10.1056/NEJM198802183180705 Peremarti A, Bassie L, Christou P, Capell T (2009) Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing Datura stramonium S-adenosylmethionine decarboxylase. Plant Mol Biol 70:253–264. https://doi.org/10.1007/ s11103-009-9470-5 Provenzano M, Mocellin S (2007) Complementary techniques: validation of gene expression data by quantitative real time PCR. In: Microarray technology and cancer gene profiling. Springer, pp 66–73. https://doi.org/10.1007/978-0-387-39978-2_7 Rajaeian S, Ehsanpour AA, Javadi M, Shojaee B (2017) Ethanolamine induced modification in glycine betaine and proline metabolism in Nicotiana rustica under salt stress. Biol Plant 61:797–800. https://doi.org/10.1007/s10535-017-0704-0 Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 37

Reddy PS, Jogeswar G, Rasineni GK, Maheswari M, Reddy AR, Varshney RK, Kishor PK (2015) Proline over-accumulation alleviates salt stress and protects photosynthetic and antioxidant enzyme activities in transgenic sorghum [Sorghum bicolor (L.) Moench]. Plant Physiol Biochem 94:104–113. https://doi.org/10.1016/j.plaphy.2015.05.014 Ren XW, Yu DW, Yang SP, Gai JY, Zhu YL (2018) Effects of StP5CS gene overexpression on nodulation and nitrogen fixation of vegetable soybean under salt stress conditions. Legume Res 41:675–680. https://doi.org/10.18805/LR-386 Ribeiro PR, Zanotti RF, Deflers C, Fernandez LG, de Castro RD, Ligterink W, Hilhorst HWM (2015) Effect of temperature on biomass allocation in seedlings of two contrasting genotypes of the oilseed crop Ricinus communis. J Plant Physiol 185:31–39. https://doi.org/10.1016/j. jplph.2015.07.005 Rickes LN, Klumb EK, Benitez LC, Jacira E, Braga B (2019) Differential expression of the genes involved in responses to water-deficit stress in peach trees cv. Chimarrita grafted onto two different rootstocks. Bragantia 78(1):60–70. https://doi.org/10.1590/1678-4499.2017372 Sagor GHM, Berberich T, Takahashi Y, Niitsu M, Kusano T (2013) The polyamine spermine protects Arabidopsis from heat stress-induced damage by increasing expression of heat shock- related genes. Transgenic Res 22:595–605. https://doi.org/10.1007/s11248-012-9666-3 Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang X, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010) Genome sequence of the palaeopolyploid soybean. Nature 463(7278):178–183 Saibi W, Feki K, Ben Mahmoud R, Brini F (2015) Durum wheat dehydrin (DHN-5) confers salinity tolerance to transgenic Arabidopsis plants through the regulation of proline metabolism and ROS scavenging system. Planta 242:1187–1194. https://doi.org/10.1007/s00425-015-2351-z Slama I, Abdelly C, Bouchereau A, Flowers T, Savouré A (2015) Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress. Annals of Botany 115(3):433–447 Sankoff D (2001) Gene and genome duplication. Current Opinion in Genetics & Development 11 (6):681–684 Saurabh S, Vidyarthi AS, Prasad D (2014) RNA interference: Concept to reality in crop improvement. Planta 239:543–564. https://doi.org/10.1007/s00425-013-2019-5 Saxena SC, Salvi P, Kaur H, Verma P, Petla BP, Rao V, Kamble N, Majee M (2013) Differentially expressed myo-inositol monophosphatase gene (CaIMP) in chickpea (Cicer arietinum L.) encodes a lithium-sensitive phosphatase enzyme with broad substrate specificity and improves seed germination and seedling growth under abiotic stresses. J Exp Bot 64:5623–5639. https:// doi.org/10.1093/jxb/ert336 Singh A, Jindal S, Longchar B, Khan F, Gupta V (2015) Overexpression of Artemisia annua sterol C-4 methyl oxidase gene, AaSMO1, enhances total sterols and improves tolerance to dehydration stress in tobacco. Plant Cell Tissue Organ Cult 121:167–181. https://doi.org/10.1007/ s11240-014-0692-0 Song C, Chung WS, Lim CO (2016) Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis. Mol Cells 39:477–483. https://doi.org/10.14348/molcells.2016.0027 Song J, Zhang R, Yue D, Chen X, Guo Z, Cheng C, Hu M, Zhang J, Zhang K (2018) Co-expression of ApGSMT2g and ApDMT2g in cotton enhances salt tolerance and increases seed cotton yield in saline fields. Plant Sci 274:369–382. https://doi.org/10.1016/j.plantsci.2018.06.007 Sun X, Xu L, Wang Y, Luo X, Zhu X, Kinuthia KB, Nie S, Feng H, Li C, Liu L (2016) Transcriptome-based gene expression profiling identifies differentially expressed genes critical for salt stress response in radish (Raphanus sativus L.). Plant Cell Rep 35:329–346. https://doi. org/10.1007/s00299-015-1887-5 38 É. A. Kido et al.

Sun Y, Fu L, Chen L, Wang X, Song Y, Li Z (2017) Characterization of two winter wheat varieties’ responses to freezing in a frigid region of the People’s Republic of China. Can J Plant Sci 97:808–815. https://doi.org/10.1139/cjps-2016-0208 Szabados L, Savoure A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97. https://doi.org/10.1016/j.tplants.2009.11.009 Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K (2002) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J 29:417–426. https://doi. org/10.1046/j.0960-7412.2001.01227.x Takabe T, Rai V, Hibino T (2006) Metabolic engineering of glycinebetaine. In: Abiotic stress toler plants. Springer, pp 137–151. https://doi.org/10.1007/1-4020-4389-9_9 Tan J, Wang C, Xiang B, Han R, Guo Z (2013) Hydrogen peroxide and nitric oxide mediated cold-and dehydration-induced myo-inositol phosphate synthase that confers multiple resistances to abiotic stresses. Plant Cell Environ 36(2):288–299. https://doi. org/10.1111/j.1365-3040.2012.02573.x Tang W, Sun J, Liu J, Liu F, Yan J (2014) RNAi-directed downregulation of betaine aldehyde dehydrogenase 1 (OsBADH1) results in decreased stress tolerance and increased oxidative markers without affecting glycine betaine biosynthesis in rice (Oryza sativa). Plant Molecular Biology 86(4-5):443–454 Tiwari LD, Mittal D, Mishra RC, Grover A (2015) Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants. Plant Physiol Biochem 92:48–55. https://doi. org/10.1016/j.plaphy.2015.03.012 Tsutsumi K, Yamada N, Cha-um S, Tanaka Y, Takabe T (2015) Differential accumulation of glycinebetaine and choline monooxygenase in bladder hairs and lamina leaves of Atriplex gmelinii under high salinity. J Plant Physiol 176:101–107. https://doi.org/10.1016/j.jplph.2014.12.009 Vaishnav A, Choudhary DK (2018) Regulation of Drought-Responsive Gene Expression in Glycine max L. merrill is mediated through Pseudomonas simiae strain AU. J Plant Growth Regul 38(1):333–342. https://doi.org/10.1007/s00344-018-9846-3 Wang T, Lu L, Zhang C, Taylor C, Thompson JE (2003) Pleiotropic effects of suppressing deoxyhypusine synthase expression in Arabidopsis thaliana. Plant Molecular Biology 52(6):1223–1235 Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics 10(1):57–63 Wang J, Sun P, Chen C, Wang Y, Fu X, Liu J (2011) An arginine decarboxylase gene PtADC from Poncirus trifoliata confers abiotic stress tolerance and promotes primary root growth in Arabidopsis. Journal of Experimental Botany 62(8):2899–2914 Wang CL, Zhang SC, Qi SD, Zheng CC, Wu CA (2016a) Delayed germination of Arabidopsis seeds under chilling stress by overexpressing an abiotic stress inducible GhTPS11. Gene 575:206–212. https://doi.org/10.1016/j.gene.2015.08.056 Wang FW, Wang ML, Guo C, Wang N, Li XW, Chen H, Dong YY, Chen XF, Wang ZM, Li HY (2016b) Cloning and characterization of a novel betaine aldehyde dehydrogenase gene from Suaeda corniculata. Genetics and Molecular Research 15(2) Wang J, Lan X, Jiang S, Ma Y, Zhang S, Li Y, Li X, Lan H (2017) CaMKK1 from Chenopodium album positively regulates salt and drought tolerance in transgenic tobacco. Plant Cell Tissue Organ Cult 130:209–225. https://doi.org/10.1007/s11240-017-1216-5 Wang J-Y, Lai L-D, Tong S-M, Li Q-L (2013) Constitutive and salt-inducible expression of SlBADH gene in transgenic tomato (Solanum lycopersicum L. cv. Micro-Tom) enhances salt tolerance. Biochem Biophys Res Commun 432:262–267. https://doi.org/10.1016/j.bbrc.2013.02.001 Wang Q, Xu W, Xue Q, Su W (2010) Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tolerance to extreme temperature and high salinity. J Zhejiang Univ Sci B 11:851–861. https://doi.org/10.1631/jzus.b1000137 Wang W, Liu JH (2016c) CsPAO4 of Citrus sinensis functions in polyamine terminal catabolism and inhibits plant growth under salt stress. Sci Rep 6:1–15. https://doi.org/10.1038/srep31384 Osmoprotectant-Related Genes in Plants Under Abiotic Stress: Expression Dynamics… 39

Wang X, Du Y, Yu D (2018) Trehalose phosphate synthase 5-dependent trehalose metabolism modulates basal defense responses in Arabidopsis thaliana. J Integrat Plant Biol 61:509. https://doi.org/10.1111/jipb.12704 Wei C, Cui Q, Zhang XQ, Zhao YQ, Jia GX (2016) Three P5CS genes including a novel one from Lilium regale play distinct roles in osmotic, drought and salt stress tolerance. J Plant Biol 59:456–466. https://doi.org/10.1007/s12374-016-0189-y Wei D, Zhang W, Wang C, Meng Q, Li G, Chen THH, Yang X (2017) Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants. Elsevier Ireland Ltd. Plant Sci 257:74–83 Wei JZ, Jerry Chatterton N, Larson SR (2001) Expression of sucrose:fructan 6-fructosyltransferase (6-SFT) and myo-inositol 1-phosphate synthase (MIPS) genes in barley (Hordeum vulgare) leaves. J Plant Physiol 158:635–643. https://doi.org/10.1078/0176-1617-00308 Wen XP, Ban Y, Inoue H, Matsuda N, Moriguchi T (2010) Spermidine levels are implicated in heavy metal tolerance in a spermidine synthase overexpressing transgenic european pear by exerting antioxidant activities. Transgenic Res 19:91–103. https://doi.org/10.1007/s11248-009-9296-6 Wu H, Zhang Y, Zhang W, Pei X, Zhang C, Jia S, Li W (2015) Transcriptomic analysis of the primary roots of Alhagi sparsifolia in response to water stress. PLoS One 10:1–25. https://doi. org/10.1371/journal.pone.0120791 Wu S, Su Q, An L (2010) Isolation of choline monooxygenase (CMO) gene from Salicornia europaea and enhanced salt tolerance of transgenic tobacco with CMO genes. Indian J Biochem Biophys 47:298–305 Xu Z, Sun M, Jiang X, Sun H, Dang X, Cong H, Qiao F (2018) Glycinebetaine Biosynthesis in response to osmotic stress depends on jasmonate signaling in watermelon suspension cells. Front Plant Sci 9:1–14. https://doi.org/10.3389/fpls.2018.01469 Yadav R, Verma OP, Padaria JC (2018) Transcript profiling and gene expression analysis under drought stress in Ziziphus nummularia (Burm.f.) Wright & Arn. Mol Biol Rep 45:163–174. https://doi.org/10.1007/s11033-018-4149-0 Yamada N, Takahashi H, Kitou K, Sahashi K, Tamagake H, Tanaka Y, Takabe T (2015) Suppressed expression of choline monooxygenase in sugar beet on the accumulation of glycine betaine. Plant Physiol Biochem 96:217–221. https://doi.org/10.1016/j.plaphy.2015.06.014 Yang C, Zhou Y, Fan J, Fu Y, Shen L, Yao Y, Li R, Fu S, Duran R, Hu X, Guo J (2015a) SpBADH of the halophyte Sesuvium portulacastrum strongly confers drought tolerance through ROS scavenging in transgenic Arabidopsis. Plant Physiol Biochem 96:377–387. https://doi. org/10.1016/j.plaphy.2015.08.010 Yang SL, Chen K, Wang SS, Gong M (2015b) Osmoregulation as a key factor in drought hardening- induced drought tolerance in Jatropha curcas. Biol Plant 59:529–536. https://doi.org/10.1007/ s10535-015-0509-y Yang SL, Lan SS, Deng FF, Gong M (2016) Effects of calcium and calmodulin antagonists on chilling stress-induced proline accumulation in Jatropha curcas L. J Plant Growth Regul 35:815–826. https://doi.org/10.1007/s00344-016-9584-3 Yang Y, Li X, Kong X, Ma L, Hu X, Yang Y (2015c) Transcriptome analysis reveals diversified adaptation of Stipa purpurea along a drought gradient on the Tibetan Plateau. Funct Integr Genomics 15:295–307. https://doi.org/10.1007/s10142-014-0419-7 Yooyongwech S, Samphumphuang T, Tisarum R, Theerawitaya C, Cha-um S (2017) Water-deficit tolerance in sweet potato [Ipomoea batatas (L.) Lam.] by foliar application of paclobutra- zol: role of soluble sugar and free proline. Front Plant Sci 8:1–13. https://doi.org/10.3389/ fpls.2017.01400 Yu C, Qiao G, Qiu W, Yu D, Zhou S, Shen Y, Yu G, Jiang J, Han X, Liu M, Zhang L, Chen F, Chen Y, Zhuo R (2018) Molecular breeding of water lily: engineering cold stress tolerance into tropical water lily. Horticulture Research 5(1) Zhai SM, Gao Q, Xue HW, Sui ZH, Yue GD, Yang AF, Zhang JR (2012) Overexpression of the phosphatidylinositol synthase gene from Zea mays in tobacco plants alters the membrane lipids composition and improves drought stress tolerance. Planta 235(1):69–84 40 É. A. Kido et al.

Zhai H, Wang F, Si Z, Huo J, Xing L, An Y, He S, Liu Q (2015) A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato. Plant Biotechnol J 14:592–602. https://doi.org/10.1111/pbi.12402 Zhang J, Yang N, Li Y, Zhu S, Zhang S, Sun Y, Zhang HX, Wang L, Su H (2018) Overexpression of PeMIPS1 confers tolerance to salt and copper stresses by scavenging reactive oxygen species in transgenic poplar. Tree Physiol 38:1566–1577. https://doi.org/10.1093/treephys/tpy028 Zhang RX, Qin LJ, Zhao DG (2017) Overexpression of the OsIMP gene increases the accumulation of inositol and confers enhanced cold tolerance in tobacco through modulation of the antioxidant enzymes’ activities. Genes (Basel) (7):8. https://doi.org/10.3390/genes8070179 Zhu J-Q, Zhang J-T, Tang R-J, Lv Q-D, Wang Q-Q, Yang L, Zhang H-X (2009) Molecular characterization of ThIPK2, an inositol polyphosphate kinase gene homolog from Thellungiella halophila, and its heterologous expression to improve abiotic stress tolerance in Brassica napus. Physiol Plant 136:407–425. https://doi.org/10.1111/j.1399-3054.2009.01235.x Proline Metabolism and Its Functions in Development and Stress Tolerance

Maurizio Trovato, Giuseppe Forlani, Santiago Signorelli, and Dietmar Funck

1 Introduction

Most green plants are fully autotrophic organisms and can produce their entire biomass from inorganic molecules with the help of light energy captured by photosynthesis. Energy from photosynthesis is thereby not only needed to reduce CO2 to carbohydrates but also to assimilate nitrogen, phosphorus, and sulfur from inorganic salts for the biosynthesis of proteins and nucleic acids (Buchanan et al. 2000; Taiz et al. 2018). In contrast, most non-photosynthetic organisms, including animals and humans, depend on the uptake of organic material both as energy source and as building material (Hill et al. 2016). This fundamental difference exists since the development of oxygenic photosynthesis by cyanobacteria, which were later converted to endosymbiotic chloroplasts in eukaryotic algae and plants (Nozaki 2005; Zimorski et al. 2014). It is therefore not surprising that despite the use of identical building blocks in all living organisms, i.e., nucleotides, amino acids, and carbohydrates, the pathways to acquire or synthesize these building blocks are not identical in distantly related groups of organisms. Knowledge about human metabolism can therefore only serve to guide investigations of regulatory and metabolic pathways in plant primary metabolism but not as a direct template.

M. Trovato (*) Department of Biology and Biotechnology, Sapienza University of Rome, Rome, Italy e-mail: [email protected] G. Forlani Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy S. Signorelli Department of Plant Biology, Universidad de la República, Montevideo, Uruguay D. Funck (*) Department of Biology, Division of Plant Physiology and Biochemistry, University of Konstanz, Konstanz, Germany e-mail: [email protected]

An additional layer of complexity is added by the fact that many primary metabolites are used for additional specific purposes in plants. This and the following two chapters focus on the amino acid proline, which is an essential constituent of most proteins but serves additionally as a compatible solute with important functions in stress defense, as a signaling molecule, and as a precursor for secondary metabolites in some plant species. The functions and regulation of proline metabolism and accumulation in stress defense are summarized in detail in the following chapter, while other chapters of this book will specifically focus on the physiological function and agronomic potential of proline uptake from external sources (Chaps. 4 and 9) and on the role of proline as a signaling molecule in stress adaptation (Chap. 11). The present chapter summarizes the current knowledge about the biochemical pathways of proline metabolism and its functions in regulating plant development and physiology both in the absence or presence of stress.

2 Proline Biosynthesis and Degradation: Enzymes and Their Subcellular Localization

The concentration of free proline in a plant cell is determined largely by four metabolic processes, namely, biosynthesis and degradation of proline as well as consumption of proline for protein biosynthesis and release of proline during protein degradation (Hildebrandt 2018). Additionally, the distribution of proline among different sub-compartments of the cell is not uniform and proline is distributed within the plant by long-distance transport along vascular bundles and locally by transport across the plasma membrane or through plasmodesmata. This section will focus on the anabolic and catabolic enzymes and their subcellular localization, whereas the next section will integrate this information with the known transport routes to establish metabolic pathways.

2.1 Proline Biosynthetic Enzymes

In prokaryotes, three enzymes have been identified that can synthesize proline: ornithine cyclodeaminase (OCD), pyrroline-2-carboxylate reductase (P2CR), and pyrroline-5-carboxylate reductase (P5CR) (Fig. 1). OCD uses NAD+ as a cofactor during the cyclization and subsequent deamination of ornithine. In the proposed reaction mechanism, NAD+ is transiently reduced and later re-oxidized during enzyme regeneration and proline release (Goodman et al. 2004). OCD is encoded by rolD on the transfer DNA (T-DNA) of Rhizobium rhizogenes (better known under its traditional name Agrobacterium rhizogenes) and an integrated copy of such a T-DNA has been found in genomic DNA of Catharanthus roseus (GenBank accession DQ852612). Endogenous proteins with homology to OCD can be Proline Metabolism and Its Functions in Development and Stress Tolerance 43

Fig. 1 Substrates, enzymes, and cofactors of proline metabolism. Blue and red circles indicate cytosolic and mitochondrial enzymes, respectively. The yellow color of OCD indicates the prokaryotic origin of this protein. Proline is depicted in its super proline dress to emphasize its many important functions in adaptation, defense, and development of plants. GSA glutamate-5- semialdehyde, OAT ornithine-δ-aminotransferase, OCD ornithine cyclodeaminase, P2C pyrroline- 2-carboxylate, P2CR P2C reductase, P5C pyrroline-5-carboxylate, P5CDH P5C dehydrogenase, P5CR P5C reductase, P5CS P5C synthetase, PLP pyridoxal phosphate, ProDH proline dehydrogenase identified in plant genomes, but an in-depth analysis of the Arabidopsis OCD homolog (At5g52810) did not yield any evidence of OCD activity or any other function in proline biosynthesis (Sharma et al. 2013). Pyrroline-5-carboxylate (P5C) and pyrroline-2-carboxylate (P2C) are reduced to proline by P5CR or P2CR, respectively, under consumption of NAD(P)H (Fichman et al. 2015). Bacterial P2CRs often have dual specificities for the conversion of P2C to proline orΔ 1-piperidine- 2-carboxylate to pipecolate and function in trans-3-hydroxy-L-proline degradation or pipecolate biosynthesis (Watanabe et al. 2014). Early biochemical studies reported or postulated P2CR activity in few plant species (Meister et al. 1957; Mestichelli et al. 1979), and in several plant genomes, hypothetical P2CRs are annotated. However, the molecular identification of a plant P2CR has not been reported so far and the hypothetical P2CRs do not align unambiguously to characterized P2CRs. Therefore, P5C is at present the only confirmed precursor for proline biosynthesis in plants. 44 M. Trovato et al.

P5CR has been cloned or purified from a number of plant species and was found to form large homo-oligomers (Delauney and Verma 1990; Forlani et al. 2015; Funck et al. 2012; Ma et al. 2008; Murahama et al. 2001; Ruszkowski et al. 2015). The first crystal structure of P5CR from rice Oryza( sativa) revealed a decameric structure consisting of a ring of five dimers, which is in agreement with most molecular mass estimates for P5CR from other plant species (Forlani et al. 2015). P5CR can use both NADH and NADPH to reduce P5C to proline, and in the absence of NAD(P)+, higher turnover rates were obtained with NADH. However, the affinity of P5CR for NADPH is much higher and even low concentrations of NADP+ inhibited the reaction of Arabidopsis (Arabidopsis thaliana) P5CR with NADH (Giberti et al. 2014). At physiological pH values, the reaction is nearly unidirectional toward proline formation, while at high pH (>9) also the reverse reaction can be detected, because P5C is highly labile at elevated pH (Rena and Splittstoesser 1975) (unpublished data by G. Forlani). Unfortunately, proline-dependent formation of NAD(P) H by soluble plant extracts at high pH is often erroneously interpreted as ProDH activity (see below). Most plant species contain a single P5CR gene, and in Arabidopsis, a P5CR:GFP fusion protein was detected exclusively in the cytosol (Funck et al. 2012). After cell fractionation, the major part of the P5CR activity was detected in the soluble protein fraction in many plant species, whereas some authors also reported P5CR activity in chloroplast-enriched fractions (Murahama et al. 2001; Noguchi et al. 1966; Rayapati et al. 1989). It remains to be clarified if P5CR, which lacks a conserved chloroplast transit peptide in all analyzed genomes, can be imported into plastids by an unconventional mechanism or can be partially attached to plastids during isolation. P5C is formed nonenzymatically by cyclization of glutamate-5-semialdehyde (GSA), which is an equilibrium reaction in aqueous solution. Two plant enzymes are known to produce GSA: ornithine-δ-aminotransferase (OAT) and P5C synthetase (P5CS). The first plantOAT gene was isolated by trans-complementation of an Escherichia coli strain unable to synthesize P5C with a cDNA clone from Vigna aconitifolia (Delauney et al. 1993). OAT is localized in the mitochondria and uses pyridoxal phosphate as cofactor in the transfer of the δ-amino group of ornithine to α-ketoglutarate, yielding GSA and glutamate (Funck et al. 2008; Roosens et al. 1998; Stránská et al. 2008). For mammalian OAT, it has been shown that it also catalyzes the reverse reaction in certain tissues, although the chemical equilibrium is far to the side of GSA and glutamate (Strecker 1965). The second enzyme, P5CS, reduces glutamate to GSA in a two-step reaction consuming ATP and NADPH. All land plants and animals have bifunctional P5CS enzymes, whereas prokaryotes and some unicellular green algae and fungi have separate γ-glutamyl kinase and glutamyl-γ-phosphate reductase enzymes (Fichman et al. 2015; Hu et al. 1992; Zhang et al. 1995). A previous report describing the occurrence of prokaryote-like γ-glutamyl kinase and glutamyl-γ-phosphate reductase genes in tomato (Solanum lycopersicum) was most likely an artifact, because no such genes are present in genomic sequences of tomato (Fujita et al. 1998). In the initial reaction, the γ-glutamyl kinase domain of P5CS uses ATP to phosphorylate the γ-carboxy group of glutamate, and in the second reaction, glutamyl-γ-phosphate Proline Metabolism and Its Functions in Development and Stress Tolerance 45 is reduced to GSA under consumption of NADPH. The γ-glutamyl kinase activity is inhibited by millimolar concentrations of proline and mutations identified in bacterial enzymes were used to engineer feedback-insensitive variants of P5CS in plants (Hu et al. 1992; Zhang et al. 1995). The first plantP5CS gene was isolated by complementation of a proline synthesis-deficientE. coli strain (Hu et al. 1992). Most plant species have at least two P5CS isoforms, of which one can be regarded as a housekeeping gene, while others are induced by stress to enable proline accumulation (Kim and Nam 2013; Signorelli and Monza 2017; Székely et al. 2008; Turchetto-Zolet et al. 2009; Wang et al. 2014). Initial characterization of the Arabidopsis P5CS proteins by GFP fusion indicated that both isoforms are cytosolic in non-stressed plants but may be imported into plastids upon osmotic stress (Székely et al. 2008), whereas our own data indicate exclusive cytosolic localization (Funck et al. 2019). Similarly, GFP fusions of two out of three P5CS isoforms from Medicago truncatula co-localized with the small subunit of ribulose bisphosphate carboxylase/oxygenase in root hairs, but like in the Arabidopsis P5CS sequences, no typical chloroplast transit peptides are present in the protein sequences (Kim and Nam 2013). By cell fractionation and Western blot, corn (Zea mays) P5CS2 was detected exclusively in the cytosol and not in the organelle fraction (Wang et al. 2014). Because glutamate, ATP, and NADPH can be used as substrates by many different enzymes (e.g., glutamine synthetase or P5C dehydrogenase, see below), a specific assay of P5CS activity in crude plant or organelle extracts has not been reported so far, and thus the subcellular localization awaits biochemical confirmation.

2.2 Proline Degradation Enzymes

For the degradation of excess proline that is not used for protein synthesis or as compatible solute, also a single enzyme is known in plants. Proline dehydrogenase (ProDH), previously also referred to as proline oxidase, is an FAD-containing enzyme at the inner mitochondrial membrane, which oxidizes proline back to P5C while transferring the obtained electrons to the mitochondrial electron transport chain and thus fueling respiratory ATP production (Elthon and Stewart 1981; Huang and Cavalieri 1979; Schertl et al. 2014). Structural studies of Put1, the ProDH of baker’s yeast (Saccharomyces cerevisiae), strongly indicate that the electrons are transferred via the tightly bound FAD cofactor to ubiquinone (Moxley et al. 2017; Wanduragala et al. 2010). A ProDH gene from Arabidopsis has been independently identified by homology searches and by screening for genes that respond rapidly to changes in the water status (Kiyosue et al. 1996; Peng et al. 1996; Verbruggen et al. 1996). Many plant genomes contain a single ProDH gene, while an early genome duplication in the Brassicaceae led to two isoforms in Arabidopsis with further multiplications occurring in Brassica species (Faes et al. 2015; Funck et al. 2010; Mani et al. 2002). ProDH activity has been exclusively detected in mitochondria unless the reverse reaction of P5CR at high pH was erroneously assigned to ProDH 46 M. Trovato et al.

(see above; (Huang and Cavalieri 1979; Schertl et al. 2014). C-terminal GFP fusion proteins of both Arabidopsis ProDH isoforms were targeted to the mitochondria in stably transformed plants, whereas a transient transformation assay provided evidence for chloroplast targeting of ProDH2 (Funck et al. 2010; Van Aken et al. 2009). P5C produced by ProDH has three potential fates: It can be converted to proline by P5CR, or it can be linearized to GSA and be converted to ornithine by OAT or to glutamate by P5C dehydrogenase (P5CDH, recently suggested to be renamed as glutamate semialdehyde dehydrogenase, GSALDH, to better reflect the actual substrate and the evolutionary relationship to the aldehyde dehydrogenase family (Tanner 2019)). The latter is the last metabolic enzyme that is discussed here in detail. P5CDH activity was first characterized in corn mitochondria, and 20 years later the firstP5CDH gene from Arabidopsis was identified by functional cloning (Deuschle et al. 2001; Elthon and Stewart 1981). In most annotated plant genomes, P5CDH is a single-copy gene except for polyploid species (Ayliffe et al. 2005; Deuschle et al. 2001; Korasick et al. 2019). Biochemical analyses of isolated corn mitochondria provided evidence for two P5CDH isoforms with distinct pH optima (Elthon and Stewart 1982), and for the single-copy P5CDH gene in the Zea mays genome, several predicted splicing variants are annotated (NCBI gene ID: 100193220). P5CDH is a soluble enzyme in the mitochondrial matrix and prefers NAD+ over NADP+ as electron acceptor during the oxidation of GSA to glutamate (Forlani et al. 1997). In many bacteria, ProDH and P5CDH activities are combined in a single enzyme that allows direct substrate channeling, and recently it was reported that also the two plant enzymes might be physically linked through interaction with the inhibitory protein DROUGHT AND FREEZING RESPONSIVE GENE 1 (DFR1; (Ren et al. 2018). Especially in fragrant rice but also in some other plant species, proline and P5C were also identified as potential precursors for the production of 2-acetyl-1- pyrroline, the main constituent of the typical flavor (Wakte et al.2017 ; Yoshihashi et al. 2002). However, a recent metabolomic and genomic study in rice proposed putrescine-derived 4-aminobutanal as immediate precursor for 2-acetyl-1-pyrroline and challenged the direct involvement of proline or P5C (Daygon et al. 2017).

3 Proline Transport and Metabolic Pathways

Proline is most likely synthesized exclusively in the cytosol but is needed for protein biosynthesis also in mitochondria and plastids. In leaves of osmotically stressed potato (Solanum tuberosum) plants, the highest concentrations of proline were reported for chloroplasts (Büssis and Heineke 1998). Additionally, a substantial part of protein degradation occurs in the vacuole, and proline degradation takes place in mitochondria. After release from stress, the high concentration of proline rapidly decreases, primarily by ProDH- and P5CDH-dependent degradation (Deuschle et al. 2004; Nanjo et al. 1999b). Therefore, efficient transport proteins for proline must exist in most intracellular membranes, but their molecular identity is only beginning to be revealed. Proline Metabolism and Its Functions in Development and Stress Tolerance 47

3.1 Intracellular Proline Transport

Isolated mitochondria from monocot seedlings can use proline and P5C/GSA as substrates for respiratory O2 consumption, but in Arabidopsis mitochondria proline- dependent respiration was only detected when the expression of ProDH1 was stimulated by proline treatment prior to the isolation of mitochondria (Boggess et al. 1978; Cabassa-Hourton et al. 2016; Elthon and Stewart 1982). No carriers for proline or P5C/GSA in mitochondria have been molecularly identified so far. However, biochemical analyses provided evidence that the import of proline into mitochondria is dependent on a proton gradient and at least two transporters, a proline uniporter and a proline/glutamate antiporter, are present in the inner mitochondrial membrane (Di Martino et al. 2006; Elthon et al. 1984). Recently, several members of the mitochondrial carrier family (MCF) were shown to mediate glutamate transport, but no evidence for proline transport activity has been obtained yet (Monne et al. 2018; Porcelli et al. 2018). Even less data is available on amino acid transport across the chloroplast membranes, where so far only a malate/glutamate antiporter (DiT2 in the inner envelope) and a transporter for neutral amino acids in the outer envelope (OEP16) have been characterized (Pohlmeyer et al. 1997; Renné et al. 2003). In the vacuolar membrane of yeast, the family of AMINO ACID VACUOLAR TRANSPORTERS (AVT) has been characterized, and recently it was shown that Arabidopsis homologues AVT3A and AVT3C complement the defects of avt3/avt4 double mutant yeast cells (Fujiki et al. 2017). These proteins are localized in the vacuolar membrane in Arabidopsis and functional studies suggested that they mediate the ATP-dependent export of several amino acids, including proline, from the vacuole. This suggestion is in agreement with evidence from potato showing that proline concentration in the cytosol can be 260-fold greater than in the vacuole, indicating the presence of an active transport system (Büssis and Heineke 1998).

3.2 Pathways for Proline Biosynthesis and Degradation

The lack of information about the activity and specificity of transport proteins in intracellular membranes makes it difficult to draw definite conclusions about the metabolic pathways of proline biosynthesis and degradation that occur in vivo. Biosynthesis of proline from glutamate by the sequential action of P5CS and P5CR appears to be the predominant pathway, especially for stress-induced proline accumulation. Accordingly, both P5CS and P5CR are essential genes in Arabidopsis, and double mutations in P5CS1 and P5CS2 are gametophytic lethal, as no fertile p5cs1/p5cs2 mutant pollen is formed, while homozygous p5cr mutant embryos were observed but aborted at a very early developmental stage (Funck et al. 2010; Mattioli et al. 2012). These observations demonstrate that no other pathway can produce sufficient amounts of proline for successful sexual reproduction. An alternative pathway of proline biosynthesis from ornithine has been assumed based on radiotracer studies, co-expression analyses, and analogy to mammals (da Rocha 48 M. Trovato et al. et al. 2012; Mestichelli et al. 1979; Roosens et al. 1998). To actually bypass P5CS activity, this pathway depends on export of GSA/P5C produced from ornithine by OAT in mitochondria, which was detectable in isolated corn mitochondria but is difficult to assess in vivo due to the high reactivity and inherent instability of GSA/ P5C at neutral pH (Elthon and Stewart 1982; Mezl and Knox 1976). When mitochondria were incubated with proline, the production of glutamate was two orders of magnitude higher than GSA/P5C production, and also in Arabidopsis plants treated with external proline, GSA/P5C content stayed below the detection limit of 50 nmol/g fresh weight unless p5cdh mutants were used (Boggess et al. 1978; Deuschle et al. 2004). Much higher GSA/P5C contents were reported in a study using plants overexpressing ProDH and, together with unchanged GSA/P5C to proline ratios, were interpreted as evidence for a proline-GSA/P5C cycle between the cytosol and mitochondria (Miller et al. 2009). However, Miller et al. (2009) did not provide evidence that the employed color reaction is specific for GSA/P5C in crude plant extracts and the production of glutamate by both OAT and P5CDH makes it difficult to exclude that ornithine only stimulates the P5CS-dependent pathway of proline biosynthesis. Similarly, there is at present no evidence that proline degradation might yield ornithine instead of glutamate in plants, as it has been proposed for certain mammalian tissues (Ginguay et al. 2017).

3.3 Intercellular Proline Transport

In contrast to organellar proline transporters, which remain elusive so far, numerous proline transporters were identified that are localized in the plasma membrane. Several members of the amino acid/auxin permease (AAAP) family mediate amino acid-proton symport (Dinkeloo et al. 2018). Among these, members of the amino acid permease (AAP) and lysine histidine transporter (LHT) subfamilies transport proline along with a rather broad range of both neutral and charged amino acids (Fischer et al. 1995; Hirner et al. 2006). Members of the proline transporter (ProT) subfamily have rather narrow substrate specificity and transport proline, glycine betaine, or γ-aminobutyric acid (GABA) (Lehmann et al. 2011). A recent analysis of Arabidopsis aap1 mutants suggested that AAP1 could contribute to the uptake of proline from the growth substrate (Perchlik et al. 2014; Wang et al. 2017). However, little is known about the concentrations of free proline in natural soils and its relevance for plant nutrition or communication, indicating that the major function of AAPs and ProTs is the redistribution of proline within the plant. Amino acids are transported in both the xylem and the phloem, and several members of the AAP family were shown to contribute to phloem loading in source leaves or retrieval of amino acids along the transport route (Tegeder and Hammes 2018). Apoplastic phloem loading requires that amino acids are released by source cells into the intercellular space and also loading of the dead xylem vessels in roots requires export of amino acids and other solutes by the surrounding living cells. In 2012, the SILIQUES ARE RED 1 (SIAR1/UMAMIT18) protein was identified in Arabidopsis as the first transporter that can mediate bidirectional amino acid transport, depending on the Proline Metabolism and Its Functions in Development and Stress Tolerance 49 electrochemical gradient across the membrane (Ladwig et al. 2012). SIAR1 is part of a protein family with 44 members in Arabidopsis, of which several members have been characterized in the meantime as broad specificity amino acid exporters and which were therefore named “usually multiple amino acids move in and out transporters” (UMAMITs) (Besnard et al. 2016, 2018; Müller et al. 2015). Also, plasmodesmata are a possible route for amino acid transport between connected cells, but to our knowledge, no specific transport mechanisms have been described so far.

4 Proline Biosynthesis and Degradation: Spatial and Temporal Regulation

The diverse functions of proline metabolism, which must sustain the variable requirements of protein synthesis, while playing multiple additional physiological functions and responding to environmental and biotic stimuli, are reflected in, and derived from, an elaborate network of gene and enzyme activity regulation (Fig. 2). Most of the known regulatory mechanisms for proline metabolism operate at the transcriptional level and appear to distinguish between stress and normal physiological conditions. Key to this distinction is the capability to detect and respond to different inputs via several signaling pathways that result in expression or activation of specific transcription factors (TFs). Accordingly, the promoters of all genes coding for proline metabolic enzymes that were characterized so far are especially rich in confirmed or predicted TF recognition elements (Fichman et al. 2015; Zarattini and Forlani 2017). Further regulatory mechanisms were found to act epigenetically or posttranscriptionally on gene expression, or allosterically on enzyme activities. Far less is known about posttranslational modifications or regulated degradation of proline metabolic enzymes and transporters. Most of the knowledge about regulatory mechanisms of proline metabolism has been obtained by analysis of Arabidopsis wild-type plants or mutants. As indicated above, duplications and functional diversification of proline metabolic genes occurred several times independently in different plant taxa. Therefore, it is at present unknown how much of the knowledge obtained in Arabidopsis can be directly transferred to different species (Mattioli et al. 2018; Signorelli and Monza 2017; Turchetto-Zolet et al. 2009). We will focus on the knowledge gained for Arabidopsis and indicate it specifically, when data from other plants are described as well.

4.1 Regulation of Genes Coding for Proline Biosynthesis Enzymes

As discussed above, the short pathway converting glutamate into GSA and P5C into proline is the most important and probably unique route of proline synthesis in higher plants. Compelling evidence indicates that P5CS, the first enzyme of glutamate-derived proline synthesis, is under most conditions the rate-limiting 50 M. Trovato et al.

Fig. 2 The regulatory network controlling proline metabolism. Enzymes are given in blue letters and metabolic fluxes as solid black arrows. The uncertain transport of P5C/GSA across the mitochondrial membrane is indicated by a dashed arrow. Green and red lines indicate induction and repression, respectively. Lines ending at an enzyme name indicate regulation of gene expression, while lines ending at the metabolic flux indicate posttranslational regulation. For reasons of sim- plicity, low water potential and high ionic strength are depicted as a single regulatory unit, although they probably use partly independent signaling cascades. ABA abscisic acid, αKG α-ketoglutarate, BR brassinosteroids, DFR1 drought and freezing regulated gene 1, GDH glutamate dehydrogenase, Glu glutamate, GSA glutamate-5-semialdehyde, OAT ornithine-δ-aminotransferase, OCD ornithine-cyclodeaminase, Orn ornithine, P5C pyrroline-5-carboxylate, P5CDH, P5C dehydrogenase, P5CR P5C reductase, P5CS P5C synthetase, Pi Phosphate, Pro proline, ProDH proline dehydrogenase, TCA tricarboxylic acid cycle, ΨW water potential enzyme of proline synthesis in higher plants. This evidence derives from the strict correlation between P5CS expression and proline accumulation (Hu et al. 1992; Peng et al. 1996; Savouré et al. 1995; Strizhov et al. 1997; Yoshiba et al. 1999) and from the effects of P5CS overexpression (Kavi Kishor et al. 1995; Per et al. 2017) and antisense inhibition (Nanjo et al. 1999b) or knockout mutations (Mattioli et al. 2008; Székely et al. 2008). Accordingly, the overall rate of proline biosynthesis is predominantly determined by the temporal and spatial regulation of P5CS gene expression. The two Arabidopsis P5CS genes are located on chromosome 2 and 3 and share the same genomic structure with 20 exons sharing a nucleotide identity ranging Proline Metabolism and Its Functions in Development and Stress Tolerance 51 from 80% to 94%. A higher degree of difference is found in the promoter regions, the 5′ and 3′ untranslated sequences and introns, including variations in putative splicing sites, which might give rise to four different P5CS1 and two different P5CS2 transcripts included in the current annotation (ARAPORT11) of the Arabidopsis genome (https://www.Arabidopsis.org). The four different /P5CS1/ transcripts are derived from two splice variants and two alternative transcription initiation sites, while in P5CS2 a single-splice variant skipping exon 3 is annotated. Skipping of exon 3 of P5CS1 produces nonfunctional transcripts and has been experimentally confirmed as potential mechanism underlying differential drought tolerance of several natural accessions from different regions (Kesari et al. 2012). The expression of P5CS1 and P5CS2 in Arabidopsis has been analyzed by northern blot, in situ hybridization, and analysis of transgenic plants carrying promoter- GUS and promoter-gene-GFP fusion constructs (Abrahám et al. 2003; Fabro et al. 2004; Mattioli et al. 2018; Mattioli et al. 2009; Strizhov et al. 1997; Yoshiba et al. 1999). There are some minor discrepancies between the results, most likely attributable to different cultivation conditions and analysis techniques. The prevailing picture is that both isoforms have partially overlapping expression patterns, whereby P5CS1 is expressed more strongly in aboveground tissues and differentiated cells, whereas P5CS2 expression levels are highest in regions of active cell division. In flowers, bothP5CS isoforms are almost exclusively expressed in developing microspores and pollen (Mattioli et al. 2018). P5CS1 and, to a lesser extent, P5CS2 transcription is rapidly induced by drought and salt stress, with light and abscisic acid (ABA) as key inducing signals (Abrahám et al. 2003; Feng et al. 2016; Strizhov et al. 1997). An independent pathway seems to mediate induction of P5CS1 expression in response to phosphate starvation (Aleksza et al. 2017). Proline-, brassinolide-, and phospholipase-dependent signaling were identified as negative regulators of P5CS expression and probably contribute to the rapid downregulation after relief from stress (Abrahám et al. 2003; Thiery et al. 2004). Bioinformatic analyses of the promoters of P5CS1 and P5CS2 showed that the promoter of P5CS1 is enriched in putative binding sites for TFs related to abiotic stress, such as ABA response elements, AP2/EREBP, ERF2, DREB/CBF, and MYB binding sites (Fichman et al. 2015). The promoter of P5CS2, on the contrary, is enriched in putative regulatory elements for TFs related to biotic stresses such as HD-HOX, AP2/EREBP, MYB, WRKY, and bZIP (Fichman et al. 2015). Additionally, the promoter of P5CS2 contains putative binding sites for TFs related to flowering time, such asSQUAMOSA PROMOTER BINDING-LIKE (SPL) and bHLH factors, and related to pollen development and function such as WRKY2 and WRKY34 (Mattioli et al. 2018). Most studies on stress-induced or developmental accumulation of proline report good correlation between P5CS transcript levels and proline content, indicating that proline biosynthesis is predominantly regulated at the level of transcription. Early studies describing proline accumulation in tomato and grapevine in the absence of increased P5CS transcript levels can now be explained by the presence of second P5CS isoforms in these species that were unknown at the time (Fujita et al. 1998; Stines et al. 1999). More direct evidence for post-transcriptional regulation of P5CS expression was obtained by computational identification of matching micro-RNAs 52 M. Trovato et al. in potato and chickpea (Shui et al. 2013; Yang et al. 2013). Expression levels of some micro-RNAs during stress were negatively correlated with P5CS transcript levels, but direct proof for their involvement in P5CS regulation is still missing. Epigenetic regulation caused by modulations of the methylation pattern of specific genes may also contribute to P5CS regulation. Changes in DNA methylation induced by environmental stresses or by developmental stimuli are known to modulate both plant stress tolerance and developmental processes, respectively (Bastow et al. 2004; Chinnusamy and Zhu 2009; Karan et al. 2012; Richards 2006). In rice, differential methylation of a P5CS gene has been proposed as a mechanism for trans-generational stress memory (Zhang et al. 2013). As mentioned above, another level of regulation is added by allosteric inhibition of the γ-glutamyl kinase activity of plant P5CS proteins by proline (Hu et al. 1992; Zhang et al. 1995). It is so far unknown if and how feedback inhibition of P5CS may be overcome in tissues or conditions where proline accumulation is desired. Immunoblot analyses of Arabidopsis P5CS1 protein levels in different protein phosphatase 2C mutants indicated the presence of posttranslational modifications or mechanisms to regulate protein stability (Bhaskara et al. 2015). The concept of P5CS catalyzing the rate-limiting step in proline biosynthesis is contested by several studies reporting higher proline content, especially under stress conditions, upon overexpression of P5CR (De Ronde et al. 2004; Ma et al. 2008; Szoke et al. 1992). The most detailed analysis of P5CR expression has again been performed in Arabidopsis. In particular, a P5CR promoter-GUS fusion construct showed ubiquitous expression with the highest expression levels in areas of active cell division, in guard cells, and in reproductive tissues, especially pollen and developing seeds (Hua et al. 1997). Similarly, a P5CR promoter-gene-GFP fusion construct was expressed ubiquitously in leaves and roots, with highest expression in the root tip (Funck et al. 2010). The 5’-UTR of P5CR was found to mediate posttranscriptional regulation by stabilizing P5CR transcripts under heat and drought stress while at the same time inhibiting translation, resulting in unchanged protein levels despite strongly increased transcript levels, thus raising the question how P5CR keeps up with increased P5CS-mediated GSA/P5C production during stress (Hua et al. 2001). The biochemical properties of P5CR might solve this apparent conflict, as the activity of purified P5CR was stimulated by high ion concentrations when NADPH was available as electron donor (Forlani et al. 2015; Giberti et al. 2014). Phosphoproteomics studies have revealed two directly adjacent phosphorylation sites at T237 and S238 of Arabidopsis P5CR, but information about the possible function of P5CR phosphorylation is not available (Schulze et al. 2015). As discussed in Sect. 3.2, it is at present unclear whether OAT-mediated production of GSA constitutes an alternative route for proline biosynthesis or whether it stimulates proline synthesis merely by increasing the level of glutamate. The spatial distribution of OAT expression has not been analyzed in Arabidopsis, while in pea the highest activity was detected in cotyledons, followed by true leaves, roots, and seeds (Taylor and Stewart 1981). In pine seedlings, OAT transcript levels were highest in the radicle and peaked transiently after germination (Canas et al. 2008). In young Arabidopsis and rice seedlings as well as in radish cotyledons and cashew Proline Metabolism and Its Functions in Development and Stress Tolerance 53 leaves, OAT activity or gene expression was induced in response to salt or drought stress (da Rocha et al. 2012; Liu et al. 2018; Roosens et al. 1998; You et al. 2012). Rice OAT expression was additionally induced by heat, ABA, brassinolide, and auxin treatment (You et al. 2012). Arabidopsis oat knockout mutants developed normally and had unchanged proline content but were unable to utilize arginine as nitrogen source for growth (Funck et al. 2008). In contrast, deletion of OAT in rice caused fertility defects and lower proline content together with general symptoms of nitrogen deficiency (Liu et al. 2018). In summary, the available data supports an essential role of OAT in recycling of nitrogen from arginine degradation, but does not demonstrate or exclude the existence of an alternative route for proline biosynthesis.

4.2 Regulation of Genes Coding for Proline Catabolic Enzymes

Since the transporters that mediate the uptake of proline into mitochondria have not been molecularly identified, we know virtually nothing about the regulation of this transport. Once cytosolic proline is imported into mitochondria, it can either be used for mitochondrial protein synthesis or it can be oxidized to glutamate by the sequential action of ProDH and P5CDH (see Sect. 2.2). Copy numbers of ProDH genes have not been thoroughly analyzed in available genomes except in Brassicaceae, where an early family-specific genome duplication produced two copies that were further multiplied in the genus Brassica (Faes et al. 2015). In Arabidopsis, the best- characterized species, it was shown that both genes, ProDH1 and ProDH2, encode functional proteins with nonredundant but partially overlapping functions (Funck et al. 2010). P5CDH is encoded by a single-copy gene in Arabidopsis and in cereals, while no systematic searches in other plant genomes were reported (Ayliffe et al. 2005; Deuschle et al. 2001). As for proline biosynthesis, most studies on temporal and spatial regulation of proline catabolism were performed in Arabidopsis and we will therefore focus on this species, being aware that this knowledge might not be readily transferred to other plants with different gene copy numbers. ProDH1, the more extensively characterized proline catabolic gene, is after a weak and transient induction repressed by dehydration but is rapidly and strongly induced by rehydration (Kiyosue et al. 1996; Peng et al. 1996; Verbruggen et al. 1996). In addition, ProDH1 expression is induced by proline and hypoosmolarity and during HR-mediated pathogen defense but repressed by hyperosmolarity (Cecchini et al. 2011; Kiyosue et al. 1996; Monteoliva et al. 2014; Verbruggen et al. 1996; Yoshiba et al. 1999). More interesting in respect to plant development is the pattern of ProDH1 expression under non-stressed conditions. Weak constitutive expression of ProDH1 was observed in most organs of Arabidopsis, while in root tips and in flowers, particularly in pollen grains, stigmata, carpels, and developing seeds, the promoter activity was higher (Nakashima et al. 1998). Analysis of orthol- 54 M. Trovato et al. ogous ProDH1 genes in Brassica species revealed a very similar expression pattern (Faes et al. 2015). These findings are particularly interesting because they imply that the molecular mechanisms that reduce proline degradation and support accumulation under stress may be quite different from those active in proline accumulation during reproductive development. Detailed analysis of the Arabidopsis ProDH1 promoter revealed an ACTCAT motif responsible for proline and hypoosmolarity-mediated induction of ProDH1 (Nakashima et al. 1998; Satoh et al. 2002). The ACTCAT motif is a typical binding site for basic leucine zipper (bZIP) TFs of the S1-group, and among these AtbZIP53 and AtbZIP1 were shown to physically interact with the promoter of ProDH1 and to mediate induction of gene expression in response to proline, hypoosmolarity, and low sugar or energy levels (Dietrich et al. 2011; Satoh et al. 2002; Weltmeier et al. 2006). The activity of the ProDH1 promoter was shown to be additionally controlled by the interaction between ARR18 and bZIP63, the former being a type-B response regulator that functions as a positive osmotic stress response regulator in Arabidopsis seeds, the latter a negative regulator of seed germination upon osmotic stress (Veerabagu et al. 2014). Furthermore, ROS- and redox-mediated signaling was reported to regulate ProDH1 expression, but the precise mechanisms remain to be determined (Shinde et al. 2016). Immunoblot analyses of ProDH1 protein levels in leaf extracts or isolated mitochondria yielded multiple bands, indicating that ProDH1 may be subject to posttranslational modifications or alternative processing during mitochondrial import (Bhaskara et al. 2015; Cabassa-Hourton et al. 2016; Schertl et al. 2014). The pattern and regulation of ProDH2 expression appear largely different from ProDH1: ProDH2 promoter activity was mainly detected in vascular tissue and in the abscission zone of sepals, petals, and stamina (Funck et al. 2010). In contrast to ProDH1, transcript levels of ProDH2 were induced during senescence and by salt stress, whereas the repression by high sugar concentrations and the induction by proline and during pathogen defense were similar for both isoforms (Cecchini et al. 2011; Funck et al. 2010). Similar to P5CS1, expression of ProDH2 was induced by phosphate starvation (Aleksza et al. 2017). Averaged over the entire seedlings or tissues, the expression level of ProDH2 was much lower compared to ProDH1, and accordingly, deletion of ProDH2 had no influence on the capacity of isolated mitochondria for proline-dependent respiration, whereas for mitochondria isolated from prodh1 mutants, proline-dependent respiration was undetectable (Cabassa-Hourton et al. 2016; Funck et al. 2010). The strong and specific expression ofProDH2 in the vascular system and its strong downregulation in the presence of sucrose are consistent with the report of Hanson et al. (2008) that identified ProDH2, along with ASPARAGINE SYNTHETASE1 (ASN1) as two of the early targets of bZIP11, a transcription factor induced by SUCROSE NON-FERMENTING1 RELATED KINASE1 (SnRK1) in response to energy deprivation (O'Hara et al. 2013; Weiste et al. 2017). SnRK1 and bZIP11 also provide a direct link between proline metabolism and trehalose signaling and metabolism, which is discussed in more detail in Chap. 8 of this book. Proline Metabolism and Its Functions in Development and Stress Tolerance 55

Arabidopsis mutants for either ProDH1 or ProDH2 (Funck et al. 2010; Nanjo et al. 2003), as well as transgenic plants with antisense-mediated repression of ProDH1 and ProDH2 (Cecchini et al. 2011; Mani et al. 2002), have been generated. While no phenotypic or developmental aberrations were observed under normal conditions, these mutants exhibited enhanced proline accumulation under stress conditions, but stress tolerance and pathogen defense were weakened (Cecchini et al. 2011; Sharma et al. 2011). An unexpected, and as yet unexplained, observation is the hypersensitivity of prodh1 mutants to exogenous proline under non-stressed conditions (Funck et al. 2010; Nanjo et al. 2003). Toxicity of proline supply was also observed in non-stressed wild-type plants but was proposed to be linked to ProDH activity, with either excess P5C production or excess electron load on the mitochondrial electron transport chain as harmful effects (Hellmann et al. 2000; Miller et al. 2009). A crucial role in preventing proline toxicity was attributed to P5CDH, either by P5C/GSA detoxification or by withdrawing P5C/GSA from the proposed P5C- proline cycle (Deuschle et al. 2004; Deuschle et al. 2001; Miller et al. 2009). P5CDH expression was observed constitutively in Arabidopsis leaves, where it increased with leaf age, and to a lesser extent in roots (Deuschle et al. 2004). In reproductive organs, P5CDH was strongly expressed in pollen, developing embryos, and aborted seeds. External proline supply stimulated P5CDH expression, although with slower kinetics compared to ProDH1, whereas no prominent changes in transcript levels were observed in response to salt stress or pathogen infection (Cecchini et al. 2011; Deuschle et al. 2001; Monteoliva et al. 2014). In contrast to Arabidopsis P5CDH, the P5CDH gene from flax Linum( usitatissimum) was strongly induced by pathogen attack, but only when the plant encountered a virulent rust strain that did not elicit a hypersensitive response (Ayliffe et al. 2002; Mitchell et al. 2006). No upstream elements of P5CDH regulation have been identified so far, but transcript levels might be regulated posttranscriptionally by double-strand RNA formation with transcripts from the overlapping SIMILAR TO RCD ONE 5 (SRO5) (Borsani et al. 2005). Recently, a posttranslational mechanism for the regulation of ProDH and P5CDH activity was identified: Binding of DFR1 to both ProDH and P5CDH was found to inhibit their enzymatic activity and could thus explain how proline accumulation and high levels of ProDH expression can occur simultaneously (Ren et al. 2018). Strong expression of DFR1 was observed in inflorescences and in response to salt, drought, and cold stress. In summary, the knowledge about the regulation of genes and enzymes involved in proline metabolism supports important contributions to stress tolerance and pathogen defense but also to sexual reproduction and other developmental processes both in the absence and presence of stress. Regulation of proline metabolism was found to occur at multiple levels, and therefore we need to be careful when inferring physiological functions from mere correlations between proline content, gene expression, and phenotypic observations. 56 M. Trovato et al.

5 Developmental Processes Influenced by Proline Metabolism

5.1 Proline and Plant Development

The idea that proline may be an active player in plant development, besides being one of the 21 amino acids used for protein synthesis, began to be accepted only at the end of the last century, when different groups detected, under non-stressed conditions, large amounts of proline in the reproductive organs of some plant species (Chiang and Dandekar 1995; Fujita et al. 1998; Mutters et al. 1989; Schwacke et al. 1999; Venekamp and Koot 1988; Walton et al. 1991). Similarly, upregulation of proline biosynthesis genes was reported in flowers, fruits, and seeds of plants not subjected to evident biotic or abiotic stress (Armengaud et al. 2004; Fujita et al. 1998; Schmidt et al. 2007; Schwacke et al. 1999; Vansuyt et al. 1979). Overall, these data indicated that proline levels could locally increase even in the absence of stress. In the vegetative Arabidopsis rosette before floral transition, for example, Chiang and Dandekar (1995) found a percentage of proline, relative to the total amino acidic pool, ranging from 1% to 3% in striking contrast to up to 26% in reproductive tissues after the floral transition (Chiang and Dandekar1995 ). A similar result was reported by Schwacke et al. (1999) who measured a proline content in tomato flowers 60 times higher than in any other organ analyzed. The striking difference in proline concentrations between vegetative and floral tissues suggested that proline might play a special role in plant reproduction while raising the problem of the origin of the accumulated proline. As described in Sect. 3, the distribution of proline in plants is subjected to a complex regulation, involving long-distance transport between tissues through vascular vessels (Girousse et al. 1996), active transport from cell to cell and between different cell compartments (Lehmann et al. 2011; Rentsch et al. 1996; Schmidt et al. 2007; Schwacke et al. 1999), direct synthesis within target tissues (Chiang and Dandekar 1995; Mattioli et al. 2018), selective catabolism (Kiyosue et al. 1996; Nanjo et al. 1999b), and the rates of protein synthesis and degradation (Hildebrandt 2018). The complexity of these regulations, by itself, was suggestive of some special importance of proline in plant development, particularly in the reproductive phase. Indeed, although proline is relatively common in plant proteins, because of the frequent occurrence of long stretches of proline and/or hydroxyproline residues in a number of cell wall proteins, such as extensins, arabinogalactan-proteins, and hybrid proline-rich (Hyp/Pro-rich) proteins (Kavi Kishor et al. 2015), it seemed unlikely that, under non-stressed conditions, such large amounts of proline would be accumulated only for the requirements of protein synthesis. However, differently from stress-induced proline accumulation, a phenomenon generally considered beneficial to plant cells, proline accumulation in the absence of stress drew little attention and was mostly attributed to some type of prior or undetected stress. Chiang and Dandekar (1995), for example, hypothesized that the high content of proline found in anthers and pollen grains of Arabidopsis could Proline Metabolism and Its Functions in Development and Stress Tolerance 57 function as a compatible osmolyte to protect pollen grains from the water stress caused by the natural process of dehydration during pollen maturation. A significant step toward the understanding of the role of proline in plant development came from the study of the hairy root syndrome induced by infection with the soil bacterium Rhizobium rhizogenes, formerly known as Agrobacterium rhizogenes (Trovato et al. 2018). The capability of R. rhizogenes to reprogram plant development and induce de novo root synthesis on differentiated tissues has been long studied as a paradigm of plant development control and relies on the integration of a transfer DNA (T-DNA) into the plant genome. It turned out that rolD, one of the four “root locus” (rol) genes in the T-DNA responsible for hairy root induction, codes for an ornithine cyclodeaminase (OCD), which converts ornithine into proline and ammonium (Trovato et al. 2001). This finding, along with the above-cited proline accumulation in floral organs of plants grown in optimal conditions, disclosed a novel role for proline in plant development, and we now know that proline is critically involved in a number of developmental processes, such as root elongation, floral transition, pollen fertility, and embryo development.

5.2 Germination

Seed germination, a developmental process of enormous physiological and economic relevance, has been sometimes reported to be positively correlated with proline accumulation, particularly under stress conditions, although the observations are rather scarce and a clear-cut demonstration of the involvement of proline in germination is still lacking. Because of the beneficial role that proline accumulation, or more probably proline metabolism, exerts on plant cells under stressful conditions, it may be difficult to distinguish a generic improvement of stress tolerance from a specific effect on seed germination. Notwithstanding this, a limited number of authors have reported that the accumulation of proline and/or the upregulation of proline biosynthesis genes can improve seed germination rates. Roosens et al. (2002) reported that overexpression of Arabidopsis OAT increased proline biosynthesis and germination rates in transgenic tobacco (Nicotiana tabacum) plants under osmotic stress conditions. Similarly, transgenic tobacco plants overexpressing a feedback-insensitive variant of Vigna aconitifolia P5CS accumulated high levels of proline and exhibited higher germination rates under stress (Zonglie et al. 2000). A few reports also described positive effects of proline pretreatment on germination rates (Hua-long et al. 2014; Kubala et al. 2015; Posmyk and Janas 2007). However, this procedure, known as “osmopriming,” can also function with different compatible solutes and might be dependent on the provision of a carbon and nitrogen source rather than a specific effect of proline. The more convincing and exhaustive report claiming a positive role of proline metabolism on Arabidopsis germination comes from a study published by Hare et al. (2003) who observed that proline biosynthesis and the oxidative pentose phosphate pathway (OPPP) were induced in parallel during Arabidopsis seed germination. 58 M. Trovato et al.

Antisense inhibition of P5CS1 (which most likely silences both P5CS1 and P5CS2 expression due to the high sequence similarity) delayed seed germination, whereas external proline supply inhibited germination and this inhibition was relieved by addition of artificial electron acceptors. Hare et al. 2003( ) proposed that proline biosynthesis served to lower the NADPH/NADP+ ratio, which is known to stimulate the OPPP in many organisms and may be needed to provide sufficient ribose for nucleotide synthesis in the geminating seed (Shetty and Wahlqvist 2004).

5.3 Root Growth

In addition to being an essential component of protein biosynthesis in any growing tissue, proline also seems to play a role as a modulator of cell division, especially in the root elongation zone (Biancucci et al. 2015; Wang et al. 2014). This novel role ascribed to proline is not completely surprising as the elongation of the hairy roots induced by transformation by R. rhizogenes was originally ascribed to the action of rolD, later recognized as a proline producing OCD (Trovato et al. 2001; White et al. 1985). A specific requirement for proline metabolism was also reported in the elongation of Arabidopsis and corn primary roots at low water potential (Verslues and Skarp 1999; Sharma, 2011). Sharma et al. (2011) proposed that proline synthesized and accumulated in leaves was transferred to the root, where it was degraded to provide energy and building blocks for sustained root growth. In non-stressed Arabidopsis seedlings, exogenous proline supplementation, at micromolar concentration, was shown to induce root elongation and branching (Mattioli et al. 2009). Contrarily, exogenous supply of proline at millimolar concentrations inhibited root growth with symptoms resembling programmed cell death (Hellmann et al. 2000). Arabidopsis mutants with strongly reduced capacity to synthesize proline (p5cs1/p5cs1;P5CS2/p5cs2) displayed reduced root growth by reduction of the area of active cell division in the root meristem (Mattioli et al. 2009). In both Arabidopsis and corn p5cs mutants, reduced root growth was correlated with decreased expression levels of cyclins and other cell cycle-related genes, suggesting a link between proline or proline biosynthesis and cell cycle regulation (Mattioli et al. 2009; Wang et al. 2014).

5.4 Flowering

After the first demonstration of the importance of the rol genes in hairy root induction (White et al. 1985), rolD/OCD from R. rhizogenes has been overexpressed in tobacco, tomato, and Arabidopsis (Bettini et al. 2003; Falasca et al. 2010; Mauro et al. 1996). The ectopic expression of rolD, driven by its own promoter, was subjected to a complex developmental regulation and eventually led to early flowering and formation of increased numbers of flowers (Trovato et al. 1997). Transgenic tobacco plants Proline Metabolism and Its Functions in Development and Stress Tolerance 59 expressing rolD under the control of its own promoter reached anthesis 60 to 75 days before untransformed plants, produced abundant and long-lasting inflorescences, and exhibited an overall altered morphology with height reduction and bract-like leaves (Mauro et al. 1996). In addition, in vitro flower formation on tissue explants was stimulated in rolD transgenic plants, presumably by RolD-mediated conversion of ornithine to proline (Mauro et al. 1996; Trovato et al. 2001). Switchgrass (Panicum virgatum) plants overexpressing a heterospecificP5CS gene flowered earlier than control plants and produced more tillers after mowing (Guan et al. 2018). In transgenic Arabidopsis plants overexpressing an additional copy of P5CS1 driven by the strong CaMV 35S promoter, the time until flowering induction was shortened and axillary coflorescences proliferated, especially in short-day conditions (Mattioli et al. 2008). The overexpression of the transgenic P5CS1 copy was only transient though, and soon after the floral transition, a downregulation of bothP5CS1 and P5CS2 took place, likely because of gene silencing (Mattioli et al. 2008). Unfortunately, most of the numerous studies on P5CS overexpression in other plant species focused on drought and salt stress tolerance and did not systematically analyze flowering. Consistent with a role of proline synthesis in flowering induction, upregulation of both proline biosynthesis (P5CS, P5CR) and transport (ProT) genes has been reported, under normo-osmotic conditions, in reproductive organs, such as flowers, inflorescences, and anthers (Savouré et al. 1997; Schwacke et al. 1999; Verbruggen et al. 1993). Intriguingly, also the expression of the proline catabolic genes (ProDH, P5CDH) was reported to increase in reproductive tissues in the absence of stress (Deuschle et al. 2001; Verbruggen et al. 1996), in contrast with the steep downregulation of these genes observed under stress conditions (Kiyosue et al. 1996; Peng et al. 1996). It is as yet unknown, whether upregulation of proline catabolic genes by high proline concentrations under non-stressed conditions causes rapid metabolic cycling or whether posttranscriptional mechanisms like the interaction with DFR1 limit the rate of proline degradation (Ren et al. 2018). The antisense expression of P5CS1 (likely affecting both P5CS1 and P5CS2, see above) has been shown to inhibit Arabidopsis bolting (Nanjo et al. 1999a). Ambiguous observations have been reported for insertional mutants: in two labs, growth and flowering of p5cs1 single mutants were not different from wild-type plants, whereas in a third lab, a delay in the onset of bolting was observed (Funck et al. 2012; Mattioli et al. 2008; Székely et al. 2008). For p5cs2 single mutants and near-double mutants (p5cs1/p5cs1, P5CS2/p5cs2), a generally slower development was observed (Funck et al. 2012; Mattioli et al. 2012). Similarly, silencing of P5CS2 expression in Lotus japonicus produced several lines with defects in flower and seed formation (S. Signorelli, unpublished observations). Altogether, these data indicate that both P5CS1 and P5CS2 can modulate flowering and suggest that proline plays a role in floral transition, bolting, and coflorescence emergence. In Arabidopsis, and probably all flowering plants, multiple signaling pathways respond to a range of environmental (photoperiod, cold, heat) and endogenous (metabolites, gibberellin, age) stimuli and converge to induce the conversion of vegetative shoot meristems into floral meristems (Khan et al. 2014; Srikanth and Schmid 2011). CONSTANS, one of the master regulators of the photoperiodic 60 M. Trovato et al. pathway of flowering induction, has been identified as an inducer ofP5CS2 in Arabidopsis (Samach et al. 2000). FLOWERING LOCUS C (FLC) was identified as inducer of P5CS1 along with ELONGATED HYPOCOTYL 5 (HY5), which was found to be a crucial factor in the light-dependent induction of P5CS1 by stress, indicating that proline may indeed contribute to the light-dependent regulation of flowering (Abrahám et al. 2003; Chen et al. 2018; Feng et al. 2016; Hayashi et al. 2000). Recently, a number of plant species, belonging to different taxonomic groups, have been reported to flower rapidly after exposure to a wide range of different stressors (Wada and Takeno 2010). Since the responses to many types of stress involve proline accumulation, it is tempting to speculate that stress-induced flowering and proline-induced flowering in non-stressed plants may rely on a common mechanism. The distribution of proline under normal physiological conditions, however, seems partly different from that found under stress conditions: In Arabidopsis, a locally and temporally confined increase of proline in the shoot apical meristem at floral transition has been reported, whereas, under stress conditions, proline is accumulated at high levels in all the tissues of the plant (Mattioli et al. 2008). Overall, the body of accumulated evidence points to proline as a modulator of floral transition, although its mechanism of action, the genes involved in this process, and the interaction with other regulatory pathways still need to be revealed in detail.

5.5 Pollen Fertility

Among floral organs, the highest proline contents have been observed in the pollen of many plant species including Arabidopsis, tomato, dandelion (Taraxacum offici- nale), willow (Salix sp.), and petunia (Petunia hybrida) (Auclair and Jamieson 1948; Chiang and Dandekar 1995; Hong-qi et al. 1982; Schwacke et al. 1999). In grass pollen, proline was the most abundant amino acid, accounting for up to 1.65% of pollen dry weight (Bathurst 1954). Proline was the most abundant amino acid in anthers of devil’s trumpet (Datura metel) and the only one found to increase during pollen development (Sangwan 1978). In addition to this correlative evidence, recently proline has been shown to be essential for pollen development and fertility by two research groups, who independently reported that in Arabidopsis p5cs1/ p5cs2 double-mutant pollen was misshaped and infertile (Funck et al. 2012; Mattioli et al. 2012). The morphological abnormalities were accompanied by lack of storage compounds and nuclei and appeared late in pollen development, starting from stage 11 of anther development. The requirement for proline biosynthesis was specific for pollen, because only the pollen failed to transmit both p5cs mutant alleles simultaneously, whereas p5cs1/p5cs2 double mutant egg cells showed almost no compromised fertility. Importantly, exogenous L-proline, supplemented in planta to developing anthers of p5cs1/p5cs1 P5CS2/p5cs2 near-double mutant plants, allowed the formation of fully developed and fertile p5cs1/p5cs2 double mutant pollen (Mattioli et al. 2012). Quite surprisingly, Arabidopsis plants carrying mutations in P5CR, the gene coding for the second and final step of proline biosynthesis, are embryo lethal, but Proline Metabolism and Its Functions in Development and Stress Tolerance 61 not male sterile, presumably because P5CR is an exceptionally long-lived protein (Funck et al. 2012). High expression of the specific proline transporter ProT1 in the pollen of tomato and Arabidopsis raised the question, whether proline is imported during pollen development or is synthesized cell autonomously (Grallath et al. 2005; Schwacke et al. 1999). By targeting P5CS expression to different tissues in Arabidopsis anthers, Mattioli et al. (2018) demonstrated that only proline synthesized within developing pollen grains can fully restore fertility of p5cs1/p5cs2 double mutant pollen. Consistently, both P5CS1 and P5CS2 genes exhibit a strong and specific expression in microspores and pollen grains but are essentially unexpressed in surrounding sporophytic tissues of the anther, as shown by β-glucuronidase (GUS) analysis, and inferred by bioinformatic analysis of P5CS1 and P5CS2 promoters (Mattioli et al. 2018).

5.6 Embryo Development

The analysis of p5cs2 knockout mutants in Arabidopsis has disclosed an essential role of proline in plant embryogenesis. Three research groups have independently isolated and characterized two p5cs2 T-DNA insertion mutants (Funck et al. 2012; Mattioli et al. 2009; Székely et al. 2008). Quite surprisingly, despite the high sequence similarity shared by the two paralogous genes, and although the same pattern of expression was detected for both P5CS1 and P5CS2 transcripts by in situ hybridisation of sections of shoot apical meristems and embryos (Mattioli et al. 2009; Székely et al. 2008), p5cs2, but not p5cs1 mutants, are embryo lethal suggesting a specific role of P5CS2 or posttranscriptional repression of P5CS1 activity during embryogenesis. P5CS2-GFP fusion proteins were uniformly distributed in the cytosol of Arabidopsis embryos, whereas P5CS1-GFP formed cytoplasmic speckles, possibly indicating that P5CS1 is inactivated by aggregation (Székely et al. 2008). Spraying flowers with proline, induction ofP5CS1 expression by salt stress, or in vitro cultivation of immature seeds allowed rescuing homozygous p5cs2 mutants, which were retarded in development but produced viable seeds under favorable conditions (Funck et al. 2012; Mattioli et al. 2009; Székely et al. 2008). The reason why homozygous p5cs2 embryos die only in the siliques of heterozygous, but not of homozygous, mutants is not yet fully understood. Potentially, the slowly developing homozygous mutants are aborted when the faster-growing wild- type and heterozygous embryos in neighboring seeds reach maturity. In addition, microscopic analysis of the malformed p5cs2 embryos revealed various aberrations typically associated with a defective cell cycle, such as anomalous orientations of cellular division planes, indicating that low proline levels may similarly inhibit cell cycle progression in embryos as in the root meristem (Mattioli et al. 2009). In corn pro1 mutants, in which the independently evolved P5CS2 of corn is inactivated, storage compounds in the seed endosperm of homozygous mutant seeds were strongly reduced, but formation of viable embryos still occurred (Wang et al. 2017). However, without exogenous proline supply, pro1 homozygous mutants were seedling lethal and successful propagation has not been reported. 62 M. Trovato et al.

In addition to P5CS2, also P5CR, the enzyme involved in the second and final step of proline synthesis, is essential for embryogenesis as shown by Funck et al. (2012), who characterized two p5cr mutants, found in the Salk collection and annotated as embryo lethal in the SeedGenes database (SeedGenes Project. http://seedgenes.org). Intriguingly, any attempt to rescue homozygous p5cr embryos by proline supplementation was ineffective, differently from p5cs2 mutants. Expression of a P5CR-GFP fusion protein under control of the endogenous P5CR promoter, which is active in developing embryos, reverted the embryo-lethal phenotype, while CaMV-35S-driven overexpression of P5CR-GFP in vegetative tissues was ineffective (Funck et al. 2012). These results indicate that, similarly to the situation in the pollen, long-distance transport of proline cannot fully substitute for local biosynthesis in tissues that critically depend on proline.

5.7 Role of Proline Metabolism in Development Under Stress

The accumulation of proline both during certain developmental processes and in response to stress is frequently regarded as two different phenomena which, accordingly, have been treated as separate chapters in this book. However, the large increase in proline content observed in reproductive tissues of most plant species is similar to that observed after many different types of stress, thus posing the question whether the function of proline may be similar in both cases. This seemingly simple question is particularly difficult to tackle because there are some hypotheses but no generally accepted idea of what the function of proline may be, neither under stress conditions nor during development. In addition, also the concept of stress is not as simple as it may seem. What is stress? According to Lichtenthaler (1998), any “unfavorable condition or substance that affects or blocks a plant’s metabolism, growth, or development is regarded as stress.” It may well be that many “normal” physiological conditions, such as seed and pollen maturation or high-intensity light, are more demanding for a plant than mild environmental stress, such as a transient period of drought or a moderate reduction of the average temperature. According to Chiang and Dandekar (1995), stronger proline accumulation was observed in tissues with low water content, such as embryos and pollen grains, which successfully entered a developmentally induced process of desiccation without loss of cellular and tissue viability. The most probable benefit of proline accumulation in these tissues is based on the kosmotropic properties of proline by which it helps to protect enzymes and membranes of plant cells with low water content (see Chap. 3). In contrast, the increase in proline concentration following osmotic stress may not be sufficient to protect cells because, as discussed in the following chapter, the amount of proline accumulated is typically not sufficient to counterbalance the decrease in the cellular osmotic potential. Another possibility is that proline in (some) reproductive tissues may be a precautionary measure in case of future adverse conditions in order to protect important plant organs and improve the fitness of the species. Consistent with this hypothesis is the observation that p5cs1 mutants have no aberrant Proline Metabolism and Its Functions in Development and Stress Tolerance 63 phenotype under normal conditions but exhibit hypersensitivity to salt and hyperosmolarity stress (Sharma et al. 2011; Székely et al. 2008). On the other hand, as described in detail in the following chapter, regardless of proline accumulation being a response to stress or part of a developmental program, it remains still unclear if its beneficial effects are mediated by accumulation per se or by increased metabolic turnover. As indicated above and discussed in more detail in the next chapter, biosynthesis and degradation of proline have the capacity to change the redox state of the cytosol and the mitochondria, respectively, and may additionally modulate the levels of reactive oxygen species. Since far less details are known about the regulation of proline metabolism during normal development, it is at present difficult to predict downstream effects, although it is tempting to speculate that the accumulation of proline under stress and its accumulation during development are two sides of the same coin. Further studies on proline-dependent signal transduction and actual flux rates of proline metabolism and of the exchange of proline between different tissues or cell types will be needed to fully understand how proline exerts its stress protective and developmental functions.

References

Abrahám E, Rigó G, Székely G, Nagy R, Koncz C, Szabados L (2003) Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis. Plant Mol Biol 51:363–372 Aleksza D, Horváth GV, Sándor G, Szabados L (2017) Proline accumulation is regulated by transcription factors associated with phosphate starvation. Plant Physiol 175:555–567. https://doi. org/10.1104/pp.17.00791 Armengaud P, Thiery L, Buhot N, Grenier-de March G, Savouré A (2004) Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features. Physiol Plant 120:442–450. https://doi.org/10.1111/j.0031-9317.2004.00251.x Auclair JL, Jamieson CA (1948) A qualitative analysis of amino acids in pollen collected by bees. Science 108:357–358. https://doi.org/10.1126/science.108.2805.357 Ayliffe MA, Mitchell HJ, Deuschle K, Pryor AJ (2005) Comparative analysis in cereals of a key proline catabolism gene. Mol Gen Genomics 274:494–505 Ayliffe MA, Roberts JK, Mitchell HJ, Zhang R, Lawrence GJ, Ellis JG, Pryor TJ (2002) A plant gene up-regulated at rust infection sites. Plant Physiol 129:169–180 Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C (2004) Vernalization requires epigenetic silencing of FLC by histone methylation. Nature 427:164. https://doi.org/10.1038/ nature02269 Bathurst NO (1954) The amino-acids of grass pollen. J Exp Bot 5:253–256. https://doi.org/10.1093/ jxb/5.2.253 Besnard J, Pratelli R, Zhao C, Sonawala U, Collakova E, Pilot G, Okumoto S (2016) UMAMIT14 is an amino acid exporter involved in phloem unloading in Arabidopsis roots. J Exp Bot 67:6385–6397. https://doi.org/10.1093/jxb/erw412 Besnard J, Zhao C, Avice JC, Vitha S, Hyodo A, Pilot G, Okumoto S (2018) Arabidopsis UMAMIT24 and 25 are amino acid exporters involved in seed loading. J Exp Bot 69:5221– 5232. https://doi.org/10.1093/jxb/ery302 Bettini P, Michelotti S, Bindi D, Giannini R, Capuana M, Buaiatti M (2003) Pleiotropic effect of the insertion of Agrobacterium rhizogenes rolD gene in tomato (Lycopersicon esculentum Mill). Theor Appl Genet 107:831–836. https://doi.org/10.1007/s00122-003-1322-0 64 M. Trovato et al.

Bhaskara GB, Yang T-H, Verslues PE (2015) Dynamic proline metabolism: importance and regulation in water limited environments. Front Plant Sci 6:484. https://doi.org/10.3389/ fpls.2015.00484 Biancucci M, Mattioli R, Moubayidin L, Sabatini S, Costantino P, Trovato M (2015) Proline affects the size of the root meristematic zone in Arabidopsis. BMC Plant Biol 15:263. https:// doi.org/10.1186/s12870-015-0637-8 Boggess SF, Koeppe DE, Stewart CR (1978) Oxidation of proline by plant mitochondria. Plant Physiol 62:22–25 Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123:1279–1291. https://doi.org/10.1016/j.cell.2005.11.035 Buchanan BB, Gruissem W, Jones RL (2000) Biochemistry and molecular biology of plants. American Society of Plant Physiologists, Rockville Büssis D, Heineke D (1998) Acclimation of potato plants to polyethylene glycol-induced water deficit II. Contents and subcellular distribution of organic solutes. J Exp Bot 49:1361–1370 Cabassa-Hourton C et al (2016) Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana. Biochem J 473:2623–2634. https://doi.org/10.1042/bcj20160314 Canas RA, Villalobos DP, Diaz-Moreno SM, Canovas FM, Canton FR (2008) Molecular and functional analyses support a role of Ornithine-{delta}-aminotransferase in the provision of glutamate for glutamine biosynthesis during pine germination. Plant Physiol 148:77–88 Cecchini NM, Monteoliva MI, Alvarez ME (2011) Proline dehydrogenase contributes to pathogen defense in Arabidopsis. Plant Physiol 155:1947–1959. https://doi.org/10.1104/pp.110.167163 Chen Q, Zheng Y, Luo L, Yang Y, Hu X, Kong X (2018) Functional FRIGIDA allele enhances drought tolerance by regulating the P5CS1 pathway in Arabidopsis thaliana. Biochem Biophys Res Commun 495:1102–1107. https://doi.org/10.1016/j.bbrc.2017.11.149 Chiang HH, Dandekar AM (1995) Regulation of proline accumulation in Arabidopsis thaliana (L) Heynh during development and in response to desiccation. Plant Cell Environ 18:1280–1290. https://doi.org/10.1111/J.1365-3040.1995.Tb00187.X Chinnusamy V, Zhu J-K (2009) Epigenetic regulation of stress responses in plants. Curr Opin Plant Biol 12:133–139. https://doi.org/10.1016/j.pbi.2008.12.006 da Rocha IM, Vitorello VA, Silva JS, Ferreira-Silva SL, Viégas RA, Silva EN, Silveira JA (2012) Exogenous ornithine is an effective precursor and the delta-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves. J Plant Physiol 169:41–49. https://doi.org/10.1016/j.jplph.2011.08.001 Daygon VD et al (2017) Metabolomics and genomics combine to unravel the pathway for the presence of fragrance in rice. Sci Rep 7:8767. https://doi.org/10.1038/s41598-017-07693-9 De Ronde JA, Cress WA, Kruger GH, Strasser RJ, Van Staden J (2004) Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress. J Plant Physiol 161:1211–1224 Delauney AJ, Hu CA, Kishor PB, Verma DP (1993) Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis. J Biol Chem 268:18673–18678 Delauney AJ, Verma DP (1990) A soybean gene encoding delta 1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmo- regulated. Mol Gen Genet 221:299–305 Deuschle K et al (2004) The role of δ1-pyrroline-5-carboxylate dehydrogenase in proline degradation. Plant Cell 16:3413–3425 Deuschle K, Funck D, Hellmann H, Däschner K, Binder S, Frommer WB (2001) A nuclear gene encoding mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase and its potential role in protection from proline toxicity. Plant J 27:345–356. https://doi. org/10.1046/j.1365-313X.2001.01101.x Di Martino C, Pizzuto R, Pallotta ML, De Santis A, Passarella S (2006) Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings. Planta 223:1123–1133 Proline Metabolism and Its Functions in Development and Stress Tolerance 65

Dietrich K, Weltmeier F, Ehlert A, Weiste C, Stahl M, Harter K, Dröge-Laser W (2011) Heterodimers of the Arabidopsis transcription factors bZIP1 and bZIP53 reprogram amino acid metabolism during low energy stress. Plant Cell 23:381–395. https://doi.org/10.1105/ tpc.110.075390 Dinkeloo K, Boyd S, Pilot G (2018) Update on amino acid transporter functions and on possible amino acid sensing mechanisms in plants. Semin Cell Dev Biol 74:105–113. https://doi. org/10.1016/j.semcdb.2017.07.010 Elthon TE, Stewart CR (1981) Submitochondrial location and electron transport characteristics of enzymes involved in proline oxidation. Plant Physiol 67:780–784 Elthon TE, Stewart CR (1982) Proline oxidation in corn mitochondria : involvement of NAD, relationship to ornithine metabolism, and sidedness on the inner membrane. Plant Physiol 70:567–572 Elthon TE, Stewart CR, Bonner WD (1984) Energetics of proline transport in corn mitochondria. Plant Physiol 75:951–955 Fabro G, Kovacs I, Pavet V, Szabados L, Alvarez ME (2004) Proline accumulation and AtP5CS2 gene activation are induced by plant-pathogen incompatible interactions in Arabidopsis. Mol Plant-Microbe Interact 17:343–350 Faes P et al (2015) Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development. Planta 241:403–419. https://doi.org/10.1007/s00425-014-2189-9 Falasca G, Altamura MM, D’Angeli S, Zaghi D, Costantino P, Mauro ML (2010) The rolD oncogene promotes axillary bud and adventitious root meristems in Arabidopsis. Plant Physiol Biochem 48:797–804. https://doi.org/10.1016/j.plaphy.2010.06.002 Feng XJ, Li JR, Qi SL, Lin QF, Jin JB, Hua XJ (2016) Light affects salt stress-induced transcriptional memory of P5CS1 in Arabidopsis. Proc Natl Acad Sci U S A 113:E8335–e8343. https:// doi.org/10.1073/pnas.1610670114 Fichman Y, Gerdes SY, Kovács H, Szabados L, Zilberstein A, Csonka L (2015) Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation. Biol Rev Camb Philos Soc 90:1065–1099. https://doi.org/10.1111/brv.12146 Fischer WN, Kwart M, Hummel S, Frommer WB (1995) Substrate specificity and expression profile of amino acid transporters (AAPs) in Arabidopsis. J Biol Chem 270:16315–16320 Forlani G, Bertazzini M, Zarattini M, Funck D, Ruszkowski M, Nocek B (2015) Functional properties and structural characterization of rice delta(1)-pyrroline-5-carboxylate reductase. Front Plant Sci 6:565. https://doi.org/10.3389/fpls.2015.00565 Forlani G, Scainelli D, Nielsen E (1997) δ1-Pyrroline-5-carboxylate dehydrogenase from cultured cells of potato (purification and properties). Plant Physiol 113:1413–1418 Fujiki Y, Teshima H, Kashiwao S, Kawano-Kawada M, Ohsumi Y, Kakinuma Y, Sekito T (2017) Functional identification of AtAVT3, a family of vacuolar amino acid transporters, in Arabidopsis. FEBS Lett 591:5–15. https://doi.org/10.1002/1873-3468.12507 Fujita T, Maggio A, Garcia-Rios M, Bressan RA, Csonka LN (1998) Comparative analysis of the regulation of expression and structures of two evolutionarily divergent genes for Delta1- pyrroline-5-carboxylate synthetase from tomato. Plant Physiol 118:661–674 Funck D, Eckard S, Müller G (2010) Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis. BMC Plant Biol 10:70. https://doi.org/10.1186/1471-2229-10-70 Funck D, Mattioli R, Biancucci M, Mosca L, Trovato M (2019) Proline biosynthesis: localisation matters. Paper presented at the 32nd Conference Molecular Biology of Plants, Dabringhausen, Germany, Funck D, Stadelhofer B, Koch W (2008) Ornithine-δ-aminotransferase is essential for arginine catabolism but not for proline biosynthesis. BMC Plant Biol 8:40. https://doi. org/10.1186/1471-2229-8-40 Funck D, Winter G, Baumgarten L, Forlani G (2012) Requirement of proline synthesis during Arabidopsis reproductive development. BMC Plant Biol 12:191. https://doi. org/10.1186/1471-2229-12-191 66 M. Trovato et al.

Giberti S, Funck D, Forlani G (2014) Δ1-Pyrroline-5-carboxylate reductase from Arabidopsis thaliana: stimulation or inhibition by chloride ions and feedback regulation by proline depend on whether NADPH or NADH acts as co-substrate. New Phytol 202:911–919. https://doi. org/10.1111/nph.12701 Ginguay A, Cynober L, Curis E, Nicolis I (2017) Ornithine aminotransferase, an important glutamate-metabolizing enzyme at the crossroads of multiple metabolic pathways. Biology 6. https://doi.org/10.3390/biology6010018 Girousse C, Bournoville R, Bonnemain J-L (1996) Water deficit-induced changes in concentrations in proline and some other amino acids in the phloem sap of alfalfa. Plant Physiol 111:109–113 Goodman JL, Wang S, Alam S, Ruzicka FJ, Frey PA, Wedekind JE (2004) Ornithine cyclodeaminase: structure, mechanism of action, and implications for the mu-crystallin family. Biochemistry 43:13883–13891. https://doi.org/10.1021/bi048207i Grallath S, Weimar T, Meyer A, Gumy C, Suter-Grotemeyer M, Neuhaus JM, Rentsch D (2005) The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns. Plant Physiol 137:117–126 Guan C, Huang YH, Cui X, Liu SJ, Zhou YZ, Zhang YW (2018) Overexpression of gene encoding the key enzyme involved in proline-biosynthesis (PuP5CS) to improve salt tolerance in switchgrass (Panicum virgatum L.). Plant Cell Rep 37:1187–1199. https://doi.org/10.1007/ s00299-018-2304-7 Hanson J, Hanssen M, Wiese A, Hendriks MMWB, Smeekens S (2008) The sucrose regulated transcription factor bZIP11 affects amino acid metabolism by regulating the expression of ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE2. Plant J 53:935–949. https://doi.org/10.1111/j.1365-313X.2007.03385.x Hare P, Cress W, van Staden J (2003) A regulatory role for proline metabolism in stimulating Arabidopsis thaliana seed germination. Plant Growth Regul 39:41–50 Hayashi F, Ichino T, Osanai M, Wada K (2000) Oscillation and regulation of proline content by P5CS and ProDH gene expressions in the light/dark cycles in Arabidopsis thaliana L. Plant Cell Physiol 41:1096–1101. https://doi.org/10.1093/pcp/pcd036 Hellmann H, Funck D, Rentsch D, Frommer WB (2000) Hypersensitivity of an Arabidopsis sugar signaling mutant toward exogenous proline application. Plant Physiol 123:779–789 Hildebrandt TM (2018) Synthesis versus degradation: directions of amino acid metabolism during Arabidopsis abiotic stress response. Plant Mol Biol 98:121–135. https://doi.org/10.1007/ s11103-018-0767-0 Hill RW, Wyse GA, Anderson M (2016) Animal physiology. Sinauer Associates, Sunderland Hirner A et al (2006) Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll. Plant Cell 18:1931–1946. https://doi.org/10.1105/ tpc.106.041012 Hong-qi Z, Croes AF, Linskens HF (1982) Protein synthesis in germinating pollen of Petunia: role of proline. Planta 154:199–203. https://doi.org/10.1007/BF00387864 Hu CA, Delauney AJ, Verma DP (1992) A bifunctional enzyme (delta 1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants. Proc Natl Acad Sci U S A 89:9354–9358 Hua XJ, van de Cotte B, Van Montagu M, Verbruggen N (1997) Developmental regulation of pyrroline-5-carboxylate reductase gene expression in Arabidopsis. Plant Physiol 114:1215–1224 Hua XJ, van de Cotte B, Van Montagu M, Verbruggen N (2001) The 5′ untranslated region of the At-P5R gene is involved in both transcriptional and post-transcriptional regulation. Plant J 26:157–169 Hua-long L, Han-jing S, Jing-guo W, Yang L, De-tang Z, Hong-wei Z (2014) Effect of seed soaking with exogenous proline on seed germination of rice under salt stress. J Northeast Aric Univ 21:1–6. https://doi.org/10.1016/S1006-8104(14)60062-3 Huang AHC, Cavalieri AJ (1979) Proline oxidase and water stress-induced proline accumulation in spinach leaves. Plant Physiol 63:531–535 Proline Metabolism and Its Functions in Development and Stress Tolerance 67

Karan R, DeLeon T, Biradar H, Subudhi PK (2012) Salt stress induced variation in DNA methylation pattern and its influence on gene expression in contrasting rice genotypes. PLoS One 7:e40203. https://doi.org/10.1371/journal.pone.0040203 Kavi Kishor P, Hong Z, Miao GH, Hu C, Verma D (1995) Overexpression of Δ1-pyrroline-5- carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 108:1387–1394. https://doi.org/10.1104/pp.108.4.1387 Kavi Kishor PB, Hima Kumari P, Sunita MSL, Sreenivasulu N (2015) Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny. Front Plant Sci 6:544. https://doi.org/10.3389/fpls.2015.00544 Kesari R et al (2012) Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation. Proc Natl Acad Sci U S A 109:9197– 9202. https://doi.org/10.1073/pnas.1203433109 Khan MR, Ai XY, Zhang JZ (2014) Genetic regulation of flowering time in annual and perennial plants. Wiley Interdiscip Rev RNA 5:347–359. https://doi.org/10.1002/wrna.1215 Kim G-B, Nam Y-W (2013) A novel Δ1-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation. J Plant Physiol 170:291–302.https://doi.org/10.1016/j.jplph.2012.10.004 Kiyosue T, Yoshiba Y, Yamaguchi-Shinozaki K, Shinozaki K (1996) A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis. Plant Cell 8:1323–1335. https://doi.org/10.1105/tpc.8.8.1323 Korasick DA et al (2019) Structural and biochemical characterization of Aldehyde Dehydrogenase 12, the last enzyme of proline catabolism in plants. J Mol Biol 431:576–592. https://doi. org/10.1016/j.jmb.2018.12.010 Kubala S, Wojtyla Ł, Quinet M, Lechowska K, Lutts S, Garnczarska M (2015) Enhanced expression of the proline synthesis gene P5CSA in relation to seed osmopriming improvement of Brassica napus germination under salinity stress. J Plant Physiol 183:1–12. https://doi. org/10.1016/j.jplph.2015.04.009 Ladwig F et al (2012) Siliques Are Red1 from Arabidopsis acts as a bidirectional amino acid transporter that is crucial for the amino acid homeostasis of siliques. Plant Physiol 158:1643–1655. https://doi.org/10.1104/pp.111.192583 Lehmann S, Gumy C, Blatter E, Boeffel S, Fricke W, Rentsch D (2011) In planta function of compatible solute transporters of the AtProT family. J Exp Bot 62:787–796. https://doi. org/10.1093/jxb/erq320 Lichtenthaler HK (1998) The stress concept in plants: an introduction. Ann N Y Acad Sci 851:187– 198. https://doi.org/10.1111/j.1749-6632.1998.tb08993.x Liu C et al (2018) Ornithine δ-aminotransferase is critical for floret development and seed setting through mediating nitrogen reutilization in rice. Plant J 96:842–854. https://doi.org/10.1111/ tpj.14072 Ma L, Zhou E, Gao L, Mao X, Zhou R, Jia J (2008) Isolation, expression analysis and chromosomal location of P5CR gene in common wheat (Triticum aestivum L.). S Afr J Bot 74:705 Mani S, Van De Cotte B, Van Montagu M, Verbruggen N (2002) Altered levels of proline dehydrogenase cause hypersensitivity to proline and its analogs in Arabidopsis. Plant Physiol 128:73–83 Mattioli R, Biancucci M, El Shall A, Mosca L, Costantino P, Funck D, Trovato M (2018) Proline synthesis in developing microspores is required for pollen development and fertility. BMC Plant Biol 18:356. https://doi.org/10.1186/s12870-018-1571-3 Mattioli R, Biancucci M, Lonoce C, Costantino P, Trovato M (2012) Proline is required for male gametophyte development in Arabidopsis. BMC Plant Biol 12:236. https://doi. org/10.1186/1471-2229-12-236 Mattioli R, Falasca G, Sabatini S, Altamura MM, Costantino P, Trovato M (2009) The proline biosynthetic genes P5CS1 and P5CS2 play overlapping roles in Arabidopsis flower transition but not in embryo development. Physiol Plant 137:72–85. https://doi. org/10.1111/j.1399-3054.2009.01261.x 68 M. Trovato et al.

Mattioli R, Marchese D, D'Angeli S, Altamura MM, Costantino P, Trovato M (2008) Modulation of intracellular proline levels affects flowering time and inflorescence architecture in Arabidopsis. Plant Mol Biol 66:277–288. https://doi.org/10.1007/s11103-007-9269-1 Mauro ML, Trovato M, De Paolis A, Gallelli A, Costantino P, Altamura MM (1996) The plant oncogene rolD stimulates flowering in transgenic tobacco plants. Dev Biol 180:693–700. https://doi.org/10.1006/dbio.1996.0338 Meister A, Radhakrishnan AN, Buckley SD (1957) Enzymatic synthesis of L-pipecolic acid and L-proline. J Biol Chem 229:789–800 Mestichelli LJ, Gupta RN, Spenser ID (1979) The biosynthetic route from ornithine to proline. J Biol Chem 254:640–647 Mezl VA, Knox WE (1976) Properties and analysis of a stable derivative of pyrroline-5-carboxylic acid for use in metabolic studies. Anal Biochem 74:430–440 Miller G, Honig A, Stein H, Suzuki N, Mittler R, Zilberstein A (2009) Unraveling δ1-pyrroline- 5-carboxylate-proline cycle in plants by uncoupled expression of proline oxidation enzymes. J Biol Chem 284:26482–26492. Epub 22009 Jul 26427 Mitchell HJ, Ayliffe MA, Rashid KY, Pryor AJ (2006) A rust-inducible gene from flax fis1( ) is involved in proline catabolism. Planta 223:213–222 Monne M et al (2018) Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate, and dicarboxylates. J Biol Chem 293:4213–4227. https://doi.org/10.1074/jbc.RA117.000771 Monteoliva MI, Rizzi YS, Cecchini NM, Hajirezaei MR, Alvarez ME (2014) Context of action of proline dehydrogenase (ProDH) in the hypersensitive response of Arabidopsis. BMC Plant Biol 14:21. https://doi.org/10.1186/1471-2229-14-21 Moxley MA, Zhang L, Christgen S, Tanner JJ, Becker DF (2017) Identification of a conserved histidine as being critical for the catalytic mechanism and functional switching of the multifunctional Proline utilization A protein. Biochemistry 56:3078–3088. https://doi.org/10.1021/ acs.biochem.7b00046 Müller B et al (2015) Amino acid export in developing Arabidopsis seeds depends on UmamiT facilitators. Curr Biol 25:3126–3131. https://doi.org/10.1016/j.cub.2015.10.038 Murahama M, Yoshida T, Hayashi F, Ichino T, Sanada Y, Wada K (2001) Purification and characterization of Delta(1)-pyrroline-5-carboxylate reductase isoenzymes, indicating differential distribution in spinach (Spinacia oleracea L.) leaves. Plant Cell Physiol 42:742–750 Mutters RG, Ferreira LGR, Hall AE (1989) Proline content of the anthers and pollen of heat- tolerant and heat-sensitive cowpea subjected to different temperatures. Crop Sci 29:1497–1500 Nakashima K, Satoh R, Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K (1998) A gene encoding proline dehydrogenase is not only induced by proline and hypoosmolarity, but is also developmentally regulated in the reproductive organs of Arabidopsis. Plant Physiol 118:1233–1241. https://doi.org/10.1104/pp.118.4.1233 Nanjo T, Fujita M, Seki M, Kato T, Tabata S, Shinozaki K (2003) Toxicity of free proline revealed in an Arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase. Plant Cell Physiol 44:541–548 Nanjo T et al (1999a) Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana. Plant J 18:185–193 Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (1999b) Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 461:205–210. https://doi.org/10.1016/ S0014-5793(99)01451-9 Noguchi M, Koiwai A, Tamaki E (1966) Studies on nitrogen metabolism in tobacco plants. Agric Biol Chem 30:452–456. https://doi.org/10.1080/00021369.1966.10858622 Nozaki H (2005) A new scenario of plastid evolution: plastid primary endosymbiosis before the divergence of the “Plantae,” emended. J Plant Res 118:247–255. https://doi.org/10.1007/ s10265-005-0219-1 Proline Metabolism and Its Functions in Development and Stress Tolerance 69

O'Hara LE, Paul MJ, Wingler A (2013) How do sugars regulate plant growth and development? New insight into the role of trehalose-6-phosphate. Mol Plant 6:261–274. https://doi. org/10.1093/mp/sss120 Peng Z, Lu Q, Verma DPS (1996) Reciprocal regulation of Δ1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants. Mol Gen Genet 253:334–341. https://doi.org/10.1007/pl00008600 Per TS, Khan NA, Reddy PS, Masood A, Hasanuzzaman M, Khan MIR, Anjum NA (2017) Approaches in modulating proline metabolism in plants for salt and drought stress tolerance: Phytohormones, mineral nutrients and transgenics. Plant Physiol Biochem 115:126–140. https://doi.org/10.1016/j.plaphy.2017.03.018 Perchlik M, Foster J, Tegeder M (2014) Different and overlapping functions of Arabidopsis LHT6 and AAP1 transporters in root amino acid uptake. J Exp Bot 65:5193–5204. https://doi. org/10.1093/jxb/eru278 Pohlmeyer K, Soll J, Steinkamp T, Hinnah S, Wagner R (1997) Isolation and characterization of an amino acid-selective channel protein present in the chloroplastic outer envelope membrane. Proc Natl Acad Sci U S A 94:9504–9509. https://doi.org/10.1073/pnas.94.17.9504 Porcelli V et al (2018) Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant mitochondria. Biochim Biophys Acta 1859:1249–1258. https://doi.org/10.1016/j.bbabio.2018.08.001 Posmyk MM, Janas KM (2007) Effects of seed hydropriming in presence of exogenous proline on chilling injury limitation in Vigna radiata L. seedlings. Acta Physiol Plant 29:509–517. https:// doi.org/10.1007/s11738-007-0061-2 Rayapati PJ, Stewart CR, Hack E (1989) Pyrroline-5-carboxylate reductase is in pea (Pisum sativum L.) leaf chloroplasts. Plant Physiol 91:581–586 Ren Y et al (2018) DFR1-mediated inhibition of proline degradation pathway regulates drought and freezing tolerance in Arabidopsis. Cell Rep 23:3960–3974. https://doi.org/10.1016/j. celrep.2018.04.011 Rena AB, Splittstoesser WE (1975) Proline dehydrogenase and pyrroline-5-carboxylate reductase from pumpkin cotyledons. Phytochemistry 14:657–661. https://doi. org/10.1016/0031-9422(75)83010-X Renné P, Dreßen U, Hebbeker U, Hille D, Flügge U-I, Westhoff P, Weber APM (2003) The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate translocator DiT2. Plant J 35:316–331. https://doi.org/10.1046/j.1365-313X.2003.01806.x Rentsch D, Hirner B, Schmelzer E, Frommer WB (1996) Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant. Plant Cell 8:1437–1446 Richards EJ (2006) Inherited epigenetic variation - revisiting soft inheritance. Nat Rev Genet 7:395. https://doi.org/10.1038/nrg1834 Roosens NH, Al Bitar F, Loenders K, Angenon G, Jacobs M (2002) Overexpression of ornithine- delta-aminotransferase increases proline biosynthesis and confers osmotolerance in transgenic plants. Mol Breed 9:73–80 Roosens NH, Thu TT, Iskandar HM, Jacobs M (1998) Isolation of the ornithine-delta- aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana. Plant Physiol 117:263–271. https://doi.org/10.1104/pp.117.1.263 Ruszkowski M, Nocek B, Forlani G, Dauter Z (2015) The structure of Medicago truncatula delta(1)-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants. Front Plant Sci 6:869. https://doi.org/10.3389/fpls.2015.00869 Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer ZS, Yanofsky MF, Coupland G (2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288:1613–1616. https://doi.org/10.1126/science.288.5471.1613 Sangwan RS (1978) Change in the amino-acid content during male gametophyte formation of Datura metel in situ. Theor Appl Genet 52:221–225. https://doi.org/10.1007/BF00273893 70 M. Trovato et al.

Satoh R, Nakashima K, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2002) ACTCAT, a novel cis-acting element for proline- and hypoosmolarity-responsive expression of the ProDH gene encoding proline dehydrogenase in Arabidopsis. Plant Physiol 130:709–719. https://doi. org/10.1104/pp.009993 Savouré A, Jaoua S, Hua X-J, Ardiles W, Van Montagu M, Verbruggen N (1995) Isolation, characterization, and chromosomal location of a gene encoding the Δ1-pyrroline-5- carboxylate synthetase in Arabidopsis thaliana. FEBS Lett 372:13–19. https://doi. org/10.1016/0014-5793(95)00935-3 Savouré A, Jaoua S, Hua XJ, Ardiles W, Van Montagu M, Verbruggen N (1997) Abscisic acid- independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana. Mol Gen Genet 254:104–109. https://doi. org/10.1007/s004380050397 Schertl P, Cabassa C, Saadallah K, Bordenave M, Savouré A, Braun HP (2014) Biochemical characterization of proline dehydrogenase in Arabidopsis mitochondria. FEBS J 281:2794–2804. https://doi.org/10.1111/febs.12821 Schmidt R, Stransky H, Koch W (2007) The amino acid permease AAP8 is important for early seed development in Arabidopsis thaliana. Planta 226:805–813. https://doi.org/10.1007/ s00425-007-0527-x Schulze WX, Yao Q, Xu D (2015) Databases for plant phosphoproteomics. Methods Mol Biol 1306:207–216. https://doi.org/10.1007/978-1-4939-2648-0_16 Schwacke R, Grallath S, Breitkreuz KE, Stransky E, Stransky H, Frommer WB, Rentsch D (1999) LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen. Plant Cell 11:377–392. https://doi.org/10.1105/tpc.11.3.377 Sharma S, Shinde S, Verslues PE (2013) Functional characterization of an ornithine cyclodeaminase-like protein of Arabidopsis thaliana. BMC Plant Biol 13:182. https://doi. org/10.1186/1471-2229-13-182 Sharma S, Villamor JG, Verslues PE (2011) Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential. Plant Physiol 157:292–304. https://doi.org/10.1104/pp.111.183210 Shetty K, Wahlqvist ML (2004) A model for the role of the proline-linked pentose-phosphate pathway in phenolic phytochemical bio-synthesis and mechanism of action for human health and environmental applications. Asia Pac J Clin Nutr 13:1–24 Shinde S, Villamor JG, Lin W, Sharma S, Verslues PE (2016) Proline coordination with fatty acid synthesis and redox metabolism of chloroplast and mitochondria. Plant Physiol 172:1074– 1088. https://doi.org/10.1104/pp.16.01097 Shui XR, Chen ZW, Li JX (2013) MicroRNA prediction and its function in regulating drought- related genes in cowpea. Plant Sci 210:25–35. https://doi.org/10.1016/j.plantsci.2013.05.002 Signorelli S, Monza J (2017) Identification of Δ1-pyrroline 5-carboxylate synthase (P5CS) genes involved in the synthesis of proline in Lotus japonicus. Plant Signal Behav 12:e1367464. https://doi.org/10.1080/15592324.2017.1367464 Srikanth A, Schmid M (2011) Regulation of flowering time: all roads lead to Rome. Cell Mol Life Sci 68:2013–2037. https://doi.org/10.1007/s00018-011-0673-y Stines AP, Naylor DJ, Høj PB, van Heeswijck R (1999) Proline accumulation in developing grapevine fruit occurs independently of changes in the levels of Δ1-Pyrroline-5-Carboxylate Synthetase mRNA or protein. Plant Physiol 120:923–923. https://doi.org/10.1104/pp.120.3.923 Stránská J, Kopecný D, Tylichová M, Snégaroff J, Sebela M (2008) Ornithine delta- aminotransferase: an enzyme implicated in salt tolerance in higher plants. Plant Signal Behav 3:929–935 Strecker HJ (1965) Purification and properties of rat liver ornithineδ -transaminase. J Biol Chem 240:1225–1230 Strizhov N et al (1997) Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. Plant J 12:557–569. https://doi.org/10.1111/j.0960-7412.1997.00557.x Proline Metabolism and Its Functions in Development and Stress Tolerance 71

Székely G et al (2008) Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. Plant J 53:11–28. https://doi. org/10.1111/j.1365-313X.2007.03318.x Szoke A, Miao GH, Hong Z, Verma DP (1992) Subcellular location of δ1-pyrroline-5-carboxylate reductase in root/nodule and leaf of soybean. Plant Physiol 99:1642–1649 Taiz L, Zeiger E, Møller IM, Murphy A (2018) Fundamentals of plant physiology. Oxford University Press, New York/Oxford Tanner JJ (2019) Structural biology of proline catabolic enzymes. Antioxid Redox Signal 30:650– 673. https://doi.org/10.1089/ars.2017.7374 Taylor AA, Stewart GR (1981) Tissue and subcellular localization of enzymes of arginine metabolism in Pisum sativum. Biochem Biophys Res Commun 101:1281–1289 Tegeder M, Hammes UZ (2018) The way out and in: phloem loading and unloading of amino acids. Curr Opin Plant Biol 43:16–21. https://doi.org/10.1016/j.pbi.2017.12.002 Thiery L, Leprince AS, Lefebvre D, Ghars MA, Debarbieux E, Savouré A (2004) Phospholipase D is a negative regulator of proline biosynthesis in Arabidopsis thaliana. J Biol Chem 279:14812– 14818. https://doi.org/10.1074/jbc.M308456200 Trovato M, Maras B, Linhares F, Costantino P (2001) The plant oncogene rolD encodes a functional ornithine cyclodeaminase. Proc Natl Acad Sci U S A 98:13449.13453. https://doi. org/10.1073/pnas.231320398 Trovato M, Mattioli R, Costantino P (2018) From A. rhizogenes RolD to plant P5CS: exploiting proline to control plant development. Plan Theory 7:108 Trovato M, Mauro ML, Costantino P, Altamura MM (1997) The rolD gene from Agrobacterium rhizogenes is developmentally regulated in transgenic tobacco. Protoplasma 197:111–120. https://doi.org/10.1007/BF01279889 Turchetto-Zolet AC, Margis-Pinheiro M, Margis R (2009) The evolution of pyrroline-5-carboxylate synthase in plants: a key enzyme in proline synthesis. Mol Gen Genomics 281:87–97 Van Aken O, Zhang B, Carrie C, Uggalla V, Paynter E, Giraud E, Whelan J (2009) Defining the mitochondrial stress response in Arabidopsis thaliana. Mol Plant 2:1310–1324. https://doi. org/10.1093/mp/ssp053 Vansuyt G, Vallee J-C, Prevost J (1979) La pyrroline-5-carboxylate réductase et la proline déhydro- génase chez Nicotiana tabacum var. Xanthi n.c. en fonction de son développement. Physiologie végétale 19:95–105 Veerabagu M et al (2014) The interaction of the Arabidopsis response regulator ARR18 with bZIP63 mediates the regulation of PROLINE DEHYDROGENASE expression. Mol Plant 7:1560–1577. https://doi.org/10.1093/mp/ssu074 Venekamp JH, Koot JTM (1988) The sources of free proline and asparagine in field bean plants, Vicia faba L., during and after a short period of water withholding. J Plant Physiol 32:102–109 Verbruggen N, Hua XJ, May M, Van Montagu M (1996) Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator. Proc Natl Acad Sci U S A 93:8787–8791. https://doi.org/10.1073/pnas.93.16.8787 Verbruggen N, Villarroroel R, Van Montagu M (1993) Osmoregulation of a pyrroline-5-carboxylate reductase gene in Arabidopsis thaliana. Plant Physiol 103:771–781 Verslues PE, Skarp RE (1999) Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. II. Metabolic source of increased proline deposition in the elongation zone. Plant Physiol 119:1349–1360 Wada KC, Takeno K (2010) Stress-induced flowering. Plant Signal Behav 5:944–947.https://doi. org/10.4161/psb.5.8.11826 Wakte K, Zanan R, Hinge V, Khandagale K, Nadaf A, Henry R (2017) Thirty-three years of 2-acetyl-1-pyrroline, a principal basmati aroma compound in scented rice (Oryza sativa L.): a status review. J Sci Food Agric 97:384–395. https://doi.org/10.1002/jsfa.7875 Walton EF, Clark CJ, Boldingh HL (1991) Effect of hydrogen cyanamide on amino acid profiles in kiwifruit buds during bud-break. Plant Physiol 97:1256–1259 72 M. Trovato et al.

Wanduragala S, Sanyal N, Liang X, Becker DF (2010) Purification and characterization of Put1p from Saccharomyces cerevisiae. Arch Biochem Biophys 498:136–142. https://doi. org/10.1016/j.abb.2010.04.020 Wang G et al (2014) Proline responding1 plays a critical role in regulating general protein synthesis and the cell cycle in Maize. Plant Cell 26:2582–2600. https://doi.org/10.1105/tpc.114.125559 Wang T, Chen Y, Zhang M, Chen J, Liu J, Han H, Hua X (2017) Arabidopsis AMINO ACID PERMEASE1 contributes to salt stress-induced proline uptake from exogenous sources. Front Plant Sci 8:2182. https://doi.org/10.3389/fpls.2017.02182 Watanabe S, Tanimoto Y, Yamauchi S, Tozawa Y, Sawayama S, Watanabe Y (2014) Identification and characterization of trans-3-hydroxy-l-proline dehydratase and Delta(1)-pyrroline-2- carboxylate reductase involved in trans-3-hydroxy-l-proline metabolism of bacteria. FEBS Open Bio 4:240–250. https://doi.org/10.1016/j.fob.2014.02.010 Weiste C et al (2017) The Arabidopsis bZIP11 transcription factor links low-energy signalling to auxin-mediated control of primary root growth. PLoS Genet 13:e1006607. https://doi. org/10.1371/journal.pgen.1006607 Weltmeier F et al (2006) Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific heterodimerisation of bZIP transcription factors. EMBO J 25:3133–3143. https://doi.org/10.1038/sj.emboj.7601206 White FF, Taylor BH, Huffman GA, Nester EW (1985) Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol 164:33–44 Yang J, Zhang N, Ma C, Qu Y, Si H, Wang D (2013) Prediction and verification of microRNAs related to proline accumulation under drought stress in potato. Comput Biol Chem 46:48–54. https://doi.org/10.1016/j.compbiolchem.2013.04.006 Yoshiba Y, Nanjo T, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Stress-responsive and developmental regulation of Delta(1)-pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Arabidopsis thaliana. Biochem Biophys Res Commun 261:766–772 Yoshihashi T, Huong NT, Inatomi H (2002) Precursors of 2-acetyl-1-pyrroline, a potent flavor compound of an aromatic rice variety. J Agric Food Chem 50:2001–2004 You J, Hu H, Xiong L (2012) An ornithine δ-aminotransferase gene OsOAT confers drought and oxidative stress tolerance in rice. Plant Sci 197:59–69. https://doi.org/10.1016/j. plantsci.2012.09.002 Zarattini M, Forlani G (2017) Toward unveiling the mechanisms for transcriptional regulation of proline biosynthesis in the plant cell response to biotic and abiotic stress conditions. Front Plant Sci 8. https://doi.org/10.3389/fpls.2017.00927 Zhang CS, Lu Q, Verma DP (1995) Removal of feedback inhibition of delta 1-pyrroline-5- carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270:20491–20496 Zhang CY, Wang NN, Zhang YH, Feng QZ, Yang CW, Liu B (2013) DNA methylation involved in proline accumulation in response to osmotic stress in rice (Oryza sativa). Genet Mol Res 12:1269–1277. https://doi.org/10.4238/2013.April.17.5 Zimorski V, Ku C, Martin WF, Gould SB (2014) Endosymbiotic theory for organelle origins. Curr Opin Microbiol 22:38–48. https://doi.org/10.1016/j.mib.2014.09.008 Zonglie H, Karuna L, Zhongming Z, Verma DPS (2000) Removal of feedback inhibition of Δ1-Pyrroline-5-Carboxylate Synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol 122:1129–1136. https://doi.org/10.1104/ pp.122.4.1129 Regulation of Proline Accumulation and Its Molecular and Physiological Functions in Stress Defence

Giuseppe Forlani, Maurizio Trovato, Dietmar Funck, and Santiago Signorelli

1 Introduction

Among the building blocks needed for protein synthesis, proline is the only cyclic amino acid, i.e. a secondary amine in which two carbons are bonded to the amine nitrogen forming a five-membered heterocyclic ring. Because of this, proline plays a unique role in determining protein structure by influencing backbone folding and stability (Ge and Pan 2009). Proline is synthesized in a two-step reduction and cyclization of glutamate (Fichman et al. 2015), although other possible routes have been hypothesized to occur in some plant species (da Rocha et al. 2012). Preliminary evidence supporting an at least partial localization of the anabolic pathway in the chloroplast (Szekely et al. 2008) has not been further supported; thus proline production is regarded as a cytosolic process. As a consequence, free proline is present in the cytoplasm, where homeostatic levels depend on the balance between the rates of its production and utilization for protein synthesis, but also on its translocation to the mitochondrion (Di Martino et al. 2006), where proline is oxidized back to glutamate via an equally short catabolic pathway. The biosynthetic route is controlled mainly through feedback regulation of the enzyme catalysing the first step, namely,

G. Forlani (*) Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy e-mail: [email protected] M. Trovato Department of Biology and Biotechnology, Sapienza University of Rome, Rome, Italy D. Funck Department of Biology, Division of Plant Physiology and Biochemistry, University of Konstanz, Konstanz, Germany S. Signorelli (*) Department of Plant Biology, Universidad de la República, Montevideo, Uruguay e-mail: [email protected]

δ1-pyrroline-5-carboxylate (P5C) synthetase (P5CS) (Hu et al. 1992; Hong et al. 2000). The turnover of amino acids into proteins and vice versa seems rapid, since similar proportions of 14C were found in unstressed cells following 14C labelling (Dong et al. 2018). Besides the involvement in protein synthesis, proline is believed to play a main role in the plant response to several (a)biotic stresses. Starting from the 1960s of the last century, hundreds of papers have reported rapid accumulation of high free proline levels in cells subjected to a variety of stress conditions (Delauney and Verma 1993; Hayat et al. 2012; Verbruggen and Hermans 2008) (Table 1). In response to stress, protein synthesis is reduced, favouring amino acid accumulation (Dong et al. 2018). Notwithstanding this, in most cases the rise of free proline content largely exceeds those of the other proteinogenic amino acids. Accordingly, genetically modified plants with increased intracellular proline content showed higher stress tolerance (Per et al. 2017). The specific increase in proline content may derive from either a reduction in its mitochondrial oxidation (Nanjo et al. 1999), an increase of its biosynthesis (Zhang et al. 1995) or import from other tissues (Lehmann et al. 2010; Wang et al. 2017) via phloem sap. The latter seems to contribute to developmentally related variations in proline homeostasis, as in reproductive organs (Biancucci et al. 2015), whereas increased biosynthesis appears the main mechanism occurring after the exposure to abiotic stress, such as drought, salinity or freezing (Yoshiba et al. 1995, 1997). Because P5CS is feedback inhibited by millimolar concentrations of proline, and homeostatic proline level results from the combination of synthesis, catabolism and transport rates, the accumulation of increased levels of proline in the cytosol requires fine regulation of its metabolism under stress. Consistently, an impressive number of putative cis-regulatory elements recognized by different classes of transcription factors were found in the promoter region of the involved genes (Fichman et al. 2015; Zarattini and Forlani 2017).

2 Regulation of Proline Accumulation Under Stress Conditions

A variety of diverse abiotic and biotic stress conditions were found to induce the accumulation of proline in plants (Szabados and Savouré 2010). Proline accumulation was suggested to be due to an increase of the de novo biosynthesis rather than lower catabolism or greater protein degradation (Chiang and Dandekar 1995; Szabados and Savouré 2010; Hildebrandt 2018). Under stress, proline is mainly biosynthesized from glutamate by two enzymatic steps consuming NADPH preferably and catalysed by P5CS and P5C reductase (P5CR) (Forlani et al. 2015c; Giberti et al. 2014). Most plants have two P5CS isoforms, one housekeeping and the other inducible, the latter being mainly responsible for proline accumulation under stress (Signorelli and Monza 2017). Proline accumulation is more significant in photosynthetic tissues (e.g. leaves) but also takes place in roots (Sharma et al. 2011; Signorelli et al. 2013a; Verslues and Sharp 1999). At the cellular level, proline accumulation is Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 75

Table 1 Stress-induced accumulation of intracellular proline levels in response to stress conditions in some selected plant species Pro content (fold Species Stressor Tissue increase) Reference Nicotiana NaCl (171 mM) Cultured cells 48 Binzel et al. (1987) tabacum NaCl (342 mM) 246 NaCl (428 mM) 445 Nicotiana 30% relative Wilted leaves 40 Sano and Kawashima tabacum humidity (1982) Solanum PEG 20% Cultured cells 38 Handa et al. (1983) lycopersicon PEG 25% 244 PEG 30% 506 Capsicum PEG 25% Cultured cells 200 del Socorro Santos- annuum Díaz and Ochoa- Alejo (1994) Arabidopsis NaCl (120 mM) Rosette leaves 8–10 Chiang and Dandekar thaliana (1995) Arabidopsis NaCl (150 mM) 14-day-old 16 Signorelli et al. thaliana seedlings (2016))

Arabidopsis Low ΨW (−1.2 MPa) Seedlings 160 Shinde et al. (2016) thaliana Oryza sativa NaCl (100 mM) Seedlings 2–10 Lv et al. (2015) Oryza sativa NaCl (200 mM) Seedlings 2–3 Bertazzini et al. (2018) Trifolium Drought (42% hydric Wilted leaves 139 Signorelli et al. pratense index reduction) (2013b) Lotus Drought (45% Leaves 18 Signorelli et al. japonicus relative water content (2013c) reduction) Drought (33% Roots 35 relative water content reduction) Zea mays Growth in medium at Apical 50 Voetberg and Sharp −1.6 MPa millimeter of (1991) corn root tips Lotus Drought (50% hydric Wilted leaves 16 Signorelli et al. corniculatus index reduction) (2013b) While several Solanaceae showed hundredfold increases in free proline content, much lower concentrations were found in other plants suggested to be greatest in the chloroplast, followed by the cytoplasm (Büssis and Heineke 1998). P5CR was shown to be induced by saline stress (Verbruggen et al. 1993); however, this was challenged by Yoshiba and co-workers (1995), who found no response of P5CR to salt stress or dehydration and attributed proline accumulation to increased P5CS1 expression. Therefore, P5CR is not believed to play a critical role in proline accumulation under osmotic stress. However, the biochemical properties 76 G. Forlani et al. of P5CR suggest that under some conditions also P5CR activity might become limiting (Giberti et al. 2014). Although proline dehydrogenase (ProDH, also known as proline oxidase or POX) is not as determinant as P5CS1 concerning proline accumulation under stress, the oscillation of gene expression in the light/dark cycles and downregulation under dehydration, saline, and osmotic stress contributes to enhanced proline accumulation (Hayashi et al. 2000; Verslues and Sharma 2010). Other factors, such as light or reactive oxygen species (ROS), also need to be present for proline accumulation to take place. Early studies demonstrated that proline content oscillated according to light/dark cycles (Hayashi et al. 2000), and even stress-induced proline accumulation was shown to be dependent on light (Sanada et al. 1995; Díaz et al. 2005; Aleksza et al. 2017). The light dependence of proline accumulation can be attributed, at least in part, to the need of reducing power for its biosynthesis, which is generated during photosynthesis and might be transferred into the cytosol by different redox shuttles. However, later reports showed that the expression of P5CS1 is also affected by light and is mediated by Elongated Hypocotyl 5 (HY5) (Feng et al. 2016; Lee et al. 2007). HY5 is a basic leucine zipper (bZIP) transcription factor, controlling a plethora of processes, such as development, abiotic stress, ROS, and hormone signalling, in a light-dependent manner (Gangappa and Botto 2016; Signorelli et al. 2018). Thus, the light dependence of proline accumulation is not just due to the requirement of NADPH but also to signalling reasons. This finding of the role of HY5 in mediatingP5CS1 expression also points out how important the coordination of proline metabolism with other processes is. Similarly, the expression of the main enzymes controlling proline accumulation, P5CS1 and ProDH, was suggested to be dependent on the Respiratory Burst Oxidase Homologue (Rboh, NADPH oxidase in animals) activity (Ben Rejeb et al. 2015). In short, the authors showed that the hydrogen peroxide (H2O2) produced as a consequence of Rboh and SOD activity is necessary for proline accumulation to occur.

The simple pharmacological scavenging of H2O2 was enough to attenuate proline accumulation under stress conditions (Ben Rejeb et al. 2015). This finding demonstrated that proline accumulation is also regulated, at least in part, by ROS signalling. ROS seem to be produced under most stress conditions. Thus, in a physiological context, while light can be a determinant factor for proline accumulation to take place or not, the role of ROS is probably more complex, and variable concentrations of different ROS species may influence proline biosynthesis differentially. The production of the phytohormone abscisic acid (ABA) is induced under stress conditions in plants, and ABA acts as a signalling molecule to mediate the adaptation of the plant to the new environment. In rice, ABA is known to mediate proline accumulation (Sripinyowanich et al. 2013); however, in arabidopsis (Arabidopsis thaliana), proline accumulation can be either ABA-dependent or ABA-independent depending on the stress condition (Savouré et al. 1997; Zarattini and Forlani 2017). Recently, a wild variety of barley was shown to accumulate higher proline levels under drought compared to the cultivated one. The authors showed that the difference between the ancestral and the cultivated variety was one nucleotide in the Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 77 sequence of the P5CS1 promoter, which modified an ABF (ABA-responsive elements (ABRE) binding factors) binding site, making the cultivated barley to be unresponsive to ABA (Muzammil et al. 2018). Likewise, in arabidopsis, the 5’ UTR region of AtP5CS1 contains ABF binding sites. Other cis-responsive elements (CRE), such as AP2/EREBP, ERF2, DREB/CBF, and MYB binding sites are also found in the promoter of P5CS1 genes of different species (for detailed reviews, see Fichman et al. 2015; Zarattini and Forlani 2017). This wide variety of CRE in the promoter of the main gene responsible for proline synthesis explains why proline accumulation is a conserved response observed in a broad range of conditions in plants.

3 Osmoprotective Role of Proline

Despite the plethora of papers describing a stress-induced increase of free proline concentration, and the evidence of a statistically significant relationship between this increase and increased stress tolerance, the mechanisms by which high levels of this amino acid may benefit the cell are far from being fully understood and conclusively proven. The early hypothesis of a central osmotic role for proline, i.e. that high levels of this compound may avoid water withdrawal to the apoplast by lowering the cellular water potential (ΨW), seems inconsistent with the relatively low absolute concentration reached in several cases and has been recently questioned (Bhaskara et al. 2015; Forlani et al. 2018; Kavi Kishor and Sreenivasulu 2014; Ben Rejeb et al. 2014; Sharma et al. 2011; Signorelli 2016). Notwithstanding this, many authors still consider a function of proline as a “compatible osmolyte” as an established fact.

3.1 Proline as Compatible Osmolyte

A famous quote says that if you repeat a lie often enough, people will believe it, and you will even come to believe it yourself. In the scientific literature, we could say that if a sound hypothesis is repeated in many papers, with citation to other previous reports, at a certain point, scientists will start to believe that it has been proven somewhere in the past, and do not verify whether it fits with their experimental system or whether their results are consistent with it. This is probably true for the “osmotic role” of proline accumulation in response to hyperosmotic stress conditions. Because of early reports describing in some plant species a striking increase of its intracellular level under drought or salt stress (Table 1), from a given moment onwards, any statistically significant increase in proline concentration has been interpreted as a genuine contribution to osmotic compensation. Is this interpretation sound? Let us make some calculations. Because in a differentiated plant tissue the measurement of both the apoplastic water volume and the external water potential 78 G. Forlani et al.

(ΨW) is not easy (Lohaus et al. 2001), the use of a cell suspension culture may facilitate the estimates. Cells growing in the widely used Murashige and Skoog (MS) −1 medium with 30 g L sucrose are in osmotic equilibrium with it, at an external ΨW of about −0.52 MPa (Fig. 1a). Since cellular concentration (ΨS) usually corresponds to an osmotic pressure (Π) ranging from −0.7 to −1.2 MPa (Handa et al. 1983; Ikeda et al. 1999), this allows cell turgidity, with a turgor pressure (ΨP) of about 0.1 to 0.7 MPa. The addition of 25% polyethylene glycol (PEG-6000) to the culture medium, a condition that has been found permissive in many cases (i.e. plant cells can adapt to these conditions [e.g. del Socorro Santos-Díaz and Ochoa-Alejo 1994]), lowers the external ΨW to −2.0 MPa (Fig. 1b). To avoid water withdrawal, the cell has to increase its internal osmolyte concentrations to obtain a Π decrease of 0.8 to

Fig. 1 Osmotic compensation in rapidly dividing cells. Suspension-cultured cells growing in MS medium are in equilibrium with an environmental water potential of about −0.52 MPa (panel a). The addition of an osmoticum, for instance, 25% PEG 6000, causes a dramatic decrease of the external water potential (ΨW) (b). To adapt, the intracellular concentration (ΨS) should increase consistently, so as to avoid loss of turgor pressure (ΨP) and the consequent occurrence of plasmoly- sis (c). Because only about 20–30% of the overall cell fresh weight is attributable to the cytoplasm (d), to obtain osmotic compensation solely by proline accumulation, an increase of cytosolic free proline concentration as high as 80–130 μmol (g fw)−1 would be required Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 79

1.3 MPa, corresponding (at 25 °C) to an increased osmolarity of about 320–520 mmol·L−1 (Fig. 1c). In most studies, proline content is measured on a fresh or dry weight (fw or dw) basis following cell extraction with sulfosalicylic acid and expressed as μmol (g fw or dw)−1 (e.g. Aleksza et al. 2017). If, as most likely, proline accumulation occurs mainly in the cytosol, the evaluation of its osmotic value would require quantitation of the cytoplasmic volume. Concerning this, we should consider that in actively proliferating cultured cells, the vacuole accounts for only 5–15% of total volume but that nucleus, mitochondria, and plastids are a significant fraction of the cell. Also, considering that the apoplastic water may represent 10–40% of water content (Binzel et al. 1987; Speer and Kaiser 1991), to a good approximation, we can consider the cytoplasmic water as 20–25% of the overall fresh weight (Fig. 1d). This would imply that if proline would be the only osmolyte produced and accumulated, to achieve osmotic compensation, its content should increase by at least 80–130 μmol (g fw)−1. In fact, in most cases, the reported increase does not exceed 5–10 μmol (g fw)−1 (e.g. Poustini et al. 2007), and often it is less than 1–2 μmol (g fw)−1 (e.g. Bertazzini et al. 2018). Consistently, in some excellent and pioneer works in which most of the main components of the cellular sap have been quantified at the same time, the contribution of proline to the required increase of osmolarity was estimated as not exceeding 3–15%, despite the fact that its concentration had shown a hundredfold increase over controls (Binzel et al. 1987; Handa et al. 1983; del Socorro Santos-Díaz and Ochoa-Alejo 1994). Only in the apical millimeter of corn roots growing at a water potential of −1.6 MPa, proline accumulation reaching about 120 μmol (g fw)−1 accounted for almost half of the osmotic adjustment (Voetberg and Sharp 1991). In wheat plants, the contribution made by proline was estimated to be equivalent to 0.07 MPa, whereas that made by K+ and Na+ was 0.21 and 0.45 MPa, respectively (Poustini et al. 2007). Of course, any significant input to the attainment of a suitably high cellularΨ s concurs to ameliorate the osmotic unbalance. However, because of the implicated values, the significance of a proline accumulation lower than 5 μmol (g fw)−1 (approximately equivalent to 0.06 MPa Ψs in the cytosol) should be regarded – in our opinion – as marginal for an effective osmotic compensation.

3.2 Proline as Stabilizer for Enzymes and Membranes

Given that proline seems unlikely to fully compensate the osmotic unbalance produced under stress, the doubtless beneficial effect of higher proline levels in the cell facing hyperosmotic conditions should also rely on other possible mechanisms. Several hypotheses have been proposed. One of the most commonly accepted is a kosmotropic (= anti-chaotropic) activity of proline. In solution, proteins, and in general all macromolecules, are surrounded by highly ordered water molecules that lower entropy, allowing the attainment of proper three-dimensional folding and, in case of an enzyme, the achievement of the catalytically active conformation (Fig. 2a). Osmotic stress-induced water withdrawal from the cell or Fig. 2 Proposed mechanism for protein protection by proline. In solution, enzymes are surrounded by water molecules that interact with the hydrophilic regions of proteins, mainly through the formation of hydrogen bonds, allowing the attainment of proper three-dimensional folding and the achievement of the catalytically active conformation (panel a). Increased concentration of ions, and/or chaotropic substances, reorders the water molecules around them and reduces the interactions with the protein, resulting in protein unfolding and activity loss (panel b). Proline is believed to exert an anti-chaotropic effect and stabilize proteins by helping to maintain a proper water solva- tion shell around them (panel c). This effect may rely, for instance, on the hydrophobic interaction of the pyrrolidine ring with hydrophobic surfaces, thereby increasing hydrophilic areas, or on stabilization of the water molecules that interact with the protein through hydrogen bonds. The hydrogen bonds are represented by dashed lines. Thicker hydrogen bonds represent stronger interactions Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 81

hyperosmolarity-driven ion influxes cause a progressive increase of chaotropic substances in the cytosol, leading to protein unfolding and activity loss (Fig. 2b). Proline and other very soluble and well-hydrated molecules with little tendency to aggregate, such as the sugar α,α-trehalose, the quaternary ammonium compound glycine betaine, and 3-dimethyl-sulfonopropionate, at high concentrations exert a kosmotropic effect and stabilize the structure of macromolecules in solution by helping to maintain a proper hydration shell around them (Fig. 2c). It was postulated that proline forms aggregates by stepwise stacking and hydrophobic interaction of the pyrrolidine ring with hydrophobic surface residues of proteins, thereby increasing their hydrophilic area (Schobert and Tschesche 1978). An increased water-binding capacity of the proline-protein solution has therefore been invoked to explain alleviation of the noxious effects of water stress effects on protein activity and stability (Arakawa and Timasheff 1983, 1985). Moreover, proline has also been reported to favour protein renaturation and avoid protein aggregation by similarly trapping the folding intermediates in a supramolecular assembly (Samuel et al. 2000). A great unknown, also in this case, is whether local concentrations high enough to ensure these beneficial effects may be reached in vivo inside the cell. On the other hand, when the phase behaviour of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine was examined in aqueous dispersions containing a range of sodium salts, the lipid phase properties exhibited a graded response analogous to that of the Hofmeister series causing protein salting out (Sanderson et al. 1991). Therefore, it is likely that at low water potential kosmotropic agents may stabilize membranes as well. Consistently, proline, betaine and trehalose were found to increase the area/molecule of three membrane phospholipids, therefore acting as stabilizer agents. In particular, data suggested that, due to its amphipathic nature, proline may interact with phosphatidylcholines through intercalation between phospholipid head groups (Rudolph et al. 1986).

4 Proline as a Potential ROS Scavenger

Abiotic and biotic stresses affect ROS homeostasis, usually resulting in an overproduction of specific ROS species. To avoid oxidative damage, plants respond by enhancing enzymatic and non-enzymatic antioxidant systems. As proline accumulates under stressful conditions, some authors hypothesized that proline protects against ROS. In good agreement with this idea, proline was shown to protect against the most potent ROS, the hydroxyl radical (•OH; Smirnoff and Cumbes 1989). Later, proline was shown to enhance photochemical activity in thylakoid membranes (Alia and Mohanty 1991). Moreover, the same group demonstrated that proline attenuates malondialdehyde (MDA) formation in cotyledons of Brassica juncea under saline stress (Alia and Mohanty 1993), zinc stress (Alia and Saradhi 1995), and UV stress (Saradhi et al. 1995). Based on the lower oxidative damage observed in plants treated with proline, the authors proposed that proline plays an essential role in non-enzymatic detoxification of free radicals, although no evidence of direct 82 G. Forlani et al. reaction between proline and oxidant molecules, or their potential products, were obtained (Alia and Saradhi 1995). Proline was also shown to reduce MDA formation in thylakoids during exposure to intense light and was suggested to either react 1 with singlet oxygen ( O2) or reduce its formation (Alia and Mohanty 1997). In 2001, the same group presented compelling evidence suggesting that proline could attenuate the singlet oxygen-mediated 2,2,6,6-tetramethylpiperidin (TEMP) oxidation, claiming they demonstrated that proline is a very effective singlet oxygen quencher (Alia and Matysik 2001). This work finished stamping the antioxidant label on proline. In an elegant work, Hamilton and Heckathorn (2001) demonstrated that the primary cause of mitochondrial electron transport disruption by saline stress is oxidative damage in complex I and Na+ toxicity in complex II. The authors showed that proline was unable to protect complex I, whereas non-enzymatic antioxidants, such as glutathione, tocopherol, and ascorbic acid, and enzymatic antioxidants, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), did it. As SOD showed the highest protection of the complex I, the authors concluded •− that superoxide (O2 ) was causing most of the oxidative damage of complex I. Altogether, these results challenge the idea that proline is an antioxidant and suggest that proline does not protect against superoxide. In addition, proline was shown to induce the activity of CAT, peroxidase, and polyphenol oxidase (Öztürk and Demir 2002), suggesting that some of the observations of proline amelioration of oxidative damage could be related to an enhancement of the enzymatic antioxidant machinery. The connection between proline accumulation and the oxidative damage/antioxidant response under different stress conditions was evaluated in many different plant species; however, a clear link between proline accumulation and oxidative stress was difficult to establish, as the results were opposite in many cases. This controversy might be explained by the fact that while proline accumulation is suggested to protect against the oxidative damage, vast evidence was also provided showing that excess proline can be a pro-oxidant, as it induces ROS production via mitochondrial electron leakage (Deuschle et al. 2004; Lv et al. 2011; Miller et al. 2009a). The latter is well established in animals (Donald et al. 2001; Elia et al. 2017; Liu et al. 2009; Polyak et al. 1997). In turn, proline-induced ROS generation can induce the antioxidant response, acting as priming agent, adding an extra layer of complexity. For instance, the exogenous treatment with proline induced the activity of antioxidant enzymes in cultured tobacco BY2 cells (Hoque et al. 2007; Hossain and Fujita 2010; Islam et al. 2009), but could not directly protect against superoxide or H2O2 (Hoque et al. 2007). Concerning endogenous proline accumulation, two arabidopsis non-proline-accumulating mutant lines, p5cs1–2 and p5cs1–4, were shown to have higher H2O2 and lipid peroxidation upon saline stress, whereas the enzymatic antioxidant activities showed no uniform results (Szekely et al. 2008). The lack of direct evidence about the putative antioxidant properties of proline was in part attributed to the difficulty to work with ROS, due to their high reactivity. Although proline was suggested to quench singlet oxygen (Alia and Matysik 2001), Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 83 the mechanism had not been described. In order to determine whether this quenching was chemical or physical, some of us investigated these putative mechanisms of quenching using direct real-time measurement of singlet oxygen by its lumines- cence at 1270 nm. Surprisingly, proline was not able to quench singlet oxygen, neither chemically nor physically (Signorelli et al. 2013b). In an attempt to reproduce the observations from Alia and Matysik (2001), we found out that the EPR signal of singlet-O2-dependent TEMP oxidation becomes less stable in the presence of proline, explaining why the authors apparently detected lower TEMP oxidation activity, which led to the misinterpretation that proline was scavenging singlet oxygen and avoiding formation of oxidised TEMP. Similarly, the reaction mechanisms of proline with hydroxyl radicals were investigated to assess whether hydroxyproline is a potential product of this interaction. Using computational chemistry, we found out that proline would rapidly react with a hydroxyl radical through hydrogen abstraction (H-abstraction). When the H-abstraction occurs on a C atom, it is more likely to yield P5C as a non-radical product than hydroxyproline (Signorelli et al. 2014). As P5C is converted back to proline by P5CR, we suggested a Pro-Pro cycle in which proline could scavenge two hydroxyl radicals and being regenerated with the consumption of one NADPH (Signorelli et al. 2014). One of the essential characteristics of antioxidants, such as glutathione and ascorbate, is that enzymes can recycle them. By the Pro-Pro cycle, proline was for the first time able to share this typical feature of antioxidants. When the H-abstraction on the nitrogen atom was evaluated, we found that this reaction was energetically preferred by hydroxyl radicals and would result in the decarboxylation of proline (Signorelli et al. 2015). The potential non-radical product was shown to be δ1-pyrroline, a ǖFE;-aminobutyric acid (GABA) precursor. This provided a link between proline and GABA involving non-enzymatic reactions under stress conditions (Signorelli et al. 2015). It is worth mentioning that, due to the non-enzymatic nature of the reaction, this link would never explain the concomitant accumulation of proline and GABA, which is most likely due to similar upstream regulators of their biosynthesis. As the second most reactive ROS, singlet oxygen, was not able to react with proline, we wondered whether proline was able to react with more stable ROS and reactive nitrogen species (RNS). In both in vivo and in vitro experimental conditions, we observed that proline was not able to scavenge • • − superoxide, nitric oxide ( NO), nitrogen dioxide ( NO2), and peroxynitrite (ONOO ; Signorelli et al. 2016). This challenged the idea that proline is accumulated under stress to act as an antioxidant. There is at present no evidence to suggest that proline is more likely to react with hydroxyl radicals than most other organic molecules. Further research is needed to understand how significant the contribution of proline to hydroxyl radical scavenging is in physiological conditions. With the current evidence, we believe that the observed beneficial effects of proline on oxidative damage are more likely to be due to its direct stabilization effect on membranes and proteins (Sect. 3.2) and/or the capacity to activate the antioxidant defence (further in Sect. 6) than to a direct role as an antioxidant. 84 G. Forlani et al.

5 Effect of Proline Accumulation on Redox Balance

Proline synthesis from glutamate requires a double equivalent amount of reducing power. When needed to accumulate to high concentrations, the increased synthetic rate is therefore capable of lowering NAD(P)H availability inside the cytosol. Conversely, the mitochondrial catabolism of proline provides reducing equivalents in the form of an FADH2 moiety bound to the ProDH enzyme (that is believed to transfer electrons directly to the respiratory chain) and an NADH molecule formed during the subsequent P5C oxidation (Fig. 3). As a consequence, a reciprocal dynamic relationship has been hypothesized between proline metabolism and the redox status of the cell, i.e. the possibility that any variation in proline homeostasis may result in a corresponding change in NAD(P)H/NAD(P)+ ratio and (more recently) that also the opposite may be true. Moreover, as the two pathways occur spatially separated in the cytosol and in mitochondria, the interconversion of

Fig. 3 The potential P5C-proline cycle. Proline synthesis in plants proceeds in the cytosol through a two-step pathway, while proline is oxidized back to glutamate in the mitochondrion. It has been hypothesized that the intermediate in both routes, P5C, may be transported through the inner mitochondrial membrane. If so, a cycle may be established in which the interconversion of P5C and proline may transfer reducing equivalents from the cytosol to the mitochondrion without the expense of cytosolic ATP, directly fuelling the respiratory chain by the activity of ProDH. Arginine catabolism might also contribute to this cycle by generating P5C through transamination between ornithine and α-ketoglutarate. GDH glutamate dehydrogenase, OAT ornithine-δ-aminotransferase, P5C δ1-pyrroline-5-carboxylate, P5CDH P5C dehydrogenase, P5CS P5C synthase, P5CR P5C reductase, ProDH proline dehydrogenase, RC respiratory chain, TCA tricarboxylic acids Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 85

glutamate and proline could be used as a mean to transfer reducing equivalents between these cell compartments.

5.1 Proline-Glutamate Interconversions in Redox Balance

The rapid activation of the oxidative pentose phosphate pathway that has been reported in response to drought, salt, and temperature stress conditions can significantly increase the cytosolic NADPH/NADP+ ratio (Esposito 2016). Moreover, as several NAD kinases were found to be calmodulin-dependent, Ca2+ fluxes early occurring following the exposure to many types of stress may alter the NADP(H)/ NAD(H) ratio (Li et al. 2018). Because of the functional properties of the enzymes involved in proline biosynthesis, these metabolic stress responses are expected to impact proline homeostasis greatly. P5CR, which can use in vitro both NADH and NADPH as the electron donor, showing higher maximal catalytic rate but a lower affinity with NADH, has been found to be highly sensitive to changes in the ratio of phosphorylated versus non-phosphorylated pyridine dinucleotides. The NADH- dependent activity of the plant P5CR is very sensitive to NADP+, being already inhibited at physiological NADP+ concentrations (Giberti et al. 2014). Moreover, high proline and salt levels were found to inhibit the enzyme when NADH was the co-factor, whereas the NADPH-dependent reaction was unaffected or even stimulated (Forlani et al. 2015b; Giberti et al. 2014; Ruszkowski et al. 2015). Concerning P5CS, which is believed to catalyse the rate-limiting step in the short anabolic pathway (Fichman et al. 2015), much less is known. However, it seems to use preferentially, if not exclusively, NADPH rather than NADH (Fichman et al. 2015; Zhang et al. 1995). Moreover, preliminary data showed an even higher sensitivity of P5CS to NADP+, with 50% inhibition at a NADPH to NADP+ ratio of 1.5 (Forlani 2017). As a consequence, increased NADPH/NADP+ and NADP(H)/ NAD(H) ratios are expected to increase the biosynthetic rate and the homeostatic level of proline inside the plant cell, without the need and before any transcriptional activation of the corresponding genes. Consistently, the Rboh inhibitor diphenyle- neiodonium was found to induce high levels of proline accumulation (Shinde et al. 2016), although Rboh is necessary for the transcriptional induction of proline accumulation (Ben Rejeb et al. 2015). An unexpected effect of the impairment of very- long-chain fatty acid synthesis on proline homeostasis was shown to be mediated by effects on redox status rather than signalling functions of lipid metabolism enzymes or intermediates (Shinde et al. 2016). A recent report showed the binding of MYB- type transcription factors Phosphate Starvation Response1 and PHR1-Like1 to P5CS1 regulatory sequences in wild-type arabidopsis plants subjected to phosphate starvation (Aleksza et al. 2017). The consequent gradual increase in proline content could also be reasonably related to a reduction in the NADP(H)/NAD(H) ratio, although this aspect was not investigated in detail. All these results showed that the cellular redox status influences proline metabolism. Nevertheless, the opposite also holds true, as increased rates of proline 86 G. Forlani et al.

synthesis (or oxidation) may influence in turn the NAD(P)H/NAD(P)+ ratio. The interconnection between high levels of proline synthesis during stress and regulation of the adenylate redox status was early hypothesized to maintain NAD(P)+/ NAD(P)H ratios at values compatible with metabolism under normal conditions and to reduce stress-induced cellular acidification (e.g. Hare and Cress 1997). This hypothesis has found more detailed substantiation in several recent studies. Although not definitely proven, proline production dissipating excess reducing equivalents was proposed to act as a compensatory strategy to sustain photosynthesis and prevent photoinhibition under excess light in arabidopsis mutants lacking a chloroplast NADP-dependent malate dehydrogenase (Hebbelmann et al. 2012). Trapping reducing power through enhanced proline biosynthesis has been proposed to limit the generation of ROS and the consequent cell damage (Ben Rejeb et al. 2014). Tissue-specific differences in proline metabolism and function in maintaining a favourable NADP+/NADPH ratio, where proline synthesis in photosynthetic tissues regenerates NADP+, while its catabolism in meristematic and expanding cells is needed to sustain growth by increased availability of energy and reducing power, were found to take place during drought adaptation in arabidopsis (Sharma et al. 2011).

5.2 The P5C-Proline Cycle

More recently, a puzzling result has been reported in an increasing number of studies: the activation under stress of genes in both the proline anabolic and the catabolic pathways. Under these conditions, only transfer of redox equivalents to mitochondria but no proline accumulation would be achieved with the expense of one cytosolic ATP per cycle, since proline is oxidized as soon as it is synthesized, and does not accumulate in the cell (or accumulates much less than expected based on the enhancement of its biosynthetic rate). In PEG-treated arabidopsis seedlings, P5CS1 was induced about 20-fold (Sharma and Verslues 2010), but a five-fold increase was evident also for P5C dehydrogenase (P5CDH, recently proposed to be renamed as glutamate semialdehyde dehydrogenase; Korasick et al. 2019), the enzyme catalysing the second and last step in the mitochondrial oxidation of proline (Forlani et al. 1997; Forlani et al. 2015a), and for ornithine aminotransferase (OAT), the enzyme deaminating ornithine to yield P5C (da Rocha et al. 2012) (Fig. 3). In some instances, microarray and RT-PCR data showed the concurrent transcriptional activation of ProDH and P5CR. During cold acclimation in arabidopsis, the steady- state mRNA levels for P5CSs markedly increased after 12 h of exposure to 4 °C but then declined to basal levels after 96 h of cold treatment, while transcript level of ProDH1 continuously increased; P5CDH mRNA level was unaffected and that for P5CR increased slightly throughout (Kaplan et al. 2007). If the product of proline oxidation in the mitochondrion, P5C, may trespass the membrane and enter the cytosol, this would cause an apparently futile cycle between glutamate and proline, feeding electrons from cytosolic NADPH to the respiratory chain (Fig. 3). Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 87

Overexpression of ProDH in tobacco and arabidopsis or impairment of P5C oxidation in the arabidopsis p5cdh mutant was reported not to change the cellular proline to P5C ratio under both normoosmotic and stress conditions, leading the authors to suggest that excess P5C produced in the mitochondrion may be reduced to proline in the cytosol (Miller et al. 2009b). This so-called P5C-proline cycle involving ProDH and P5CR, conclusively demonstrated in mammalian cells where both enzymes are localized in mitochondria (Liu and Phang 2012), has therefore been hypothesized to play a role also in plants. However, as long as P5CDH is active in mitochondria, it is very difficult to distinguish whether a glutamate-proline or a P5C-proline cycle is operative in vivo. Hyperactivity of either cycle, for instance, following the exogenous supply of proline, enhances ROS production in the mitochondrion. Consistently, p5cdh mutants showed hypersensitivity to exogenous proline (Deuschle et al. 2004). Metabolic cycling between glutamate and proline or P5C and proline could potentially benefit the cell and play a critical role for plant survival under stress through maintenance of the cellular redox balance, regulation of the NAD(P)H/NAD(P)+ ratio, and enhancement of the oxidative pentose phosphate pathway (Hayat et al. 2012; Kavi Kishor and Sreenivasulu 2014; Lv et al. 2011; Miller et al. 2009b). Moreover, an unbalanced activity of ProDH and P5CDH could lead to direct electron transfer to O2 and production of ROS or unspecific damage to mitochondrial components by reaction with P5C (Liang et al. 2013). At low levels, ROS act as a signal for reinstating metabolic homeostasis during stress situations (Türkan and Demiral 2009), whereas at high levels ROS can play a role in the hypersensitive reaction to pathogens (see Sect. 5.3). Conclusive evidence for the occurrence of a P5C transporter in the mitochondrial membrane has not been obtained, yet, and in arabidopsis p5cs1-p5cs2 double mutants, arginine and ornithine could not substitute glutamate as precursor for proline (Funck et al. 2012; Mattioli et al. 2012). Therefore, the P5C-proline cycle has still to be regarded as a hypothesis in plants, and further work is required to confirm its occurrence and physiological role. However, an increasing number of data point at the activation of proline metabolism, more than the resulting homeostatic level of the free amino acid inside the cell, as the determinant for an effective stress response of the plant (e.g. Signorelli et al. 2016; Forlani et al. 2018).

5.3 Proline Catabolism and ROS Generation Under Stress

Under this perspective, some early experimental evidence about the expression of the catabolic pathway in rust-infected plants may be reconsidered and suggest a role for proline metabolism also in the plant response to biotic stress conditions. Early induction of the gene coding for P5CDH was shown in several crops following penetration of virulent, but not of avirulent fungal strains (Ayliffe et al. 2002; Mitchell et al. 2006). Moreover, induction of proline oxidation was reported in arabidopsis during incompatible plant-pathogen interactions (Cecchini et al. 2011). Therefore, the possibility that proline metabolism may be part of the process leading 88 G. Forlani et al. to programmed cell death (PCD) during the hypersensitive defence reaction was proposed (Senthil-Kumar and Mysore 2012). However, it is still unclear which may be the active molecule, whether proline itself, P5C, or ROS produced during proline catabolism. The early activation of ProDH during pathogen attack was accompanied by an increase in P5CR but not in P5CDH transcripts, apparently with few changes occurring in proline and P5C levels (Cecchini et al. 2011). Therefore, also in this case, the whole picture strengthened the possible occurrence of the P5C- proline cycle, leading to ROS production. Enhanced proline oxidation in the mitochondrion leads to sustained ROS generation (Cecchini et al. 2011; Servet et al. 2012), which in turn act as second messengers in various signalling cascades and induce the expression of defence pathways conferring tolerance to either abiotic or biotic stress conditions (Miller et al. 2011; Ben Rejeb et al. 2014). Indeed, the analysis of wild-type arabidopsis plants and p5cdh mutants showed that the absence of P5CDH does not reduce ROS production, cell death, or pathogen resistance and suggested that the enzyme does not act synergistically with ProDH in the potentia- tion of such defence responses (Monteoliva et al. 2014). The whole picture is made even more complex by the presence of another pathway yielding P5C (and possibly proline), the mitochondrial catabolism of arginine via ornithine (Fig. 3). Upon treatment with exogenous proline or pathogen infection, arabidopsis wild-type and p5cdh plants consecutively induced the expression of ProDH and Pro biosynthetic genes, but while the former seemed to induce both routes, p5cdh mutant plants may primarily activate the ornithine route and sustain ProDH induction without reducing the Pro content but rather increasing it (Rizzi et al. 2015). Whatever the way to fuel the P5C-proline cycle, the concurrent induction of P5CDH could make the difference between compatible and incompatible plant-pathogen interactions. In incompatible interactions, low levels of P5CDH activity increases the rate of proline-P5C interconversion, which in turn leads to increased ROS production by ProDH and (directly or indirectly) to PCD. In the former, on the contrary, high levels of P5CDH lower substrate availability for the cycle, delaying PCD induction and allowing a systemic spread of the pathogen. In any case, the exact mechanisms underlying such a role of proline metabolism under biotic stress conditions still await full elucidation.

6 Effect of Proline Metabolism on Antioxidant Enzymes

Notwithstanding the role of proline catabolism in generating ROS in the mitochondrion, different reports have shown that proline accumulation correlates with an enhanced antioxidant enzymatic activity (Hoque et al. 2007, 2008; Islam et al. 2009; Kaushal et al. 2011). This effect has been mainly inferred from the capacity of proline to act as a protectant for enzymes (Ben Rejeb et al. 2014; Szabados and Savouré 2010) or from the transient ROS signals induced by proline catabolism, which result in increased expression of antioxidant enzymes (Ben Rejeb et al. 2014; Zarse et al. 2012). As mentioned before, proline accumulation was suggested to be dependent Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 89 on Rboh activity (Ben Rejeb et al. 2015). This enzyme produces superoxide in the apoplast, which is then converted to H2O2, and it is considered to be one of the main sources of ROS accumulation under stress and to mediate a systemic ROS signal throughout the plant (Miller et al. 2009b). Thus, it is not surprising that proline accumulation and induction of the levels of antioxidant enzymes occur in parallel, while at present it is unclear whether there is a direct effect of proline on the expression of antioxidant enzymes. In the arabidopsis mutants p5cs1–2 and p5cs1–4, unable to accumulate proline, some of the antioxidant enzymes showed higher activity (CAT, glutathione peroxidase), whereas others showed lower activity (SOD, APX, and glutathione reductase) (Szekely et al. 2008). These opposite effects questioned how important the endogenous proline accumulation is for the overexpression- protection of antioxidant enzymes in vivo. Moreover, when the transcriptomic data of one of these mutants (p5cs1–4) was analysed and compared to wild-type plants, none of the genes coding for the aforementioned antioxidant enzymes were differentially expressed under both stress and control conditions (Shinde et al. 2016). This suggests that proline anabolism does not contribute to the regulation of antioxidant response; however, it does not exclude the effect of its catabolism. To establish whether proline catabolism regulates antioxidant response in plants, the study of the expression of antioxidant enzymes under stress conditions in wild-type and pdh mutant lines would be beneficial.

7 Proline as a Source of C and N During Recovery

Because of the high consumption of reducing power needed for its synthesis, the oxidation of proline to glutamate and the subsequent channelling of the latter into the TCA cycle can yield as many as 30 ATP equivalents (Atkinson 1977). Proline accumulation may therefore represent an efficient method for energy storage. Consistently, honeybees and other nectar-foraging insects preferentially utilize proline as a fuel during the initial phases of flight (Micheu et al. 2000), and experimental evidence supports the preference of bees and butterflies for nectars or sugar solutions enriched with proline (Bertazzini et al. 2010). In plants, proline oxidation was shown to be required to sustain growth even at low external water potential, since high ProDH expression was maintained in the root apex and shoot meristem under stress rather than being repressed (Sharma et al. 2011). A fortiori, the use of proline to fuel cell metabolism and as a source of organic nitrogen and carbon to resume growth should be highly valuable after coming back to non-stressful conditions. In fact, a rapid reactivation of ProDH transcription to high levels has been reported in many cases during recovery (Yoshiba et al. 1997). However, modulation of proline metabolism during recovery and its role in plant survival are still largely unexplored. Some data showed that the post-drought response is dependent on drought severity, suggesting that sustained synthesis and accumulation of proline can promote plant damage reparability by up-regulating antioxidant activity also during the recovery from stress (An et al. 2013). If not required, as following the 90 G. Forlani et al. exogenous supply in the absence of stress, proline is promptly utilized, and its intracellular concentration rapidly lowers to homeostatic levels (e.g. Forlani et al. 2015a). Therefore, there is a need to distinguish between different cases, in order to avoid that proline may be oxidized when its accumulation is functional to withstand stress conditions. This goal could be accomplished by differential signals regulating ProDH transcription. Additionally, recent results in arabidopsis described the identification of a mitochondrial protein, Drought and Freezing Responsive gene 1 (DFR1), involved in the inhibition of proline degradation during drought and cold stresses. Two alternatively spliced isoforms of DFR1 were detected that are strongly induced by stress and specifically interact with ProDH and P5CDH, thereby inhibiting their activities (Ren et al. 2018).

8 Conclusions

Over the last decades, many studies have shown that proline accumulation in plants correlates with greater stress tolerance. The broad variety of cis-responsive elements present in the promoter of the plant P5CS1 gene explains why proline accumulation is a conserved response observed in a wide range of conditions. Furthermore, the fact that a master regulator of light-dependent processes such as HY5 mediates P5CS1 expression shows that proline biosynthesis is important to be coordinated with other light-dependent processes. However, the main mechanism by which proline accumulation contributes to stress tolerance remains largely elusive. It is unlikely that proline accumulation exerts its protective function just by preventing water withdrawal from the plant cells, as in many cases its contribution maximally reached 15% of the required osmotic adjustment. Instead, proline can act as a kosmotropic agent, stabilizing proteins and membranes under unfavourable conditions. More fundamental research needs to be done to understand the kosmotropic properties of proline. Recent findings showed that proline accumulation is also dependent on Rboh activity, suggesting that ROS signalling is involved in the regulation of its accumulation. This response could be important if proline accumulation attenuates oxidative damage. Currently, the most likely ways in which this can be achieved is directly by proline, protecting the antioxidant enzymes from denaturation, or by its catabolism, inducing the antioxidant response. Yet, recent findings showed that proline is unable to directly protect against most ROS and RNS. In addition, proline can contribute to regulation of the NAD(P)H/NAD(P)+ ratio, and the activation of a P5C-proline (or glutamate-proline) cycle in plants appears as an effective stress response, in which cytosolic reducing equivalents can be converted into mitochondrial reducing equivalents to fuel the respiratory chain. Regarding biotic stress, proline catabolism was suggested to lead to programmed cell death during the hypersensitive defence reaction. Overall, the latest research in the field has contributed to limit some speculations about the role of proline and its metabolism during abiotic and biotic stress in plants, Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 91 but also several new questions have arisen. Thus, future research on proline metabolism in stressed plants needs to be supported to finally understand its molecular and physiological function under stress.

References

Aleksza D, Horváth GV, Sándor G, Szabados L (2017) Proline accumulation is regulated by transcription factors associated with phosphate starvation. Plant Physiol 175:555–567 Alia MP, Matysik J (2001) Effect of proline on the production of singlet oxygen. Amino Acids 21:195–200 Alia SPP, Mohanty P (1991) Proline enhances primary photochemical activities in isolated thylakoid membranes of Brassica juncea by arresting photoinhibitory damage. Biochem Biophys Res Commun 181:1238–1244 Alia SPP, Mohanty P (1993) Proline in relation to free radical production in seedlings of Brassica juncea raised under sodium chloride stress. Plant Soil 155:497–500 Alia PKVSK, Saradhi PP (1995) Effect of zinc on free radicals and proline in Brassica and Cajanus. Phytochemistry 39:45–97 Alia SPP, Mohanty P (1997) Involvement of proline in protecting thylakoid membranes against free radical-induced photodamage. J Photochem Photobiol B Biol 38:253–257 An Y, Zhang M, Liu G, Han R, Liang Z (2013) Proline accumulation in leaves of Periploca sepium via both biosynthesis up-regulation and transport during recovery from severe drought. PLoS One 17:e69942 Arakawa T, Timasheff SN (1983) Preferential interactions of proteins with solvent components in aqueous amino acid solutions. Arch Biochem Biophys 224:169–177 Arakawa T, Timasheff SN (1985) The stabilization of proteins by osmolytes. Biophys J 47:411–414 Atkinson DE (1977) Cellular energy metabolism and its regulation. Academic Press, New York Ayliffe MA, Roberts JK, Mitchell HJ, Zhang R, Lawrence GJ, Ellis JG, Pryor TJ (2002) A plant gene up-regulated at rust infection sites. Plant Physiol 129:169–180 Ben Rejeb K, Abdelly C, Savouré A (2014) How reactive oxygen species and proline face stress together. Plant Physiol Biochem 80:278–284 Ben Rejeb K, Lefebvre-De Vos D, Le Disquet I, Leprince AS, Bordenave M, Maldiney R, Jdey A, Abdelly C, Savouré A (2015) Hydrogen peroxide produced by NADPH oxidases increases proline accumulation during salt or mannitol stress in Arabidopsis thaliana. New Phytol 208:1138–1148 Bertazzini M, Medrzycki P, Bortolotti L, Maistrello L, Forlani G (2010) Amino acid content and nectar choice by forager honeybees (Apis mellifera L.). Amino Acids 39:315–318 Bertazzini M, Sacchi G, Forlani G (2018) A differential tolerance to mild salt stress conditions among six Italian rice genotypes does not rely on Na+ exclusion from shoots. J Plant Physiol 226:145–153 Bhaskara GB, Yang T-H, Verslues PE (2015) Dynamic proline metabolism: importance and regulation in water limited environments. Front Plant Sci 6:484 Biancucci M, Mattioli R, Forlani G, Funck D, Costantino P, Trovato M (2015) Role of proline and GABA in sexual reproduction of angiosperms. Front Plant Sci 6:484 Binzel M, Hasegawa P, Rhodes D, Handa S, Handa A, Bressan R (1987) Solute accumulation in tobacco cells adapted to NaCl. Plant Physiol 84:1408–1415 Büssis D, Heineke D (1998) Acclimation of potato plants to polyethylene glycol-induced water deficit. II. Contents and subcellular distribution of organic solutes. J Exp Bot 49:1361–1370 Cecchini NM, Monteoliva MI, Alvarez ME (2011) Proline dehydrogenase contributes to pathogen defense in Arabidopsis. Plant Physiol 155:1947–1959 92 G. Forlani et al.

Chiang H, Dandekar M (1995) Regulation of proline accumulation in Arabidopsis thaliana (L.) Heynh during development and in response to desiccation. Plant Cell Environ 18:1280–1290 da Rocha IMA, Vitorello VA, Silva JS, Ferreira-Silva SL, Viégas RA, Silva EN, Silveira JAG (2012) Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves. J Plant Physiol 169:41–49 del Socorro Santos-Díaz M, Ochoa-Alejo N (1994) PEG-tolerant cell clones of chili pepper: growth, osmotic potentials and solute accumulation. Plant Cell Tissue Organ Cult 37:1–8 Delauney A, Verma D (1993) Proline biosynthesis and osmoregulation in plants. Plant J 4:215–223 Deuschle K, Funck D, Forlani G, Stransky H, Biehl A, Leister D, van der Graaff E, Kunze R, Frommer WB (2004) The role of δ1-pyrroline-5-carboxylate dehydrogenase in proline degradation. Plant Cell 16:3413–3425 Di Martino C, Pizzuto R, Pallotta ML, De Santis A, Passarella S (2006) Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings. Planta 223:1123–1133 Díaz P, Borsani O, Márquez M, Monza J (2005) Osmotically induced proline accumulation in Lotus corniculatus leaves affected by light and nitrogen source. Plant Growth Regul 46:223–232 Donald SP, Sun XY, Hu CAA, Yu J, Mei JM, Valle D, Phang JM (2001) Proline oxidase, encoded by p53-induced gene-6, catalyzes the generation of proline-dependent reactive oxygen species. Cancer Res 61:1810–1815 Dong S, Zhang J, Beckles DM (2018) A pivotal role for starch in the reconfiguration of 14C-partitioning and allocation in Arabidopsis thaliana under short-term abiotic stress. Sci Rep 8:9314 Elia I, Broekaert D, Christen S, Boon R, Radaelli E, Orth MF, Verfaillie C, Grünewald TGP, Fendt SM (2017) Proline metabolism supports metastasis formation and could be inhibited to selec- tively target metastasizing cancer cells. Nat Commun 8:15267 Esposito S (2016) Nitrogen assimilation, abiotic stress and glucose 6-phosphate dehydrogenase: the full circle of reductants. Plants 5:24 Feng XJ, Li JR, Qi SL, Lin QF, Jin JB, Hua XJ (2016) Light affects salt stress-induced transcriptional memory of P5CS1 in Arabidopsis. Proc Natl Acad Sci U S A 113:E8335–E8343 Fichman Y, Gerdes SY, Kovács H, Szabados L, Zilberstein A, Csonka LN (2015) Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation. Biol Rev 90:1065–1099 Forlani G (2017) Post-translational regulation of proline synthesis in rice. In: Proceedings of the SIBV-SIGA Joint Meeting Sustainability of Agricultural Environment: Contributions of Plant Genetics and Physiology, p 5.62. Pisa, Italy Forlani G, Scainelli D, Nielsen E (1997) Δ1-pyrroline-5-carboxylate dehydrogenase from cultured cells of potato (Purification and properties). Plant Physiol 113:1413–1418 Forlani G, Bertazzini M, Zarattini M, Funck D (2015a) Functional characterization and expression analysis of rice δ1-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism. Front Plant Sci 6:591 Forlani G, Bertazzini M, Zarattini M, Funck D, Ruszkowski MJ, Nocek B (2015b) Functional properties and preliminary structural characterization of rice δ1-pyrroline-5-carboxylate reductase. Front Plant Sci 6:565 Forlani G, Makarova KS, Ruszkowski M, Bertazzini M, Nocek B (2015c) Evolution of plant δ1- pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives. Front Plant Sci 6:567 Forlani G, Bertazzini M, Cagnano G (2018) Stress-driven increase in proline levels, and not proline levels themselves, correlates with the ability to withstand excess salt in a group of 17 Italian rice genotypes. Plant Biol 21:336–342 Funck D, Winter G, Baumgarten L, Forlani G (2012) Requirement of proline synthesis during Arabidopsis reproductive development. BMC Plant Biol 12:191 Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 93

Gangappa SN, Botto JF (2016) The multifaceted roles of HY5 in plant growth and development. Mol Plant 9:1353–1365 Ge M, Pan XM (2009) The contribution of proline residues to protein stability is associated with isomerization equilibrium in both unfolded and folded states. Extremophiles 13:481–489 Giberti S, Funck D, Forlani G (2014) Δ1-pyrroline-5-carboxylate reductase from Arabidopsis thaliana: stimulation or inhibition by chloride ions and feedback regulation by proline depend on whether NADPH or NADH acts as co-substrate. New Phytol 202:911–919 Hamilton EW, Heckathorn SA (2001) Mitochondrial adaptations to NaCl. Complex I is protected by anti-oxidants and small heat shock proteins, whereas complex II is protected by proline and betaine. Plant Physiol 126:1266–1274 Handa S, Bressan RA, Handa AK, Carpita NC, Hasegawa PM (1983) Solutes contributing to osmotic adjustment in cultured plant cells adapted to water stress. Plant Physiol 73:834–843 Hare PD, Cress WA (1997) Metabolic implications of stress-induced proline accumulation in plants. Plant Growth Regul 21:79–102 Hayashi F, Ichino T, Osanai M, Wada K (2000) Oscillation and regulation of proline content by P5CS and ProDH gene expressions in the light/dark cycles in Arabidopsis thaliana L. Plant Cell Physiol 41:1096–1101 Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A (2012) Role of proline under changing environments: a review. Plant Signal Behav 7:1456–1466 Hebbelmann I, Selinski J, Wehmeyer C, Goss T, Voss I, Mulo P, Kangasjärvi S, Aro EM, Oelze ML, Dietz KJ, Nunes-Nesi A, Do PT, Fernie AR, Talla SK, Raghavendra AS, Linke V, Scheibe R (2012) Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase. J Exp Bot 63:1445–1459 Hildebrandt TM, (2018) Synthesis versus degradation: directions of amino acid metabolism during Arabidopsis abiotic stress response. Plant Molecular Biology 98 (1-2):121–135 Hong Z, Lakkineni K, Zhang Z, Verma DP (2000) Removal of feedback inhibition of δ1-pyrroline- 5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol 122:1129–1136 Hoque MA, Banu MNA, Okuma E, Amako K, Nakamura Y, Shimoishi Y, Murata Y (2007) Exogenous proline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells. J Plant Physiol 164:1457–1468 Hoque MA, Banu MNA, Nakamura Y, Shimoishi Y, Murata Y (2008) Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl- induced damage in cultured tobacco cells. J Plant Physiol 165:813–824 Hossain MA, Fujita M (2010) Evidence for a role of exogenous glycinebetaine and proline in antioxidant defense and methylglyoxal detoxification systems in mung bean seedlings under salt stress. Physiol Mol Biol Plants 16:19–29 Hu CA, Delauney AJ, Verma DP (1992) A bifunctional enzyme (δ1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants. Proc Natl Acad Sci U S A 89:9354–9358 Ikeda T, Nonami H, Fukuyama T, Hashimoto Y (1999) Water potential associated with cell elongation and cell division of tissue-cultured carnation plants. Plant Biotechnol 16:115–121 Islam MM, Hoque MA, Okuma E, Banu MNA, Shimoishi Y, Nakamura Y, Murata Y (2009) Exogenous proline and glycinebetaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells. J Plant Physiol 166:1587–1597 Kaplan F, Kopka J, Sung DY, Zhao W, Popp M, Porat R, Guy CL (2007) Transcript and metabolite profiling during cold acclimation of Arabidopsis reveals an intricate relationship of cold- regulated gene expression with modifications in metabolite content. Plant J 50:967–981 Kaushal N, Gupta K, Bhandhari K, Kumar S, Thakur P, Nayyar H (2011) Proline induces heat tolerance in chickpea (Cicer arietinum L.) plants by protecting vital enzymes of carbon and antioxidative metabolism. Physiol Mol Biol Plants 17:203–213 94 G. Forlani et al.

Kavi Kishor PB, Sreenivasulu N (2014) Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant Cell Environ 37:300–311 Korasick DA, Kon R, Kope M, Hájková E, Vigouroux A, Moréra S, Becker DF, Marek Š, Tanner JJ, Kope D (2019) Structural and biochemical characterization of aldehyde dehydrogenase 12, the last enzyme of proline catabolism in plants. J Mol Biol 431:576–592 Lee J, He K, Stolc V, Lee H, Figueroa P, Gao Y, Tongprasit W, Zhao H, Lee I, Deng XW (2007) Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development. Plant Cell 19:731–749 Li B-B, Wang X, Tai L, Ma T-T, Shalmani A, Liu W-T, Li W-Q, Chen K-M (2018) NAD kinases: metabolic targets controlling redox co-enzymes and reducing power partitioning in plant stress and development. Front Plant Sci 9:379 Liang X, Zhang L, Natarajan SK, Becker DF (2013) Proline mechanisms of stress survival. Antioxid Redox Signal 19:998–1011 Liu W, Phang JM (2012) Proline dehydrogenase (oxidase), a mitochondrial tumor suppressor, and autophagy under the hypoxia microenvironment. Autophagy 8:1407–1409 Liu Y, Borchert GL, Donald SP, Diwan BA, Anver M, Phang JM (2009) Proline oxidase functions as a mitochondrial tumor suppressor in human cancers. Cancer Res 69:6414–6422 Lohaus G, Pennewiss K, Sattelmacher B, Hussmann M, Muehling KH (2001) Is the infiltration- centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species. Physiol Plant 111:457–465 Lv WT, Lin B, Zhang M, Hua X-J (2011) Proline accumulation is inhibitory to Arabidopsis seedlings during heat stress. Plant Physiol 156:1921–1933 Lv BS, Ma HY, Li XW, Wei LX, Lv HY, Yang HY, Jiang CJ, Liang ZW (2015) Proline accu mulation is not correlated with saline-alkaline stress tolerance in rice seedlings. Agron J 107:51–60 Lehmann S, Funck D, Szabados L, Rentsch D (2010) Proline metabolism and transport in plant development. Amino Acids 39(4):949–962 Mattioli R, Biancucci M, Lonoce C, Costantino P, Trovato M (2012) Proline is required for male gametophyte development in Arabidopsis. BMC Plant Biol 12:236 Micheu S, Crailsheim K, Leonhard B (2000) Importance of proline and other amino acids during honeybee flight Apis( mellifera carnica POLLMANN). Amino Acids 18:157–175 Miller G, Honig A, Stein H, Suzuki N, Mittler R, Zilberstein A (2009a) Unraveling δ1-pyrroline- 5-carboxylate-proline cycle in plants by uncoupled expression of proline oxidation enzymes. J Biol Chem 284:26482–26492 Miller G, Schlauch K, Tam R, Cortes D, Torres MA, Shulaev V, Dangl JL, Mittler R (2009b) The plant NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli. Sci Signal 2:ra45 Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2011) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 33:453–467 Mitchell HJ, Ayliffe MA, Rashid KY, Pryor AJ (2006) A rust-inducible gene from flax fis1( ) is involved in proline catabolism. Planta 223:213–222 Monteoliva MI, Rizzi YS, Cecchini NM, Hajirezaei MR, Alvarez ME (2014) Context of action of proline dehydrogenase (ProDH) in the hypersensitive response of Arabidopsis. BMC Plant Biol 14:21 Muzammil S, Shrestha A, Dadshani S, Pillen K, Siddique S, Léon J, Naz A (2018) An ancestral allele of Pyrroline-5-carboxylate synthase1 promotes proline accumulation and drought adaptation in cultivated barley. Plant Physiol 78:771–782 Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (1999) Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 461:205–210 Öztürk L, Demir Y (2002) In vivo and in vitro protective role of proline. Plant Growth Regul 38:259–264 Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 95

Per TS, Khan NA, Reddy PS, Masood A, Hasanuzzaman M, Khan MIR, Anjum NA (2017) Approaches in modulating proline metabolism in plants for salt and drought stress tolerance: phytohormones, mineral nutrients and transgenics. Plant Physiol Biochem 461:205–210 Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B (1997) A model for p53-induced apoptosis. Nature 389:300–305 Poustini K, Siosemardeh A, Ranjbar M (2007) Proline accumulation as a response to salt stress in 30 wheat (Triticum aestivum L.) cultivars differing in salt tolerance. Genet Resour Crop Evol 54:925–934 Ren Y, Miao M, Meng Y, Cao J, Fan T, Yue J, Xiao F, Liu Y, Cao S (2018) DFR1-Mediated inhibition of proline degradation pathway regulates drought and freezing tolerance in Arabidopsis. Cell Rep 23:3960–3974 Rizzi YS, Monteoliva MI, Fabro G, Grosso CL, Laróvere LE, Alvarez ME (2015) P5CDH affects the pathways contributing to pro synthesis after ProDH activation by biotic and abiotic stress conditions. Front Plant Sci 6:572 Rudolph AS, Crowe JH, Crowe LM (1986) Effects of three stabilizing agents -proline, betaine, and trehalose- on membrane phospholipids. Arch Biochem Biophys 245:134–143 Ruszkowski M, Nocek B, Forlani G, Dauter Z (2015) The structure of Medicago truncatula δ1- pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants. Front Plant Sci 6:869 Samuel D, Kumar TK, Ganesh G, Jayaraman G, Yang PW, Chang MM, Trivedi VD, Wang SL, Hwang KC, Chang DK, Yu C (2000) Proline inhibits aggregation during protein refolding. Protein Sci 9:344–352 Sanada Y, Ueda H, Kuribayashi K, Andoh T, Hayashi F, Tamai N, Wada K (1995) Novel light-dark change of proline levels in halophyte (Mesembryanthemum crystallinum L.) and glycophytes (Hordeum vulgare L. and Triticum aestivum L.) leaves and roots under salt stress. Plant Cell Physiol 36:965–970 Sanderson PW, Lis LJ, Quinn PJ, Williams WP (1991) The Hofmeister effect in relation to membrane lipid phase stability. Biochim Biophys Acta Biomembr 1067:43–50 Sano M, Kawashima N (1982) Water stress induced proline accumulation at different stalk positions and growth stages of detached tobacco leaves. Agric Biol Chem 46:647–653 Saradhi PP, Alia AS, Prasad KVSK (1995) Proline accumulates in plants exposed to UV radiation and protects them against UV induced peroxidation. Biochem Biophys Res Commun 209:1–5 Schobert B, Tschesche H (1978) Unusual solution properties of proline and its interaction with proteins. Biochim Biophys Acta 541:270–277 Senthil-Kumar M, Mysore KS (2012) Ornithine-δ-aminotransferase and proline dehydrogenase genes play a role in non-host disease resistance by regulating pyrroline-5-carboxylate metabolism-induced hypersensitive response. Plant Cell Environ 35:1329–1343 Servet C, Ghelis T, Richard L, Zilberstein A, Savouré A (2012) Proline dehydrogenase: a key enzyme in controlling cellular homeostasis. Front Biosci 17:607–620 Sharma S, Verslues PE (2010) Mechanisms independent of abscisic acid (ABA) or proline feedback have a predominant role in transcriptional regulation of proline metabolism during low water potential and stress recovery. Plant Cell Environ 33:1838–1851 Sharma S, Villamor JG, Versules PE (2011) Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential. Plant Physiol 157:292–304 Shinde S, Villamor JG, Lin W-D, Sharma S, Verslues PE (2016) Proline coordination with fatty acid synthesis and redox metabolism of chloroplast and mitochondria. Plant Physiol 172:1074–1088 Signorelli S (2016) The fermentation analogy: a point of view for understanding the intriguing role of proline accumulation in stressed plants. Front Plant Sci 7:1339 Signorelli S, Monza J (2017) Identification ofδ 1-pyrroline 5-carboxylate synthase (P5CS) genes involved in the synthesis of proline in Lotus japonicus. Plant Signal Behav 12:e1367464 Signorelli S, Arellano JB, Melø TB, Borsani O, Monza J (2013a) Proline does not quench singlet oxygen: evidence to reconsider its protective role in plants. Plant Physiol Biochem 64:80–83 96 G. Forlani et al.

Signorelli S, Casaretto E, Sainz M, Díaz P, Monza J, Borsani O (2013b) Antioxidant and photosystem II responses contribute to explain the drought-heat contrasting tolerance of two forage legumes. Plant Physiol Biochem 70:195–203 Signorelli S, Corpas FJ, Borsani O, Barroso JB, Monza J (2013c) Water stress induces a differential and spatially distributed nitro-oxidative stress response in roots and leaves of Lotus japonicus. Plant Sci 201–202:137–146 Signorelli S, Coitiño EL, Borsani O, Monza J (2014) Molecular mechanisms for the reaction between ˙OH radicals and proline: insights on the role as reactive oxygen species scavenger in plant stress. J Phys Chem B 118:37–47 Signorelli S, Dans PD, Coitiño EL, Borsani O, Monza J (2015) Connecting proline and γ-aminobutyric acid in stressed plants through non-enzymatic reactions. PLoS One 10:e0115349 Signorelli S, Imparatta C, Rodríguez-ruiz M, Borsani O, Corpas FJ, Monza J (2016) In vivo and in vitro approaches demonstrate proline is not directly involved in the protection against superoxide, nitric oxide, nitrogen dioxide and peroxynitrite. Funct Plant Biol 43:870–879 Signorelli S, Agudelo-Romero P, Considine MJ, Foyer CH (2018) Roles for light, energy and oxygen in the fate of quiescent axillary buds. Plant Physiol 176:1171–1181 Smirnoff N, Cumbes QJ (1989) Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28:1057–1060 Speer M, Kaiser WM (1991) Ion relations of symplastic and apoplastic space in leaves from Spinacia oleracea L. and Pisum sativum L. under salinity. Plant Physiol 97:990–997 Sripinyowanich S, Klomsakul P, Boonburapong B, Bangyeekhun T, Asami T, Gu H, Buaboocha T, Chadchawan S (2013) Exogenous ABA induces salt tolerance in indica rice (Oryza sativa L.): the role of OsP5CS1 and OsP5CR gene expression during salt stress. Environ Exp Bot 86:94–105 Szabados L, Savouré A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97 Szekely G, Abraham E, Cseplo A, Rigo G, Zsigmond L, Csiszar J, Ayaydin F, Strizhov N, Jasik J, Schmelzer E, Koncz C, Szabados L (2008) Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. Plant J 53:11–28 Savouré A (1997) Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana. Molecular and General Genetics MGG 254(1):104–109 Türkan I, Demiral T (2009) Recent developments in understanding salinity tolerance. Environ Exp Bot 67:2–9 Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35:753–759 Verbruggen N, Villarroel R, Van Montagu M (1993) Osmoregulation of a pyrroline-5-carboxylate reductase gene in Arabidopsis thaliana. Plant Physiol 103:771–781 Verslues PE, Sharma S (2010) Proline metabolism and its implications for plant-environment interaction. Arabidopsis Book 8:e0140 Verslues P, Sharp R (1999) Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. II Metabolic source of increased proline deposition in the elongation zone. Plant Physiol 119:1349–1360 Voetberg GS, Sharp RE (1991) Growth of the maize primary root at low water potentials III. Role of increased proline deposition in osmotic adjustment. Plant Physiol 96:1125–1130 Wang T, Chen Y, Zhang M, Chen J, Liu J, Han H, Hua X (2017) Arabidopsis AMINO ACID PERMEASE1 contributes to salt stress-induced proline uptake from exogenous sources. Front Plant Sci 8:2182 Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi T, Yamaguchi-Shinozaki K, Wada K, Harada Y, Shinozaki K (1995) Correlation between the induction of a gene for δ1-pyrroline-5- carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J 7:751–760 Regulation of Proline Accumulation and Its Molecular and Physiological Functions… 97

Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K (1997) Regulation of levels of proline as an osmolyte in plants under water stress. Plant Cell Physiol 38:1095–1102 Zarattini M, Forlani G (2017) Toward unveiling the mechanisms for transcriptional regulation of proline biosynthesis in the plant cell response to biotic and abiotic stress conditions. Front Plant Sci 8:927 Zarse K, Schmeisser S, Groth M, Priebe S, Beuster G, Kuhlow D, Guthke R, Platzer M, Kahn CR, Ristow M (2012) Impaired insulin/IGF1 signaling extends life span by pro moting mitochondrial L-proline catabolism to induce a transient ROS signal. Cell Metab 15:451–465 Zhang CS, Lu Q, Verma DPS (1995) Removal of feedback inhibition of δ1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270:20491–20496 Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms

Mohamed Zouari, Ameni Ben Hassena, Lina Trabelsi, Bechir Ben Rouina, Raphaël Decou, and Pascal Labrousse

1 Introduction

Plants are constantly exposed to abiotic stresses such as drought, salinity, metal toxicity and extreme temperatures. One of the stress responses in plants is the stimu- . lated production of reactive oxygen species (ROS) such as superoxide (O2 ), . hydroxyl radical (OH ) and hydrogen peroxide (H2O2) (Hayat et al. 2012; You and Chan 2015). ROS overproduction directly damages cellular biomolecules such as proteins, amino acids, purine nucleotides and nucleic acids and causes the peroxidation of the membrane lipids (Osman 2015; Choudhury et al. 2017). Cells have developed and adapted different mechanisms to maintain low intracellular ROS level. These ROS are scavenged by antioxidative metabolites like glutathione (GSH), ascorbic acid (AsA), α-tocopherol (vitamin E) as well as antioxidative enzymes such as catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) (Gill and Tuteja 2010; de Freitas et al. 2018). In addition to these antioxidants, osmotic regulators like proline also protect plant cell against abiotic stress. They are characterized by low molecular weight and high solubility. Proline accumulation is known to occur under water deficit, salinity, extreme temperature and heavy metal (Ashraf and Foolad 2007; Hayat et al. 2012; Hossain et al. 2014; Aslam et al. 2017, De Freitas et al. 2019). In addition to act as an osmolyte for osmotic adjustment, proline contributes to the stabilization of subcellular structures

M. Zouari · L. Trabelsi · B. B. Rouina Laboratory of Improvement of Olive Productivity and Fruit Trees, Olive Tree Institute of Sfax, University of Sfax, Sfax, Tunisia A. B. Hassena Laboratory of Amelioration and Protection of Olive Genetic Resources, Olive Tree Institute of Sfax, University of Sfax, Sfax, Tunisia R. Decou · P. Labrousse (*) University of Limoges, Limoges, France e-mail: [email protected]

(membranes and proteins), to the scavenging of free radicals, and to buffering of cellular redox potential under stress conditions (Heuer 2010; Hossain et al. 2014). It may also act as protein-compatible hydrotrope, alleviating cytoplasmic acidosis and maintaining appropriate NADP+/NADPH ratios compatible with metabolism (Hare and Cress 1997; Gholami Zali and Ehsanzadeh 2018). In many plant species, proline accumulation under abiotic stress has been correlated with stress tolerance, and its concentration has been shown to be generally higher in tolerant plants than in salt-sensitive plants (Hayat et al. 2012). The level of proline accumulation in plants varies from species to species and can be 100 times greater than in control situation. Thus, the exogenous use of proline is considered as a simple technique to provoke abiotic stress tolerance in plants.

2 Proline and Proline Metabolism in Plants

Proline, abbreviated as Pro or P, is a nonessential proteinogenic amino acid with formula C5H9NO2 and a molecular mass of 115.13. Proline is encoded by the codon CCU, CCC, CCA and CCG and is the only proteinogenic amino acid including a secondary amine group called an imine leading to name proline an imino acid (Bhagavan and Ha 2015). The fusion of the three-carbon R-group of proline to the alpha-nitrogen group confers to this compound a rotationally constrained rigid ring structure and thus an exceptional conformational rigidity. Accumulation of proline under abiotic stress can be mediated by the increase in proline synthesis or a decrease in proline degradation. A diagrammatic representation of proline metabolic pathway and interconnection with polyamine (PA) and gamma-aminobutyric acid (GABA) metabolic pathways is presented in Fig. 1. Proline could be synthesized by two pathways, and, even if glutamate pathway is predominant, ornithine pathway also occurs. The glutamate pathway accounts for major proline accumulation during osmotic stress. Proline is synthesized from glutamic acid via glutamate semialdehyde (GSA) and Δ1-pyrroline-5-carboxylate (P5C). The glutamate to GSA reaction is catalyzed by Δ1-pyrroline-5-carboxylate synthetase (P5CS, E.C. 2.7.2.11). GSA is spontaneously converted to P5C, and Δ1- pyrroline-5-carboxylate reductase (P5CR, E.C. 1.5.1.2) catalyze the transformation of P5C to proline. Proline catabolism occurs in mitochondria in several steps involving proline dehydrogenase (PDH, E.C. 1.5.5.2) producing P5C from proline and P5C dehydrogenase (P5CDH, E.C. 1.2.1.88) converting P5C to glutamate. As previously said, proline can be also synthesized from ornithine in an alternative pathway. Ornithine (Orn) is transaminated to P5C (and GSA) by ornithine delta- aminotransferase (δOAT, E.C. 2.6.1.13), a mitochondrial located enzyme (Hayat et al. 2012; Hossain et al. 2014). P5C is then converted into proline by P5CDH. It has been suggested that the ornithine pathway is important during seedling development and in some plants for stress-induced proline accumulation. Proline biosynthesis occurs in the cytosol and in the chloroplasts, while proline degradation takes place in mitochondria (de Freitas et al. 2019). Indeed, biosynthetic Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 101

Cytosol PyrDH

SSA GABA

GluDC

NADH+H+ Glutamate P5CDH NADPH+H+ ATP NADPH+H+ NADP+ P5CS NADP+ ATP P5CS ADP+Pi NADP+ GSA GSA ADP+Pi Chloroplas i

r KG GSA d Krebs cycle n GABA o KG h c

o SA

t OAT P5C i P5C Orn +

NADPH+H t P5C Ma + P5CR NADPH+H Pyr FADH2 + PDH NADP P5CR FAD+ + Spd Proline NADP Put Urea Ar Arg Arg

Fig. 1 Proline metabolic pathway in higher plants and possible interconnection with gamma- aminobutyric acid and polyamines pathways. (Adapted from Pál et al. 2018, Huang et al. 2008, Szabados and Savouré 2010, Signorelli et al. 2015). Proline biosynthetic pathways appear with green arrows, catabolic pathway appears with red arrow, and the ornithine pathway is represented with blue arrow. ADP adenosine diphosphate, Ar arginase, Arg arginine, ATP adenosine triphos- phate, FAD flavin adenine dinucleotide, FADH2 flavin adenine dinucleotide reduced, GABA gamma-aminobutyric acid, GSA glutamate-semialdehyde, GluDC glutamate decarboxylase, KG apha-ketoglutarate, NADP+ nicotinamide adenine dinucleotide phosphate, NADPH nicotinamide adenine dinucleotide phosphate reduced, OAT ornithine-delta-aminotransferase, Orn ornithine, P5C pyrroline-5-carboxylate, P5CR pyrroline-5-carboxylate reductase, P5CS pyrroline-5- carboxylate synthetase, P5CDH pyrroline-5-carboxylate dehydrogenase, PDH proline dehydrogenase, Put putrescine, Pyr pyrroline, PyrDH pyrolline dehydrogenase, SA succinic acid, Spd spermidine, SSA succinic semiadlehyde enzymes are preferentially located in cytosol (PCS and PCR), whereas enzymes of proline catabolism are preferentially located in the mitochondria (PDH, PCDH, and OAT) (Szbados and Savouré 2010). This compartmentalization of proline metabolism suggests the occurrence of intracellular proline transport between the cytosol, the chloroplast and the mitochondria. If some proline carriers have been identified like mitochondrial proline uniporter and proline/glutamate antiporter, the involvement of basic amino acid transporters is also needed to transfer arginine and ornithine through mitochondrial membrane. Moreover, the preferential localization of proline catabolic enzyme in the mitochondria and the involvement of glutamate and alpha- ketoglutarate (KG) in the ornithine pathway suggest the interconnection with Krebs cycle (or tricarboxylic acid cycle) (Rana et al. 2017). Through glutamate and pyrroline, proline pathway could also be connected to GABA. Indeed, Δ1-pyrroline is converted to GABA thanks to pyrroline dehydrogenase (PyrDH, E.C. 1.2.1.19) even if GABA is mainly produced from glutamate by glutamate decarboxylase (GluDC, E.C. 4.1.1.15). GABA accumulation occurs during several stresses leading to attri- 102 M. Zouari et al.

Photo period Light

Mitochondria Cytosol Chloroplast Water stress Glutamate P5CDH Salt stresss Ca2 P5CS + P5CS BR GSA GSA GSA H2O2 ABA

Light Water P5C P5C stress P5C P5CR Re PDH P5CR hydration Proline

Fig. 2 Possible regulation ways of proline metabolic pathway in higher plants by abiotic factors. (Adapted from Szabados and Savouré 2010). ABA abscisic acid, BR brassinolides, GSA glutamate- semialdehyde, KG alpha-ketoglutarate, P5C pyrroline-5-carboxylate, P5CR pyrroline-5- carboxylate reductase, P5CS pyrroline-5-carboxylate synthetase, P5CDH pyrroline-5-carboxylate dehydrogenase, PDH proline dehydrogenase bute to this molecule several protective roles (like proline, GABA could be involved in osmoregulation, cell signaling, and protection against oxidative stress, cytosolic pH regulation). Recently, Signorelli et al. (2015) proposed an alternative pathway to connect proline to GABA via pyrroline through nonenzymatic reactions that would explain the simultaneous accumulation of GABA and proline under oxidative stress. Moreover, Pál et al. (2018) suggested the existence of an interconnection between proline pathways and polyamine (putrescine, spermidine) pathways. Indeed, putrescine is synthetized by ornithine decarboxylase (E.C. 4.1.1.17) from ornithine or indirectly by arginine decarboxylase (E.C. 4.1.1.19) from arginine via agmatine. Thus, complex interactions between proline, polyamines, GABA synthesis pathways, and ROS balance exist, and new connections must be probably deciphered in the future as abscisic acid plays also an important role in these stress responses. Proline metabolism is regulated by multiple factors (Fig. 2), and the regulation processes are still poorly known. Proline biosynthesis is stimulated during dehydration while its catabolism is reduced. At the contrary, the process is reversed during rehydration. Proline biosynthesis is stimulated by light and osmotic stress, whereas proline catabolism is stimulated in dark and during stress relief. Proline accumulation is also reported to be repressed by brassinosteroïds, whereas it was stimulated during salt stress. Under stress, proline metabolism is regulated by multiple and complex pathways that can drastically influence cell death and survival of the organism. Indeed, the coupling of proline metabolic pathways with the mitochondrial and chloroplastic + + + electron transport chain (through NADPH/NADP , NADH/NAD , FADH2/FAD ) Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 103

Table 1 Summary of proline functions in plant at different organization levels from cell to whole plant Organization Proline compounds and level pathways Function Cell wall PRPs Wall component Drought stress Plasma RPRPs Roots: sensitivity to ABA membrane HRGPs Links between plasma membrane and cytoskeleton Cell PRPs Cell elongation Root hair development HRGPs Cell wall assembly Cell wall remodeling Intercellular communications Callus HRGPs Somatic embryogenesis Germination of somatic embryos HyPRPs Cell elongation Size increase Phloem PRPs Expression during drought stress Embryo Proline biosynthesis Embryo death impairment Impaired seed development Leaf Normal proline level Flavor compounds Floral buds PRPs Style structural integrity Proline accumulation Bud break Pollen and style HRGPs Pollen tube growth Style growth Floral nectars Proline accumulation Pollinators attraction Flower Normal proline level Flavor compounds PRPs Flower development Cotton fiber development Fruit Proline accumulation Enhanced fermentability (grapevine) Seed/grain Normal proline levels Seed germination Flavor compounds Plant PRPs Abscission, senescence Development, abiotic stress tolerance Proline transporters Xylogenesis Proline biosynthesis Reduced protein synthesis impairment Cyclin genes downregulated Adapted from Kavi Kishor et al. (2015) ABA abscisic acid, HRGPs hydroxyproline-rich glycoproteins, HyPRP hybrid proline-rich proteins, PRPs proline-rich proteins induces an opportunity to balance the redox state by regulating the generation of ROS. For example, Zhang and Becker (2015) indicated that proline metabolism may influence ROS signaling pathways to delay the senescence. Proline functions in plants are complex, are not entirely deciphered, and depend on the organization level (Table 1). Proline is a main element of the cell wall matrix 104 M. Zouari et al.

Metabolism (carbohydrate, amino acids)

NADPH/NADP+ Redox balance

ROS

Proline Proteins Osmoprotection Translation (Proline rich proteins)

Photosynthesis enzymesGST, CAT, APX Cell wall N2 assimilation in Fabaceae

Signaling

Mitochondrial Plant development functions (ROS, PCD) (embryo, root growth, flowering)

Fig. 3 Proline role in plant functioning. (Adapted from Szabados and Savouré 2010, Verdoy et al. 2006) through proteins like hydroxyproline-rich glycoproteins (HRGP) or proline-rich proteins (PRPs), thus giving to proline a key role in the plant development (Fig. 3). For example, proline is vital for proper seed development and for producing viable seeds. In in vitro culture, proline via HRGPs is necessary to embryo for their regeneration and their germination during somatic embryogenesis. HRGPs are also necessary to pollen tube and style development. Proline is a key actor in root elongation and in flower initiation but also in the further reproductive tissue development (Kavi Kishior et al. 2015). Thus, proline is not only involved in protein synthesis but regulates also key functions like osmotic adjustment or protein protection. It should be noted that a positive correlation probably exists between proline and glycine betaine, another molecule playing a highly beneficial role in plants exposed to stress (Murmu et al. 2017). Through its involvement in cell wall synthesis, root growth, embryo formation, and germination, proline becomes therefore a major stakeholder during all the plant life cycle.

3 Genetic Features of Proline Metabolism and Regulation

As described above, the proline metabolism occurs through two pathways intercon- necting various organelles in plants (Fig. 1). This metabolism appears conserved between prokaryote and eukaryote organisms, and various genes are involved in the Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 105

Table 2 Genetics features of genes involved in the main network of proline biosynthesis Gene E.C Gene/protein Exons Response to abiotic stress name Plant number Chrom. length (bp/aa) (number) in plants P5CS1 A. 2.7.2.11 2 2154/717 20 Salinity/drought /oxidative thaliana stress /Light/phosphate starvation/cold/heat

P5CS2 3 2181/726 20 NaCl (weak)/cold/H2O2 P5CS3 L. regale ? 2139/712 ? Salinity/drought P5CR A. 1.5.1.2 5 831/276 7 Salinity/drought/heat/cold thaliana P5CDH A. 1.2.1.88 5 1671/556 16 Salinity/cold/drought/dark thaliana PDH1 A. 1.5.5.2 3 1500/499 4 Salinity/drought/ thaliana hypo-osmolarity/ phosphate starvation/ABA PDH2 5 1431/476 4 Salinity/sucrose

δOAT A. 2.6.1.13 5 1428/475 10 Salinity/drought/H2O2 thaliana

biosynthesis of the different enzymes (Table 2). P5CS, the eukaryotic key fusion enzyme exhibiting the two conserved domains glutamate 5-kinase (GK, EC: 2.7.2.11; N-terminal) and γ-glutamyl phosphate reductase (GPR, EC: 1.2.1.41; C-terminal) (Pérez-Arellano et al. 2010; Fichman et al. 2015), is synthetized by two duplicated P5CS genes in most plants (P5CS1 and P5CS2). From several studies, P5CS revealed to play distinct roles according to the stress, in an organ-specific manner and following cell spatiotemporal expression patterns (thoroughly reviewed by Rai and Penna 2013, Amini et al. 2015, and Rana et al. 2017). For example, P5CS1 is mediated by hyperosmotic stress and regulated by abscisic acid, while P5CS2 appears as a constitutive and ubiquitous gene in plants (Savouré et al. 1997; Székely et al. 2008; Verslues and Sharma 2010). Recently, a third P5CS gene (P5CS3) was found in the dicot Medicago truncatula (Kim and Nam 2013) and in the monocot Lilium regale (Wei et al. 2016). These genes contribute also to proline accumulation and abiotic stress tolerance. At the contrary, the second reduction step leading to proline from P5C is managed by only one gene of the plant genome. However, two P5CR isoforms were identified from pea and spinach allowing a lin- gering doubt on the exact number of P5CR genes in these plants (Murahama et al. 2001; Lehmann et al. 2010). In addition, although P5CS represent a rate-limiting step, the absence of a functional P5CR prevents both routes for proline biosynthesis what raise the P5CR gene to a paramount converging point of the two anabolic pathways. Therefore, the unique P5CR supposed fine transcriptional regulation although a post-translational regulation is highly suggested even more evident (Forlani et al. 2015; Anwar et al. 2018). As mentioned above, δOAT and P5CDH constitute another pathway for proline metabolism although δOAT is involved in the anabolism route contrary to P5CDH that corresponds more precisely to the proline 106 M. Zouari et al. catabolism pathway (cf. Fig. 1). Whatever, both genes are described for having only one copy in the nuclear plant genomes, and AtP5CDH exhibits a ubiquitous low basal level but can be upregulated by proline as shown in Fig. 2 (Deuschle et al. 2001). Concurrently, catabolism of proline to Glu is performed through PDH and P5CDH gene transcription. PDH is represented by two copies in the Arabidopsis thaliana genome (forming two isoforms, PDH1/PDH2), and their suppression leads to Pro accumulation. Indeed, as for P5CDH, proline cellular level insures the post- transcriptional regulation on PDH (Verbruggen and Hermans 2008). However, the two protein isoforms were shown to be differentially expressed (Funck et al. 2010). Overall, many transcription factors (TFs) revealed to be involved in the regulation of the proline metabolism genes. Several TFs gene families like MYC/MYB, bZIP, AP2/EREBP, RAV, PHR1, PHL, etc. participate to the abiotic stress tolerance in plants as already demonstrated or reported (Aleksza et al. 2017; Fichman et al. 2015; Roychoudhury et al. 2015; Anwar et al. 2018). In addition, various binding sites were predicted or demonstrated like the ACTCAT cis-acting element of the PDH1 promoter (Satoh et al. 2004; Weltmeier et al. 2006) or the HD-HOX, bZIP- DOF, AP2/EREBP, and P1BS-binding sites of AtP5CS1, AtP5CS2, AtP5CR, and AtOAT promoters (Fichman et al. 2015). Owing to the loss of crop productivity and the role of proline in the plant tolerance to abiotic stresses, engineering strategies using plant mutants for proline anabolism/catabolism allow to improve the knowledge on the molecular factors modulating these biological pathways (cf. reviews of Kavi Kishor et al. 2015, Singh et al. 2017, and Hasanuzzaman et al. 2019). Biotechnologies could therefore give substantial advantage for developing new food crop cultivars tolerant to multiple abiotic stresses. Moreover, parallel to plant molecular enhancements, researchers proposed another tool for a higher crop productivity as described hereinafter.

4 Application of Exogenous Proline on Plants Grown Under Abiotic Stresses

4.1 Effect of Exogenous Proline Application on Plant Water Status

In an analysis of the beneficial effect of exogenous proline in plants exposed to abiotic stress (Table 3 and Fig. 4), it is important to consider the role of proline supplementation in plant water status. Drought stress is known to induce a decline in water content in plants tissues. For example, in cowpea (Vigna unguiculata L.) grown under three levels of water deficit (60, 40, and 20% of soil water holding capacity), Merwad et al. (2018) reported that water stress induced a significant decrease of leaf relative water content (LRWC). In Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 107 ) ) ) ) 2018 ) 2016 2013 (continued) 2015 2014 ) ) 2013 2019 Abdelhamid et al. ( Butt et al. ( De Freitas et al. ( Reference Orsini et al. ( Hasanuzzaman et al. ( Ali et al. ( Osman ( free radical scavenging activity free radical scavenging . Increased the content of seed sugar, oil, protein, Increased the content of seed sugar, and ash, increased the oil oleic moisture, fiber, linoleic acid contents, increased the concentrations of antioxidant compounds in the seed oil, enhanced oil DPPH Increased the activities of antioxidant enzymes and Increased the activities concentrations of carotenoids, ascorbic acid, and endogenous proline, increased the concentrations of P and K+, decreased Na+ ion concentrations, enhanced the growth Alleviated oxidative damages by enhancing the oxidative Alleviated antioxidant and glyoxalase systems the best concentration 0.8 mM was ionic, and physiological, growth, Improved (biomass, photosynthetic biochemical attributes rate, transpiration and antioxidant enzyme activities) proline Decreased membrane damage and regulated levels Response Increased photosynthetic rate and total yield Increased the yield and soluble protein concentration, increase nonenzymatic antioxidant defense system, enhanced the production and translocation of assimilates from source to sink Maize Common bean Chili Sorghum Species Lettuce Rice Pea ) 1 − Drought stress Salt stress (75 mM) Salt stress (saline soil with EC = 1.84, 6.03, and 8.97 dS m Salt stress (50 mM) Salt stress (150 and 300 mM NaCl) Salt stress (15 mM) Type and level and level Type of stress Drought stress M μ 30 mm 30 mM 5 mM (0.4, 0.6, 0.8, 1.0, and 1.2 mM) 5 mM 5 Concentration 4 mM Improvement in growth and regulation of various physiological and biochemical processes in different plant species by exogenous application of plant species by exogenous and biochemical processes in different physiological of various and regulation in growth Improvement

Foliar spray Foliar Foliar spray Foliar Foliar spray Foliar Foliar spray Foliar Foliar spray Foliar Foliar spray Foliar Mode of application Foliar spray Foliar Table 3 Table proline under abiotic stress 108 M. Zouari et al. ) ) ) ) 2018 2004 2018 2014 ) ) 2012 2011 Noreen et al. ( Oukarroum et al. ( Shahid et al. ( et al. ( Merwad Ben Ahmed et al. ( Gleeson et al. ( Reference leakage + content in leaves and roots of content in leaves + Improved growth, total chlorophyll content, total chlorophyll growth, Improved and membrane RWC, photosynthetic attributes, of (MSI), increased activity stability index enzymatic antioxidants, enhanced osmolytes, lipid peroxidation, and polyamine metabolism criteria, yield characteristics, growth Improved a and b, total carotenoids, contents of chlorophylls membrane stability shoot and seed nutrients, RWC, of leaf antioxidant enzymes, and activity index, content of leaf proline the best concentration 50 mM was LRWC, Increased photosynthetic activity, and carotenoid, starch contents, chlorophyll reduced the Na Reduced the generation of reactive oxygen species Reduced the generation of reactive and enhanced accumulation of proline protein contents, enhanced the plant height and shoot and root fresh dry weight, enhanced the photosynthetic capacity Increased the tolerance of photosystem II through (OEC) complex protection of the oxygen evolving stressed plants rate and reduced K Increased growth Response Cowpea Olive Wheat Barley Pea and ray grass Larch, sitka spruce, and oak Species M) M, μ μ O) 2 .5H 4 Salt stress (100 and 200 mM) Water deficit Water Salt stress (100 mM)/nickel stress (100 Heat stress (45 °C) Cold stress (4 °C) Heavy metal Heavy stress (400 CuSO Type and level and level Type of stress 2, 4, and 6 mM 25 and 50 mM 60 mM 10 mM 1, 10, and 100 mM 80 mM Concentration (continued)

Rooting medium Foliar spray Foliar Foliar spray Foliar Foliar spray Foliar Rooting medium Foliar spray Foliar Mode of application Table 3 Table Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 109 ) ) ) ) 2011 2016a 2016b 2007 ) 2011 Zouari et al. ( Zouari et al. ( Kaushal et al. ( Aggarwal et al. Aggarwal ( Hoque et al. ( content 2 O 2 M was the best concentration M was μ Alleviated the oxidative damage, increased the the oxidative Alleviated of antioxidant enzymes in roots and leaves activity nutritional status, Enhanced photosynthetic activity, fruit and oil content of olive plant growth, Reduced oxidative injury by elevating enzymatic injury by elevating Reduced oxidative and nonenzymatic antioxidants, improved enhanced the content and LRWC, chlorophyll of enzymes carbon metabolism activities 50 Increased the endogenous proline content and increased the enzymatic and enhanced the growth, nonenzymatic antioxidants, stimulated components reduced lipid of the ascorbate-glutathione cycle, peroxidation and H Increased fresh mass and the activities of enzymatic Increased fresh mass and the activities antioxidant Date palm Olive Chickpea Mung bean Tobacco soil) soil) 1 1 − − Cadmium stress (10 and 30 mg Cd kg Heavy metal Heavy stress (1, 2, 4, and 6 ppm selenium in hydroponic medium) Heat stress (30/25 °C, 35/30 °C, 40/35 °C, and 45/40 °C) Salt stress (200 mM) Cadmium stress (10 and 30 mg Cd kg M M μ μ 20 mM 25, 50, and 100 10 10 and 20 mM 20 mM Rooting medium Rooting medium Rooting medium Rooting medium Rooting medium 110 M. Zouari et al.

Abiotic stresses

Drought Heat/Cold

Salinity Heavy metals

Secondary stress Ionic stress Osmotic stresses

Oxydative stress

Adaptive strategies

Osmolyte Antioxidant

Proline Enzymatic antioxidant Glycine betaine (SOD, APX, CAT...) Solubles sugar Non-enzymatic antioxidant (glutathione, α-tocopherol, ascorbic acid...)

Stress tolerance

Fig. 4 Plant responses to abiotic stresses Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 111 this study, the decline in LRWC due to water deficit stress can be explained by the decrease in the ability of osmotic adjustment due to the reduced absorption of nutrients, especially K+. The same authors reported that, when proline (6 mM) was applied as foliar spray treatments, LRWC increased. This enhancement was attributed to the significant accumulation of proline in cowpea that proves the important adjusting role of this osmolyte under unfavorable conditions. Its contribution to osmotic adjustment is considered as a mechanism to maintain water relations and postpone dehydration under osmotic stress. In addition, Iqbal (2018) observed that under drought conditions, the exogenous application of proline increased its endogenous level that decreased the water potential in cells to a level lower to the one in soil. This may facilitate the uptake of water by roots and therefore maintain the turgor pressure within cells. Salinity stress affects also plant-water relations. De Freitas et al. (2019) studied the impact of proline supply to sorghum (Sorghum bicolor L.) exposed to salt stress (75 mM NaCl) and observed a significant increase in LRWC of stressed plants sprayed with 30 mM proline solution. The beneficial role of exogenous proline was also obtained in salt-stressed plants such as rice (Oryza sativa L.) (Hasanuzzaman et al. 2014) and searocket (Cakile maritima L.) (Messedi et al. 2016). Referring to these authors, lowering of leaf osmotic potential by proline supplementation might be the result of higher accumulation of endogenous proline, which enhances the osmoregulation ability of plants under salt stress conditions. The same authors suggest that exogenous proline supplementation can restore water use efficiency, leaf water status, production of free proline, and membrane damage during salinity stress. Proline can enhance water influx and decrease water efflux to restore water content in plant exposed to stress. Other environmental stress conditions like extreme temperatures similarly account for a significant reduction in plant water status (Kaushal et al. 2011; Oukarroum et al. 2012). In these studies, exogenous proline application maintained the leaf water status, whereas it was reduced in non-treated plants. According to these authors, the maintained leaf water status in proline-treated plants may be attributed to higher accumulation of compatible solutes like proline that possibly improved the turgor content. Plant-water relations are also affected by heavy metal stress. Zouari et al. (2016a) showed that LRWC and water potential (WP) were decreased in the leaves of date palm (Phoenix dactylifera L.) exposed to cadmium stress. The same authors reported that exogenous supply of proline improved the water status of Cd-stressed plants and attributed this enhancement to the interactive effect of proline on osmotic adjustment. Similarly, Shahid et al. (2014) demonstrated that proline application significantly mitigated the alteration of water status of pea (Pisum sativum L.) induced by the phytotoxic effect of nickel. According to Aggarwal et al. (2011), exogenous application of proline (50 μM) increased its endogenous levels that antagonized the toxic effects of selenium by improving water status of bean (Phaseolus vulgaris L.) seedlings. 112 M. Zouari et al.

4.2 Effect of Exogenous Proline Application on Nutrient Status

Absorption of mineral elements is a key process for plants to survive and grow. However, it is well known that several abiotic stresses result in decreased nutrient uptake and consequently reduced mineral nutrients content in plant tissues. Several studies reported that the exogenous supplementation of proline can ameliorate the uptake and accumulation of inorganic nutrients in stressed plants. Ali et al. (2008) reported that maize plants (Zea mays L.) subjected to drought stress by maintaining moisture content at 60% field capacity presented a decrease in N, P, K+, Ca2+, and Mg2+ contents in the shoots and roots. In the same study, exogenously proline (applied at 30 and 60 mM) increased endogenous proline and promoted the uptake of all the macronutrients under water stress conditions. According to these authors, 30 mM proline concentration was more beneficial than 60 mM as this concentration appeared more effective to increase the transpiration rate. Leaf transpiration creates the water tension necessary to the root absorption of essential nutrients from the soil solution. Similar findings were reported by Merwad et al. (2018) who noticed lower nutrient contents (shoots and seed N, P, and K+ contents) in cowpea plant submitted to drought stress than in control ones. These authors reported that exogenous proline has maintained nutrient status by promoting the uptake of N, P, and K+ under water stress. Salt stress causes also ion imbalance. Abdelhamid et al. (2013) reported that highly saline soil (EC = 8.97 dS m−1) resulted in an increase of Na+ and in a decrease of P and K+ content in bean plants. In the same study, spraying bean plants with 5 mM proline significantly increased the content of P and +K and the K+/Na+ ratio and decreased Na+ levels in salt-affected plants. Butt et al. (2016) also found similar results. These authors grown two chili genotypes under 50 mM NaCl saline condition with and without various concentrations of proline (0.4, 0.6, 0.8, 1 and 1.2 mM) applied as a foliar spray and concluded that proline supply had increased the K+ concentration in leaves of stressed plants. In this study, authors reported that K+ efflux was significantly reduced by the application of proline and ionic homeostasis was maintained by enhancing the H+ATPase activity. In the same way, Sobahan et al. (2009) reported that exogenous proline application reduces the Na+-enhanced apoplastic flow to reduce Na+ uptake and transport by plants, suggesting that proline interact with macromolecules in the Na+ diffusion pathways. It has been demonstrated that heavy metal stress may result in disturbance of ionic homeostasis. According to Noreen et al. (2018), the uptake of Ca2+, Mg2+, and K+ ions by root, shoot, and leaf organs of wheat (Triticum aestivum L.) was reduced by copper stress. On the other hand, copper content substantially increased in root organs compared to shoot and leaf organs under copper stress environment. The foliar spray of proline increased the uptake of Ca2+, Mg2+, Na+, and K+ by root, shoot, and leaf organs, while the copper uptake was reduced in all parts. Ashraf and Foolad (2007) reported that ion uptake by plants was regulated by proline spray under stress. In young olive plants (Olea europaea L.) treated with 30 mg CdCl2 Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 113 kg−1 soil, Zouari et al. (2016b) demonstrated that the content of Ca2+, Mg2+, and K+ was strongly reduced, while Cd2+ content was increased in leaf and root tissues. According to these authors, this perturbation of mineral nutrient status could be due to competitions between Cd2+ and essential elements via common transporters. In the same study, exogenous addition of proline to growth medium resulted in increased Ca2+, Mg2+, and K+ contents and in reduced Cd2+ content.

4.3 Effect of Exogenous Proline on Photosynthetic Performance

Abiotic stresses generally affect the plant performance and development by altering the photosynthetic machinery (Hayat et al. 2012). Salinity stress is one of the most common abiotic factors that inhibit crop growth and productivity by reducing the photosynthetic capacity of plants. De Freitas et al. (2019) reported that under NaCl stress, photosynthesis rate, stomatal conductance, transpiration rate, and internal CO2 concentration of sorghum were significantly decreased as compared to the control. Salt toxic effects on photosynthesis can be generated by stomatal factors, including restrictions for CO2 diffusion, and by nonstomatal limitations such as decreased Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity or damage on photosynthetic apparatus by photosystem II performance impairment. The same authors indicated that stressed plants treated with proline presented higher CO2 assimilation in comparison to proline-untreated stressed plants, a response closely related to increases in stomatal conductance and transpiration rate. These responses indicate that proline supplementation might play a key role for CO2 assimilation and photosynthesis recovery in plants against salt stress. Kaushal et al. (2011) studied the comportment of chickpea (Cicer arietinum L.) grown under heat stress and investigated the effects of exogenous proline on total chlorophyll content. Proline-treated plants improved their chlorophyll content by 18% at 40/35 °C and by 44% at 45/40 °C in comparison to untreated plants. According to these authors, proline application significantly reduced the decrease in chlorophyll contents due to heat stress, and such physiological enhancement could result from leaf water status improvement and in possibly reduced photooxidation. The same authors suggested that proline may play an important role in maintaining respiratory metabolism and membrane structure of cells and organelles like chloroplast. Hayat et al. (2012) and Hossain et al. (2014) reported that proline application under drought conditions may maintain the photosynthetic capacity not only through increasing stomatal conductance but also by protecting the subcellular structures such as the chloroplast ultrastructure, the electron transport complex II in mitochondria, as well as the activity of many enzymes like Rubisco which thereby improved the photosynthetic capacity. Referring to Hare and Cress (1997) and Gholami Zali and Ehsanzadeh (2018), proline biosynthesis is a reductive pathway that require 114 M. Zouari et al.

NADPH (for the reduction of glutamate to P5C and P5C to proline) and generate NADP+ which can be used further as electron acceptor and dissipate electron pressure in thylakoid electron transport chain thus avoiding the photoinhibition and thereby the alteration of photosynthetic machinery.

4.4 Effect of Exogenous Proline on Antioxidant Defense System

Plants naturally synthesize ROS as byproducts of cellular oxidative metabolism. The role of proline as ROS scavenger was firstly observed in vivo by Smirnoff and Cumbes (1989) on Arabidopsis P5CS insertion mutants. Then, Ashraf and Foolad (2007) confirmed that proline was an effective scavenger of hydroxyl (OH.) and peroxide ion. Hong et al. (2000) concluded that the role of proline as a free radical scavenger is more important in alleviating stress than its role as a simple osmolyte.

Reduced lipid peroxidation and H2O2 contents, along with the upregulation of the antioxidant defense system, were reported in rice seedlings under salt stress conditions when treated with proline (Hasanuzzaman et al. 2014; Wutipraditkul et al. 2015). Similar patterns were observed also by Butt et al. (2016) in chili genotypes subjected to salt stress and treated with various concentrations of proline. The results proved that both genotypes can cope with salt stress conditions by reducing lipid peroxidation and through the modulation of antioxidant enzymes (SOD and CAT) with exogenous application of proline. It has been reported that proline activates defense mechanisms in response to salt stress, such as activation of antioxidant enzymes. Proline also plays an important role in stress-induced phenolic synthesis, which exhibit antioxidant activities. It has been suggested that proline synthesis stimulates biosynthesis of phenolics via shikimate and phenylpropanoid pathways (Shetty 1997). Drought stress induces a severe oxidative stress in pea leading to oxidative damages as the antioxidant defense system was unable to cope with this stress (Osman 2015). In this study foliar applied proline (4 mM) enhanced the tolerance of peas to oxidative damage by enhancing ROS detoxification systems. These findings suggest that proline has protective effects against drought-induced oxidative stress by reducing H2O2 content and by increasing the enzymatic antioxidant defense system (SOD, CAT, and APX). Ghaffari et al. (2019) noticed in sugar beet (Beta vulgaris L.) exposed to drought stress (50% water requirement of plant) that foliar proline applications (low, 5 mM; high, 10 mM) increased enzymatic antioxidant activities and then reduced levels of MDA (malondialdehyde) and H2O2. Referring to these authors, proline foliar application might induce the drought tolerance in plants by up-regulating the antioxidant enzymatic activities, quenching the ROS and improving cellular membrane stability. In regard to heat stress, Kaushal et al. (2011) reported that elevated temperature causes significant reduction in proline content and antioxidant enzymes and resulted Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 115 in severe membrane lipid peroxidation in chickpea plants. In this respect, a syn- chronic increase in some components of the antioxidative system would be necessary in order to obtain an improvement in heat stress tolerance. In this connection, exogenous application of proline increased enzymatic (SOD and APX) and nonenzymatic antioxidants (AsA and GSH) to a significant level comparing with control. According to these authors, proline has been shown to function as a molecular chaperone able to protect protein integrity and enhance the activities of different enzymes.

4.5 Effect of Exogenous Proline on Growth and Yield Quantity and Quality

Several studies reported that different abiotic stresses reduced cell division and cell expansion, resulting in substantial growth reduction. Inhibition of stem and leaf development negatively affects plant height and leaf area and consequently reduces photosynthesis and crop productivity (Dawood et al. 2014; Osman 2015; Zouari et al. 2016a). Proline regulates many aspects of growth and development, particularly under abiotic stresses. Transgenic rice overexpressing P5C genes presented increased root and shoot growth and increased biomass production under drought conditions. Transgenic plant accumulated more proline than the control (Su and Wu 2004). Therefore, it has been postulated that exogenous application of proline can effectively stimulate growth and yield attributes. Ali et al. (2013) reported that foliar applied proline significantly increased the seed oil content of maize under well irrigated and water-deficit conditions. Furthermore, exogenous application of proline increased the oil oleic and linoleic acid contents. In a similar study, Teh et al. (2016) reported that proline supplementation significantly increased the plant height and the number of roots of rice under salt stress. More recently, Merwad et al. (2018) reported that foliar application of proline ameliorated growth criteria (shoot dry weight, plant height, leaf area, and number of branches per plant) and yield characteristics (dry seed weight, biological yield per plant, and 100-seed weight) of cowpea submitted to water stress. Amelioration of plant growth and yield attributes due to proline application might be due to (i) the improved synthesis of compatible solutes leading to better osmotic adjustment (Dawood et al. 2014); (ii) the enhanced accumulation of total soluble phenolics, thus protecting the tridimensional structure of proteins and enzymes (Ashraf and Foolad 2007; Rasheed et al. 2014); (iii) the improvement in chlorophyll contents (Zouari et al. 2016b); (iv) the reduced oxidative damages (Shahid et al. 2014); (v) the increased antioxidant system activities (Osman 2015); (vi) the stabilization of biological membranes (lipids, protein, plasma membrane) (Hayat et al. 2012); (vii) the enhancement of Rubisco activity (Kaushal et al. (2011); and (viii) the improved photosynthesis (De Freitas et al. 2019). The growth- promoting effect of proline application could be also attributed to its role in protein synthesis. 116 M. Zouari et al.

5 Effective Concentrations of Exogenous Proline

Exogenous application of proline to abiotic-stressed plants generally provides a stress preventing or recovering effect. Despite the beneficial effects of exogenous proline application, proline has toxic effects if over-accumulated and/or applied at excessive concentrations (Ashraf and Foolad 2007). Therefore, it is essential to determine optimal concentrations of proline that provide beneficial effects for each plant species. In maize plants, for example, it was determined that foliar applied proline at 30 mM mitigated the adverse effects of NaCl stress, but, at 60 mM, proline inhibited the growth of salt-stressed and non-stressed plants (Ali et al. 2008). In lettuce (Lactuca sativa L.), exogenous proline spraying at 10 μM was very effective in alleviating the effects of salt stress, while higher concentrations (15 μM) were not beneficial (Orsini et al.2018 ). Butt et al. (2016) applied various concentrations of proline (0.4, 0.6, 0.8, 1.0, and 1.2 mM) as a foliar spray on chili seedlings submitted to salt stress. Among all proline concentrations, 0.8 mM proved to be the best concentration regarding growth, physiological, ionic, and biochemical attributes. Proline application in high concentrations has shown to present harmful effects, such as an inhibition of growth and cellular metabolism (Ashraf and Foolad 2007). Thus, in spite of its protective role, the toxicity effect of proline at high concentrations may be a problem. This toxicity could be due to the repression of genes involved in key functions of the plant metabolism like photosynthesis or synthesis of cell wall-associated proteins (Verbruggen and Hermans 2008). The available information from different studies suggest that optimal concentrations of proline may be species- or genotype-dependent, which need to be determined a priori before commercial application of exogenous proline to improve crop stress tolerance.

6 Conclusion and Future Perspectives

Abiotic environmental stresses remain the major obstacle in plant growth, development, and global crop productivity. However, understanding the physiological and biochemical responses of plants to stress remains necessary to plant science researchers around the world. Scientists are constantly developing new strategies to improve plant stress physiology. In this regard, many studies have provided the notion that the exogenous application of proline provided better protection against different abiotic stresses such as salinity, drought, metal toxicity and extreme temperatures, etc. Under these stressful environmental conditions, exogenous applications of proline have been shown to: (i) Increase the endogenous levels of proline and compatible solute which provide protection to cells through osmotic adjustment. (ii) Help to maintain cellular ionic homeostasis. Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 117

Proline application

Foliar spray

Root medium

Beneficial effects of proline

Osmotic ROS Protection Reduction adjustment Scavenging of cellular of toxic ion structures uptake

Fig. 5 Beneficial effects of exogenous proline application on plants under abiotic stresses

(iii) Act as an antioxidative defense which efficiently scavenge toxic ROS, confer detoxification processes, and reduce oxidative damages through stabilizing antioxidant enzymes. (iv) Affect plant-water relations by maintaining turgidity of cells under stress and increase the photosynthesis rate. (v) Enhance plant growth and final crop yield (Fig. 5). Deciphering proline metabolic pathways and their interconnections with TCA cycle, GABA, polyamine pathway, etc. is of major interest to develop future applications of proline-mediated stress abiotic tolerance. GMO crop fully benefiting from these future breakthroughs are probably not ready before several decades and will be probably not accepted by the public, as they are not biological and environmental friendly. In that sense, combination of proline, glycine betaine, and polyamine exogenous application could constitute a main key to help plant coping with many stresses induced through climate change and global warming even if the exact effects of these applications must be elucidated and their effect on soil microbiota clarified. For plants, climate change leads to increased drought stress, salinity, and heavy metal stresses linked with the fast growing of water reuse techniques occurring currently. Assisting plants in their adaptation to this changing environment is probably the key to maintain crop production at an acceptable level to insure human survival and “proline engineering” is certainly one of the possible solutions. 118 M. Zouari et al.

References

Abdelhamid MT, Rady MM, Osman AS, Abdalla MA (2013) Exogenous application of proline alleviates salt-induced oxidative stress in Phaseolus vulgaris L. plants. J Hortic Sci Biotechnol 88:439–446 Aggarwal M, Sharma S, Kaur N, Pathania D, Bhandhari K, Kaushal N, Kaur R, Singh K, Srivastava A, Nayyar H (2011) Exogenous proline application reduces phytotoxic effects of selenium by minimising oxidative stress and improves growth in bean (Phaseolus vulgaris L.) seedlings. Biol Trace Elem Res 140:354–367 Aleksza D, Horváth GV, Sándor G, Szabados L (2017) Proline accumulation is regulated by transcription factors associated with phosphate starvation. Plant Physiol 175(1):555–567 Ali Q, Ashraf M, Shahbaz M, Humera H (2008) Ameliorative effect of foliar applied proline on nutrient uptake in water stressed maize (Zea mays L.) plants. Pak J Bot 40:211–219 Ali Q, Anwar F, Ashraf M, Saari N, Perveen R (2013) Ameliorating effects of exogenously applied proline on seed composition, seed oil quality and oil antioxidant activity of maize (Zea mays L.) under drought stress. Int J Mol Sci 14:818–835 Amini S, Ghobadi C, Yamchi A (2015) Proline accumulation and osmotic stress: an overview of P5CS gene in plants. J Plant Mol Breed 3(2):44–55 Anwar A, She M, Wang K, Riaz B, Ye X (2018) Biological roles of ornithine aminotransferase (OAT) in plant stress tolerance: present progress and future perspectives. Int J Mol Sci 19(11):3681 Ashraf M, Foolad M (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot 59:206–216 Aslam M, Saeed MS, Sattar S, Sajad S, Sajjad M, Adnan M, Iqbal M, Sharif MT (2017) Specific role of proline against heavy metals toxicity in plants. Int J Pure Appl Biosci 5(6):27–34 Ben Ahmed C, Magdich S, Ben Rouina B, Sensoy S, Boukhris M, Ben Abdullah F (2011) Exogenous proline effects on water relations and ions contents in leaves and roots of young olive. Amino Acids 40(2):565–573 Bhagavan NV, Ha CE (2015) Chapter 3 - Amino acids. In: Essentials of medical biochemistry (Second edition) with clinical cases. Academic Press, Cambridge, USA, pp 21–29 Butt M, Ayyub CM, Amjad M, Ahmad R (2016) Proline application enhances growth of chilli by improving physiological and biochemical attributes under salt stress. Pak J Agric Sci 53:43–49 Choudhury FK, Rivero RM, Blumwald E, Mittler R (2017) Reactive oxygen species, abiotic stress and stress combination. Plant J 90:856–867 Dawood MG, Taie HAA, Nassar RMA, Abdelhamid MT, Schmidhalter U (2014) The changes induced in the physiological, biochemical and anatomical characteristics of Vicia faba by the exogenous application of proline under seawater stress. S Afr J Bot 93:54–63 De Freitas PAF, de Souza MR, Marques EC, Prisco JT, Gomes-Filho E (2018) Salt tolerance induced by exogenous proline in maize is related to low oxidative damage and favorable ionic homeostasis. J Plant Growth Regul 37:911–924 De Freitas PAF, De Carvalho HH, Costa JH, De Souza MR, Da Cruz Saraiva KD, De Oliveira FDB, Gomes Coelho D, Tarquinio Prisco J, Gomes-Filho E (2019) Salt acclimation in sorghum plants by exogenous proline: physiological and biochemical changes and regulation of proline metabolism. Plant Cell Rep 38:403–416 Deuschle K, Funck D, Hellmann H, Däschner K, Binder S, Frommer WB (2001) A nuclear gene encoding mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase and its potential role in protection from proline toxicity. Plant J 27(4):345–356 Fichman Y, Gerdes SY, Kovács H, Szabados L, Zilberstein A, Csonka LN (2015) Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation. Biol Rev 90(4):1065–1099 Forlani G, Bertazzini M, Zarattini M, Funck D (2015) Functional characterization and expression analysis of rice δ1-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism. Front Plant Sci 6:591 Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 119

Funck D, Eckard S, Müller G (2010) Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis. BMC Plant Biol 10(1):70 Ghaffari H, Tadayon MR, Nadeem M, Cheema M, Razmjoo J (2019) Proline-mediated changes in antioxidant enzymatic activities and the physiology of sugar beet under drought stress. Acta Physiol Plant 41:23 Gholami Zali A, Ehsanzadeh P (2018) Exogenous proline improves osmoregulation, physiological functions, essential oil, and seed yield of fennel. Ind Crop Prod 111:133–140 Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery abiotic stress tolerance crop plants. Plant Physiol Biochem 48:909–930 Gleeson D, Lelu-Walter MA, Parkinson M (2004) Influence of exogenous L-proline on embryogenic cultures of larch (Larix leptoeuropaea Dengler), sitka spruce (Picea sitchensis (Bong.) Carr.) and oak (Quercus robur L.) subjected to cold and salt stress. Ann For Sci 61:125–128 Hare PD, Cress WA (1997) Metabolic implications of stress-induced proline accumulation in plants. Plant Growth Regul 21:79–102 Hasanuzzaman M, Alam M, Rahman A, Hasanuzzama M, Nahar K, Fujita M (2014) Exogenous proline and glycine betaine mediated upregulation of antioxidant defense and glyoxalase systems provides better protection against salt-induced oxidative stress in two rice (Oryza sativa L.) varieties. Biomed Res Int 2014:Article ID 757219, 17 pages Hasanuzzaman M, Fujita M, Oku H, Islam MT (eds) (2019) Plant tolerance to environmental stress: role of phytoprotectants, 1st edn. CRC Press, Boca Raton, USA, p 448 Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A (2012) Role of proline under changing environments: a review. Plant Signal Behav 7:1456–1466 Heuer B (2010) Role of proline in plant response to drought and salinity. In: Pessarakli M (ed) Handbook of plant and crop stress. CRC Press, Boca Raton, pp 213–238 Hong Z, Lakkineni K, Zhang Z, Verma DPS (2000) Removal of feedback inhibition of delta(1)- pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol 122:1129–1136 Hoque MA, Banu MN, Okuma E, Amako K, Nakamura Y, Shimoishi Y (2007) Exogenous proline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells. J Plant Physiol 64:1457–1468 Hossain MA, Hoque MA, Burritt DJ, Fujita M (2014) Proline protects plants against abiotic oxidative stress: biochemical and molecular mechanisms. In: Ahmad P (ed) Oxidative damage to plants. Academic press, Cambridge, USA, pp 477–522 Huang TC, Teng CS, Chang JL, Chuang HS, Ho CT, Wu ML (2008) Biosynthetic mechanism of 2-acetyl-1-pyrroline and its relationship with Δ1-pyrroline-5-carboxylic acid and methylglyoxal in aromatic rice (Oryza sativa L.) callus. J Agric Food Chem 56:7399–7404 Iqbal MJ (2018) Role of osmolytes and antioxidant enzymes for drought tolerance in wheat. In: Fahad S (ed) Global wheat production. IntechOpen. https://doi.org/10.5772/intechopen.75926. Available from: https://www.intechopen.com/books/global-wheat-production/ role-of-osmolytes-and-antioxidant-enzymes-for-drought-tolerance-in-wheat Kaushal N, Gupta K, Bhandhari K, Kumar S, Thakur P, Nayyar H (2011) Proline induces heat tolerance in chickpea (Cicer arietinum L.) plants by protecting vital enzymes of carbon and antioxidative metabolism. Physiol Mol Biol Plants 17(3):203–213 Kavi Kishor PB, HimaKumari P, Sunita MSL, Sreenivasulu N (2015) Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny. Front Plant Sci 6:544 Kim GB, Nam YW (2013) A novel Δ1-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation. J Plant Physiol 170(3):291–302 Lehmann S, Funck D, Szabados L, Rentsch D (2010) Proline metabolism and transport in plant development. Amino Acids 39(4):949–962 Merwad ARM, Desoky ESM, Rady MM (2018) Response of water deficit-stressed Vigna unguiculata performances to silicon, proline or methionine foliar application. Sci Hortic 228:132–144 120 M. Zouari et al.

Messedi D, Farhani F, Hamed KB, Trabelsi NAJLA, Ksouri R, Habib-Ur-Rehman Athar CA (2016) Highlighting the mechanisms by which proline can confer tolerance to salt stress in Cakile maritima. Pak J Bot 48:417–427 Murahama M, Yoshida T, Hayashi F, Ichino T, Sanada Y, Wada K (2001) Purification and characterization of δ1-pyrroline-5-carboxylate reductase isoenzymes, indicating differential distribution in spinach (Spinacia oleracea L.) leaves. Plant Cell Physiol 42(7):742–750 Murmu K, Murmu S, Kumar Kundu C, Sekhar Bera P (2017) Exogenous proline and glycine betaine in plants under stress tolerance. Int J Curr Microbiol App Sci 6(9):901–913 Noreen S, Akhter MS, Yaamin T, Arfan M (2018) The ameliorative effects of exogenously applied proline on physiological and biochemical parameters of wheat (Triticum aestivum L.) crop under copper stress condition. J Plant Interact 13:221–230 Orsini F, Pennisi G, Mancarella S, Al Nayef M, Sanoubar R, Nicola S, Gianquinto G (2018) Hydroponic lettuce yields are improved under salt stress by utilizing white plastic film and exogenous applications of proline. Sci Hortic 233:283–293 Osman HS (2015) Enhancing antioxidant–yield relationship of pea plant under drought at different growth stages by exogenously applied glycine betaine and proline. Ann Agric Sci 60:389–402 Oukarroum A, El Madidi S, Strasser RJ (2012) Exogenous glycine betaine and proline play a protective role in heat-stressed barley leaves (Hordeum vulgare L.): a chlorophyll a fluorescence study. Plant Biosyst 146:1037–1043 Pál M, Tajti J, Szalai G, Peeva V, Végh B, Janda T (2018) Interaction of polyamines, abscisic acid and proline under osmotic stress in the leaves of wheat plants. Sci Rep 8:12839 Pérez-Arellano I, Carmona-Álvarez F, Martínez AI, Rodríguez-Díaz J, Cervera J (2010) Pyrroline- 5-carboxylate synthase and proline biosynthesis: from osmotolerance to rare metabolic disease. Protein Sci 19(3):372–382 Rai AN, Penna S (2013) Molecular evolution of plant P5CS gene involved in proline biosynthesis. Mol Biol Rep 40(11):6429–6435 Rana V, Ram S, Nehra K (2017) Review proline biosynthesis and its role in abiotic stress. IJAIR 6(3):473–478 Rasheed R, Ashraf MA, Hussain I, Haider MZ, Kanwal U, Iqbal M (2014) Exogenous proline and glycinebetaine mitigate cadmium stress in two genetically different spring wheat (Triticum aestivum L.) cultivars. Braz J Bot 37:399–406 Roychoudhury A, Banerjee A, Lahiri V (2015) Metabolic and molecular-genetic regulation of proline signaling and itscross-talk with major effectors mediates abiotic stress tolerance in plants. Turk J Bot 39(6):887–910 Satoh R, Fujita Y, Nakashima K, Shinozaki K, Yamaguchi-Shinozaki K (2004) A novel subgroup of bZIP proteins functions as transcriptional activators in hypoosmolarity-responsive expression of the ProDH gene in Arabidopsis. Plant Cell Physiol 45:309–317 Savouré A, Hua XJ, Bertauche N, Van Montagu M, Verbruggen N (1997) Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana. Mol Gen Genet 254(1):104–109 Shahid MA, Balal RM, Pervez MA, Abbas T, Aqeel MA, Javaid MM, Garcia-Sanchez F (2014) Exogenous proline and proline-enriched Lolium perenne leaf extract protects against phytotoxic effects of nickel and salinity in Pisum sativum by altering polyamine metabolism in leaves. Turk J Bot 38:914–926 Shetty K (1997) Biotechnology to harness the benefits of dietary phenolics; focus on Lamiaceae. Asia Pac J Clin Nutr 6:162–171 Signorelli S, Dans PD, Coitiño EL, Borsani O, Monza J (2015) Connecting proline and γ-aminobutyric acid in stressed plants through non-enzymatic reactions. PLoS One 10(3):e0115349 Singh A, Sharma MK, Sengar RS (2017) Osmolytes: Proline metabolism in plants as sensors of abiotic stress. JANS 9(4):2079–2092 Smirnoff N, Cumbes QJ (1989) Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28:1057–1060 Exogenous Proline-Mediated Abiotic Stress Tolerance in Plants: Possible Mechanisms 121

Sobahan MA, Arias CR, Okuma E, Shimoishi Y, Nakamura Y, Hirai Y, Mori IC, Murata Y (2009) Exogenous proline and glycinebetaine suppress apoplastic flow to reduce Na+ uptake in rice seedlings. Biosci Biotechnol Biochem 73:2037–2042 Su J, Wu R (2004) Stress-inducible synthesis of proline in transgenic rice confers faster growth under stress conditions than that with constitutive synthesis. Plant Sci 166:941–948 Szabados L, Savouré A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15(2):89–97 Székely G, Ábrahám E, Cséplő Á, Rigó G, Zsigmond L, Csiszár J, Ayaydin F, Strizhov N, Jásik J, Schmelzer E, Koncz C, Szabados L (2008) Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. Plant J 53(1):11–28 Teh CY, Shaharuddin NA, Ho CL, Mahmood M (2016) Exogenous proline significantly affects the plant growth and nitrogen assimilation enzymes activities in rice (Oryza sativa) under salt stress. Acta Physiol Plant 38:151 Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35:753–759 Verdoy D, Coba De La Peña T, Redondo FJ, Lucas MM, Pueyo JJ (2006) Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress. Plant Cell Environ 29:1913–1923 Verslues PE, Sharma S (2010) Proline metabolism and its implications for plant-environment interaction. Arabidopsis Book/American Society of Plant Biologists 8:e0140 Wei C, Cui Q, Zhang XQ, Zhao YQ, Jia GX (2016) Three P5CS genes including a novel one from Lilium regale play distinct roles in osmotic, drought and salt stress tolerance. J Plant Biol 59(5):456–466 Weltmeier F, Ehlert A, Mayer CS, Dietrich K, Wang X, Schutze K, Alonso R, Harter K, Vicente- Carbajosa J, Droge-Laser W (2006) Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific heterodimerisation of bZIP transcription factors. EMBO J 25:3133–3143 Wutipraditkul N, Wongwean P, Buaboocha T (2015) Alleviation of salt-induced oxidative stress in rice seedlings by proline and/or glycinebetaine. Biol Plant 59:547–553 You J, Chan Z (2015) ROS regulation during abiotic stress responses in crop plants. Front Plant Zhang L, Becker DF (2015) Connecting proline metabolism and signaling pathways in plant senescence. Front Plant Sci 6:552 Zouari M, Ben Ahmed C, Zorrig W, Elloumi N, Rabhi M, Delmail D, Ben Rouina B, Labrousse P, Ben Abdallah F (2016a) Exogenous proline mediates alleviation of cadmium stress by promoting photosynthetic activity, water status and antioxidative enzymes activities of young date palm (Phoenix dactylifera L.). Ecotoxicol Environ Saf 128:100–108 Zouari M, Ben Ahmed C, Elloumi N, Bellassoued K, Delmail D, Labrousse P, Ben Abdallah F, Ben Rouina B (2016b) Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress. Ecotoxicol Environ Saf 128:195–205 Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant Growth and Development

Elisa M. Valenzuela-Soto and Ciria G. Figueroa-Soto

1 Introduction

Plant growth and development is affected by abiotic stress which results in important losses in plant yield and in the money spent in agriculture. The abiotic stress that has a strong impact on plant yield is the hydric stress (drought, salinity, and cold). Plants have developed strategies to contend with hydric stress, between them one of the most important is the synthesis and accumulation of osmolytes (Yancey et al. 1982). In plants, one of the most studied osmolytes is glycine betaine followed by proline and trehalose (Singh et al. 1972; Stewart and Lee 1974; Hare et al. 1998; Iordachescu and Imai 2008; Chen and Murata 2008; Paul et al. 2008; Krasenky and Jonak 2012). Glycine betaine (N,N,N-trimethyl glycine, GB) is a quaternary amine, isolated for the first time from sugar beet (Scheibler1869 ). In mammals, GB participates in homocysteine/methionine cycle [Hcy/Met cycle] as methyl donor to homocysteine to produce methionine, reaction catalyzed by betaine homocysteine methyl transferase [BHMT] (du Vigneaud et al. 1946; Finkelstein and Martin 1984; Pajares and Pérez-Sala 2006). As a consequence of GB participation in the Hcy/Met cycle, a wide set of physiological roles of GB has been found (Craig 2004; Olthof and Verhoef 2005; Lawson-Yuen and Levy 2006; Lever and Slow 2010; Ueland 2011; Figueroa-Soto and Valenzuela-Soto 2018). Physiological functions of GB are as an osmolyte to contribute to maintaining cellular volume, as an osmoprotector to protect cells under stress, and/or as a source of methyl groups through transmethylation reactions (Takabe et al. 2006; Craig 2004; Chen and Murata 2011). However, not all plants accumulate GB in response to stress; in fact, the vast majority of plants of agricultural importance are not accumulators of GB. For this reason, attempts have been made to genetically transform

E. M. Valenzuela-Soto (*) · C. G. Figueroa-Soto Centro de Investigación en Alimentación y Desarrollo A.C, Hermosillo, Sonora, México e-mail: [email protected]

© Springer Nature Switzerland AG 2019 123 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_5 124 E. M. Valenzuela-Soto and C. G. Figueroa-Soto those plants with the genes of GB synthesis enzymes (Takabe et al. 2006; Giri 2011; Chen and Murata 2011; Wani et al. 2013). In plants, two routes of GB synthesis have been proposed, one from choline and the other from glycine. The first route requires two choline oxidation steps catalyzed by the choline monooxygenase [CMO] and betaine aldehyde dehydrogenase [BADH]; the second route involves two methylation steps catalyzed by glycine sarcosine methyltransferase [GSMT] and sarcosine dimethylglycine transferase [SDMT] (Weretilnyk and Hanson 1989; Rathinasabapathi et al. 1997; Valenzuela-Soto and Muñoz-Clares 1994; Nyyssola et al. 2000; Waditee et al. 2005; Chen et al. 2008). There are several studies about the GB accumulation in different plant species as a response to abiotic stress [drought, salinity, cold, heat, etc.] (Rhodes and Hanson 1993; Sakamoto and Murata 2002; Giri 2011; Chen and Murata 2011; Kurepin et al. 2015). These studies have demonstrated that GB plays a role in different processes in plant metabolism, e.g., there is evidence of GB direct and/or indirect participation in protein stability, protein synthesis, enzyme activity, photosynthesis, oxidative stress response, and plant growth and development (Chen et al. 2008; Khan et al. 2009; Giri 2011; Chen and Murata 2011; Wani et al. 2013). However, less is known about the enzymes that synthesize GB or transform it into other compounds, e.g., what are the structural and kinetic characteristics of that enzymes or how they are regulated. The aim of this review is to summarize the knowledge garnered about GB’s metabolism and how it impacts the growth and development of plants under abiotic stress conditions.

2 Glycine Betaine Metabolism

2.1 Synthesis Pathways

In GB accumulator plants, it is synthesized from choline, choline is oxidized to betaine aldehyde by choline monooxygenase [E.C. 1.14.15.7], and betaine aldehyde dehydrogenase [BADH EC 1.2.1.8] catalyzes the betaine aldehyde oxidation to GB (Fig. 1a) (Rathinasabapathi et al. 1997; Ling et al. 2001; Hibino 2002; Wang and Showalter 2004; Park et al. 2007; Muñoz-Clares and Valenzuela-Soto 2008). Extremely halophilic plants and microorganisms, also methanogenic organisms, synthesize GB from glycine; it is methylated by glycine sarcosine methyl transferase [GSMT] to N,N-dimethylglycine and sarcosine; furthermore, N,N- dimethylglycine is methylated by the sarcosine dimethylglycine transferase [SDMT] to GB; thus, both enzymes use S-adenosylmethionine (SAM) as methyl donor (Fig. 1b) (Nyyssola et al. 2000; Waditee et al. 2005). Choline is synthesized in the cytosol, and there are three described possible choline synthesis routes, all of them start with ethanolamine [EA], which can be N-methylated by SAM as free bases, phosphorylethanolamine bases, or phosphatidylethanolamine bases; each methylation step is catalyzed by phosphoethanolamine methyltransferase [PEAMT] (Fig. 2) (Hanson and Rhodes 1983; Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… 125

NAD+ NADH a Choline Betaine aldehyde Glycine betaine CMO BADH

SAM SAH SAM SAH SAMSAH b Glycine Sarcosine N,N-Dimethyl Glycine betaine GSMT GSMT/ glycine SDMT SDMT

c Glycine betaine+Homocysteine Dimethyl glycine + Methionine BHMT

Fig. 1 Glycine betaine synthesis and degradation pathways. (a) GB synthesis in plants. (b) GB synthesis in extremely halophylic plants and microorganisms or metanogenics organisms. (c) GB catabolism in animals, some bacteria and in the cyanobacteria Aphanothece halophytica. CMO choline monooxygenase, BADH betaine aldehyde dehydrogenase, GSMT glycine sarcosine methyl transferase, and SDMT dimethylglycine transferase, BHMT betainehomocysteine methyltransferase

Fig. 2 Interplay between choline, GB, ethylene, and polyamine synthesis pathways. SAMS S-adenosylmethionine synthase, PEAMT phosphoethanolamine methyltransferase, CK choline kinase, CMO choline monooxygenase, BADH betaine aldehyde dehydrogenase, ACC aminocyclopropane carboxylic acid, ACCS aminocyclopropane carboxylic acid synthase, EA ethanolamine, PEA phosphoethanolamine, Pd-EA phosphatidylethanolamine. Pink circle, choline transporter; green ellipse, chloroplast; red arrows indicate inhibition of PEAMT by P-choline 126 E. M. Valenzuela-Soto and C. G. Figueroa-Soto

Datko and Mudd 1988; Nuccio et al. 1998, 2000). The three pathways can be used by plants; however, preference by one of them has been found, e.g., in Chenopodiaceae plants, choline comes from phosphoethanolamine (P-EA); in tobacco, choline originates from phosphatidyl-EA; in soy bean instead, the first step is the methylation of P-EA to phosphomonomethyl ethanolamine [P-MME] followed by a conversion to phosphatidylmonomethylethanolamine [Ptd-MME] via a cytidyl intermediate (Mudd and Datko 1989a; McNeil et al. 2000a). Later Ptd-MME is methylated to phosphatidyldimethylethanolamine [Ptd-DME], which is converted to Ptd-choline and later to P-choline (Hanson and Rhodes 1983; McNeil et al. 2000a, b; Nuccio et al. 2000). The last step in choline synthesis is the dephosphorylation of P-choline by choline phosphatase or choline kinase [CK] (Summers and Weretilnyk 1993; McNeil et al. 2000b). Choline synthesis is regulated by P-choline and S-adenosylhomocysteine [SAH]; both are inhibitors of PEAMT activity (Fig. 2) (Mudd and Datko 1989b; Nuccio et al. 2000; Sahu and Shaw 2009). Choline is transported to the chloroplast and used as a substrate by CMO, the first enzyme in the GB synthesis. CMO is unique in plants and catalyzes the BA synthesis, its crystallographic structure has not been determined yet, the molecular mass of the monomer is ≈ 45 kDa, and it contains a Rieske-type [2Fe-2S] center and requires ferredoxin to be active (Rathinasabapathi et al. 1997). Hibino et al. (2002) found that Cys-181 is essential to the spinach CMO function, and as found in other oxygenases, a histidine [Hys-283] participates in the coordination of the [2Fe-2S] center. The genes coding CMO have been studied in plant accumulator species and plant non-accumulator species. CMO gene sequences from spinach and sugar beet share 78% identity between them, while the sequence of CMO from Arabidopsis shares 51% identity with that of spinach and sugar beet (Hibino et al. 2002). Instead, Amaranthus tricolor CMO shares 69.4% and 69.5% identity with spinach and sugar beet CMOs and Atriplex prostrate, while rice shares 82.9% and 63% identity with deduced amino acid sequence of spinach and sugar beet, respectively (Ling et al. 2001; Wang and Showalter 2004; Luo 2007). Amino acid sequence analysis of the CMO from Amaranthus tricolor, Arabidopsis, barley, rice, sugar beet, and spinach showed that all of them contained consensus sequences for coordination of the Rieske-type [2Fe-2S] cluster, CXHX15–17CX2H, and for coordination of mononuclear non-heme Fe, G/DX3–4 DX2HX4–5H [X equal to any amino acid] (Russell et al. 1998; Rathinasabapathi et al. 1997; Meng et al. 2001; Ling et al. 2001; Hibino et al. 2002; Wang and Showalter 2004; Luo et al. 2007; Mitsuya et al. 2011). In addition, the modeling of Spinacia oleracea CMO showed that in the active site there is an aromatic box con- formed by Tyr281, Tyr295, and Phe301 and by a Glu residue [Glu346] (Carrillo- Campos et al. 2018). Aromatic box is involved in the choline’s trimethylammonium group, while the side chain carboxyl group of Glu346 participates in an ionic interaction with that group (Carrillo-Campos et al. 2018). An analysis of the promoter of Amaranthus tricolor CMO gene allowed identifying a fragment of 410 pb upstream of the translation start codon that contains the sequence responsive to salt stress (Bhuiyan et al. 2007). In addition, Xu et al. (2018) Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… 127 found that the CMO gene from watermelon [Citrullus lanatus] suspension cells contained responsive elements to light, plant hormone-responsive cis-elements, and cis-elements responsive to biotic and abiotic stresses. To this date, it seems that plants that do not accumulate GB possess the CMO genes, but those genes were proposed as not functional, as it has been found in rice and maize (Peel et al. 2010; Luo et al. 2012). However, there is other possible explanation to that results, for a recent phylogenetic study in Amaranthaceae plants showed that plant CMO evolved to two kinds of CMO proteins grouped in two clades called CMO1 and CMO2 and CMO2 diverged from CMO1 (Carrillo-Campos et al. 2018). From 167 plant CMO sequences analyzed, Carrillo et al. (2018) found that CMO1 and CMO2 proteins share 30% identity, CMO1 proteins share 50% identity between them, and otherwise CMO2 proteins share more than 85% identity. CMO1 and CMO2 modelling results showed that neither the CMO1 active site nor the Glu346 as found in CMO2 has the aromatic box; this would explain why CMO1 does not catalyze the oxidation of choline to betaine aldehyde (Carrillo-Campos et al. 2018). In addition, the chloroplast signal peptide is not conserved in CMO1 amino acid sequences (Carrillo-Campos et al. 2018). The second step in GB synthesis is catalyzed by betaine aldehyde dehydrogenase [BADH]. Plant BADHs belong to ALDH superfamily, and they are grouped in the family ten [ALDH10] (Sophos and Vasiliou 2003). Within the ALDH10 family, there are proteins that use as substrate ω-aminoaldehydes [3-amino propionalde- hyde or 4-aminobutyraldehyde] and ω-quaternary amino group [trimethylammonium] and as betaine aldehyde, trimethylaminobutiraldehyde or dimethylsulfoniopropionaldehyde (Trossat et al. 1997; Vojtechová et al. 1997; Ŝebela et al. 2000; Brauner et al. 2003; Livingstone et al. 2003; Oishi and Ebina 2005; Bradbury et al. 2008; Fujiwara et al. 2008). It has been proposed that BADH activity depends on only one amino acid residue at position 441 [SoBADH number- ing] (Muñoz-Clares et al. 2014). The ability to oxidize betaine aldehyde by the BADH is related to the presence of an Ala or Cys in the 441 position in the protein (Muñoz-Clares et al. 2014). The first plant BADH crystal structure obtained was from spinach, which showed that there are four aromatic residues Tyr160, Trp167, Trp285, and Trp456 at the active site (Díaz-Sanchez et al. 2012). By using in silico model building, kinetic studies, and site-directed mutagenesis of SoBADH, it was found that the aromatic ring of Tyr160 is of great importance for BA binding, followed by Trp285 and Trp167 (Díaz-Sanchez et al. 2012). The position that occupies in the active site pocket Trp456 is determined by the conformation adopted by the side chain of the amino acid residue in position 441 [Ile, Ala or Cys], to allow or not the proper posi- tioning of the trimethylammonium group of BA, so Ile size would push Trp456 to such a position that there would be no adequate space for the binding of trimethylammonium group (Díaz-Sanchez et al. 2012; Muñoz-Clares et al. 2014). Interestingly, a great number of BADH from GB accumulators’ plants possess an Ala or Cys in position 441 (Muñoz-Clares et al. 2014). ALDH10 isoenzymes evolved from the gene coding to an Ile in position 441 as a consequence of environmental pressure; however, all plants conserved isoenzymes 128 E. M. Valenzuela-Soto and C. G. Figueroa-Soto with one of three amino acids in the 441 position (Ile, Ala, and/or Cys), which allow isoenzymes to perform other metabolic functions in plants (Muñoz-Clares et al. 2014). Different studies have demonstrated that BADH in plants is a homodimer of ≈ 120 kDa, except in wild amaranth and pea which are heterodimeric and homotetra- meric, respectively (Weretilnyk and Hanson 1989; Valenzuela-Soto and Muñoz- Clares 1994; Figueroa-Soto and Valenzuela-Soto 2001; Ŝebela et al. 2000; Livingstone et al. 2002; Oishi and Ebina 2005; Fujiwara et al. 2008). Plant BADHs show an acidic pI, an optimum pH ranging from 8.0 to 8.5, and they exhibit preference to use NAD+ as coenzyme (Weretilnyk and Hanson 1989; Trossat et al. 1997; Valenzuela-Soto and Muñoz-Clares 1994; Incharoensakdi et al. 2000; Hibino et al. 2001; Fujiwara et al. 2008). Similar to cis-acting regulatory elements described before for CMO, the BADH is also regulated at the genetic level. Analysis of the BADHs’ gene promoter sequence from Suaeda liaotungensis revealed regulatory elements, such as a TATA- box, a CAAT-box, a GC-motif, EIRE, MRE, WUNmotif, a heat shock element, ABRE, methyl jasmonate-responsive element, and ethylene-responsive element [ERE] (Zhang et al. 2008; Xu et al. 2018).

2.2 Glycine Betaine Degradation Routes

In animals and some bacteria, GB is catabolized to methionine and glycine by betaine homocysteine methyl transferase [BHMT], it removes a methyl group from GB to produce dimethylglycine, and the methyl group is transferred to homocysteine for methionine synthesis (Fig. 1c) (Pajares and Perez-Salas 2006). A glycine betaine transmethylase was proposed in Rhizobium meliloti as the enzyme to convert GB to dimethylglycine (Smith et al. 1988), whereas in the cyanobacteria Aphanothece halophytica, GB was catabolized by BHMT under hyperosmotic conditions (Incharoensakdi and Waditee 2000; Waditee and Incharoensakdi 2001). After a deep search about BHMT in plants, no information was found. This being the reason why is not possible to relate methionine synthesis with GB degradation. However, it is possible to speculate that BHMT has not been searched and therefore identified.

2.3 Cellular Compartment of Glycine Betaine Synthesis

GB synthesis has been localized in chloroplasts, peroxisomes, and cytoplasm. It has been suggested that in dicotyledons GB synthesis takes place in the chloroplast, while in monocotyledons it occurs in the peroxisome (Nakamura et al. 1997; Mitsuya et al. 2011). BADH isoenzyme localization differs between plants, e.g., in spinach, one of them is targeted to chloroplast and the other to cytosol; in barley, one isoenzyme is directed to the peroxisome and the other to the cytosol; and in rice, both isoenzymes are targeted to the peroxisome, whereas in Avicennia marina, one of them is delivered to the chloroplast and the other to peroxisome (Weigel et al. 1986; Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… 129

Nakamura et al. 1997; Hibino et al. 2001; Nakamura et al. 2001; Shirasawa et al. 2006). BADH isoenzymes targeted to chloroplast or to peroxisome possess a short signal peptide [seven or three residues, respectively]; in barley, the signal peptide is located in the C-terminus, whereas in spinach it is in the N-terminus (Weretilnyk and Hanson 1990; Nakamura et al. 2001). To this date, it is known the BADHs with high BA affinity are located in the chloroplast (Weigel et al. 1986; Hibino et al. 2001).

2.4 GB Synthesis in Plant Tissue

In plants capable of synthesizing and accumulating GB, it has been found that GB is distributed throughout the whole plant under stress conditions (Yamada et al. 2009). The leaf is the tissue with the highest content of GB, but it is influenced by the leaf age; in barley and sugar beet, it was found that GB is synthesized mainly in old leaves where CMO activity was detected (Nakamura et al. 1996; Hattori et al. 2009; Yamada et al. 2009). The root has the ability to synthesize GB; however, the expression of CMO and BADH is lower compared to the leaf (Bhuiyan et al. 2007; Yamada et al. 2009). On the other hand, BADH was detected in old and young leaves and roots of sugar beet, so it is concluded that the synthesis of GB is limited by the availability of CMO (Bhuiyan et al. 2007; Fujiwara et al. 2008; Yamada et al. 2009). Since GB has been found in tissues that do not contain CMO activity, the mobilization of GB has been investigated. Two transporters have been found: one in sugar beet and another in barley called BvBet/ProT1 and HvProT2, respectively; they transport proline and GB with the highest affinity detected for GB (Yamada et al. 2009; Fujiwara et al. 2010). BvBet/Pro1 and HvProT2 were localized in plasma membrane: BvBet/Pro1 was more abundant in old than in young leaves, while HvProT2 is distributed in old leaves and roots (Yamada et al. 2009; Fujiwara et al. 2010).

3 GB Synthesis and Control of Plant Growth and Development

GB synthesis requires choline in any cellular compartment where it is carried out; at the date, the important aspects that limit the synthesis of GB are the availability of choline and the structural characteristics to carry out the union of the substrates and the catalysis of the CMO and BADH isoenzymes (Nuccio et al. 1998; Díaz- Sanchez et al. 2012; Muñoz-Clares et al. 2014; Carrillo-Campos et al. 2018). Synthesis of P-choline is strongly favored in the cytosol; however, it depends on the dephosporylation of choline because only choline can be transported to chloroplast or vacuole (Bligny et al. 1989; McNeil et al. 2000a). On the other hand, choline produced is distributed between vacuole, chloroplast, and cytosol which limit the 130 E. M. Valenzuela-Soto and C. G. Figueroa-Soto availability of choline to GB synthesis (Nuccio et al. 1998; McNeil et al. 2000a; Sahu and Shaw 2009). Considering that the concentration of choline is not limiting, the synthesis of GB would require a high concentration of SAM which would immediately cause a decrease in the synthesis of ethylene and polyamines (Ravanel et al. 1998; Sahu and Shaw 2009; Wang et al. 2010; Khan et al. 2014). In addition, SAM is required for chlorophyll synthesis, DNA replication, cell wall synthesis, etc.; therefore, GB synthesis cannot be very high, which corresponds with GB concentrations found in plants (Huang et al. 2000; Holstrom et al. 2000; Sakamoto and Murata 2001; Quan et al. 2004; Tabuchi et al. 2005; Wei et al. 2017). Tabuchi et al. (2005) suggested a co-regulation between the levels of S-adenosyl-L-methionine synthetase [SAMS] transcript with those of CMO and PEAMT; this would allow sustain active GB production without significantly diminishing the synthesis of other metabolites dependent on SAM. Plants capable of synthesizing and accumulating GB show tolerance to stress mainly to drought, salinity, and extreme temperature [cold and heat] stress; the growth and development of those plants are affected depending on the stage of development of the plant, as well as the type and species of the plant. It has been demonstrated that drought, salinity, and low and high temperature decrease root and shoot growth and development, but GB’s synthesizing plants manage to reduce the effect of stress on both parameters. The degree or level of protection varies between species and even between varieties of the same species. Under stress conditions, GB participates in maintaining of fundamental processes for growth and development such as (a) photosynthesis, energy production [ATP], and carbon skeletal, (b) conservation of the cell-reducing environment, and (c) enzyme functionality. Since GB can be present in all parts of the plant [either by synthesis or transport], its effect can occur in the entire plant. With all the information generated, it can be said that GB effects on plants under stress conditions are related to its ability to stabilize protein structure and regulate gene transcription and enzyme activities; those functions are the ways on how GB can play a role in plant growth and development. Photosynthesis is inhibited by heat, chilling, salinity, and drought stress; however, GB contributes to maintain the photosynthesis activity through the PSII damaged reparation increasing the expression of D1 protein and increasing its degradation when it is damaged (Fig. 3) (Onishi and Murata 2006; Murata et al. 2007; Yang et al. 2008; Fan et al. 2012). In addition, PSII oxygen-evolving complex structure, Mn cluster, and PSII association with extrinsic polypeptides 18, 23, and 33 kDa are strongly stabilized by GB in plants under stress (Murata et al. 1992; Papageorgiou and Murata 1995; Allakhverdiev et al. 1996; Allakhverdiev et al. 1999). An adequate electron transport in the thylakoid maintains adequate levels of photosynthetic parameters as photosynthetic rate [A], intercellular CO2 [Ci], transpiration rate [E], stomatal conductance [gs], and maximal efficiency of PSII [Fv/Fm] (Fig. 3) (Zhao et al. 2007; Yang et al. 2008; Guha et al. 2010; Wei et al. 2017).

The other face of photosynthesis is the CO2 fixation by the RUBISCO and the flux of carbon skeletal through the Calvin cycle enzymes. RUBISCO, Rubisco acti- Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… 131

Fig. 3 Schematic model of glycine betaine synthesis effects and changes induced to explain its mode of action. The scheme includes plant hormones involved in the induction of GB synthesis. Orange ellipse enclosed proteins involved in photosynthesis; proteins are enclosed in a yellow circle vase, fructose biphosphatase [FBPase], fructose biphosphatase aldolase [FBPaldolase], and phosphoribulose kinase [PRKase] are activated by GB by stabilizing their structure under stress conditions (Fig. 3) (Makela et al. 2000; Yang et al. 2005; Murata et al. 2007; Konrad and Zvi 2008; Fan et al. 2012). Interestingly, Yang et al. (2005) found that under heat stress conditions, Rubisco activase is associated with thylakoid membrane, which is avoided by GB. A suitable CO2 fixation has been proposed by Murata et al. (2007) as an important factor to decrease the

PSII damage, because suppression of CO2 fixation drives to oxidative stress which inhibits D1 protein synthesis and the repair of PSII. To contend with oxidative stress, transgenic plants or wild-type plants able to synthesize GB increase the expression of enzymes of antioxidant system; an increase in mRNA of the enzymes superoxide dismutase [SOD], catalase [CAT], ascorbate peroxidase [APX], glutathione reductase [GR], glutathione peroxidase [GPX], and dehydroascorbate reductase [DHAR] has been found in different plant species (Fig. 3) (Hoque et al. 2008; Islam et al. 2009; Fan et al. 2012; Hasanuzzaman et al. 2014; Zhang et al. 2016; Yao et al. 2018). Increases in antioxidant enzyme activity decrease the lipid peroxidation and protein carbonylation protecting cell survival (Hoque et al. 2008; Islam et al. 2009; Karabudak et al. 2014). Likewise, increases in the concentration of metabolites with antioxidant activity have been found, e.g., increases in glutathione reduced [GSH], ascorbate reduced [ASA], phe- 132 E. M. Valenzuela-Soto and C. G. Figueroa-Soto nolic compounds, and flavonoids (Hoque et al.2008 ; Islam et al. 2009; Ahmed et al. 2013; Wang et al. 2019). Changes in the activity of enzymes involved in Calvin cycle, antioxidant system (enzymatic and nonenzymatic), or proline synthesis are consequence of changes in their gene expression or changes in the enzyme activity induced by GB. In animals, GB induces changes in DNA methylation status and interacts with transcription factors to modify gene expression (Song et al. 2007; Zhang et al. 2013; Deminice et al. 2015; Idriss et al. 2017); in plants, there is no information about it. However, it is tempting to propose that something similar to what happens in animals may be hap- pening in plants. Synthesis of ATP under stress conditions is less studied; Jin et al. (2015) found a high ATP/ADP ratio induced by GB in loquat fruit submitted to a low-temperature conditioning. It has been proposed that GB improves the lipid composition of cell membranes, that is, thylakoid membranes of wheat were protected by GB application, which provoked changes in the fat acid composition (Zhao et al. 2007; Tiang et al. 2017). Changes in lipid composition of thylakoid membranes increased the membrane fluidity, improving their function (Zhao et al.2007 ; Tiang et al. 2017). Therefore, if GB maintains the functionality of thylakoid membranes and positively modulates photosynthesis, then there is a good proton gradient to carry out the ATP synthesis (Yang et al. 2008; Zhao et al. 2007; Guha et al. 2010; Ogbaba et al. 2014; Wei et al. 2017; Tiang et al. 2017). Drought, salinity, heat, and cold stress increase GB synthesis both in GB natural synthesizing and in transgenic plants; this GB increase has a strong impact in the plant growth. Several works have been demonstrated that GB increases the growth of root, shoot, hypocotyl, and plant measured as high, biomass [fresh weight or dry weight], or leaf area under stress conditions and relative of the control plant (Kishitani et al. 2000; Quan et al. 2004; Yang et al. 2005; Park et al. 2007; Yang et al. 2008; Guha et al. 2010; Goel et al. 2011; Fan et al. 2012; Karabudak et al. 2014; Ke et al. 2016; Manaf 2016). The spiking time in transgenic maize plants under stress was less affected compared with wild-type plants (Quan et al. 2004). GB increased the number of anthers, pistils, and petals in transgenic Arabidopsis plants (Sulpice et al. 2003). In transgenic maize, the reproductive development is promoted by GB under drought stress (Quan et al. 2004). The percentage and time of germination of seeds of transgenic rice, tomato, and tobacco plants are promoted by GB under salt and drought stress (Park et al. 2007; Kathuria et al. 2009; Goel et al. 2011; Li et al. 2011). Plant productivity of plants under stress conditions is also promoted in those plants capable of synthesizing GB. Based on maintaining the growth and development of the plants synthesizing GB, the productivity of them is also positively affected. Sulpice et al. (2003) found in transgenic Arabidopsis plants a greater number of flowers and seeds per plant, whereas in transgenic maize, Quan et al. (2004) reported a greater number of seeds per plant and a greater weight per grain. Despite all the positive effects of GB on growth and development of plants, there is no evidence that GB is directly promoting growth, so it has been proposed that GB could be interacting with plant hormones like auxins and ABA, since they are involved in the control of growth (Kurepin et al. 2015). To date it has been found Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… 133 that ABA and methyl jasmonate increase the synthesis of GB (Fig. 3) (Ishitani et al. 1995; Jagendorf and Takabe 2001; Xing and Rajashekar 2001; Xu et al. 2018). Salicylic acid seems to be playing a role of increasing the methionine content to support the SAM production used to GB synthesis and decreasing the ethylene production (Khan et al. 2014). Barley and poplar transgenic plants overexpressing CodA gene showed increased expression levels of auxin-responsive IAA genes (Li et al. 2014; Ke et al. 2016).

4 Conclusion and Future Perspectives

The growth of plants primarily requires sugars, protein synthesis, ATP, reducing power, and a reducing cellular environment, all of which are influenced directly or indirectly by GB. All those aspects are influenced by GB synthesis, transport, and accumulation, by which it has a positive influence in plant growth and development under stress conditions. The mechanism by which GB influences the expression of genes is much less known and is an important aspect to study. The capacity of GB to stabilize proteins and to induce their synthesis explains in part changes in the activity of enzymes studied up to now; however, it remains to be defined if GB interacts directly as activator or inhibitor of enzymes. A great advance has been reached in the knowledge of the impact that GB synthesis has on the growth and development of the plants, as well as in the structural and evolutionary characteristics of the enzymes that catalyze its synthesis. The role of plant hormones in the induction of GB synthesis also begins to be clearer, as well as the impact that the synthesis of GB has on the ethylene and polyamine synthesis pathways. There are still important aspects of the GB synthesis that need to be defined to increase agricultural productivity through plants with the ability to synthesize

GB. Photosynthesis requires all proteins involved in H2O hydrolysis and in the transport of electrons and protons to remain functional, just as the enzymes that participate in ATP synthesis and carbon skeletal synthesis, as well as the chloroplast and thylakoid membranes. However, GB synthesis requires the availability of choline whose synthesis demands a high content of SAM, methionine, and ethanolamine. All these points must be taken into account for the improvement or genetic engineering of plants that synthesize and accumulate GB.

References

Ahmed IM, Cao F, Zhang M, Chen X, Zhang G, Wu F (2013) Difference in yield and physiological features in response to drought and salinity combined stress during anthesis in tibetan wild and cultivated barleys. PLoS One 8(10):e77869 Allakhverdiev SI, Feyziev YM, Ahmed A, Hayashi H, Alie JA, Klimov VV, Murata N, Carpentier R (1996) Stabilization of oxygen evolution and primary electron transport reactions in photosystem II against heat stress with glycinebetaine and sucrose. Photochem Photobiol 34:149–157 134 E. M. Valenzuela-Soto and C. G. Figueroa-Soto

Allakhverdiev YM, Mamedov MD, Ferimazova N, Papageorgiou GC, Gasanov RA (1999) Glycinebetaine stabilizes photosystem 1 and photosystem 2 electron transport in spinach thylakoid membranes against heat inactivation. Photosynthetica 37:423–432 Bhuiyan NH, Hamada A, Yamada N, Rai V, Hibino T, Takabe T (2007) Regulation of betaine synthesis by precursor supply and choline monooxygenase expression in Amaranthus tricolor. J Exp Bot 58(15):4203–4212 Bligny R, Foray M-F, Roby C, Douce R (1989) Transport and phosphorylation of choline in higher plant cells: phosphorus-31 nuclear magnetic resonance studies. J Biol Chem 264:4888–4895 Bradbury LMT, Gillies SA, Brushett DJ, Waters DLE, Henry RJ (2008) Inactivation of an aminoaldehyde dehydrogenase is responsible for fragrance in rice. Plant Mol Biol 68:439–449 Brauner F, Šebela M, Snégaroff J, Peč P, Meunier JC (2003) Pea seedling aminoaldehyde dehydrogenase: primary structure and active site residues. Plant Physiol Biochem 41(1):1–10 Carrillo-Campos J, Riveros-Rosas H, Rodríguez-Sotres R, Muñoz-Clares RA (2018) Bona fide choline monoxygenases evolved in Amaranthaceae plants from oxygenases of unknown function: evidence from phylogenetics, homology modeling and docking studies. PLoS One 13(9):e0204711 Chen TH, Murata N (2008) Glycinebetaine: an effective protectant against abiotic stress in plants. Trends Plant Sci 13(9):499–505 Chen TH, Murata N (2011) Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 34(1):1–20 Craig SAS (2004) Betaine in human nutrition. Am J Clin Nutr 80:539–549 Datko AH, Mudd SH (1988) Phosphatidylcholine synthesis. Plant Physiol 88(3):854–861 Deminice R, da Silva RP, Lamarre SG, Kelly KB, Jacobs RL, Brosnan ME, Brosnan JT (2015) Betaine supplementation prevents fatty liver induced by a high-fat diet: effects on one-carbon metabolism. Amino Acids 47:839–846 Díaz-Sánchez AG, González-Segura L, Mújica-Jiménez C, Rudiño-Piñera E, Montiel C, Martínez- Castilla LP, Muñoz-Clares RA (2012) Amino acid residues critical for the specificity for betaine aldehyde of the plant ALDH10 isoenzyme involved in the synthesis of glycine betaine. Plant Physiol 158(4):1570–1582 Du Vigneaud V, Simmonds JP, Chandler, Cohn CM (1946) A further investigation of the role of betaine in transmethylation reactions in vivo. J Biol Chem 165(2):639–648 Fan W, Zhang M, Zhang H, Zhang P (2012) Improved tolerance to various abiotic stresses in transgenic sweet potato (Ipomoea batatas) expressing spinach betaine aldehyde dehydrogenase. PLoS One 7(5):e37344 Finkelstein JD, Martin JJ (1984) Methionine metabolism in mammals. Distribution of homocysteine between competing pathways. J Biol Chem 259(15):9508–9513 Fujiwara T, Hori K, Ozaki K, Yokota Y, Mitsuya S, Ichiyanagi T, Hattori T, Takabe T (2008) Enzymatic characterization of peroxisomal and cytosolic betaine aldehyde dehydrogenases in barley. Physiol Plant 134:22–30 Fujiwara T, Mitsuya S, Miyake H, Hattori T, Takabe T (2010) Characterization of a novel glycinebetaine/proline transporter gene expressed in the mestome sheath and lateral root cap cells in barley. Planta 232(1):133–143 Figueroa-Soto CG, Valenzuela-Soto EM (2001) Purification of a heterodimeric betaine aldehyde dehydrogenase from wild amaranth plants subjected to water deficit. Biochem Biophys Res Commun 285(4):1052–1058 Figueroa-Soto CG, Valenzuela-Soto EM (2018) Glycine betaine rather than acting only as an osmolyte also plays a role as regulator in cellular metabolism. Biochimie 147:89–97 Giri J (2011) Glycinebetaine and abiotic stress tolerance in plants. Plant Signal Behav 6(11):1746–1751 Goel D, Singh AK, Yadav V, Babbar SB, Murata N, Bansal KC (2011) Transformation of tomato with a bacterial CodA gene enhances tolerance to salt and water stresses. J Plant Physiol 168(11):1286–1294 Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… 135

Guha A, Sengupta D, Reddy AR (2010) Physiological optimality, allocation trade-offs and antioxidant protection linked to better leaf yield performance in drought exposed mulberry. J Sci Food Agric 90(15):2649–2659 Hanson AD, Rhodes D (1983) 14C-tracer evidence for synthesis of choline and betaine via phos- phoryl base intermediates in salinized sugarbeet leaves. Plant Physiol 71:692–700 Hare PD, Cress WA, Van Staden J (1998) Dissecting the roles of osmolyte accumulation during stress. Plant Cell Environ 21(6):535–553 Hasanuzzaman M, Alam M, Rahman A, Hasanuzzaman M, Nahar K, Fujita M (2014) Exogenous proline and glycine betaine mediated upregulation of antioxidant defense and glyoxalase systems provides better protection against salt-induced oxidative stress in two rice (Oryza sativa) varieties. Biomed Res Int 2014:ID 757219 Hattori T, Mitsuya S, Fujiwara T, Jagendorf AT, Takabe T (2009) Tissue specificity of glycinebetaine synthesis in barley. Plant Sci 176:112–118 Hibino T, Meng YL, Kawamitsu Y, Uehara N, Matsuda N, Tanaka Y, Ishikawa H, Baba S, Takabe T, Wada K, Ishii T, Takabe T (2001) Molecular cloning and functional characterization of two kinds of betaine-aldehyde dehydrogenase in betaine-accumulating mangrove Avicennia marina (Forsk,) Vierh. Plant Mol Biol 45(3):353–363 Hibino T, Waditee R, Araki E, Ishikawa H, Aoki K, Tanaka Y, Takabe T (2002) Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants. J Biol Chem 277(44):41352–41360 Holmström KO, Somersalo S, Mandal A, Palva ET, Welin B (2000) Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine. J Exp Bot 51(543):177–185 Hoque MA, Banu MN, Nakamura Y, Shimoishi Y, Murata Y (2008) Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification system and reduce NaCl- induced damage in cultured tobacco cells. J Plant Physiol 165:813–824 Huang J, Hirji R, Adam L, Rozwadowski KL, Hammerlin JK, Keller WA, Selvaraj G (2000) Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations. Plant Physiol 122(3):747–756 Idriss AA, Hu Y, Sun Q, Jia L, Jia Y, Omer NA, Abobaker H, Zhao R (2017) Prenatal betaine exposure modulates hypothalamic expression of cholesterol metabolic genes in cockerels through modifications of DNA methylation. Poult Sci 96:1715–1724 Incharoensakdi A, Matsuda N, Hibino T, Meng YL, Ishikawa H, Hara A, Funaguma T, Takabe T, Takabe T (2000) Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type mutants and E. coli enzymes. Eur J Biochem 267(24):7015–7023 Incharoensakdi A, Waditee R (2000) Degradation of glycinebetaine by betaine-homocysteine methyltransferase in Aphanothece halophytica: effect of salt downshock and starvation. Curr Microbiol 41:227 Iordachescu M, Imai R (2008) Trehalose biosynthesis in response to abiotic stresses. J Integr Plant Biol 50(10):1223–1229 Ishitani M, Nakamura T, Han SY, Takabe T (1995) Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid. Plant Mol Biol 27:307–315 Islam MM, Hoque MA, Okuma E, Banu MN, Shimoishi Y, Nakamura Y, Murata Y (2009) Exogenous proline and glycinebetaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells. J Plant Physiol 166(15):1587–1597 Jagendorf AT, Takabe T (2001) Inducers of glycinebetaine synthesis in barley. Plant Physiol 127:1827–1835 Jin P, Zhang Y, Shan T, Huang Y, Xu J, Zheng Y (2015) Low-temperature conditioning alleviates chilling injury in loquat fruit and regulates glycine betaine content and energy status. J Agric Food Chem 63(14):3654–3659 136 E. M. Valenzuela-Soto and C. G. Figueroa-Soto

Karabudak T, Bor M, Özdemir F, Türkan I (2014) Glycine betaine protects tomato (Solanum lycopersicum) plants at low temperature by inducing fatty acid desaturase7 and lipoxygenase gene expression. Mol Biol Rep 41(3):1401–1410 Kathuria H, Giri J, Nataraja KN, Murata N, Udayakumar M, Tyagi AK (2009) Glycinebetaine- induced water-stress tolerance in CodA-expressing transgenic indica rice is associated with upregulation of several stress responsive genes. Plant Biotechnol J 7:512–526 Ke Q, Wang Z, Ji CY, Jeong JC, Lee HS, Li H, Xu B, Deng X, Kwak SS (2016) Transgenic poplar expressing CodA exhibits enhanced growth and abiotic stress tolerance. Plant Physiol Biochem 100:75–84 Khan MS, Yu X, Kikuchi A, Asahina M, Watanabe KN (2009) Genetic engineering of glycine betaine biosynthesis to enhance abiotic stress tolerance in plants. Plant Biotechnol 26(1):125–134 Khan MI, Asgher M, Khan NA (2014) Alleviation of salt-induced photosynthesis and growth inhibition by salicylic acid involves glycinebetaine and ethylene in mungbean (Vigna radiata L.). Plant Physiol Biochem 80:67–74 Kishitani S, Takanami T, Suzuki M, Oikawa M, Yokoi S, Ishitani M, Alvarez-Nakase AM, Takabe T, Takabe T (2000) Compatibility of glycinebetaine in rice plants: evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley. Plant Cell Environ 23(1):107–114 Konrad Z, Bar-Zvi D (2008) Synergism between the chaperone-like activities of the stress regulated ASR1 protein and the osmolyte glycine-betaine. Planta 227(6):1213–1219 Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic rearrange- ments and regulatory networks. J Exp Bot 63(4):1593–1608 Kurepin LV, Ivanov AG, Zaman M, Pharis RP, Allakhverdiev SI, Hurry V, Hüner NP (2015) Stress- related hormones and glycinebetaine interplay in protection of photosynthesis under abiotic stress conditions. Photosynth Res 126(2–3):221–235 Lawson-Yuen A, Levy HL (2006) The use of betaine in the treatment of elevated homocysteine. Mol Genet Metab 88(3):201–207 Lever M, Slow S (2010) The clinical significance of betaine, an osmolyte with a key role in methyl group metabolism. Clin Biochem 43(9):732–744 Li S, Li F, Wang J, Zhang W, Meng Q, Chen TH, Murata N, Yang X (2011) Glycinebetaine enhances the tolerance of tomato plants to high temperature during germination of seeds and growth of seedlings. Plant Cell Environ 34(11):1931–1943 Li H, Wang Z, Ke Q, Ji CY, Jeong JC, Lee HS, Lim YP, Xu B, Deng XP, Kwak SS (2014) Overexpression of CodA gene confers enhanced tolerance to abiotic stresses in alfalfa. Plant Physiol Biochem 85:31–40 Ling MY, Wang YM, Zhang DA, Nii N (2001) Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions. Cell Res 11(3):187–193 Livingstone JR, Yoshida I, Tarui Y, Hirooka K, Yamamoto Y, Tsutui N, Hirasawa E (2002) Purification and properties of aminoaldehyde dehydrogenase from Avena sativa. J Plant Res 115(5):393–400 Livingstone JR, Maruo T, Yoshida I, Tarui Y, Hirooka K, YamamotoY TN, Hirasawa E (2003) Purification and properties of betaine aldehyde dehydrogenase from Avena sativa. J Plant Res 116(2):133–140 Luo D, Niu X, Wang Y, Zheng W, Chang L, Wang Q, Wei X, Yu G, Lu BR, Liu Y (2007) Functional defect at the rice choline monooxygenase locus from an unusual post-transcriptional processing is associated with the sequence elements of short-direct repeats. New Phytol 175(3):439–447 Luo D, Niu X, Yu J, Yan J, Gou X, Lu BR, Liu Y (2012) Rice choline monooxygenase (OsCMO) protein functions in enhancing glycine betaine biosynthesis in transgenic tobacco but does not accumulate in rice (Oryza sativa L ssp. Japonica). Plant Cell Rep 31:1625–1635 Makela P, Karkkainen J, Somersalo S (2000) Effect of glycinebetaine on chloroplast ultrastructure, chlorophyll and protein content, and RuBPCO activities in tomato grown under drought or salinity. Biol Plant 43:471–475 Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… 137

Manaf HH (2016) Beneficial effects of exogenous selenium, glycine betaine and seaweed extract on salt stressed cowpea plant. Ann Agric Sci 61(1):41–48 McNeil SD, Nuccio ML, Rhodes D, Shachar-Hill Y, Hanson AD (2000a) Radiotracer and computer modeling evidence that phospho-base methylation is the main route of choline synthesis in tobacco. Plant Physiol 123(1):371–380 McNeil SD, Rhodes D, Russell BL, Nuccio ML, Shachar-Hill Y, Hanson AD (2000b) Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco. Plant Physiol 124(1):153–162 Meng YL, Wang YM, Zhang DB, Nii N (2001) Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions. Cell Res 11(3):187–193 Mitsuya S, Kuwahara J, Ozaki K, Saeki E, Fujiwara T, Takabe T (2011) Isolation and characterization of a novel peroxisomal choline monooxygenase in Barley. Planta 234(6):1215–1226 Mudd SH, Datko AH (1989a) Synthesis of methylated ethanolamine moieties. Plant Physiol 90(1):306–310 Mudd SH, Datko AH (1989b) Synthesis of methylated ethanolamine moieties. Regulation by choline in Lemna. Plant Physiol 90(1):296–305 Muñoz-Clares RA, Valenzuela-Soto EM (2008) Betaine aldehyde dehydrogenases: evolution, physiological functions, mechanism, kinetics, regulation, structure, and stability. In: Advances in protein physical chemistry. Research Signpost, Kerala, pp 279–302 Muñoz-Clares RA, Riveros-Rosas H, Garza-Ramos G, Gonzales-Segura L, Mújica-Jiménez C, Julián-Sanchez A (2014) Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALD10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine. BMC Plant Biol 14:149 Murata N, Mohanty PS, Hayashi H, Papageorgiou GC (1992) Glycinebetaine stabilizes the association of extrinsic proteins with the photosynthetic oxygen-evolving complex. FEBS Lett 296:187–189 Murata N, Takahashi S, Nishiyama Y, Allakhverdiev SI (2007) Photoinhibition of photosystem II under environmental stress. Biochem Biophys Acta 1767(6):414–421 Nakamura T, Ishitani M, Harinasut P, Nomura M, Takabe T, Takabe T (1996) Distribution of glycinebetaine in old and young leaf blades of salt-stressed barley plants. Plant Cell Physiol 37(6):873–877 Nakamura T, Yokota S, Muramoto Y, Tsutsui K, Oguri Y, Fukui K, Takabe T (1997) Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes. Plant J 11(5):1115–1120 Nakamura T, Nomura M, Mori H, Jagendorf AT, Ueda A, Takabe T (2001) An isozyme of betaine aldehyde dehydrogenase in barley. Plant Cell Physiol 42(10):1088–1092 Nyyssola A, Kerovuo J, Kaukinen P, von Weymarn N, Reinikainen T (2000) Extreme halophiles synthesize betaine from glycine by methylation. J Biol Chem 275:22196–22201 Nuccio ML, Russell BL, Nolte KD, Rathinasabapathi B, Gage DA, Hanson A (1998) The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J 16:487–496 Nuccio ML, Ziemak MJ, Henry SA, Weretilnyk EA, Hanson AD (2000) cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme. J Biol Chem 275:14095–11410 Oishi H, Ebina M (2005) Isolation of cDNA and enzymatic properties of betaine aldehyde dehydrogenase from Zoysia tenuifolia. J Plant Physiol 162(10):1077–1086 Ohnishi N, Murata N (2006) Glycinebetaine counteracts the inhibitory effects of salt stress on the degradation and synthesis of D1 protein during photoinhibition in Synechococcus sp. PCC 7942. Plant Physiol 141(2):758–765 Ogbaga CC, Stepien P, Johnson GN (2014) Sorghum (Sorghum bicolor) varieties adopt strongly contrasting strategies in response to drought. Physiol Plant 152:389–401 Olthof MR, Verhoef P (2005) Effects of betaine intake on plasma homocysteine concentrations and consequences for health. Curr Drug Metab 6(1):15–22 138 E. M. Valenzuela-Soto and C. G. Figueroa-Soto

Pajares MA, Pérez-Sala D (2006) Betaine homocysteine S-methyltransferase: just a regulator of homocysteine metabolism? Cell Mol Life Sci 63(23):2792–2803 Papageorgiou GC, Murata N (1995) The unusually strong stabilizing effects of glycine betaine on the structure and function of the oxygen-evolving photosystem II complex. Photosynth Res 25:243–252 Park EJ, Jeknic Z, Chen TH, Murata N (2007) The codA transgene for glycinebetaine synthesis increases the size of flowers and fruits in tomato. Plant Biotechnol J 5(3):422–430 Paul MJ, Primavesi LF, Jhurreea D, Zhang YH (2008) Trehalose metabolism and signaling. Annu Rev Plant Biol 59:417–441 Peel GJ, Mickelbart MV, Rhodes D (2010) Choline metabolism in glycinebetaine accumulating and non-accumulating near-isogenic lines of Zea mays and Sorghum bicolor. Phytochemistry 71(4):404–414 Quan R, Shang M, Zhang H, Zhao Y, Zhang J (2004) Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize. Plant Biotechnol J 2:477–486 Rathinasabapathi B, Burnet M, Russell BL, Gage DA, Liao PC, Nye GJ, Scott P, Golbeck JH, Hanson AD (1997) Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning. Proc Natl Acad Sci U S A 94(7):3454–3458 Ravanel S, Gakière B, Job DR (1998) The specific features of methionine biosynthesis and metabolism in plants. Proc Natl Acad Sci U S A 95(13):7805–7812 Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Ann Rev Plant Biol 44:357–384 Russell BL, Rathinasabapathi B, Hanson AD (1998) Osmotic stress induces expression of choline monooxygenase in sugar beet and amaranth. Plant Physiol 116(2):859–865 Sahu BB, Shaw BP (2009) Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization. BMC Plant Biol 9(1):69 Sakamoto A, Murata N (2001) The use of bacterial choline oxidase, a glycine betaine-synthesizing enzyme, to create stress-resistant transgenic plants. Plant Physiol 125(1):180–188 Sakamoto A, Murata N (2002) The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant Cell Environ 25(2):163–171 Scheibler C (1869) Ueber das betain, eine im Safte der Zuckerrüben (Beta vulgaris) vorkommende Pflanzenbase. Ber Dtsch Chem Ges 2:292–295 Šebela M, Brauner F, Radová A, Jacobsen S, Havliš J, Galuszka P, Peč P (2000) Characterisation of a homogeneous plant aminoaldehyde dehydrogenase. Biochem Biophys Acta 1480(1–2):329–341 Shirasawa K, Takabe T, Takabe T, Kishitani C (2006) Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation on their tolerance to abiotic stress. Ann Bot 98(3):565–571 Singh TN, Aspinall D, Paleg LG (1972) Proline accumulation and varietal adaptability to drought in barley: potential metabolic measure of drought resistance. Nat New Biol 236:188–190 Smith LT, Pocard JA, Bernard T, Le Rudulier D (1988) Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti. J Bacteriol 170(7):3142–3149 Summers PS, Weretilnyk EA (1993) Choline synthesis in spinach in relation to salt stress. Plant Physiol 103:1269–1273 Song Z, Deaciuc I, Zhou Z, Song M, Chen T, Hill D, McClain CJ (2007) Involvement of AMP- activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis. Am J Physiol Gastrointest Liver Physiol 293(4):G894–G902 Sophos NA, Vasiliou V (2003) Aldehyde dehydrogenase gene superfamily: the 2002 update. Chem Biol Interact 143:5–22 Stewart GR, Lee JA (1974) Role of proline accumulation in halophytes. Planta 120(3):279–289 Biosynthesis and Degradation of Glycine Betaine and Its Potential to Control Plant… 139

Sulpice R, Tsukaya H, Nonaka H, Mustardy L, Chen TH, Murata N (2003) Enhanced formation of flowers in salt-stressed Arabidopsis after genetic engineering of the synthesis of glycine betaine. Plant J 36(2):165–176 Tabuchi T, Kawaguchi Y, Azuma T, Nanmori T, Yasuda T (2005) Similar regulation patterns of choline monooxygenase, phosphoethanolamine N-methyltransferase and S-adenosyl-L- methionine synthetase in leaves of the halophyte Atriplex nummularia L. Plant Cell Physiol 46(3):505–513 Takabe T, Rai V, Hibino T (2006) Metabolic engineering of glycinebetaine. In: Abiotic stress tolerance in plants. Springer, Dordrecht, pp 137–151 Tiang F, Wang W, Liang C, Wang X, Wang G, Wang W (2017) Overaccumulation of glycine betaine makes the function of the thylakoid membrane better in wheat under salt stress. Crop J 5(1):73–82 Trossat C, Rathinasabapathi B, Hanson AD (1997) Transgenically expressed betaine aldehyde dehydrogenase efficiently catalyzes oxidation of dimethylsulfoniopropionaldehyde and ω-Aminoaldehydes. Plant Physiol 113(4):1457–1461 Ueland PM (2011) Choline and betaine in health and disease. J Inherit Metab Dis 34(1):3–15 Valenzuela-Soto EM, Muñoz-Clares RA (1994) Purification and properties of betaine aldehyde dehydrogenase extracted from detached leaves of Amaranthus hypochondriacus L. subjected to water deficit. J Plant Physiol 143(2):145–152 Vojtechová M, Rodríguez-Sotres R, Valenzuela-Soto EM, Muñoz-Clares RA (1997) Substrate inhibition by betaine dehydrogenase from leaves of Amaranthus hypochondriacus L. Biochim Biophys Acta 1341:49–5700 Waditee R, Incharoensakdi A (2001) Purification and kinetic properties of betaine-homocysteine methyltransferase from Aphanothece halophytica. Curr Microbiol 43(2):107–111 Waditee R, Bhuiyan MNH, Rai V, Aoki K, Tanaka Y, Hibino T, Suzukim S, Takano J, Jagendorf AT, Takabe T, Takabe T (2005) Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc Natl Acad Sci U S A 102(5):1318–1323 Wani SH, Singh NB, Haribhushan A, Mir JI (2013) Compatible solute engineering in plants for abiotic stress tolerance-role of glycine betaine. Curr Genomics 14(3):157–165 Wang LW, Showalter AM (2004) Cloning and salt-induced, ABA-independent expression of choline mono-oxygenase in Atriplex prostrata. Physiol Plant 120:405–412 Wang GP, Li F, Zhang J, Zhao MR, Hui Z, Wang W (2010) Overaccumulation of glycine betaine enhances tolerance of the photosynthetic apparatus to drought and heat stress in wheat. Photosynthetica 48:30–41 Wang L, Shan T, Xie B, Ling C, Shao S, Jin P, Zheng Y (2019) Glycine betaine reduces chilling injury in peach fruit by enhancing phenolic and sugar metabolisms. Food Chem 272:530–538 Wei D, Zhang W, Wang C, Meng Q, Li G, Chen THH, Yang X (2017) Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants. Plant Sci 257:74–83 Weigel P, Weretilnyk EA, Hanson AD (1986) Betaine aldehyde oxidation by spinach chloroplast. Plant Physiol 82(3):753–759 Weretilnyk EA, Hanson AD (1989) Betaine aldehyde dehydrogenase from spinach leaves: purification in vitro translation of the mRNA, and regulation by salinity. Arch Biochem Biophys 271(1):56–63 Weretilnyk A, Hanson AD (1990) Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought. Proc Natl Acad Sci 87:2745–2749 Xing W, Rajashekar CB (2001) Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana. Environ Exp Bot 46(1):21–28 Xu Z, Sun M, Jiang X, Sun H, Dang X, Cong H, Qiao F (2018) Glycinebetaine biosynthesis in response to osmotic stress depends on jasmonate signaling in watermelon suspension cells. Front Plant Sci 9:1469 140 E. M. Valenzuela-Soto and C. G. Figueroa-Soto

Yamada N, Promden W, Yamane K, Tamagake H, Hibino T, Tanaka Y, Takabe T (2009) Preferential accumulation of betaine uncoupled to choline monooxygenase in young leaves of sugar beet – importance of long-distance translocation of betaine under normal and salt-stressed conditions. J Plant Physiol 166(18):2058–2070 Yancey PH, Clark ME, Hand C, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217:1214–1222 Yang X, Liang Z, Lu C (2005) Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants. Plant Physiol 138(4):2299–2309 Yang X, Liang Z, Wen X, Lu C (2008) Genetic engineering of the biosynthesis of glycinebetaine leads to increased tolerance of photosynthesis to salt stress in transgenic tobacco plants. Plant Mol Biol 66(1–2):73–86 Yao W, Xu T, Farooq SY, Jin P, Zheng Y (2018) Glycine betaine treatment alleviates chilling injury in zucchini fruit (Cucurbita pepo L.) by modulating antioxidant enzymes and membrane fatty acid metabolism. Postharvest Biol Technol 144:20–28 Zhao X-X, Ma Q-Q, Liang C, Fang Y, Wang YQ, Wang W (2007) Effect of glycine betaine on function of thylakoid membranes in wheat flag leaves under drought stress. Biol Plant 51:584–588 Zhang Y, Yin H, Li D, Zhu W, Li Q (2008) Functional analysis of BADH gene promoter from Suaeda liaotungensis K. Plant Cell Rep 27(3):585–592 Zhang W, Wang LW, Wang LK, Li X, Zhang H, Luo LP, Song JC, Gong ZJ (2013) Betaine protects against high-fat-diet-induced liver injury by inhibition of high-mobility group box 1 and Toll- like receptor 4 expression in rats. Dig Dis Sci 58(1):3198–31206 Zhang Y, Jin P, Huang Y, Shan T, Wang L, Li Y, Zheng Y (2016) Effect of hot water combined with glycine betaine alleviates chilling injury in cold-stored loquat fruit. Postharvest Biol Technol 118:141–147 Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants: Possible Mechanisms

Tianpeng Zhang and Xinghong Yang

1 Introduction

Because of their sessile character, in the natural environment, plants are most frequently subjected to various types of abiotic stresses, such as salinity, drought, flooding, extreme temperatures, nutrient deficiency, heavy metals and high light intensities, all of which frequently exhibit decreased vegetative growth and negative impacts on crop production and reproductive capabilities (Tuteja et al. 2011; Kurepin et al. 2015; Kumar et al. 2017). One of the best-documented and most important abiotic stress-responsive mechanisms in plants is the biosynthesis and accumulation of compatible solutes (Kumar et al. 2017), such as proline, glycinebetaine, trehalose and polyols (Khan et al. 2009; Jewell et al. 2010; Giri 2011; Kumar and Khare 2015). Glycinebetaine (GB), a fully N-methyl-substituted derivative of glycine found in a large variety of microorganisms, higher plants, and animals, is one of the best-studied compatible solutes that enables plants to tolerate abiotic stress (e.g. Rhodes and Hanson 1993; Chen and Murata 2002, 2008, 2011; Takabe et al. 2006; Masood et al. 2016). GB belongs to a group of compounds collectively known as ‘compatible solutes’, small organic metabolites that are very soluble in water and nontoxic at high concentrations. Both the exogenous application and the genetically engineered biosynthesis of GB increase the tolerance of plants to abiotic stress (Chen and Murata 2002, 2008, 2011). The exogenous application of GB can improve the tolerance of numerous plant species to various types of abiotic stresses, and it can enhance subsequent growth and yield. The GB applied to roots is usually taken up and accumulated in the cytosol, and only a small amount is translocated to chloroplasts. When applied to leaves, GB is translocated to meristematic tissues, in particular, flower buds and shoot

T. Zhang · X. Yang (*) College of Life Science, State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, Taian, China e-mail: [email protected]

apices, and then translocated to actively growing and expanding tissues (Mäkelä et al. 1996; Park et al. 2006). In plants, even if GB is applied to old or mature tissues, this solute reallocates to young actively growing tissues, where its protective functions are mainly required (Ladyman et al. 1980; Annunziata et al. 2019). Due to the beneficial effects of GB, numerous experiments on the exogenous application of this compatible compound on low accumulator and non-accumulator plant species have been performed (Annunziata et al. 2019). In this chapter, we summarize and discuss the current understanding of the physiological and molecular mechanisms of exogenous GB, including the regulation of reactive oxygen species (ROS) scavenging and detoxification under stress, protection of the photosynthetic machinery, interactions and synergistic physiological effects of GB with plant hormones and metabolites and the induction of specific genes involved in stress tolerance. The future perspective of the exogenous application of GB is also discussed.

2 Exogenous Glycinebetaine Enhances Abiotic Oxidative Stress Tolerance

All forms of abiotic stress, including drought, salinity, heat, cold, nutrient deficiency, heavy metals, high light intensities and UV radiation, can cause an excessive accumulation of ROS, leading to various types of deterioration, irreparable dysfunc- tion and cell death in plants (Ashraf 2009; Chen and Murata 2011; Ahmad et al. 2013; Kumar et al. 2017). At present, many studies have shown that the exogenous application of GB on plants enhances oxidative stress tolerance (e.g. Park et al. 2006; Hoque et al. 2007, 2008; Farooq et al. 2008a, b; Hossain et al. 2010, 2011a, b, 2014; Anjum et al. 2012; Hu et al. 2012; Hasanuzzaman et al. 2014; Yildirim et al. 2015; Kumar et al. 2017). Ma et al. (2004) found that exogenous GB application ameliorated the water status of and improved the antioxidant enzyme activities in water-stressed wheat (Triticum aestivum L.) seedlings. Moreover, in fine rice Oryza( sativa), the exogenous application of GB significantly enhanced drought tolerance by altering the level of ROS and malondialdehyde (MDA), increasing the activities of enzyme antioxidants and promoting seedling growth (Farooq et al. 2008b). Additionally, in two maize (Zea mays L.) cultivars, prolonged drought stress increased lipid peroxidation, whereas GB treatment significantly reduced oxidative damage, as indicated by lower MDA levels. Importantly, GB-treated plants maintained higher antioxidant enzyme activity than did non-GB-treated plants in the course of drought stress, which ultimately enhanced the growth and yield of maize (Anjum et al. 2012). Furthermore, Molla et al. (2014) also demonstrated that the exogenous application of GB resulted in a significant increase in the glutathione (GSH) content and maintenance of the high activities of glutathione S-transferase (GST) and glyoxalase I (Gly I) enzymes, with a simultaneous reduction in glutathione disulfide (GSSG) and hydrogen peroxide Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants… 143

(H2O2) levels in lentil (Lens culinaris) seedlings compared to control plants under drought stress, indicating that exogenous GB application enhances drought stress tolerance by limiting H2O2 accumulation and increasing the activities of the antioxidant and glyoxalase systems (Molla et al. 2014). Additionally, the protective roles of GB in modulating cold-, heat- and salinity- induced oxidative stress tolerance have also been well documented in plants. Park et al. (2006) showed that after the exogenous application of GB on tomato (Solanum lycopersicum) plants, the level of catalase activity and expression of the catalase gene (CAT1) were higher, and the H2O2 levels were lower in GB-treated plants than in control plants during 2 days of chilling treatment, indicating that GB may participate in the induction of H2O2-detoxifying antioxidant systems, namely, enhanced catalase expression and catalase activity, when the plants were exposed to chilling stress (Park et al. 2006). Moreover, seed treatments with GB in hybrid maize reduced membrane electrolyte leakage (EL) and maintained higher tissue water contents, antioxidant enzyme activities and carbohydrate metabolism (Farooq et al. 2008a). Furthermore, under cold stress, the exogenous application of GB showed a protective effect on tea buds by regulating the formation of methylglyoxal (MG) and lipid peroxidation and by activating or protecting some antioxidant and glyoxalase pathway enzymes (Kumar and Yadav 2009). Similar effects were also observed in plants under heat stress. Sorwong and Sakhonwasee (2015) reported that the foliar application of GB enhanced heat stress tolerance in marigold (Calendula officinalis) cultivars by reducing the levels of

H2O2, superoxide and MDA, indicating that GB may be involved in the induction of ROS detoxification, thereby mitigating the effect of heat stress on marigolds. Similarly, salinity stress inhibited the growth and development of most plants as a result of the overproduction of ROS, whereas exogenous GB ameliorated the detrimental effect of salinity stress on plants. Hoque et al. (2007) revealed that exogenous GB enhances salinity-induced oxidative stress tolerance in cultured tobacco (Nicotiana tabacum) (BY-2) cells by modulating the activities of ascorbate- glutathione (AsA-GSH) cycle enzymes and GST, glutathione peroxidase (GPX) and glyoxalase system enzymes activities and reducing protein oxidation (Hoque et al. 2008). In addition, compared with control plants, in mung bean seedlings under salinity stress, the exogenous application of GB resulted in a conspicuous increase in the GSH content and the maintenance of a high glutathione redox state and higher activities of correlative enzymes involved in the ROS and methylglyoxal (MG) detoxification system, with a simultaneous decrease in the GSSG content and the levels of H2O2 and lipid peroxidation, suggesting that GB provides a protective action against salt-induced oxidative damage by activating antioxidant defence and

MG detoxification systems and reducing the levels of 2H O2 and lipid peroxidation (Hossain and Fujita 2010). Moreover, Nawaz and Ashraf (2010) found that compared with control maize plants, in two maize genotypes, the exogenous application of GB, as a modulator of salt tolerance, prominently enhanced the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). Furthermore, compared with control plants, in perennial ryegrass (Lolium perenne) under salinity stress, the exogenous application of GB enhances salinity stress tolerance by 144 T. Zhang and X. Yang

reducing the content of EL, MDA, and proline and increasing the vertical shoot growth rate (VSGR), relative water content (RWC), relative transpiration rate (Tr), chlorophyll (Chl) content and activities of SOD, CAT and ascorbate peroxidase (APX) (Hu et al. 2012). Recently, Kotb and Elhamahmy (2014) showed that long- term exogenous GB application at a suitable concentration (50 mM) on bread wheat under saline soil conditions significantly increased enzymatic antioxidant activities, total chlorophylls, leaf osmotic potential and the K+ contents in leaves and grain, thereby alleviating the oxidative stress damage of salinity stress, reflected by improving the growth and productivity of bread wheat plants. In addition, Hasanuzzaman et al. (2014) found that the exogenous application of GB on rice seedlings enhanced salinity-induced oxidative stress tolerance by the upregulation of the ROS and MG detoxification pathways. Similarly, in lettuce Lactuca( sativa L.) plants, exogenous foliar applications of GB mitigated the deleterious effects of salt stress by reducing membrane permeability and the MDA and H2O2 content (Yildirim et al. 2015). The protective roles of GB have also been reported in plants subjected to nitrogen deficiency and cadmium (Cd) stress. Under nitrogen stress conditions, the exogenous application of GB was beneficial for improving the endogenous nitrogen status (Bowman and Rohringer 1970) and thereby enhancing photosynthesis and activating the antioxidant defence system in plants (Ashraf and Foolad 2007; Hoque et al. 2008). Additionally, the exogenous application of varying doses of GB on maize plants resulted in a significant decrease in lipid peroxidation and the intercellular CO2 concentration (Ci), while an increase in the content of leaf total nitrogen and endogenous GB, net photosynthetic rate (Pn), and SOD, CAT, phosphoenolpyruvate carboxylase (PEPCase) and ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities were observed under nitrogen stress (Zhang et al. 2014). Cd is a highly toxic environmental pollutant that can produce excessive ROS, resulting in cellular damage through the oxidation of membrane lipids, proteins and nucleic acids (Flora 2009; De Maria et al. 2013; Lou et al. 2015). Nevertheless, many researchers have demonstrated that exogenous GB ameliorates the adverse effect of Cd stress on plants. Hossain et al. (2010) showed that compared to control plants, the exogenous application of GB on mung bean (Vigna radiata L.) seedlings enhanced Cd tolerance by decreasing H2O2 and MDA levels and enhancing the activities of the relative enzymes involved in ROS and MG detoxification systems. Moreover, Duman et al. (2011) concluded that the use of exogenous GB on an aquatic plant (Lemna gibba L.) relieved the deleterious effects of Cd stress by reducing both ROS and MDA levels, as well as enhancing photosynthetic activity, endogenous proline accumulation and antioxidant enzyme activities. Furthermore, compared with control plants, exogenous applications of GB on perennial ryegrass resulted in alleviating the detrimental effect of Cd stress by elevating SOD, CAT and POD activities and higher stress-responsive gene expression (Lou et al. 2015). From the above reports, it has become clear that GB performs a pivotal function in maintaining ROS levels by modulating the activities of correlative enzymes involved in ROS scavenging and detoxification and the glyoxalase system under various abiotic stresses. Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants… 145

3 Exogenous Glycinebetaine Protects Photosynthetic Machinery Under Abiotic Stress

One of the physiological processes greatly affected by abiotic stress in plants is photosynthesis, and within the photosynthetic machinery, photosystem II (PSII) is the most vulnerable and crucial component that bears the brunt of abiotic stress (Nishiyama et al. 2006; Takahashi and Murata 2008; Nishiyama and Murata 2014; Gururani et al. 2015). Under no-stress conditions, exogenous glycinebetaine can improve the growth,

CO2 assimilation and PSII photochemistry of maize plants, and the enhanced CO2 assimilation rate may be explained by the increased stomatal conductance (Yang and Lu 2006). It is now evident that exogenous glycinebetaine can also play a pivotal role in protecting the photosynthetic machinery in plants under various stressful conditions, which is considered to be one of the major mechanisms of attaining relief from abiotic stress (Chen and Murata 2011; Masood et al. 2016; Kurepin et al.

2017). Exogenous GB application improves CO2 assimilation under drought stress (Mäkelä et al. 1998, 1999; Xing and Rajashekar 1999) and salinity stress (Mäkelä et al. 1998, 1999; Lopez et al. 2002). The exogenous application of GB increased the relative area of starch granules in salt-stressed tomato leaflets and the relative area of plastoglobuli in GB-treated tomato plants under drought stress (Mäkelä et al. 2000). Furthermore, the application of GB on spinach leaves alleviated the photodamage of photosystem I (PSI) submembrane particles by minimizing the alteration in photochemical activity and chlorophyll-protein complexes under cold stress conditions (Rajagopal and Carpentier 2003). Foliar-applied GB also prevented photoinhibition in wheat under freezing (Allard et al. 1998) and drought stresses (Ma et al. 2006). Yang and Lu (2005) observed that the exogenous application of GB on maize plants improved photosynthesis by improving stomatal conductance and PSII efficiency. Similarly, in salt-stressed wheat plants, the application of GB mitigated the adverse effects on photosynthetic capacity by favouring the net CO2 fixation rate, increasing stomatal conductance and protecting the photosynthetic pigments in wheat cultivars (Raza et al. 2006). In another study, the foliar application of GB increased chlorophyll content, gas exchange and photosynthesis, alleviated the deleterious effect of drought on Hill reaction activities and improved the modified lipid composition of the thylakoid membranes in drought-stressed wheat cultivars (Zhao et al. 2007). When tobacco is subjected to low-temperature stress, the exogenous application of GB to plant roots could protect violaxanthin de-epoxidase and enhance non-radiative energy dissipation (NPQ), thereby improving the function of the thylakoid membrane (Wang et al. 2008). Moreover, pretreating rice plants with GB maintained a higher net photosynthetic rate and CO2 assimilation rate compared with those of control plants during drought stress (Farooq et al. 2008b). Analogously, foliar-applied GB maintained water-use efficiency and pigments and increased plant height and the net photosynthetic rate when rice plants were exposed to salt stress (Cha-um and Kirdmanee 2010). Under heat stress, Oukarroum et al. (2012) reported that the foliar application 146 T. Zhang and X. Yang of GB on barley (Hordeum vulgare L.) plants mitigated thermal stress by protecting the oxygen-evolving complex and increasing the energy connectivity between the PSII antennae to increase the stability of the system PSII, thereby reinforcing the heat tolerance in GB-treated plants. In salt-stressed canola plants, foliar-applied GB improved the water-use efficiency, photosynthetic CO2 fixation and stomatal conductance while protecting the oxygen-evolving centre of PSII and maintaining the activity of PSII (Athar et al. 2015). Furthermore, Gupta and Thind (2015) found that the exogenous application of GB on bread wheat plants prominently improved their photosynthetic performance due to more utilization of glutathione and high levels of ascorbic acid in wheat flag leaves under drought stress, indicating the role of nonenzymatic antioxidants in sustaining photosynthetic efficiency and yield stability under prolonged field drought stress conditions. Stepien et al. 2016( ) clearly demonstrated that the foliar application of exogenous GB could significantly mitigate the adverse effects of aluminium (Al) stress in cucumber (Cucumis sativus L. cv. Wisconsin) seedlings by protecting the photosynthetic apparatus components, leading to improved electron transport, gas exchange and enzymatic CO2 fixation. The exogenous application of GB plays a role in protecting the photosynthetic machinery in plants via improving the CO2 assimilation rate and chlorophyll content, as well as ameliorating the negative effect of photodamage and maintaining thylakoid membrane stabilization during various types of abiotic stresses.

4 Interactions of Exogenous Glycinebetaine with Plant Hormones and Metabolites Under Abiotic Stress

During the life span of plants, plant hormones are synthesized in very minute quantities, and these compounds regulate the development and growth of plants and play pivotal roles under various types of abiotic stresses (Masood et al. 2012, 2016; Khan et al. 2013, 2015; Khan and Khan 2014; Asgher et al. 2014, 2015). Many scientists have suggested that the interactions of exogenous GB, plant hormones, and metabolites can be beneficial to plants in abiotic stress tolerance (Yang et al. 2012; Aldesuquy et al. 2012; Yildirim et al. 2015; Gupta and Thind 2019). The foliar application of either GB or abscisic acid (ABA) on creeping bentgrass (Agrostis stolonifera) and Kentucky bluegrass (Poa pratensis) similarly suppresses membrane EL and the accumulation of MDA and increases the activities of APX, POD, and SOD during prolonged periods of drought or salinity stress, indicating that the foliar application of ABA or GB could mitigate physiological damage in turfgrass under drought or salt stress (Yang et al. 2012). Similarly, in a recent study of wheat under water deficit conditions, the application of either salicylic acid (SA) or GB similarly increased grain yield; nevertheless, their co-application was more pronounced than the application of either applied alone due to the repairing effect of the provided chemicals on the growth and metabolism of wheat plants under drought stress (Aldesuquy et al. 2012). Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants… 147

Yildirim et al. (2015) reported that compared to control plants, the foliar application of GB on lettuce plants mitigated the deleterious effect of salt stress by alleviating stomatal conductance, water status, plant nutrient uptake and soluble sugar content and elevating the concentrations of gibberellin (GA), SA and indole acetic acid (IAA) . Additionally, another study demonstrated that the foliar application of 100 mM GB on 19 wheat genotypes resulted in a higher total soluble sugar content under drought stress, whereas the starch content was reduced in GB-treated plants during anthesis. Furthermore, GB application also led to a decline in the activity of leaf sucrose phosphate synthase and sucrose synthase at both tillering and anthesis stages, confirming that exogenous applications of GB could alter the levels of the various sugar components that coordinate the drought response of selected wheat genotypes, resulting in grain yield benefit under prolonged field drought stress (Gupta and Thind 2019).

5 Exogenous Glycinebetaine Induces Specific Gene Expression

GB in the micromolar range, either after the uptake of exogenous GB in plants or as a result of its genetically engineered synthesis, can confer tolerance to several types of stresses (Einset et al. 2007; Chen and Murata 2008, 2011). Allard et al. (1998) demonstrated that the exogenous application of GB enhanced the freezing tolerance of wheat plants. Immunoblot analysis revealed that WCOR410, a low-temperature-inducible protein, was accumulated in the presence of GB, and the ultimate level depended on the concentration of GB. Similarly, northern blotting analysis also illustrated that GB treatment resulted in the induction of a subset of low-temperature-responsive genes, such as WCOR410 and WCOR413, indicating that GB elevated the freezing tolerance of plants by inducing the expression of low-temperature-responsive genes. In tomato plants, after the exogenous application of GB, the level of catalase activity and expression of the catalase gene (CAT1) were higher than those in control plants during 2 days of chilling treatment, suggesting that GB may increase catalase expression and catalase activity when the plants were exposed to chilling stress (Park et al. 2006). Additionally, the exogenous application of GB on both the leaves and roots of Arabidopsis thaliana resulted in the upregulated expression of the genes in roots, including those for membrane- trafficking components, NADP-dependent ferric reductase, transcription factors and ROS-scavenging enzymes, suggesting that GB may confer chilling tolerance to plants by activating the expression of a number of stress-tolerance genes (Einset et al. 2007, 2008). Furthermore, compared to controls, exogenous GB application on tomato seeds under high-temperature stress resulted in elevated levels of heat- shock genes, such as MT-sHSP, HSP70 and HSC70, and accumulated HSP70 protein (Li et al. 2011). 148 T. Zhang and X. Yang

Consequently, based on previous research on the concentrations of GB and testi- mony of its effects on gene expression, it is reasonable to postulate that, at least in part, the effects of GB might be ascribed to the induction and activation of the expression of stress-tolerance genes (Einset et al. 2007; Chen and Murata 2008, 2011). Further studies on the identification of GB-inducible genes and the functions of their products will advance our understanding of the GB-enhanced tolerance in plants under abiotic stress.

6 Conclusion and Future Perspectives

The exogenous application of GB can improve the tolerance of numerous plant species to various types of abiotic stresses, and due to its multiple functions, the possible mechanisms of the exogenous GB-induced tolerance of plants to various types of abiotic stresses include but are not limited to (i) the regulation of ROS scavenging and detoxification under stress, (ii) the protection of the photosynthetic machinery, (iii) interactions with plant hormones and metabolites and (iv) the induction of specific genes whose products are involved in stress tolerance. Although research on the improvement of plant resistance by GB has made great progress, more in-depth studies are needed to reveal subtler regulatory roles for GB in modulating abiotic stress tolerance. For instance, why specific genes are direct targets of GB and whether GB could modulate the tolerance of plants under biotic stress, as well as how to use GB more effectively to develop crops with enhanced tolerance to multiple environmental stresses in the field.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31470341, 31870216) and the State Key Basic Research and Development Plan of China (2015CB150105).

References

Ahmad R, Lim CJ, Kwon SY (2013) Glycine betaine: a versatile compound with great potential for gene pyramiding to improve crop plant performance against environmental stresses. Plant Biotechnol Rep 7:49–57 Aldesuquy HS, Abbas MA, Abo-Hamed SA, Elhakem AH, Alsokari SS (2012) Glycine betaine and salicylic acid induced modification in productivity of two different cultivars of wheat grown under water stress. J Stress Physiol Biochem 8:72–89 Allard F, Houde M, Krol M, Ivanov A, Huner NPA, Sarhan F (1998) Betaine improves freezing tolerance in wheat. Plant Cell Physiol 39:1194–1202 Anjum SA, Saleem MF, Wang LC, Bilal MF, Saeed A (2012) Protective role of glycinebetaine in maize against drought-induced lipid peroxidation by enhancing capacity of antioxidative system. Aust J Crop Sci 6:576–583 Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants… 149

Annunziata MG, Ciarmiello LF, Woodrow P, Aversana ED, Carillo P (2019) Spatial and temporal profile of glycinebetaine accumulation in plants under abiotic stresses. Front Plant Sci 10:230 Asgher M, Khan NA, Khan MIR, Fatma F, Masood A (2014) Ethylene production is associated with alleviation of cadmium-induced oxidative stress by sulfur in mustard types differing in ethylene sensitivity. Ecotox Environ Safe 106:54–61 Asgher M, Khan MIR, Anjum NA, Khan NA (2015) Minimising toxicity of cadmium in plants- role of plant growth regulators. Protoplasma 252:399–413 Ashraf M (2009) Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnol Adv 27:84–93 Ashraf M, Foolad MR (2007) Roles of glycinebetaine and proline in improving plant abiotic stress resistance. Environ Exp Bot 59:206–216 Athar HUR, Zafar ZU, Ashraf M (2015) Glycinebetaine improved photosynthesis in canola under salt stress: evaluation of chlorophyll fluorescence parameters as potential indicators. J Agron Crop Sci 201:428–442 Bowman MS, Rohringer R (1970) Formate metabolism and betaine formation in healthy and rust- affected wheat. Can J Bot 48:803–811 Cha-um S, Kirdmanee C (2010) Effect of glycinebetaine on proline, water use, and photosynthetic efficiencies, and growth of rice seedlings under salt stress. Turk J Agric For 34:517–527 Chen THH, Murata N (2002) Enhancement of tolerance to abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol 5:250–257 Chen THH, Murata N (2008) Glycinebetaine: an effective protectant against abiotic stress in plants. Trends Plant Sci 13:499–505 Chen THH, Murata N (2011) Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 34:1–20 De Maria S, Puschenreiter M, Rivelli AR (2013) Cadmium accumulation and physiological response of sunflower plants to Cd during the vegetative growing cycle. Plant Soil Environ 59:254–261 Duman F, Aksoy A, Aydin Z, Temizgul R (2011) Effects of exogenous glycinebetaine and trehalose on cadmium accumulation and biological responses of an aquatic plant (Lemna gibba L.). Water Air Soil Poll 217:545–556 Einset J, Nielsen E, Connolly EL, Bones A, Sparstad T, Winge P, Zhu JK (2007) Membrane- trafficking RabA4c involved in the effect of glycine betaine on recovery from chilling stress in Arabidopsis. Physiol Plant 130:511–518 Einset J, Winge P, Bones AM, Connolly EL (2008) The FRO2 ferric reductase is required for glycine betaine’s effect on chilling tolerance in Arabidopsis roots. Physiol Plant 134:334–341 Farooq M, Aziz T, Hussain M, Rehman H, Jabran K, Khan MB (2008a) Glycinebetaine improves chilling tolerance in hybrid maize. J Agron Crop Sci 194:152–160 Farooq M, Basra S, Wahid A, Cheema Z, Cheema M, Khaliq A (2008b) Physiological role of exogenously applied glycinebetaine to improve drought tolerance in fine grain aromatic rice (Oryza sativa L.). J Agron Crop Sci 194:325–333 Flora SJS (2009) Structural, chemical and biological aspects of antioxidants for strategies against metal and metalloid exposure. Oxidative Med Cell Longev 2:191–206 Giri J (2011) Glycinebetaine and abiotic stress tolerance in plants. Plant Signal Behav 6:1746–1751 Gupta N, Thind S (2015) Improving photosynthetic performance of bread wheat under field drought stress by foliar applied glycine betaine. J Agric Sci Technol 17:75–86 Gupta N, Thind SK (2019) Foliar application of glycine betaine alters sugar metabolism of wheat leaves under prolonged field drought stress. Proc Natl Acad Sci India Sect B Biol Sci 89:877–884 Gururani MA, Venkatesh J, Tran LSP (2015) Regulation of photosynthesis during abiotic stress- induced photoinhibition. Mol Plant 8:1304–1320 Hasanuzzaman M, Alam MM, Rahman A, Hasanuzzaman M, Nahar K, Fujita M (2014) Exogenous proline and glycinebetaine mediated upregulation of antioxidant defence and glyoxalase sys- 150 T. Zhang and X. Yang

tems provides better protection against salt-induced oxidative stress in two rice (Oryza sativa L.) varieties. Biomed Res Int 2014:1–17 Hoque MA, Banu MNA, Okuma E, Amako K, Nakamura Y, Shimoishi Y, Murata Y (2007) Exogenous proline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells. J Plant Physiol 164:1457–1468 Hoque MA, Banu MNA, Nakamura Y, Shimoishi Y, Murata Y (2008) Proline and glycinebetaine enhance antioxidant defence and methylglyoxal detoxification systems and reduce NaCl- induced damage in cultured tobacco cells. J Plant Physiol 165:813–824 Hossain MA, Fujita M (2010) Evidence for a role of exogenous glycinebetaine and proline in antioxidant defense and methylglyoxal detoxification systems in mung bean seedlings under salt stress. Physiol Mol Biol Pla 16:19–29 Hossain MA, Hasanuzzaman M, Fujita M (2010) Up-regulation of antioxidant and glyoxalase systems by exogenous glycinebetaine and proline in mung bean confer tolerance to cadmium stress. Physiol Mol Biol Pla 16:259–272 Hossain MA, Hasanuzzaman M, Fujita M (2011a) Coordinate induction of antioxidant defense and glyoxalase system by exogenous proline and glycinebetaine is correlated with salt tolerance in mung bean. Front Agric China 5:1–14 Hossain MA, Teixeira da Silva JA, Fujita M (2011b) Glyoxalase system and reactive oxygen species detoxification system in plant abiotic stress response and tolerance: an intimate relationship. In: Shanker AK, Venkateswarlu B (eds) Abiotic stress/book 1. INTECH Open Access Publisher, Rijeka, pp 235–266 Hossain MA, Mostofa MG, Burritt DJ, Fujita M (2014) Modulation of reactive oxygen species and methylglyoxal detoxification systems by exogenous glycinebetaine and proline improves drought tolerance in mustard (Brassica juncea L.). Int J Plant Biol Res 2:1014 Hu L, Hu T, Zhang X, Pang H, Fu J (2012) Exogenous glycinebetaine ameliorates the adverse effect of salt stress on perennial ryegrass. J Am Soc Hortic Sci 137:38–46 Jewell MC, Campbell BC, Godwin ID (2010) Transgenic plants for abiotic stress resistance. In: Kole C, Michler C, Abbott AG, Hall TC (eds) Transgenic crop plants. Springer, Berlin, pp 67–132 Khan MIR, Khan NA (2014) Ethylene reverses photosynthetic inhibition by nickel and zinc in mustard through changes in PS II activity, photosynthetic nitrogen use efficiency, and antioxidant metabolism. Protoplasma 251:1007–1019 Khan MS, Yu X, Kikuchi A, Asahina M, Watanabe K (2009) Genetic engineering of glycine betaine biosynthesis to enhance abiotic stress tolerance in plants. Plant Biotechnol 26:125–134 Khan MIR, Iqbal N, Masood A, Per TS, Khan NA (2013) Salicylic acid alleviates adverse effects of heat stress on photosynthesis through changes in proline production and ethylene formation. Plant Signal Behav 8:e26374 Khan MIR, Nazir F, Asgher M, Per TS, Khan NA (2015) Selenium and sulfur influence ethylene formation and alleviate cadmium-induced oxidative stress by improving proline and glutathione production in wheat. J Plant Physiol 173:9–18 Kotb MA, Elhamahmy MA (2014) Improvement of wheat productivity and their salt tolerance by exogenous glycinebetaine application under saline soil condition for long-term. Zagazig J Agric Res 41:1127–1143 Kumar V, Khare T (2015) Individual and additive effects of Na+ and Cl− ions on rice under salinity stress. Arch Agron Soil Sci 61:381–395 Kumar V, Yadav SK (2009) Proline and betaine provide protection to antioxidant and methylglyoxal detoxification systems during cold stress inCamellia sinensis (L.) O. Kuntze. Acta Physiol Plant 31:261–269 Kumar V, Shriram V, Hoque TS, Hasan MM, Burritt DJ, Hossain MA (2017) Glycinebetaine- mediated abiotic oxidative-stress tolerance in plants: physiological and biochemical mechanisms. In: Stress signaling in plants: genomics and proteomics perspective, vol 2. Springer, Cham, pp 111–133 Exogenous Glycinebetaine-Mediated Modulation of Abiotic Stress Tolerance in Plants… 151

Kurepin LV, Ivanov AG, Zaman M, Pharis RP, Allakhverdiev SI, Hurry V, Hüner NPA (2015) Stress-related hormones and glycinebetaine interplay in protection of photosynthesis under abiotic stress conditions. Photosynth Res 126:221–235 Kurepin LV, Ivanov AG, Zaman M, Pharis RP, Hurry V, Hüner NP (2017) Interaction of glycine betaine and plant hormones: protection of the photosynthetic apparatus during abiotic stress. In: Photosynthesis: structures, mechanisms, and applications. Springer, Cham, pp 185–202 Ladyman JAR, Hitz WD, Hanson AD (1980) Translocation and metabolism of glycine betaine by barley plants in relation to water stress. Planta 150:191–196 Li SF, Li F, Wang JW, Zhang W, Meng QW, Chen THH, Murata N, Yang XH (2011) Glycinebetaine enhances the tolerance of tomato plants to high temperature during germination of seeds and growth of seedlings. Plant Cell Environ 34:1931–1943 Lopez CML, Takahashi H, Yamazaki S (2002) Plant-water relations of kidney bean plants treated with NaCl and foliarly applied glycinebetaine. J Agron Crop Sci 188:73–80 Lou Y, Yang Y, Hy L, Liu H, Xu Q (2015) Exogenous glycinebetaine alleviates the detrimental effect of Cd stress on perennial ryegrass. Ecotoxicology 24:1330–1340 Ma QQ, Zou Q, Li Y, Li DQ, Wang W (2004) Amelioration of the water status and improvement of the anti-oxidant enzyme activities by exogenous glycinebetaine in water-stressed wheat seedlings. Acta Agron Sin 30:321–328 Ma QQ, Wang W, Lib YH, Lib DQ, Zou Q (2006) Alleviation of photoinhibition in drought-stressed wheat (Triticum aestivum) by foliar-applied glycinebetaine. J Plant Physiol 163:165–175 Mäkelä P, Mantila J, Hinkkanen R, Pehu E, Peltonen-Sainio P (1996) Effect of foliar applications of glycinebetaine on stress tolerance, growth, and yield of spring cereals and summer turnip rape in Finland. J Agron Crop Sci 176:223–234 Mäkelä P, Munns R, Colmer TD, Condon AG, Peltonen-Sainio P (1998) Effects of foliar applications of glycinebetaine on stomatal conductance, abscisic acid and solute concentrations in leaves of salt- or drought-stressed tomato. Aust J Plant Physiol 25:655–663 Mäkelä P, Konttur M, Pehu E, Somersalo S (1999) Photosyntehtic response of drought- and salt- stressed tomato and turnip rape plants to foliar-applied glycinebetaine. Physiol Plant 105:45–50 Mäkelä P, Kärkkäinen J, Somersalo S (2000) Effect of glycinebetaine on chloroplast infrastructure, chlorophyll and protein content, and RuBPCO activities in tomato grown under drought or salinity. Biol Plant 43:471–475 Masood A, Iqbal N, Khan NA (2012) Role of ethylene in alleviation of cadmium-induced photosynthetic capacity inhibition by sulphur in mustard. Plant Cell Environ 35:524–533 Masood A, Per TS, Asgher M, Fatma M, Khan MIR, Rasheed F, Hussain SJ, Khan NA (2016) Glycine betaine: role in shifting plants toward adaptation under extreme environments. In: Osmolytes and plants acclimation to changing environment: emerging omics technologies. Springer, New Delhi, pp 69–82 Molla MR, Ali MR, Hasanuzzaman M, Almamun MH, Ahmed A, Nazimuddowla MAN, Rohman MM (2014) Exogenous proline and betaine-induced upregulation of glutathione transferase and glyoxalase I in lentil (Lens culinaris) under drought stress. Not Bot Horti Agrobo 42:73–80 Nawaz K, Ashraf M (2010) Exogenous application of glycinebetaine modulates activities of antioxidants in maize plants subjected to salt stress. J Agron Crop Sci 196:28–37 Nishiyama Y, Murata N (2014) Revised scheme for the mechanism of photoinhibition and its application to enhance the abiotic stress tolerance of the photosynthetic machinery. Appl Microbiol Biot 98:8777–8796 Nishiyama Y, Allakhverdiev SI, Murata N (2006) A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II. Biochim Biophys Acta 1757:742–749 Oukarroum A, El Madidi S, Strasser RJ (2012) Exogenous glycine betaine and proline play a protective role in heat-stressed barley leaves (Hordeum vulgare L.): a chlorophyll a fluorescence study. Plant Biosyst 146:1037–1043 Park EJ, Jeknic Z, Chen THH (2006) Exogenous application of glycinebetaine increases chilling tolerance in tomato plants. Plant Cell Physiol 47:706–714 152 T. Zhang and X. Yang

Rajagopal S, Carpentier R (2003) Retardation of photo-induced changes in photosystem I submembrane particles by glycinebetaine and sucrose. Photosynth Res 78:77–85 Raza SH, Athar H, Ashraf M (2006) Influence of exogenously applied glycinebetaine on the photosynthetic capacity of two differently adapted wheat cultivars under salt stress. Pak J Bot 38:341–351 Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Mol Biol 44:357–384 Sorwong A, Sakhonwasee S (2015) Foliar application of glycinebetaine mitigates the effect of heat stress in three marigold (Tagetes erecta) cultivars. Hort J 84:161–171 Stepien P, Gediga K, Piszcz U, Karmowska K (2016) Effects of the exogenous glycinebetaine on photosynthetic apparatus in cucumber leaves challenging Al stress. In Proceedings of the 18th International Conference on Heavy Metals in the Environment Takabe T, Rai V, Hibino T (2006) Metabolic engineering of glycinebetaine. In: Rai A, Takabe T (eds) Abiotic stress tolerance in plants: toward the improvement of global environment and food. Springer, Dordrecht, pp 137–151 Takahashi S, Murata N (2008) How do environmental stresses accelerate photoinhibition? Trends Plant Sci 13:178–182 Tuteja N, Gill SS, Tuteja R (2011) Plant responses to abiotic stresses: shedding light on salt drought cold heavy metal stress. In: Tuteja N, Gill SS, Tuteja R (eds) Omics and plant abiotic stress tolerance. Bentham Science Publishers Ltd., Beijing, pp 39–64 Wang C, Ma XL, Hui Z, Wang W (2008) Glycine betaine improves thylakoid membrane function of tobacco leaves under low-temperature stress. Photosynthetica 46:400–409 Xing W, Rajashekar CB (1999) Alleviation of water stress in beans by exogenous glycine betaine. Plant Sci 148:185–195 Yang XH, Lu CM (2005) Photosynthesis is improved by exogenous glycinebetaine in salt-stressed maize plants. Physiol Plant 124:343–352

Yang XH, Lu CM (2006) Effects of exogenous glycinebetaine on growth, CO2 assimilation, and photosystem II photochemistry of maize plants. Physiol Plant 127:593–602 Yang Z, Yu J, Merewitz E, Huang B (2012) Differential effects of abscisic acid and glycine betaine on physiological responses to drought and salinity stress for two perennial grass species. J Am Soc Hortic Sci 137:96–106 Yildirim E, Ekinci M, Turan M, Dursun A, Kul R, Parlakova F (2015) Roles of glycinebetaine in mitigating deleterious effect of salt stress on lettuce (Lactuca sativa L.). Arch Agron Soil Sci 61:1673–1689 Zhang LX, Lai JH, Gao M, Ashraf M (2014) Exogenous glycinebetaine and humic acid improve growth, nitrogen status, photosynthesis, and antioxidant defense system and confer tolerance to nitrogen stress in maize seedlings. J Plant Interact 9:159–166 Zhao XX, Ma QQ, Liang C, Fang Y, Wang YQ, Wang W (2007) Effect of glycinebetaine on function of thylakoid membranes in wheat flag leaves under drought stress. Biol Plant 51:584–588 Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses

Pirjo S. A. Mäkelä, Kari Jokinen, and Kristiina Himanen

1 Introduction

Abiotic stresses, the most common of which are water deficit (Boyer 1982) followed by water logging, high and low temperature, and salinity, annually restrict not only plant growth but also global crop yield. It has been estimated that during the period 1961–2014, drought and heat spells caused a global production loss of US$ 237 billion (Mehrabi and Ramankutty 2017). According to an IPCC report in 2017, occurrences and damages caused by weather extremes will increase in the future due to climate change. The impact of global warming differs regionally, and it is envisaged that developing countries will be affected to a greater extent, resulting in increased food insecurity (Rosenzweig and Parry 1994). Changes in ambient temperature occur more rapidly than changes in stress factors such as water deficit and salinity. Furthermore, temperature extremes aggravate the adverse effects of other stresses, including water deficit and salinity, on crop production and quality. For example, heat stress adversely affects grain quality and final crop yield in 40% of the global irrigated wheat growing area (Fischer and Byerlee 1991). Cold stress, although seasonal, has some similarities to water deficit. As water freezes, it creates concentrated solutions of solutes, thereby subjecting plants to a shortage of liquid water (Sakai and Larcher 1987). Global agricultural land area is approximately 4.86 billion ha (FAO 2019). It is estimated that less than 10% of the world’s agricultural land may be free of major environmental stresses (Dudal 1976). As much as 45% of agricultural land is subject to different kinds of water deficit, and 38% of the world’s human population resides

P. S. A. Mäkelä (*) · K. Himanen Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland e-mail: [email protected] K. Jokinen Luke Natural Resources Institute Finland, Helsinki, Finland

© Springer Nature Switzerland AG 2019 153 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_7 154 P. S. A. Mäkelä et al. in those areas (Bot et al. 2000). In relation, the proportion of irrigated field area is approximately 20%, concentrating mostly in Asia (271 Mha) (FAO 2019). In 2015, approximately 510 Mha of total land area, and 19.5% of irrigated agricultural land, was considered saline (FAO and ITPS 2015). Each year a further 2 million ha (about 1%) of the world’s agricultural land deteriorates due to salinity, leading to reduced or no crop productivity (reviewed in Ashraf and Foolad 2007). Apart from irrigation, other major contributors to the increasing area of saline soils are poor management practices, low precipitation, high surface evaporation, and weathering of native rocks. However, secondary salinization causes further problems as productive agricultural land is becoming unsuitable for cultivation due to low quality of irrigation water (Munns 2010). To minimize the effects of abiotic stresses on crop yield, solutions have been actively sought and investigated. These include improving crop tolerance by means of crop management – for example, by the utilization of exogenous and endogenous compounds, including glycinebetaine (GB) – as well as by traditional and molecular plant breeding. Many of the traits resulting in increased abiotic stress tolerance are an interplay of several genes, which make them difficult to modify via traditional and modern plant breeding. Moreover, different abiotic stress factors may provoke osmotic stress, oxidative stress, and protein denaturation in plants. These lead to similar cellular adaptive responses in plants, such as accumulation of compatible solutes, induction of stress proteins, and acceleration of reactive oxygen species (ROS)-scavenging systems (Zhu 2002). Further complexity is associated with phenology as well as species- and cultivar-specific responses to abiotic stresses. Exposure to a single abiotic stress factor can lead to plants obtaining tolerance against a wide range of future abiotic stress events, which is referred to as priming, acclimation, conditioning, hardening, or cross-stress tolerance (Li and Gong 2011; Walter et al. 2013; Antoniou et al. 2016). This involves a memory phase that sepa- rates the primary stress event from the following stress events (Bäurle 2016). During the primary stress phase, changes take place at the physiological, biochemical, molecular, and epigenetic levels. These changes can be transient or maintained throughout the lifetime of a plant and, in some cases, can even be inherited by subsequent generations, for example, in seeds (Mauch-Mani et al. 2017). Over the last 10 years, significant steps have been taken in understanding the biology of osmolytes and especially GB in plants. New associations and insights between GB, genes, and ROS and plant hormones, for example, have been discovered. This chapter provides an update on the most recent research related to osmolytes with special emphasis on endogenous GB and on the transgenesis approach for GB.

2 Osmoprotectants in Plants Under Stress Conditions

Identifying the mechanisms involved in plant adaptation to multiple abiotic stresses such as drought, salinity, nutrient imbalances, extreme temperatures, and light is essential for breeding new crop varieties. In addition, understanding the role of Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 155

factors resulting in increased plant abiotic stress tolerance may assist in developing novel management practices. In this respect, the early dispersion of stress signals, the successive activation of stress-responsive pathways, and finally the responses of plant yield formation are of primary interest to plant biologists, breeders, and agronomists. Within the last few years, several comprehensive reviews on plant stress and the roles of osmoprotectants in improving plant stress tolerance have been published (Singh et al. 2015; Verma et al. 2016; Zhu 2016; Hossain et al. 2018). Here we summarize the increasing amount of literature on osmoprotection in relation to plant stress tolerance. In response to different stresses, plants have developed several mechanisms that involve changes at the morphological, physiological, and molecular level. The sensing of various stresses initiates several complex signaling pathways in plants (Hossain et al. 2018 and cited literature). At first, plants recognize the external stress by using multiple sensors present in the plasma membrane or cell wall. Early signaling events usually include changes to intracellular calcium (Ca2+) concentration followed by an increase in secondary messengers, like reactive nitrogen species (such as nitric oxide), ROS (such as hydrogen peroxide), reactive carbonyl species (such as methylglyoxal), cytosolic calcium ions (Ca2+), hydrogen sulfide, and kinases. In addition, groups of plant hormones (auxins, gibberellins, cytokinins, abscisic acid, ethylene, salicylic acid, jasmonates, brassinosteroids, and strigolactones) participate in plant defense responses (Kurepin et al. 2015; Verma et al. 2016; Xu et al. 2018). Their signaling pathways are interconnected to assist the generation of an efficient stress response. Currently, the fundamental molecules in plant cells and tissues for the acquisition of stress tolerance are considered to be plant hormones. The compounds collaborate with each other to regulate gene expression, resulting in the modification of membrane rigidity and fluidity, changes in the levels of ROS and methylglyoxal detoxifying enzymatic and nonenzymatic antioxidants, and an increase in the synthesis of osmolytes and stress-related proteins. The complex set of responses at the cellular level is also considered to lead to the cross-stress tolerance discussed recently by Hossain et al. (2018). To improve plant tolerance to abiotic stresses such as excess light, water deficit, extreme environmental temperatures, or salinity, the osmotic potential of plant cells must increase. This occurs by the enhancement of cell solutes (reviewed in Singh et al. 2015, Stadmiller et al. 2017), which can be inorganic or organic. In general, inorganic solutes are energetically less expensive but may interfere with metabolism. Organic solutes are energetically more expensive but usually have only minor or no effect on metabolism. In addition, salts in the soil negatively affect water absorption by roots and may result in ion toxicity due to the accumulation of sodium (Na+) and chloride (Cl−) ions in the plant. Under stress conditions, a significant enhancement of extracellular salt concentration results in water efflux, which decreases cell volume and increases the concentration of macromolecules inside the cytoplasm. Accordingly, an increase of common solutes alone, such as organic acids and inorganic ions, may lead to ionic and nutritional imbalance and may prevent the activity of important plant enzymes. Therefore, the localization of common solutes 156 P. S. A. Mäkelä et al. is mainly in the vacuoles, where their increased concentration does not lower the metabolic activity of the cell. In contrast to common solutes, plants can produce different types of compatible organic solutes in response to various stresses (Burg and Ferraris 2008, Singh et al. 2015 and cited literature). In many cases, these solutes seem to accumulate in low concentrations when considered from the whole-plant perspective. However, they typically accumulate in the cytoplasm with high concentrations and do not adversely affect metabolic activity in the cell. Compatible solutes are highly soluble compounds, usually nontoxic at high cellular concentrations, and typically have low molecular weight. Compatible solutes protect plant cells and tissues from stress through several ways. These include contributing to cellular osmotic adjustment, protecting membrane integrity, stabilizing enzymes and proteins, and the detoxification of ROS (Burg and Ferraris 2008 and cited literature, Stadmiller et al. 2017, Hossain et al. 2018 and cited literature). Some compatible solutes can also act as antioxidants. Moreover, they may play a role in stress tolerance by regulating gene replication and transcription (reviewed in Giri 2011 and Hossain et al. 2018). Because some compatible solutes also protect cellular components from dehydration injury, they are called osmoprotectants. Recently, Singh et al. (2015) categorized osmoprotectants into three different groups: osmoprotectants containing ammonium compounds (polyamines, GB, β-alanine betaine, dimethylsulfonio propionate, and choline-O-sulfate), osmoprotectants containing sugars and sugar alcohols (trehalose, fructan, mannitol, D-ononitol, and sorbitol), and osmoprotectants containing amino acids (proline and ectoine). The specific role of different osmoprotectants in plant metabolism and stress tolerance has recently been reviewed by Singh et al. (2015) and Hossain et al. (2018). The majority of osmoprotectants avoid participation in biochemical reactions and are stored in the cytosol. In addition to the conventional osmoprotective role of the compatible solutes, osmoprotectants also detoxify the adverse impacts of stress (e.g., from salinity, water deficit, and cold stress) through two different mechanisms. The first mechanism improves the antioxidant defense system, whereas the second one improves the sustainability of ion homeostasis (reviewed in Singh et al. 2015). In terms of the antioxidant defense system, several studies (Singh et al. 2015; Hossain et al. 2018; Wei et al. 2017; Razavi et al. 2018; Rady et al. 2018) have indicated that under various stress circumstances, osmoprotectants such as polyamines, GB, sugar alcohols, and proline upregulate antioxidant enzyme activities and increase the concentration of nonenzymatic antioxidants to reduce the adverse effects of oxidative stress. Well-known antioxidant enzymes include superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase and some other nonenzymatic low-molecular-weight antioxidants, like glutathione, ascorbate, and carotenoids. Both enzymes and antioxidants have the capability of providing protection via reducing the toxicity of ROS. In a series of detoxifying mechanisms, plants enhance the production of the metalloenzyme superoxide dismutase, which is responsible for the conversion of superoxide to hydrogen peroxide. The breakdown Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 157 of hydrogen peroxide is then catalyzed by CAT and peroxidases. The modulation of the glyoxalase (Gly 1 and Gly 2) and antioxidant defense systems by heat, cold, or osmo-priming has also shown the importance of osmoprotectants for induced cross- stress tolerance. Accordingly, osmoprotectants are promising compounds for improving crop abiotic stress tolerance through the enhancement of the antioxidant system. During stress caused by salinity and water deficit, the sustainability of ion homeostasis is affected by the accumulation of osmoprotectants providing osmotic adjustment via specific ion exchange activity (Singh et al.2015 and cited literature, Wei et al. 2017). Under salinity stress, the most common effect is a reduction of plant growth due to specific ion toxicity, such as from Na+ and Cl−. This also reduces the uptake of essential nutrients like phosphorus (P), potassium (K+), nitrogen (N), and calcium (Ca). The toxic ions negatively impact intracellular K+ influx, reducing the uptake of K+ by cells. Some osmoprotectants may maintain low cytoplasmic Na+ concentration in the cell by decreasing K+ efflux and increasing Na+ efflux, resulting in an optimal K+/Na+ ratio. In addition, osmoprotectants may increase efflux of Na+ from the roots to the environment, leading to less Na+ transfer to plant leaves. Thus, it has been proposed that some osmoprotectants also regulate ion channels and transporters in plants (Wei et al. 2017).

3 Endogenous Glycinebetaine and Plant Abiotic Stress Responses

GB is usually classified as an osmolyte, an osmoprotectant, and a compatible solute. GB could also be regarded as a biostimulant, i.e., a non-fertilizer compound applied in low concentrations that promotes either plant growth, abiotic stress tolerance, or crop quality. Osmolytes and osmoprotectants have gained increased attention over the last two decades. A search in Google Scholar for articles related to GB found 338 published before 1979 and 25,800 published in the decade up to February 2019 (Fig. 1). GB (2-N,N,N-trimethylammonio acetate or N,N′,N″-trimethylglycine), earlier known as lycine or oxyneurine, is a quaternary amine derived from glycine with an average molecular mass of 117.15 (Fig. 2). Due to its zwitterionic nature, it is highly soluble and has low viscosity (Yancey et al. 1982; Yancey 2005). GB is a nontoxic, colorless, tasteless, and odorless compound that accumulates in many plant species, especially in halophytes, when grown under abiotic stresses (see comprehensive list of plant species available in Paleg and Aspinall (1981)). In higher plants, GB is synthesized as a result of the two-step oxidation of choline (Cromwell and Rennie 1954). The first step is catalyzed by choline monooxygenase (CMO), and the second step is mediated by betaine aldehyde dehydrogenase (BADH). The gene expression of CMO and BADH is induced by salinity, water deficit, and temperature stresses in various organisms (for a review, see Hashemi et al. (2018)). Under osmotic stress, changes of turgor may initiate the signal trans- 158 P. S. A. Mäkelä et al.

Fig. 1 The number of scientific articles containing the word “glycinebetaine” published in different decades based on a search in Google Scholar

Fig. 2 Chemical structure of GB. GB has a zwitterionic nature as it possesses both negative (−) and positive (+) charges duction (Xu et al. 2018 and cited literature). Accordingly, under abiotic stresses, increased ion concentration (e.g., Ca2+ and Na+) can be detected by mitogen- activated protein kinase (MAPK), phospholipase D, and some proteins bound to the plasma membrane. MAPK signaling pathways transduce the stress signals which subsequently activate BADH and ROS-scavenging enzymes, such as peroxidase, catalase, superoxide dismutase, ascorbate peroxidase, and lipoxygenase. Finally, BADH accelerates the oxidation of betaine aldehyde to glycinebetaine. Within 24 h, GB is translocated via the phloem throughout the plant, especially to the youngest and developing plant parts (Mäkelä et al. 1996). BADH gene expression can also be regulated by abscisic acid (ABA) (Kurepin et al. 2015 and cited literature). Kurepin et al. (2015) suggested that the close interaction and synergistic physiological effects of GB and ABA, resulting in increased freezing tolerance and a dwarf phenotype, are the major factors leading to effective cold acclimation of higher plants. However, Xu et al. (2018) concluded that the expression of BADH may also be ABA-independent. Instead, they proposed that jasmonate biosynthesis plays a dominant role in the activation of BADH and CMO under osmotic stress. Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 159

3.1 Endogenous Glycinebetaine and Osmotic Stress

Soil salinity is among the main abiotic stresses restricting crop production, and thus major efforts have been made to improve the salinity tolerance of crops. At first, the effect of soil salinity on plants is comparable to water deficit due to low water potential, and the effects of ion-specific toxicity only appear later, in the second phase (Munns 2010). Accumulation of osmolytes, such as GB, allows additional water uptake and therefore buffers the immediate effects of water deficit. While some crops, especially Amaranthaceae and Poaceae, accumulate GB, in the majority of cases the accumulated concentrations for the whole plant might not be physiologically significant. Red beet (Beta vulgaris L.) is salt tolerant and one of the crops which accumulate GB as a response to increasing cell Na+ concentration, among other triggers (Subbarao et al. 2001). In red beet subjected to salt stress, the leaf water content did not vary markedly even though the Na concentration increased up to 400 mol m−3 in the leaves and leaf osmotic potential increased. This was due to a simultaneous increase in GB concentration, contributing 50–60% to the leaf osmotic potential in the cytoplasm. Increasing GB concentration also correlates with maintenance of photosynthesis and chlorophyll fluorescence (Subbarao et al. 2001). According to Leigh et al. (1981), in red beet 26–84% of GB is localized in the cytoplasm, and the concentration in the cytoplasm varies between 46 and 467 mol m−3, whereas the concentration in the vacuole ranges between 2.7 and 17.8 mol m−3. Furthermore, Robinson and Jones (1986) showed that in salt-stressed spinach (Spinacia oleracea L.), at least 40% of GB is localized in chloroplasts, contributing 36% of the leaf osmotic potential. Thus, when GB concentration is calculated according to cytoplasm volume, its physiological role becomes significant. Grumet and Hanson (1986) stated that GB has a marked role in osmoregulation of barley (Hordeum vulgare L.) by maintaining osmotic potential. Later, it was found that GB is the main compatible solute accumulating specifically in young barley leaves (Hattori et al. 2009). GB synthesis is localized in the vascular tissues of leaves and in the pericycle of roots. This is based on the finding that signal transcripts of BBD2 gene increased in the vascular parenchyma cells of leaves and in the root pericycle. BBD2, more abundant in barley, has a 2000-fold affinity for betaine aldehyde in comparison to BBD1. In durum wheat (Triticum durum Desf.), GB is one of the major osmolytes accumulating under prolonged salinity, accumulating especially in young leaves (Carillo et al. 2008). Interestingly, GB accumulation has been shown to correlate positively with glutamate synthase activity in young leaves, though it was independent of nitrogen nutrition of the plant. According to Khan et al. (2012), GB accumulation in salt-stressed bread wheat (Triticum aestivum L.) is linked to both increased salt tolerance and ethylene evolution. These changes are related to the maintenance of photosynthesis fluorescence and lower hydrogen peroxide content. Accumulation of GB can also be cultivar or genotype specific. In cereals, the species and cultivar differences in GB accumulation are marked. For example, some 160 P. S. A. Mäkelä et al. genotypes of sorghum (Sorghum bicolor (L.) Moench) and maize (Zea mays L.) accumulate GB, whereas others do not (Grote et al. 1994; Saneoka et al. 1995). However, even cereal cultivars that do not accumulate detectable concentrations of GB have active BADH and BADH protein in leaves (Ishitani et al. 1993). Peel et al. (2010) compared the GB metabolism in GB-accumulating and non-accumulating maize and sorghum. They concluded that GB deficiency in non-accumulating cereals could result either due to limited availability of choline or lack of choline transporter. The presence of genotypic differences in GB accumulation may explain at least partly the occurrence of stress-tolerant and stress-susceptible genotypes within individual plant species. Some legumes, including mung bean (Vigna radiata (L.) R. Wilczek), also accumulate GB as a response to abiotic stresses. Misra and Gupta (2005) showed a salt- tolerant mung bean cultivar accumulating a higher concentration of GB under salt treatment in comparison to a salt-sensitive cultivar. Similarly, chlorophyll remained higher in the salt-tolerant cultivar. Khan et al. (2014) found that under salinity, GB accumulation in mung beans was induced by salicylic acid, which increased methionine production and suppressed ethylene production, opposite to the results of their barley study (Khan et al. 2012). When salicylic acid inhibits ethylene production, the metabolite of methionine and precursor of ethylene, s-adenosyl methionine, donates a methyl group to GB synthesis and promotes GB synthesis.

3.2 Endogenous Glycinebetaine and Temperature Stress

Yang et al. (1996) tested the high temperature (45 °C) tolerance of near-isogenic maize lines which differ in their ability to accumulate GB. The leaves of GB accumulators had less membrane damage, and the temperature threshold difference between the lines was 2 °C. Furthermore, the GB accumulators showed better ther- mostability of the PSII electron chain. These results indicate that GB might play a role in the protection of plasma membranes. At the other extreme, Kishitani et al. (1994) studied the role of GB on the freezing tolerance of barley leaves by using near-isogenic lines whose ability to accumulate GB ranges from 10 to 90 μmol g−1 DM. After acclimation at 5 °C and freezing at −5 °C, the youngest leaves with the highest GB concentration survived, whereas the oldest leaves with the lowest concentration of GB died. Thus, it was concluded that GB plays a marked role in cold acclimation against freezing injury in young barley leaves. Cooling is a useful storage method commonly employed to prolong postharvest life of plant produce. It reduces postharvest decay of tissues during transportation to distant markets and assures the availability of good quality produce to consumers for an extended period. However, many fruits and vegetables are chilling sensitive and highly vulnerable to chilling injury during cold storage at low temperatures, e.g., below 8 °C. The severe development of chilling injury decreases produce quality, for example, in appearance, texture, flavor, and nutrition. Unfavorable chilling Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 161 temperature directly promotes membrane phase transition from fluid liquid crystal- line to rigid solid gel, leading to a decline in the membrane selective permeability. In addition, chilling temperature as an oxidative stress factor indirectly promotes ROS accumulation, resulting in the peroxidation of unsaturated fatty acids in plant membranes. Recent reports, summarized here, indicate that GB is a useful molecule for reducing chilling injuries in several fruits. The mechanisms seem to be similar to those found in whole-plant studies and in their response to common stresses. Jin et al. (2015) studied the influence of low-temperature conditioning treatment (at 10 °C for 6 days) on chilling injury, GB concentration, and energy metabolism in loquat fruit (Eriobotrya japonica (Thunb.) Lindl) stored at 1 °C. Their results indicate that low-temperature conditioning treatment significantly reduces chilling injury, ion leakage, and malondialdehyde content in loquat fruit. BADH activity and endogenous GB content in loquats treated with low-temperature conditioning were significantly higher than in control fruit. Moreover, low-temperature conditioning treatment induced activities of energy metabolism-associated enzymes, including H+-adenosine triphosphatase, Ca2+-adenosine triphosphatase, succinic dehydrogenase, and cytochrome c oxidase. The low-temperature conditioning treatment clearly triggered higher levels of ATP content and energy charge, and together these results show that low-temperature conditioning may alleviate chilling injury and improve chilling tolerance of loquat fruit by enhancing endogenous GB accumulation and energy status. Yao et al. (2018) suggested that GB can ameliorate the chilling injury in zucchini (Cucurbita pepo L.) fruit. The effects of GB treatment were associated with an accumulation of proline and a reduction in lipid peroxidation. In addition, GB-treated fruit also showed lower levels of palmitic acid and stearic acid, and lower lipoxygenase and plant phospholipase D activities, but higher activity levels of enzymes related to proline metabolism. The gene expression and antioxidant enzyme activities of superoxide dismutase, catalase, and ascorbate peroxidase in GB-treated fruit were significantly higher than that of control fruit. Thus, GB could alleviate chilling injury in cold-stored zucchini fruit through improved antioxidant enzymatic mechanisms in addition to the involvement of fatty acid metabolism. Recently, Razavi et al. (2018) reported that in hawthorn (Crataegus monogyna Jacq.) fruits, GB applied by immersion for 15 min at 20 °C resulted in a steady increase of endogenous GB accumulation during storage at 1 °C for 20 days. This accumulation was then associated with delayed fruit pitting development. They also found that higher endogenous GB accumulation correlated with higher activity of antioxidant enzymes, such as superoxide dismutase, catalase, and ascorbate peroxidase, leading to lower buildup of hydrogen peroxide. In addition, fruits treated with GB exhibited significantly higher content of phenols, flavonoids, and anthocyanins, which was due to the higher activity of phenylalanine ammonia lyase enzyme. Furthermore, the observed higher ascorbic acid accumulation in GB-treated fruits resulted in higher 1,1-diphenyl-2-picrylhydrazyl-scavenging capacity during storage at 1 °C for 20 days. The authors propose that GB treatment is a useful strategy for attenuating chilling injury of hawthorn fruit due to lower ROS accumulation. Moreover, the application of GB could be favorable in terms of maintaining nutri- 162 P. S. A. Mäkelä et al. tional quality of hawthorn fruit because it increases the level of antioxidant molecules, beneficial for human health. Wang et al. (2019) also showed that GB could enhance the chilling tolerance of peach (Prunus persica (L.) Batsch) fruits through the regulation of phenolic and sugar metabolism, leading to the maintenance of high levels of individual phenolic and sucrose content.

4 Glycinebetaine and Transgenesis Approaches to Improve Plant Stress Tolerance

Plants cope with abiotic stresses by activating response pathways that result in redi- rection of resources from growth toward resistance. Abiotic stress tolerance is often manifested in the accumulation of protective enzymes and metabolites. Primary metabolites are conserved molecules required for normal growth and development, while secondary metabolites are related more to signaling and are more diverse among different species. Understanding metabolic fluxes in plant cells in response to many environmental factors requires genome-wide systems approaches. Plant metabolomics addresses the biochemistry and molecular mechanisms of plant responses to cope with osmotic stress. It combines sample separation by liquid or gas chromatography and the detection of metabolites based on their ion mass and charge. In general, metabolomic analysis is less dependent on genomic information than many other molecular omics studies, such as transcriptomics or proteomics. Therefore, this technology is accessible for a wide range of species. With regard to the accumulation of osmolytes, such as GB, plant species are recognized as GB accumulators or non-accumulators. Transgenesis has introduced the GB pathway into many non-accumulator species and increased GB levels in GB-accumulating species. In this chapter, we summarize the current understanding of the challenges in genetically engineering GB accumulation in plants.

4.1 Transgenesis for Improved GB Levels

In plants, biosynthesis of GB is a simple two-step reaction cascade involving choline oxidation reaction by CMO followed by oxidation of the resulting BADH. In Escherichia coli, the BetA and BetB enzymes mediate these two reactions. The COD (Arthrobacter globiformis) and COX (Arthrobacter pascens) pathways represent prokaryotic choline oxidases that mediate direct conversion of choline to GB (Sakamoto and Murata 2001). Despite these straightforward reaction cascades, transgenesis approaches have proven challenging to optimize for obtaining physiologically relevant GB osmolyte levels. Transgenesis approaches in plant species lacking a functional GB biosynthesis pathway have utilized both prokaryotic and eukaryotic genes. Utilizing genes from a prokaryotic origin reduces considerations of translational and posttranslational modifications. Standard overexpression of one Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 163 of the biosynthetic enzymes aims to increase levels of gene expression in the cell. Overexpression vectors usually harbor a 35S promoter and terminators together with antibiotic selection. Physiologically relevant levels for GB to act as an osmotic regulator range between tens of μM to hundreds of μM (Annunziata et al. 2019). GB accumulation at the level of 5 μmol g−1 DM, or down to 1 μmol g−1 FW, has also been suggested as promoting stress resistance as summarized in Khan et al. (2009) and Chen and Murata (2011). As stated earlier, this activity depends on the compartmentation of GB in cells. In tobacco, overexpression of E. coli BetA (CDH) alone or together with BetB (BADH) conferred the transgenic plants with increased resistance to salt stress compared to wild-type plants (Holmström et al. 2000). Overexpression resulted in functional enzymes and enhanced the plant’s ability to process betaine aldehyde, the toxic intermediate of the GB synthesis pathway. The GB levels, however, remained at a low level (40–80 nmol g−1 FW), suggesting that the stress-protective effect was not due to osmoregulation. Mild accumulation of GB might still be adequate to protect protein complexes and membranes, for example, in chloroplasts. Cotton cv. Luyuan890 has been engineered to constitutively overexpress the betA gene from E. coli (Lv et al. 2007). In wild-type plants, the GB levels were already physiologically relevant, with high levels of approximately 100 μmol g−1 DM. The betA transgenic lines accumulated GB at over 130 μmol g−1 DM, and their drought resistance and physiological performance were analyzed. Four out of five of the lines were shown to perform better for maintenance of osmotic potential and relative water content. In overexpression approaches, codA from Arthrobacter globiformis has been most popular, although the resulting GB levels usually remain moderate (Khan et al. 2009; Chen and Murata 2011). In tomato (Solanum lycopersicum L.) transgenesis, codA from Arthrobacter globiformis was used to mediate direct choline conversion to GB, in contrast to two-step biosynthesis (Wei et al. 2017; Khan et al. 2009). Overexpression in tomato cv. Moneymaker resulted in L1, L2, and L3 lines with minor increases in GB accumulation of up to 2 μmol g−1 DM. Following NaCl treatment, GB accumulation reached 5–6 μmol g−1 DM and was shown during stress to increase photosynthetic rate and antioxidant enzyme activity and to reduce ROS accumulation (Wei et al. 2017). Changes in Na+/K+ ion balances were observed in the transgenic lines, resulting from increased Na+ exclusion and decreased K+ efflux. These effects were mediated through ion channel gene expression. It is proposed that GB could promote salt tolerance through regulation of the respective channels and transporters. In addition, GB may enhance antioxidant enzyme activities and thereby alleviate ROS responses and damage to photosynthesis in the leaves. Salt stress is known to impair photosynthesis, and it has been suggested that the positive impact of GB on photosynthesis results from better osmotic adjustment and prevention of stomatal closure (Lv et al. 2007). A second study on the tomato cv. Moneymaker codA transgenic lines (codA Arthrobacter globiformis) with relatively low GB accumulation (up to 2.5 μmol g−1 FW) addressed the role of GB in abiotic stress resulting from phosphate starvation + (Li et al. 2019). The transgenics were able to maintain Pi/H co-transport, and the 164 P. S. A. Mäkelä et al. gene expression of the PHO regulon was also modified, and photosynthetic rates remained high. In the transgenic lines, growth was enhanced as indicated by increased fresh weight and shoot and root size, while stress responses such as anthocyanin accumulation were lower compared to wild type. Here, moderate GB accumulation mediated physiological and biochemical changes so that environmental adaptation processes were impacted. GB biosynthesis by COD/COX results in side product hydrogen peroxide accumulation, which operates in redox sensing, signaling, and regulation in eukaryotic cells (Sies 2017). In Arabidopsis (Arabidopsis thaliana L.) transformed with the codA gene for choline oxidase, accumulation of steady-state hydrogen peroxide was detected at the level of 960 nmol g−1 FW compared to 750 nmol g−1 FW in wild type (Hayashi et al. 1997; Sakamoto and Murata 2001). Part of the observed effects from COD/COX transgenesis thus might be due to such alternative responses. Transgenic wheat line (T6) has been generated to overexpress the Atriplex hor- tensis L. BADH gene in the shi4185 line. In the wild-type wheat line, GB concentration is already at a high level of 75 μmol g−1; BADH overexpression caused this to increase to 100 μmol g−1 DM (Wang et al. 2010). A similar increase was seen in the wild type after drought treatment. In the study, drought, heat, and their combination were tested in the wild-type and overexpressing line. The responses in the T6 line appeared milder compared to wild type for most of the parameters measured for the three replicates. The heat stress effects on transpiration and stomatal conductance deviated from drought and combination responses. Interestingly, most transgenic plants can utilize exogenously applied choline, and GB levels remain stress-inducible in transgenic lines even if transgenes are driven by a constitutive 35S promoter (Lv et al. 2007). This suggests that GB biosynthesis is further promoted by the stress condition. This regulation can be at the transcript level or at the post-translational level. Conversely, this also suggests that transgenesis approaches have not addressed all the components involved. In transgenesis of non-accumulators that lack all functional GB biosynthesis enzymes, overexpression of only one component often leaves the GB accumulation levels moderate. Unbalanced expression of biosynthetic enzymes from the GB pathway can create different cellular and metabolic imbalances (Hare et al. 1998; Gage et al. 2003; Chen and Murata 2011). For example, BADH is not a substrate-specific enzyme and has been associated with diverse aldehydes (Trossat et al. 1997; Muñoz- Clares et al. 2014). The alternative reaction cascades of the GB biosynthesis enzymes can result in competition between substrates and cause side effects, for example, in polyamine metabolism, possibly resulting in new phenotypes (Trossat et al. 1997). Taken together, transgenesis of only one gene from a biosynthetic pathway is usually not enough to achieve the intended outcome. Limiting factors for GB biosynthesis can be the availability of choline, activity of the biosynthetic enzymes and their specificities toward the substrates, as well as the subcellular localization of the enzymes and their respective substrates (Huang et al. 2000; Nuccio et al. 1998, 2000; Kumar et al. 2004; Muñoz-Clares et al. 2014; Carrillo-Campos et al. 2018). Modifications to the single-gene overexpression approaches are represented by Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 165 gene stacking, a transgenesis method in which combinations of constructs harbor more than one gene and can be transferred under one selection (Zorrilla-López et al. 2013). In principle, gene stacking would allow transferring all the limiting factors from a biosynthesis pathway in one or consecutive events. Hence, gene stacking could solve some of the bottlenecks in transgenesis for GB accumulation.

4.2 Considerations for CMO and BADH Isoenzymes

Significant sequence-specific differences have been discovered in the GB biosynthesis isoenzymes. Phylogenetic studies show that all land plant species have genes encoding for CMO enzymes (Carrillo-Campos et al. 2018). The CMO genes are present in two clades, CMO1 and CMO2, whereby CMO2 has diverged from the CMO1 after genome duplication. CMO2-type enzymes have evolved at a fast rate and are present in GB-accumulating plant species, such as spinach (Fig. 3). Homology modeling and docking simulations have shown that the CMO2 active site has three aromatic residues and a glutamate that allow efficient interaction with the substrate, choline. The four critical amino acids of CMO2 that confer substrate specificity for choline are indicated in Fig. 3. Such binding capacity toward choline is lacking from the CMO1-type isoenzymes, the isozymes that prevail in GB non- accumulators. Spinach also has CMO1-type enzymes that don’t utilize choline but act as oxygenases on different substrates. It would be interesting to verify which spinach CMO form was used in the transgenesis approaches that resulted in low GB accumulation (Shirasawa et al. 2006). Functional isoenzyme differences have also been discovered for the second step of GB biosynthesis, in the BADH isozymes (Muñoz-Clares et al. 2014). BADH isoenzymes belong to the family 10 of aldehyde dehydrogenases, but only certain ALDH10 enzymes appear to have BADH activity on BAL. Phylogenetic analysis has shown that in spinach, a GB accumulator, the BADH enzyme has a particular amino acid at position 441 (alanine A441), while GB non-accumulators, such as Arabidopsis, have isoleucine at this position (Fig. 4). The amino acid in position 441 (painted gray in Fig. 3) appears to determine if enzymes are able to oxidize BAL into GB. These structure functional discoveries in GB biosynthesis enzymes are likely to influence the success of future transgenesis approaches for enhancing GB production in plants.

4.3 Chloroplast Targeted Transgenesis for Optimized GB Production

Endogenous GB biosynthesis is compartmentalized within the chloroplast. Targeting GB accumulation directly in the chloroplast can facilitate correct enzyme conformation in the correct subcellular compartment. Chloroplast genetic engineering has 166 P. S. A. Mäkelä et al.

Spinacia_CMO2 MMAASASATTMLLKYPTTVCG-IPNPSSNNNNDPSNNIASIPQNTTNPTLKSRTPNKITT 59 Spinacia_CMO1 MS------ITHSIT---QNPLTNHVTLQSFGNNFIPK------IERFPNRIHQ38 Arabidopsis_CMO1 MMTT------LTATVPEFLPPSLKSTRGYFNSHSEFGVS------ISKFSRRRFH43 * :: .. . . .: .:

Spinacia_CMO2 NAVAAPSFPSLTTTTPSSIQSLVHEFDPQIPPEDAHTPPSSWYTEPAFYSHELERIFYKG 119 Spinacia_CMO1 APIKLTKC-LSNSSSIQSTHKIAHEFDPNIPIEEAQTPPCSWYSDPEFYSHEIDRVFYSG97 Arabidopsis_CMO1 NPTR------VFAVSDISKLVTEFDPKIPLERASTPPSSWYTDPQFYSFELDRVFYGG 95 : .. .:. ****:** * * ***.***::* ***.*::*:** *

Spinacia_CMO2 WQVAGISDQIKEPNQYFTGSLGNVEYLVSRDGEGKVHAFHNVCTHRASILACGSGKKSCF 179 Spinacia_CMO1 WRVVGCVDQIKNAHDYFTGRLGNVEYVICRDGVGKIHAFHNVCRHHASILAYGSGRKTCF 157 Arabidopsis_CMO1 WQAVGYSDQIKESRDFFTGRLGDVDFVVCRDENGKIHAFHNVCSHHASILASGNGRKSCF 155 *:..* ****: .::*** **:*::::.** **:******* *:***** *.*:*:**

Spinacia_CMO2 VCPYHGWVYGMDGSLAKASKAKPEQNLDPKELGLVPLKVAVWGPFVLISLDRSLEEG--- 236 Spinacia_CMO1 VCPYHGWTYGLEGNLLKAPRITGLRNFNPKEYGLVPINVATWGPFVVVNLSSSEEE---V 214 Arabidopsis_CMO1 VCLYHGWTYSLSGSLVKATRMSGIQNFSLSEMGLKPLRVAVWGPFVLLKVTAATSRKGEV 215 ** ****.*.:.*.* ** : . :*:. .* ** *:.**.*****::.: : ..

Spinacia_CMO2 ---GDVGTEWLGTSAEDVKAHAFDPSLQFIHRSELPMESNWKIFSDNYLDSSYHVPYAHK 293 Spinacia_CMO1 -DYGNMENDWLGGSADLLSINGVDTSLSYICRREYTLECNWKVFCDNYLDGGYHVPYAHK 273 Arabidopsis_CMO1 ETDELVASEWLGTSVGRLSQGGVDSPLSYICRREYTIDCNWKVFCDNYLDGGYHVPYAHK 275 : .:*** *. :. ..* *.:* * * ::.***:*.*****..********

Spinacia_CMO2 YYATELNFDTYDTQMIENVTIQRVEGSS-NKPDGFDRVGIQAFYAFAYPNFAVERYGPWM 352 Spinacia_CMO1 NLASGLNLDSYSTEMFEKVSIQRCASSSTETGEDFDRLGSKALYAFVYPNFMINRYGPWM 333 Arabidopsis_CMO1 GLMSGLDLETYSTTIFEKVSIQECGGGSKVGEDGFDRLGSEALYAFVYPNFMINRYGPWM 335 : *::::*.* ::*:*:**. ..* :.***:* :*:***.**** ::******

Spinacia_CMO2 TTMHIHPLGPRKCKLVVDYYIENSMLDDKDYIEKGIAINDNVQREDVVLCESVQRGLETP 412 Spinacia_CMO1 DTNLVIPLGPRKCQVVFDYFLDASLKDDKAFIERSLKDSEEVQIEDIMLCEGVQRGLESP 393 Arabidopsis_CMO1 DTNLVLPLGPRKCKVVFDYFLDPSLKDDEAFIKRSLEESDRVQMEDVMLCESVQRGLESQ 395 * : *******::*.**::: *: **: :*::.: .:.** **::***.******:

Spinacia_CMO2 AYRSGRYVMPIEKGIHHFHCWLQQTLK------439 Spinacia_CMO1 AYNTGRYAPTLEKPMHHFHCLLYRNLTEQTLQF 426 Arabidopsis_CMO1 AYDKGRYAL-VEKPMHHFHCLLHHNLKL----- 422 ** .***. :** :***** * :.*.

Fig. 3 Spinacia CMO1 (XP_021866412.1) and CMO2 (ABN43460.1) amino acid sequence alignment with Arabidopsis CMO1 amino acid sequence by Clustal Omega tool (Madeira et al. 2019). Spinacia CMO2 functional motives for choline oxidation as indicated by Carrillo-Campos et al. (2018) are underlined and painted in grey. Stars under sequences indicate shared amino acids, single and double dot indicates semi- and conservative amino acids, respectively, no sign indicates non-conservative amino acid many benefits over nuclear transgenesis (Kumar et al.2004 ). Direct chloroplast genome transgenesis and efficient transgene expression have been achieved with appropriate regulatory sequences for both selection and the gene of interest. Homologous recombination in the chloroplast genome requires extensive flanking sequences around the gene of interest. The carrot (Daucus carota subsp. sativus (Hoffm.) Schübl. & G. Martens)-specific transformation vector, pDD-DC-aadA/ badh, harbored aadA and badh sequences regulated by the 5’ribosome-binding site region of the bacteriophage T7 gene 10 leader to facilitate expression in green and non-green tissues. Similarly, the promoter sequence was designed to harbor binding sites for both plastid- and nuclear-encoded RNA polymerases. Transgenesis was performed by particle bombardment of a yellow carrot cell culture. The untransformed cell remained yellow in color while transformed cells turned green, allowing selection without a selectable marker. The method was completed with the successful regeneration of mature plants through somatic embryogenesis. Directing Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 167

Spinacia_BADH MAFPIPARQLFIDGEWREPIKKNRIPVINPSTEEIIGDIPAATAEDVEVAVVAARRAFRR 60 Arabidopsis_ALDH MAIPMPTRQLFIDGEWREPILKKRIPIVNPATEEVIGDIPAATTEDVDVAVNAARRALSR 60 **:*:*:************* *:***::**:***:********:***:*** *****: *

Spinacia_BADH N---NWSATSGAHRATYLRAIAAKITEKKDHFVKLETIDSGKPFDEAVLDIDDVASCFEY 117 Arabidopsis_ALDH NKGKDWAKAPGAVRAKYLRAIAAKVNERKTDLAKLEALDCGKPLDEAVWDMDDVAGCFEF 120 * :*: : ** **.********:.*:* .:.***::*.***:**** *:****.***:

Spinacia_BADH FAGQAEALDGKQKAPVTLPMERFKSHVLRQPLGVVGLISPWNYPLLMATWKIAPALAAGC 177 Arabidopsis_ALDH YADLAEGLDAKQKAPVSLPMESFKSYVLKQPLGVVGLITPWNYPLLMAVWKVAPSLAAGC 180 :*. **.**.******:**** ***:**:*********:*********.**:**:*****

Spinacia_BADH TAVLKPSELASVTCLEFGEVCNEVGLPPGVLNILTGLGPDAGAPLVSHPDVDKIAFTGSS 237 Arabidopsis_ALDH TAILKPSELASVTCLELADICREVGLPPGVLNVLTGFGSEAGAPLASHPGVDKIAFTGSF 240 **:*************:.::*.**********:***:* :*****.***.*********

Spinacia_BADH ATGSKVMASAAQLVKPVTLELGGKSPIVVFEDVDIDKVVEWTIFGCFWTNGQIXCSATSR 297 Arabidopsis_ALDH ATGSKVMTAAAQLVKPVSMELGGKSPLIVFDDVDLDKAAEWALFGCFWTNGQI-CSATSR 299 *******::********::*******::**:***:**..**::********** ******

Spinacia_BADH LLVHESIAAEFVDKLVKWTKNIKISDPFEEGCRLGPVISKGQYDKIMKFISTAKSEGATI 357 Arabidopsis_ALDH LLVHESIASEFIEKLVKWSKNIKISDPMEEGCRLGPVVSKGQYEKILKFISTAKSEGATI 359 ********:**::*****:********:*********:*****:**:*************

Spinacia_BADH LYGGSRPEHLKKGYYIEPTIVTDISTSMQIWKEEVFGPVLCVKTFSSEDEAIALANDTEY 417 Arabidopsis_ALDH LHGGSRPEHLEKGFFIEPTIITDVTTSMQIWREEVFGPVLCVKTFASEDEAIELANDSHY 419 *:********:**::*****:**::******:*************:****** ****:.*

Spinacia_BADH GLAAAVFSNDLERCERITKALEVGAVWVNCSQPCFVQAPWGGIKRSGFGRELGEWGIQNY 477 Arabidopsis_ALDH GLGAAVISNDTERCDRISEAFEAGIVWINCSQPCFTQAPWGGVKRSGFGRELGEWGLDNY 479 **.***:*** ***:**::*:*.* **:*******.******:*************::**

Spinacia_BADH LNIKQVTQDISDEPWGWYKSP-498 Arabidopsis_ALDH LSVKQVTLYTSNDPWGWYKSPN 501 *.:**** *::********

Fig. 4 Amino acid sequence alignment of Spinacia BADH protein (ACM67311.1) with Arabidopsis non-BAL form ALDH (10A8) enzyme. The critical amino acid (441A or 441C) for BADH BAL activity as shown by Muñoz-Clares et al. (2014) is underlined and painted in grey. Stars under sequences indicate shared amino acids, single and double dot indicates semi- and conservative amino acids, respectively, no sign indicates non-conservative amino acid

BADH gene overexpression in carrot chloroplasts resulted in the highest salt tolerance of up to 400 mM NaCl (Kumar et al. 2004). While enzyme activities and substrate specificities are fundamental for biosynthetic pathways, subcellular localization of the biosynthesis enzymes also plays a significant role. In GB accumulators, all functional biosynthesis enzymes are present in the correct subcellular localization, and the substrate choline is also available. Biosynthetic enzymes of GB are encoded in the plant genome while GB biosynthesis takes place in chloroplasts. The substrate choline is transported into the chloroplasts via nuclear pores. Successful transgenesis for enhanced GB production would thus require enhanced levels of both the transgene products and the substrate choline (Nuccio et al. 2000; McNeil et al. 2000). Gene expression levels and organ-specific expression patterns of biosynthetic enzymes can be regulated by promoter elements, but localization of the gene products is also affected by signaling peptides, called transit signals. Overexpression of biosynthetic enzyme-encoding genes can be accompanied by signal sequences to translocate the gene products into chloroplasts. The study of Nuccio et al. (2000) represents a rigorous effort to optimize the expression, localization, and posttranslational modifications of GB biosynthesis enzymes. 168 P. S. A. Mäkelä et al.

In the study, a 100-fold higher CMO activity was achieved in tobacco chloroplasts, yet the levels of GB remained at a low level. The availability of the substrate, choline, was shown to be the limiting factor. Plants engineered to express CMO in chloroplasts failed to produce GB even at high gene expression levels, while transgenic lines expressing CMO in the cytoplasm accumulated significantly more GB. It was shown that poor choline transport into chloroplasts caused the lack of GB accumulation in the chloroplast-targeted CMO line. These studies are a reminder of the importance of assessing all the components along the pathway. To further promote choline availability for GB biosynthesis, choline biosynthesis could also be enhanced through transgenesis. Recently, a newly identified factor, GB1, was shown to promote GB accumulation at high levels in different maize cultivars (Castiglioni et al. 2018). Overexpression of this fatty acid hydroxylase superfamily protein was speculated to be involved in choline biosynthesis and/or transported into chloroplasts. Future work will confirm GB1 function, but the availability of choline clearly represents a critical limiting factor for GB accumulation.

5 Conclusions and Future Perspectives

The amount of scientific literature related to GB is accumulating quickly, yet our knowledge of the mechanisms by which GB affects crop stress tolerance remain partly unknown. It is proposed that GB acts as a compatible solute in plants with two major roles. The first role of GB involves the regulation of osmotic balance via acting as a conventional osmolyte. The second one includes the maintenance of normal cell metabolism under stress conditions and thus acting on ROS scavenging, macromolecule protection, and carbon and N reserves. Some of the proposed effects of GB might be the result of alternative metabolic routes caused by imbalanced metabolic engineering. Integrated omics analysis combining transcriptomic, proteomic, and metabolomic studies on the transgenic lines could shed light on the complete picture of the GB accumulation profiles of the different transgenesis approaches. There are many limiting factors that seemingly influence GB accumulation in transgenic plants. Gene stacking as a transgenesis strategy could solve some of the bottlenecks in improving GB accumulation. The significant structure function discoveries in the GB biosynthesis isoenzymes are especially likely to drive the success of future GB transgenesis approaches in plants. It could also be considered whether marker-assisted selection could prove useful in the isoenzyme approach. In future, more attention should be paid to investigating the mechanisms by which GB affects plant growth and metabolism instead of simply testing new plant species. Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 169

References

Annunziata MG, Ciarmiello LF, Woodrow P, Dell’Aversana E, Carillo P (2019) Spatial and temporal profile of glycine betaine accumulation in plants under abiotic stresses. Front Plant Sci 10:230. https://doi.org/10.3389/fpls.2019.00230 Antoniou C, Savvides A, Christou A, Fotopoulos V (2016) Unravelling chemical priming machinery in plants: the role of reactive oxygen–nitrogen–sulfur species in abiotic stress tolerance enhancement. Curr Opin Plant Biol 33:101–107. https://doi.org/10.1016/j.pbi.2016.06.020 Ashraf M, Foolad MR (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot 59:206–216. https://doi.org/10.1016/j.envexpbot.2005.12.006 Bäurle I (2016) Plant heat adaptation: priming in response to heat stress. F1000Res 5:pii: F1000 Faculty Rev-694. https://doi.org/10.12688/f1000research.7526.1 Bot AJ, Nachtergaele FO, Young A (2000) Land resource potential and constraints at regional and country levels. World Soil Resources Reports 90. Land and Water Development Division, FAO, Rome Boyer JS (1982) Plant productivity and environment. Science 218:443–448. https://doi. org/10.1126/science.218.4571.443 Burg MB, Ferraris JD (2008) Intracellular organic osmolytes: function and regulation. J Biol Chem 283:7309–7313. https://doi.org/10.1074/jbc.R700042200 Carillo P, Mastrolonardo G, Nacca F, Parisi D, Verlotta A, Fuggi A (2008) Nitrogen metabolism in durum wheat under salinity: accumulation of proline and glycine betaine. Funct Plant Biol 35:412–426. https://doi.org/10.1071/FP08108 Carrillo-Campos J, Riveros-Rosas H, Rodríguez-Sotres R, Muñoz-Clares RA (2018) Bona fide choline monoxygenases evolved in Amaranthaceae plants from oxygenases of unknown function: evidence from phylogenetics, homology modeling and docking studies. PLoS One 13:e0204711. https://doi.org/10.1371/journal.pone.0204711 Castiglioni P, Bell E, Lund A, Rosenberg AF, Galligan M, Hinchey BS, Bauer S, Nelson D, Bensen RJ (2018) Identification of GB1, a gene whose constitutive overexpression increases glycinebetaine content in maize and soybean. Plant Direct 2:e00040. https://doi.org/10.1002/pld3.40 Chen THH, Murata N (2011) Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 34:1–20. https://doi. org/10.1111/j.1365-3040.2010.02232.x Cromwell BT, Rennie SD (1954) The biosynthesis and metabolism of betaines in plants. 2. The biosynthesis of glycinebetaine (betaine) in higher plants. Biochem J 58:318–322. https://doi. org/10.1042/bj0580318 Dudal R (1976) Inventory of major soils of the world with special reference to mineral stress. In: Wright M (ed) Plant adaptation of mineral stress in problems. Cornell University, Agriculture Experiment Station, Ithaca FAO (2019) AQUASTAT. http://www.fao.org/nr/water/aquastat/sets/index.stm. Accessed 8 Apr 2019 FAO, ITPS (2015) Status of the world’s soil resources: main report. FAO, Rome Fischer RA, Byerlee DR (1991) Trends of wheat production in the warmer areas: major issues and economic considerations. In: Saunders DA (ed) Wheat for nontraditional, warm areas. CIMMYT, Mexico, pp 3–27 Gage DA, Nolte KD, Russell BL, Rathinasabapathi B, Hanson AD, Nuccio ML (2003) The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J 16:487–496. https://doi.org/10.1046/j.1365-313x.1998.00316.x Giri J (2011) Glycinebetaine and abiotic stress tolerance in plants. Plant Signal Behav 6:1746– 1751. https://doi.org/10.4161/psb.6.11.17801 Grote EM, Ejeta G, Rhodes D (1994) Inheritance of glycinebetaine deficiency in sorghum. Crop Sci 34:1217–1220. https://doi.org/10.2135/cropsci1994.0011183X003400050013x Grumet R, Hanson AD (1986) Genetic evidence for an osmoregulatory function of glycinebetaine accumulation in barley. Aust J Plant Physiol 13:353–364. https://doi.org/10.1071/PP9860353 170 P. S. A. Mäkelä et al.

Hare PD, Cress WA, van Staden J (1998) Dissecting the roles of osmolyte accumulation during stress. Plant Cell Environ 21:535–553. https://doi.org/10.1046/j.1365-3040.1998.00309.x Hashemi FSG, Ismail MR, Rafii MY, Aslani F, Miah G, Muharam FM (2018) Critical multifunctional role of the betaine aldehyde dehydrogenase gene in plants. Biotechnol Biotechnol Equip 32:815–829. https://doi.org/10.1080/13102818.2018.1478748 Hattori T, Mitsuya S, Fujiwara T, Jagendorf AT, Takabe T (2009) Tissue specificity of glycinebetaine synthesis in barley. Plant Sci 176:112–118. https://doi.org/10.1016/j.plantsci.2008.10.003 Hayashi H, Alia, Mustardy L, Deshnium P, Ida M, Murata N (1997) Transformation of Arabidopsis thaliana with the codA gene for choline oxidase, accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J 12:133–142. https://doi. org/10.1046/j.1365-313X.1997.12010133.x Holmström K, Somersalo S, Mandal A, Palva TE, Welin B (2000) Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine. J Exp Bot 51:177–185. https://doi.org/10.1093/jexbot/51.343.177 Hossain MA, Li Z-G, Hoque TS, Burritt DJ, Fujita M, Munné-Bosch S (2018) Heat or cold priming-induced cross-tolerance to abiotic stresses in plants: key regulators and possible mechanisms. Protoplasma 255:399–412. https://doi.org/10.1007/s00709-017-1150-8 Huang J, Hirji R, Adam L, Rozwadowski KL, Hammerlindl JK, Keller WA, Selvaraj G (2000) Genetic engineering of gycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations. Plant Physiol 122:747–756. https://doi.org/10.1104/pp.122.3.747 IPCC (2017) Climate updates. What have we learnt since the IPCC 5th assessment report? The Royal Society, London Ishitani M, Arakawa K, Mizuno K, Kishitani S, Takabe T (1993) Betaine aldehyde dehydrogenase in the graminae: levels in leaves of both betaine-accumulating and non accumulating cereal plants. Plant Cell Physiol 34:493–495. https://doi.org/10.1093/oxfordjournals.pcp.a078445 Jin P, Zhang Y, Shan T, Huang Y, Xu J, Zheng Y (2015) Low-temperature conditioning alleviates chilling injury in loquat fruit and regulates glycine betaine content and energy status. J Agric Food Chem 63:3654–3365. https://doi.org/10.1021/acs.jafc.5b00605 Khan MS, Yu X, Kikuchi A, Asahina M, Watanabe KN (2009) Genetic engineering of glycine betaine biosynthesis to enhance abiotic stress tolerance in plants. Plant Biotechnol 26:125–134. https://doi.org/10.5511/plantbiotechnology.26.125 Khan MIR, Oqbal N, Masood A, Khan AN (2012) Variation in salt tolerance of wheat cultivars: role of glycinebetaine and ethylene. Pedosphere 22:746–754. https://doi.org/10.1016/ S1002-0160(12)60060-5 Khan MIR, Asgher M, Khan NA (2014) Alleviation of salt-induced photosynthesis and growth inhibition by salicylic acid involves glycinebetaine and ethylene in mungbean (Vigna radiata L.). Plant Physiol Biochem 80:67–74. https://doi.org/10.1016/j.plaphy.2014.03.026 Kishitani S, Watanabe K, Yasuda S, Arakawa K, Takabe T (1994) Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants. Plant Cell Environ 17:89–95. https://doi.org/10.1111/j.1365-3040.1994.tb00269.x Kumar S, Dhingra A, Daniell H (2004) Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance. Plant Physiol 136:2843– 2854. https://doi.org/10.1104/pp.104.045187 Kurepin LV, Ivanov AG, Zaman M, Pharis RP, Allakhverdiev SI, Hurry V, Huner NPA (2015) Stress- related hormones and glycinebetaine interplay in protection of photosynthesis under abiotic stress conditions. Photosynth Res 126:221–235. https://doi.org/10.1007/s11120-015-0125-x Leigh RA, Ahmad N, Wyn Jones RG (1981) Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles. Planta 153:34–41. https://doi.org/10.1007/ BF00385315 Li ZG, Gong M (2011) Mechanical stimulation-induced cross-adaptation in plants: an overview. J Plant Biol 54:358–364. https://doi.org/10.1007/s12374-011-9178-3 Li D, Zhang T, Wang M, Liu Y, Brestic M, Chen T, Yang X (2019) Genetic engineering of the biosynthesis of glycine betaine modulates phosphate homeostasis by regulating phosphate acquisition in tomato. Front Plant Sci 9:1995. https://doi.org/10.3389/fpls.2018.01995 Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 171

Lv S, Yang A, Zhang K, Wang L, Zhang J (2007) Increase of glycinebetaine improves drought tolerance in cotton. Mol Breed 20:233–248. https://doi.org/10.1007/s11032-007-9086-x Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Bsutkar P, Tivey ARN, Potter SC, Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acid Res 47(W1):W636–W641. https://doi.org/10.1093/nar/gkz268 Mäkelä P, Peltonen-Sainio P, Jokinen K, Pehu E, Setälä H, Hinkkanen R, Somersalo S (1996) Uptake and translocation of foliar-applied glycinebetaine in crop plants. Plant Sci 121:221– 230. https://doi.org/10.1016/S0168-9452(96)04527-X Mauch-Mani B, Baccelli I, Luna E, Flors V (2017) Defense priming: an adaptive part of induced resistance. Annu Rev Plant Biol 68:485–512. https://doi.org/10.1146/ annurev-arplant-042916-041132 McNeil SD, Rhodes D, Russell BL, Nuccio ML, Shachar-Hill Y, Hanson AD (2000) Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco. Plant Physiol 124:153–162. https://doi.org/10.1104/pp.124.1.153 Mehrabi Z, Ramankutty N (2017) The cost of heat waves and droughts for global crop production. https://doi.org/10.1101/188151. Accessed 8 Apr 2019 Misra N, Gupta AK (2005) Effect of salt stress on proline metabolism in two high yielding genotypes of green gram. Plant Sci 169:531–539. https://doi.org/10.1016/j.plantsci.2005.02.013 Munns R (2010) Strategies for crop improvement in saline soils. In: Asraf M, Ozturk M, Athar HR (eds) Salinity and water stress. Improving crop efficiency. Tasks for Vegetation Science, vol 44. Springer, Dordrecht, pp 99–110. https://doi.org/10.1007/978-1-4020-9065-3_11 Muñoz-Clares RA, Riveros-Rosas H, Garza-Ramos G, González-Segura L, Mújica-Jiménez C, Julián-Sánchez A (2014) Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine. BMC Plant Biol 14:149. https://doi.org/10.1186/1471-2229-14-149 Nuccio ML, Russell BL, Moite KD, Rathinasabapathi B, Gage DA, Hanson AD (1998) The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J 16:487–496. https://doi.org/10.1046/j.1365-313x.1998.00316.x Nuccio ML, McNeil SD, Ziemak MJ, Hanson AD, Jain RK, Selvaraj G (2000) Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway. Metab Eng 2:300–311. https://doi.org/10.1006/ mben.2000.0158 Paleg LG, Aspinall D (1981) The physiology and biochemistry of drought resistance in plants. Academic Press, Sydney Peel GJ, Mickelbart MV, Rhodes D (2010) Choline metabolism in glycinebetaine accumulating and non-accumulating near-isogenic lines of Zea mays and Sorghum bicolor. Phytochemistry 71:404–414. https://doi.org/10.1016/j.phytochem.2009.11.002 Rady MOA, Semida WM, El-Mageed TAA, Hemida KA, Rady MM (2018) Up-regulation of antioxidative defense systems by glycine betaine foliar application in onion plants confer tolerance to salinity stress. Sci Hortic 240:614–622. https://doi.org/10.1016/j.scienta.2018.06.069 Razavi F, Mahmoudi R, Rabiei V, Soleimani Aghdam M, Soleimani A (2018) Glycine betaine treatment attenuates chilling injury and maintains nutritional quality of hawthorn fruit during storage at low temperature. Sci Hortic 233:188–194. https://doi.org/10.1016/j.scienta.2018.01.053 Robinson SP, Jones GP (1986) Accumulation of glycinebetaine in chloroplasts provides osmotic adjustment during salt stress. Aust J Plant Physiol 13:659–668. https://doi.org/10.1071/ PP9860659 Rosenzweig C, Parry ML (1994) Potential impact of climate change on world food supply. Nature 367:133–138. https://doi.org/10.1038/367133a0 Sakai A, Larcher W (1987) Frost survival of plants. Springer, New York. https://doi. org/10.1007/978-3-642-71745-1 Sakamoto A, Murata N (2001) The use of bacterial choline oxidase, a glycinebetaine-synthesizing enzyme, to create stress-resistant transgenic plants. Plant Physiol 125:180–188. https://doi. org/10.1104/pp.125.1.180 172 P. S. A. Mäkelä et al.

Saneoka H, Nagasaka C, Hahn DT, Yang WJ, Premachandra GS, Joly RJ, Rhodes D (1995) Salt tolerance of glycinebetaine-deficient and -containing maize lines. Plant Physiol 107:631–638. https://doi.org/10.1104/pp.107.2.631 Shirasawa K, Takabe T, Takabe T, Kishitani S (2006) Accumulation of Glycinebetaine in Rice Plants that Overexpress Choline Monooxygenase from Spinach and Evaluation of their Tolerance to Abiotic Stress. Annals of Botany 98(3):565–571 Sies H (2017) Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biology 11:613–619. https://doi.org/10.1016/j. redox.2016.12.035 Singh M, Kumar J, Singh S, Singh VP, Prasad SM (2015) Roles of osmoprotectants in improving salinity and drought tolerance in plants: a review. Rev Environ Sci Biotechnol 4:407–426. https://doi.org/10.1007/s11157-015-9372-8 Stadmiller SS, Gorensek-Benitez AH, Guseman AJ, Pielak GJ (2017) Osmotic shock induced protein destabilization in living cells and its reversal by glycine betaine. J Mol Biol 429:1155– 1161. https://doi.org/10.1016/j.jmb.2017.03.001 Subbarao GV, Wheeler RM, Levine LH, Stutte GW (2001) Glycine betaine accumulation, ionic and water relations of red-beet at contrasting levels of sodium supply. J Plant Physiol 158:767– 776. https://doi.org/10.1078/0176-1617-00309 Trossat C, Rathinasabapathi B, Hanson AD (1997) Transgenically expressed betaine aldehyde dehydrogenase efficiently catalyses ozidation of dimethylsulfoniopropionaldehyde and omega- aminoaldehydes. Plant Physiol 113:1457–1461. https://doi.org/10.1104/pp.113.4.1457 Verma V, Ravindran P, Kumar PP (2016) Plant hormone-mediated regulation of stress responses. BMC Plant Biol 16:86. https://doi.org/10.1186/s12870-016-0771-y Walter J, Jentsch A, Beierkuhnlein C, Kreyling J (2013) Ecological stress memory and cross stress tolerance in plants in the fact of climate extremes. Environ Exp Bot 94:3–8. https://doi. org/10.1016/j.envexpbot.2012.02.009 Wang GP, Zhang XY, Li F, Luo Y, Wang W (2010) Overaccumulation of glycine betaine enhances tolerance to drought and heat stress in wheat leaves in the protection of photosynthesis. Photosynthetica 48:117–126. https://doi.org/10.1007/s11099-010-0016-5 Wang L, Shan T, Xie B, Ling C, Shao S, Jin P, Zheng Y (2019) Glycine betaine reduces chilling injury in peach fruit by enhancing phenolic and sugar metabolisms. Food Chem 272:530–538. https://doi.org/10.1016/j.foodchem.2018.08.085 Wei DD, Zhang W, Wang CC, Meng QW, Li G, Chen THH, Yang XH (2017) Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants. Plant Sci 257:74–83. https://doi.org/10.1016/j. plantsci.2017.01.012 Xu Z, Sun M, Jiang X, Sun H, Dang X, Cong H, Qiao F (2018) Glycinebetaine biosynthesis in response to osmotic stress depends on jasmonate signaling in watermelon suspension cells. Front Plant Sci 9:1469. https://doi.org/10.3389/fpls.2018.01469 Yancey PH (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotec- tants in high osmolarity and other stresses. J Exp Biol 208:2819–2830. https://doi.org/10.1242/ jeb.01730 Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217:1214–1222. https://doi.org/10.1126/science.7112124 Yang G, Rhodes D, Joly RJ (1996) Effects of high temperature on membrane stability and chlorophyll fluorescence in glycinebetaine-deficient and glycinebetaine-containing maize lines. Aust J Plant Physiol 23:437–443. https://doi.org/10.1071/PP9960437 Yao W, Xu T, Farooq SU, Jin P, Zheng Y (2018) Glycine betaine treatment alleviates chilling injury in zucchini fruit (Cucurbita pepo L.) by modulating antioxidant enzymes and membrane fatty acid metabolism. Postharvest Biol Technol 144:20–28. https://doi.org/10.1016/j. postharvbio.2018.05.007 Zhu J-K (2002) S D S S T P. Annual Review of Plant Biology 53(1):247–273 Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses 173

Zhu J-K (2016) Abiotic stress signalling and responses in plants. Cell 167:313–324. https://doi. org/10.1016/j.cell.2016.08.029 Zorrilla-López U, Masip G, Arjó G, Bai C, Banakar R, Bassie L, Berman J, Farre G, Miralpeiz B, Perez-Massot E, Sabalza M, Sanahuja G, Vamvaka E, Twyman R, Christou P, Zhu C, Capell T (2013) Engineering metabolic pathways in plants by multigene transformation. Int J Dev Biol 57:565–576. https://doi.org/10.1387/ijdb.130162pc Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth, Development, and (A)biotic Stress Tolerance

Le Cong Huyen Bao Tran Phan and Patrick Van Dijck

1 Trehalose Biosynthesis and Degradation

Trehalose is a nonreducing disaccharide of two glucose monomers which are linked in a 1,1-glycosidic bond. There are three possible anomers of trehalose (α,β-1,1-; β,β-1,1-; and α,α-1,1-), but only the latter has been isolated from living organisms so far. This sugar can be synthesized by a wide range of organisms such as bacteria, fungi, nematodes, arthropods, and plants (Elbein 2003) but not by vertebrates. In 1913, trehalose was for the first time reported in plants (Selaginella lepidophylla) (Anselmino ad Gilg 1913). Interestingly, trehalose levels are negligible in most higher plants, except for some resurrection plant species, for example, S. lepidophylla, where its high trehalose content was correlated with high stress tolerance (Iturriaga 2000). More recently, the origin of this trehalose was questioned as S. lepidophylla hosts many endophytes, which could be the real source for the high trehalose levels (Pampurova and Van Dijck 2014; Pampurova et al. 2014). Due to the presence of trace amounts of trehalose in the majority of angiosperms, it was suggested that trehalose had an unimportant role in plants. However, as described below, it is now clear that trehalose and the intermediates in its biosynthesis have an important role in plants.

L. C. H. B. T. Phan VIB-KU Leuven Center for Microbiology, Heverlee, Belgium Laboratory of Molecular Cell Biology, KU Leuven, Leuven, Belgium Department of Biology, College of Natural Sciences, Can Tho University, Can Tho, Vietnam P. Van Dijck (*) VIB-KU Leuven Center for Microbiology, Heverlee, Belgium Laboratory of Molecular Cell Biology, KU Leuven, Leuven, Belgium e-mail: [email protected]

© Springer Nature Switzerland AG 2019 175 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_8 176 L. C. H. B. T. Phan and P. Van Dijck

UDP-glc TPS TPS/TPP + T6P G6P TPP TreS Maltose TreS

TRE TreY Malto- TreZ Maltooligosaccharides/ 2Glc TreY/TreZ oligosyltrehalose Trehalose Starch TreT ADP-glc TreT + Glc TreP

Glc TreP + G1P

Fig. 1 The trehalose biosynthesis pathways. Abbreviations: Uridine diphosphate glucose (UDP- glc), glucose-6-phosphate (G6P), trehalose-6-phosphate (T6P), adenosine diphosphate glucose (ADP-glc), glucose (glc), glucose-1-phosphate (G1P), trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), trehalose synthase (TreS), maltooligosyl trehalose synthase (TreY), maltooligosyl trehalose trehalohydrolase (TreZ), trehalose glycosyl transferring synthase (TreT), trehalose phosphorylase (TreP), trehalase (TRE). (Figure based on Fernandez et al. (2010))

There are at least five trehalose biosynthesis pathways in nature (Fig. 1) (Avonce 2006). The most common pathway (the TPS-TPP pathway) is found in a wide range of organisms from eubacteria, archaea, fungi, insects, and plants. In this pathway, trehalose-6-phosphate synthase (TPS) (OtsA in Escherichia coli) catalyzes the transfer of glucose from UDP-glucose to glucose-6-phosphate (G6P), to produce the intermediate trehalose-6-phosphate (T6P). Trehalose-6-phosphate phosphatase (TPP) (OtsB in E. coli) dephosphorylates T6P to trehalose (Elbein 2003). The second pathway (the TreZ-TreY pathway) is distributed in thermophilic archaea of the genus Sulfolobus. Trehalose biosynthesis in this pathway involves the conversion of maltooligosaccharides or starch to trehalose under the catalysis of maltooligosyl trehalose synthase (TreY) and maltooligosyl trehalose trehalohydrolase (TreZ) subsequently (Maruta 1996). The trehalose synthase (TreS) isomerizes the α1-α4 linkage of maltose into the α1-α1 linkage of trehalose. The TreS pathway was first reported in Pimelobacter sp. (Elbein 2003). In fungi such as Agaricus bisporus and protista such as Euglena gracilis, trehalose phosphorylase (TreP) catalyses the reversible synthesis of trehalose from glucose-1-phosphate and glucose (Wannet 1998; Avonce 2006). The TreT pathway involves trehalose glycosyl-transferring synthase (TreT) which catalyzes the conversion of ADP-glucose and glucose into trehalose. This pathway occurs in extremophiles such as Thermococcus litoralis and Thermotoga maritima (Qu 2004). Some species, such as the genus Mycobacterium, use multiple distinct pathways (the TPS-TPP, TreY-TreZ, and TreS pathways) to Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 177 synthesize trehalose. This allows the accumulation of trehalose without substrate depletion in response to abiotic stresses (De Smet 2000). Trehalose is hydrolyzed into two glucose units by trehalase (TRE). This has been observed in many organisms from bacteria, fungi, plants, and animals, including mammals that do not produce trehalose (Elbein 2003). Some species contain various isoforms of trehalase. For example, there are three trehalase genes in Saccharomyces cerevisiae. Two cytosolic trehalases are encoded by the neutral trehalase genes Nth1 and Nth2, respectively, while an acid trehalase (Ath1) is functional in the extracellular space and vacuoles (Parrou 2005). In E. coli, a periplasmic trehalase is encoded by TreA, and TreF encodes a cytosolic trehalase (Schluepmann 2003). However, there is only one single gene (AtTRE1) that encodes a functional trehalase in Arabidopsis thaliana as the Attre1-1 knockout mutant showed no detectable trehalase activity.

2 Gene Families Involved in Trehalose Metabolism and Functional Characterization of Those Genes in Plants

In A. thaliana, based on genome sequencing, it has been revealed that there are 11 genes encoding TPS proteins and 10 genes encoding TPP proteins and only a single trehalase-encoding gene. The TPS and TPP genes were also detected in other plant genome sequences, including monocotyledonous and eudicotyledonous angiosperms, suggesting that trehalose synthesis occurs widely in the plant kingdom (Lunn 2014).

2.1 Plant TPS Proteins

The A. thaliana TPS proteins are encoded by 11 genes (AtTPS1–AtTPS11) which are divided into class I and class II proteins. The class I proteins (AtTPS1–AtTPS4) with fused TPS and TPP domains are more complex than ScTPS1 (Fig. 2). It has been shown that donor (UDP-glc) and acceptor (G6P) binding sites are highly conserved in the TPS domain when doing alignment of AtTPS1, AtTPS2, and AtTPS4 with OtsA sequence. AtTPS3 is likely a pseudogene as it contains a premature translational stop codon (Vandesteene 2010). Among the A. thaliana class I proteins, only AtTPS1 exhibits an N-terminal extension. Truncation of this N-terminal region in AtTPS1 led to a sharp increase in plant TPS activity upon expression of the different alleles in yeast. This result suggests that the plant-specific N-terminal extension might function as an autoinhibitory domain which mediates TPS activity (Van Dijck 2002). This domain is present throughout the plant kingdom, from single-celled green organisms (e.g., Ostreococcus tauri), over moss plants (Physcomitrella patens), to higher plants, which indicates that it may have an important role in the regulation of trehalose metabolism, as it was con- 178 L. C. H. B. T. Phan and P. Van Dijck

OtsA E. coli OtsB

ScTPS1 S. cerevisiae ScTPS2

AtTPS1 Class I AtTPS2-4 A. thaliana

AtTPS5-11 Class II

AtTPPA-J Class III

Fig. 2 Domain structure of bacterial, yeast, and plant trehalose metabolism proteins. E. coli and S. cerevisiae have active TPS and TPP proteins. In plants, all TPS enzymes contain TPS- and TPP- like domains. An extra N-terminal domain (green) is present in AtTPS1. The class III proteins lack the TPS-like domain, and all TPP enzymes are active phosphatases. Three conserved HAD motifs in the TPP domain are indicated by black boxes. Different shades of colors imply different levels of similarity in sequences. (Figure based on Vandesteene et al. (2010)) served during plant evolution (Van Dijck 2002; Avonce 2010; Lunn 2007). However, the real function in planta needs to be further investigated (Lunn 2014). In contrast to AtTPS1, the other two class I proteins AtTPS2 and AtTPS4 lack the N-terminal extension region. Previously, AtTPS2 and AtTPS4 were considered as catalytically inactive proteins as they were unable to complement the growth defect of the yeast tps1Δ mutant on glucose (Vandesteene 2010). Nonetheless, it has been found recently that both AtTPS2 and AtTPS4 can rescue the growth defect on glucose when expressed in yeast tps1Δtps2Δ mutant. Moreover, expression of these genes in the double yeast mutant also resulted in the accumulation of high levels of T6P when grown on glucose (Delorge 2015). These results have shown that AtTPS2 and AtTPS4 are also active enzymes. Interestingly, AtTPS2 and AtTPS4 produced much more T6P when compared to AtTPS1, when expressed in this double yeast mutant. In addition, expression of AtTPS2 and AtTPS4 also resulted in the production of trehalose in this strain background. The reason for the higher amount of T6P and the production of trehalose has most probably something to do with the yeast Tps2. To synthesize trehalose, a trehalose biosynthesis complex, consisting of ScTps1, ScTps2, and two regulatory subunits encoded by either ScTSL1 or ScTPS3, is required (Trevisol 2014). It is not unlikely that the plant TPS proteins participate in the enzyme complex formation in yeast cells and that the plant TPS proteins occupy the space of ScTps1 and ScTps2, resulting in an optimized situation to produce T6P and trehalose, which may not be the case for ScTps2 is still present (in the Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 179

Sctps1∆ mutant). In addition, AtTPS2 and AtTPS4 genes are specifically expressed in developing seeds and siliques, which suggests that they might serve particular functions in reproductive tissues (Schmid 2005). None of class I proteins can complement the heat stress-sensitive phenotype of the yeast tps2Δ mutant (Vandesteene 2010). This result obtained was as expected since the TPP-like domain is very poorly conserved. Nevertheless, as mentioned above, expression of AtTPS2 and AtTPS4 does result in the biosynthesis of trehalose in the double yeast mutant, which indicates that when ScTps1 is present, the TPS complex formation may not be optimal. In contrast to A. thaliana which contains four class I TPS proteins, most plants only harbor one or two class I genes. For example, class I family in rice has only one gene, and in poplar, two genes were identified PtTPS1( and PtTPS2) (Lunn 2007). In Poaceae, sorghum harbors a single class I gene, whereas maize contains two genes. Also, the moss P. patens contains two active class I proteins (Delorge et al., unpublished). The A. thaliana class II proteins (AtTPS5-AtTPS11) (Fig. 2) are mostly similar to ScTPS2, especially in the C-terminal TPP-like domain (Leyman 2001). As compared to the class I proteins, the TPS catalytic sites in the class II proteins are less well conserved (Lunn 2007). No TPS or TPP activity could be detected in class II proteins upon their expression in S. cerevisiae. As the T6P binding sites are very well conserved, but no TPP activity can be observed, one hypothesis that can be stipulated is that these class II proteins are sensors for T6P. This may be supported by the fact that these 7 genes have a conditional and tissue-specific expression. Their expression is regulated by hormones, light, and nutrient supply (Ramon 2009; Vandesteene 2010). The expression of class II TPSs might be regulated by the nutritional status of the plants/tissues as, for instance, expression of AtTPS8-10 is induced by carbon deprivation and suppressed by sugars, whereas the opposite has been shown for AtTPS5 (Osuna 2007; Schluepmann 2004). In addition, several of the encoded proteins are phosphorylated by SnRK1 and CDPK, allowing them to be bound by 14-3-3 proteins (Glinski and Weckwerth 2005; Harthill 2006). These findings further suggest that the class II proteins may play important regulatory functions rather than a metabolic control mechanism in Arabidopsis (Ramon 2009). Recently, several studies have been done to support this opinion. For example, the silencing of PvTPS9, which is the major transcript of the class II TPS genes (PvTPS4-PvTPS10) in root nodules of common bean (Phaseolus vulgaris), reduced the expression of PvTPS9 by approximately 85% in the PvTPS9-RNAi transgenic nodules (Barraza 2016). Moreover, other class II genes such as PvTPS4, PvTPS6, PvTPS7, and PvTPS10 were also downregulated in the transgenic root nodules. The reduction in expression of these transcripts was correlated with a significant decrease in trehalose levels in plant biomass. It was suggested that the accumulation of trehalose in the root nodules most probably originates from the symbiont, rather than from the host plant (Müller 2001b; Suárez 2008; López 2009; Vauclare 2010). Hence, the silencing of PvTPS9, which leads to variations in the expression of some other class II TPS genes, might interfere with the biosynthesis of trehalose in rhizobia, resulting in the decrease in trehalose levels in the PvTPS9-RNAi transgenic root nodules. As data have shown above, it indicates that PvTPS9 plays an important role 180 L. C. H. B. T. Phan and P. Van Dijck in regulation of trehalose metabolism in root nodules and, consequently, in the whole plant (Barraza 2016). Another evidence of regulatory functions of the class II TPS proteins was presented in a study of Zang et al. (2011). There are ten class II TPS proteins (OsTPS2-11) in rice, and none of them exhibit the TPS or TPP activity when expressed in the yeast tps1∆ or tps2∆ mutants, respectively. By yeast two- hybrid analysis and bimolecular fluorescence complementation assay, strongly direct interactions of OsTPS1 and OsTPS5/OsTPS8 were detected. Furthermore, two possible isoforms of OsTPS1 (OsTPS1a and OsTPS1b) were identified by gel filtration assay. In addition, it was observed that either OsTPS1a or OsTPS1b and OsTPS8 are incorporated into the TPS complex(es). These results suggest that the class II TPS proteins, particularly OsTPS8 in rice, might regulate the activity of OsTPS1 by forming the TPS complex(es), in order to modulate the concentration of intracellular T6P to control plant growth and development.

2.2 Plant TPP Proteins

The Arabidopsis TPP proteins consist of ten isoforms (AtTPPA–AtTPPJ) with three well-conserved HAD (L-2-haloacid dehalogenase) phosphatase motifs (Fig. 2) (Vandesteene 2012). Similar to A. thaliana, the rice and poplar genomes contain 10 TPP genes. Some other species have less TPP isoforms, for example, P. patens has two, S. moellendorffii has three, and O. taurus has only one TPP protein. These proteins are likely derived from the endosymbiotic ancestor of mitochondria because TPP homologues exist in proteobacteria such as Rhodoferax ferrireducens (Lunn 2007; Avonce 2010). In addition, collinearity analysis indicated that all the TPP proteins of Arabidopsis originate from whole-genome duplication (Vandesteene 2012). Beside the conserved HAD domain, the plant TPP isoforms also contain a highly variable N-terminal domain with uncharacterized function. This might be involved in subcellular protein localization (Lunn 2007). During heat stress, T6P is accumulated in the yeast tps2Δ mutant, resulting in a thermosensitive growth phenotype. When complemented by each of the ten A. thaliana TPP proteins, growth deficiency of the yeast tps2Δ mutant at 38.6 °C was rescued, which indicates that all ten proteins have TPP activity (Vandesteene 2012). Similar results were observed for the TPP homologues from rice (Oryza sativa), maize (Zea mays), and grapevine (Vitis vinifera) (Pramanik and Imai 2005; Satoh-Nagasawa 2006; Fernandez 2012). Moreover, expression of the TPP genes is tightly controlled in cell- and tissue-specific manner, which suggests that TPP proteins strictly regulate T6P levels in plants (Vandesteene 2012).

2.3 Trehalase

The plant TPS and TPP proteins are encoded by multiple genes, while only one single gene encodes for trehalase (TRE) enzyme in most plants. Interestingly, a high degree of natural variations was identified in the catalytic domain ofA. thaliana Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 181

TRE1 when doing SNP analysis in 81 sequenced accessions of Arabidopsis (Schluepmann 2012). There are a few exceptions in which Populus trichocarpa and P. patens display three and four trehalase genes, respectively (Lunn 2007; Van Houtte et al., our unpublished results). AtTRE1 expression is abundant in flowers and siliques, whereas much less expression was observed in roots and shoots (Müller 2001a; van Dijken 2004). AtTRE1 activity might be important for the development of flowers, siliques, and seeds as addition of validamycin A, a trehalose analog and competitive inhibitor of trehalase, caused defects in fruiting and a poor seed set in Arabidopsis plants (Müller 2001a). AtTRE1 can fulfill the function of ScAth1 in yeast ath1Δ cells as AtTRE1 complemented the growth defect of the yeast ath1Δ mutant on the trehalose-containing growth medium, adjusted at pH 4.8. This indicates that A. thaliana trehalase has an extracellular activity. There are five putative N-glycosylation sites in the AtTRE1 sequence, indicating that trehalase might be secreted. It has been shown that AtTRE1 is a plasma membrane-bound protein with its catalytic domain facing the apoplast (Frison 2007). AtTRE1 anchors to the plasma membrane through a putative N-terminally transmembrane domain (residues 46–63). When plants were infected by Plasmodiophora brassicae, trehalase activity was increased even before pathogen-produced trehalose increased in the host plant. This indicates that trehalase is unlikely to be induced/activated by trehalose (Brodmann 2002). These findings suggest that trehalase might be a sensor to detect extracellular pathogen-secreted trehalose and thereby prevents an accumulation of trehalose which could interfere with metabolic signaling inside the cells (Schluepmann 2004; Brodmann 2002; Gravot 2011). However, the question of how the extracellular AtTRE1 regulates intracellular trehalose levels is still unclear. It is also possible that trehalase is a sensor for stress and that it may be involved in closing stomata to prevent pathogens from entering the plant. This hypothesis is supported by the fact that trehalase is specifically expressed in guard cells and that more trehalase expression results in fast closing of stomata (Van Houtte 2013).

3 Role of Trehalose-6-Phosphate/Trehalose in Plant Growth and Development

Trehalose acts as a stress protectant which stabilizes proteins and lipid membranes upon various abiotic stresses such as dehydration, cold, heat, and oxidative stress (Elbein 2003). This disaccharide is stable at high temperature (up to 100 °C) and in a broad pH range for 24 h (Richards 2002). Anhydrobiotic organisms such as yeast cells, fungal spores, resurrection plants, nematodes, rotifers, and the cysts of brine shrimp generally have high amounts of trehalose. The survival ability of these creatures is strongly correlated with the synthesis of trehalose in the absence of water. However, in most plants, except in certain resurrection plants (e.g., Selaginella lepidophylla), trehalose is hardly detectable. Therefore, trehalose was not considered as an osmoprotectant in plants (Schluepmann 2004). 182 L. C. H. B. T. Phan and P. Van Dijck

Interestingly, trehalose accumulation is most likely toxic for plant growth by interfering with cell wall biosynthesis (O’Hara 2013; Veluthambi 1982a). In Cuscuta reflexa, inhibition of trehalase by validamycin A or trehalose supplementation was associated with a decrease in sucrose and starch contents, leading to growth inhibition (Veluthambi 1982b). A similar pattern was detected in soybean plantlets and Arabidopsis when plants were treated with validamycin A and/or addition of trehalose (Müller 1995, 2001a). Nonetheless, the growth defect caused by trehalose feeding was not observed in plant species with high trehalase activity (Veluthambi 1981). Trehalose-6-phosphate (T6P), a metabolic precursor of trehalose, has emerged as a signaling metabolite, regulating plant metabolism, growth, and development (Schluepmann 2004; Lunn 2006). Schluepmann et al. (2004) figured out that trehalose-mediated growth inhibition is due to a rapid accumulation of T6P in seedlings of Arabidopsis. When levels of trehalose are high, it might lead to a reduction of T6P dephosphorylation, resulting in an increase in T6P contents. Remarkably, the growth inhibition of T6P was rescued when metabolizable sugars were added to the trehalose containing growth medium. The first reports where the effects of T6P/trehalose on plant development were described were released in 1997. Transgenic tobacco plants expressing a heterologous TPS gene with accumulated trehalose showed lancet-shaped leaves and stunted growth phenotypes (Romero 1997; Goddijn 1997). In addition, tobacco plants that expressed E. coli TPP with lower T6P levels displayed an increase in leaf size. Another example is that the cell shape phenotype-1 (csp-1) mutant displayed a dramatic cellular effect in the leaf epidermis, which resulted in an altered pavement cell morphology, arrested development, as well as changes in plant architecture such as reduced trichome branching, altered stem branching, and increased stomatal density (Chary 2008). Mapping of the csp-1 locus revealed that the mutated gene in the csp- 1 mutant encodes AtTPS6. As expected, AtTPS6 complemented the defects in morphology (e.g., pavement cells, trichomes) and overall growth phenotypes of the csp-1 mutant. These results indicate that the trehalose metabolism plays a role in plant development. Although T6P is a causative metabolite for the growth defect of plants, when the addition of exogenous trehalose (100 mM) led to an accumulation of T6P levels, causing the growth inhibition of Arabidopsis seedlings (Schluepmann 2004), T6P is also critical for normal growth, with a main defect in root growth (Schluepmann 2003). In A. thaliana, the null tps1 mutant is embryo lethal. The mutant failed to germinate. Addition of trehalose cannot rescue the growth deficiency oftps1 mutants (Eastmond 2002), but this defect can be rescued by the heterologous expression of E. coli TPS (Schluepmann 2003). These results suggest that T6P plays an important role in embryo development. Besides that, T6P affects the vegetative growth of plants. For instance, TILLING mutants with weak TPS1 alleles displayed delayed vegetative growth phenotypes. In addition, Arabidopsistps1 loss-of- function alleles exhibited a delay in flowering as compared to the wild type (Gómez 2010). Furthermore, Arabidopsis seedlings expressing heterologous TPP or TPH from E. coli contain low levels of T6P, leading to a failure in the use of supplied sugars. Transgenic plants with reduced T6P levels experienced growth inhibition Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 183

Catabolism Anabolism (e.g.respiration) Sucrose (e.g.photosynthesis)

UDPG TPS TPP + T6P Trehalose Glc6P

SnRK1

Fig. 3 The interaction network of T6P, sucrose, and SnRK1. An increase in T6P level represents an accumulation in sucrose level. This potentially triggers reprogramming in the cell to reduce the levels of sucrose to the optimal concentration range, by activating catabolism processes (e.g., sucrose consumption, respiration, etc.) and/or inhibiting sucrose synthesis via anabolism. When sucrose levels go back to the desired range, T6P also decreases. SnRK1, a key metabolic sensor which maintains energy homeostasis for cell growth and survival, is inhibited by T6P. Dashed lines with arrows and bars show activation and inhibition with unknown molecular mechanism, respectively. Lines with arrows indicate metabolite conversions. (Figure based on Lunn et al. (2014)) when sugars were applied, while plants with enhanced T6P levels demonstrated the opposite effect (Schluepmann 2003). From these experiments it is clear that adequate T6P levels are required for carbon utilization during normal growth. When T6P levels are not in balance with sugar availability, growth is hampered. Addition of sucrose to the medium can rescue trehalose-dependent growth inhibition, which indicates that T6P improves plant growth when carbon supply is high (Schluepmann 2004). Surprisingly, T6P is not only related to regulating the use of sucrose, but the amounts of T6P are also elevated dramatically when sucrose is supplied (Lunn 2006). The intracellular T6P levels are inversely proportional to the amounts of hexose phosphates and UDP-glucose (UDPG), which are downstream products of sucrose breakdown (Pellny 2004; Schluepmann 2003). Hence, when sucrose is added, a constitutive expression of TPS1 happens, leading to the rapid accumulation of T6P in order to response to a rise in the pool size of G6P and UDPG (Paul 2008). Therefore, T6P amounts might reflect the levels of hexose phosphates, UDPG, and sucrose. The sucrose-T6P interaction network is discussed in Fig. 3. Another example of metabolic regulation by the trehalose pathway is an increase of starch synthesis upon trehalose feeding (Wingler 2000). It was found that T6P activates AGP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis (Kolbe 2005). A strong accumulation of starch by trehalose feeding is not only due to an increase in starch synthesis but also comes from an inhibition of starch breakdown through suppressing the expression of SEX1 and β-amylase (Ramon 2007). These observations support the idea that the trehalose pathway 184 L. C. H. B. T. Phan and P. Van Dijck affects the plant growth via regulation of the flux from central metabolism metabolites to starch. Sucrose non-fermenting-related kinase-1 (SnRK1) is a key kinase involved in plant starvation signaling (Baena-González 2007). During low sugar conditions, KIN10 (a catalytic α-subunit of SnRK1) induces genes involved in catabolism, while sugar feeding triggers an elevation in the expression of genes that are related to biosynthetic processes (Baena-González and Sheen 2008). Interestingly, downregulation of genes involved in photosynthesis and incatabolic processes was observed in Arabidopsis seedlings with accumulated T6P levels. In contrast, genes associated with biosynthetic processes, which are normally downregulated by SnRK1, were upregulated by T6P. These results indicate that T6P inhibits the catalytic activity of SnRK1 (Zhang 2009) (Fig. 3). Moreover, it was revealed that SnRK1-overexpressing seedlings with low T6P levels show a glucose-hypersensitive phenotype in rice (Oryza sativa) and A. thaliana (Cho 2012). These results are consistent with T6P inhibition of SnRK1 and the sugar hypersensitivity of seedlings with low T6P (Schluepmann 2003). However, the inhibition of SnRK1 by T6P may only occur in young, growing tissues such as seedlings, but not in mature leaves. T6P did not show any inhibitory effect on SnRK1 activity when the catalytic α-subunits of SnRK1 (KIN10 and KIN11) purified by anion-exchange chromatography were assayed. Nonetheless, when the supernatant of a seedling crude extract, from which KIN10 and KIN11 had been removed, was added back to the purified KIN10 and KIN11, the inhibition of SnRK1 by T6P was restored (Zhang 2009). This suggests that (an) unknown factor(s) that is (are) present in seedlings is (are) necessary for inhibition of SnRK1 by T6P. The factors could be (a) protein(s) because they show a heat-labile property as boiling destroys the activity. In senescing leaves of A. thaliana, there is a strong accumulation of T6P, in combination with an increase in sugar levels (Wingler 2012). Moreover, when sugar levels are high, the anthocyanin biosynthetic pathway is stimulated. Mature otsB- expressing plants with decreased T6P exhibited a reduction of anthocyanin synthesis and delayed senescence phenotype (Wingler 2012). Furthermore, expression of senescence-associated genes is lower in otsB-expressing plants. These observations support the fact that T6P plays a role in senescence. On the other hand, altered T6P metabolism in otsB-expressing plants led to less sensitivity to sugar supply. For instance, in wild-type and otsA-expressing plants, senescence was promoted when metabolizable sugars such as glucose, fructose, or sucrose are supplied, but this effect was delayed in transgenic plants with lower T6P. T6P also controls flowering time and inflorescence architecture. Deletion of AtTPS1 prevents floral transition (van Dijken2004 ), but overexpression of TPS1 postponed the time of flowering (Avonce 2004). The latter phenotype was also detected in KIN10-overexpressing plants (Baena-González 2007), indicating that the effect of T6P on flowering time might be mediated by SnRK1. Tobacco plants (Nicotiana tabacum) expressing otsA produced more axillary shoots (Goddijn 1997), which suggests that T6P might control meristem activity. The role of T6P on meristem development was confirmed in maize. A mutation in Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 185 the RA3 gene, encoding a functional TPP protein, led to changes in flowering archi- tectures such as improved branching of the male and the female inflorescences (Satoh-Nagasawa 2006). It is likely that most proteins involved in the trehalose biosynthetic pathway might have evolved to function as signaling sensors rather than catalytic enzymes since a point mutation in AtTPS6, a protein with inactive TPS activity, increased the branching of inflorescences in A. thaliana (Chary 2008). Likewise, a direct interaction between class II TPS proteins (inactive OsTPS5 and OsTPS8) and the active class I TPS enzyme (OsTPS1) in rice could mediate endogenous T6P concentration in a feedback loop (Zang 2011). Therefore, proteins with no catalytic activity in the trehalose biosynthetic pathway may sense the intracellular T6P levels in response to sugar availability to control meristem development. Regulation of plant growth and development by T6P/trehalose is a noncontrover- sial fact. Thus, researches on trehalose metabolism in crop plants need to be studied more to unravel the fundamental molecular mechanisms involved in trehalose signaling, providing opportunities for improving crop yield.

4 Role of Trehalose-6-Phosphate/Trehalose in Abiotic Stress Tolerance

During the whole life cycle, plants face multiple stresses which cause severe effects on their growth and development. Among the two major types of stresses, abiotic and biotic stresses, abiotic stresses including cold, heat, wind, drought, oxidation, radiation, etc. can change physiological processes, leading to disruption of important signaling pathways and finally complete tissue damage (Hirt and Shinozaki 2004). Recently, trehalose and its precursor T6P have been shown that they are involved in plant stress responses (Fernandez 2010). In order to understand how trehalose metabolism may react to abiotic stresses, several studies were performed under various stress conditions.

4.1 Salt Stress

Salinity stress brings a big challenge to agriculture, and trehalose alters many processes that may improve plant survival upon salt stress. For instance, an increase in trehalose levels was observed in the roots of rice and Medicago truncatula during NaCl stress (Shima 2007; López 2008a, b; Garcia 1997). Moreover, treatment of trehalose prevented chlorophyll loss in leaf blades and facilitated aerenchyma formation in roots of rice seedlings grown in a medium containing 1% NaCl (Garcia 1997). Although exogenous trehalose feeding did not prevent plants from excess NaCl uptake, it reduced Na+ ion accumulation in the leaf blade of NaCl-stressed 186 L. C. H. B. T. Phan and P. Van Dijck rice. The protective effect of trehalose on plants could be explained by protection of ion pumps which prevent an uptake of sodium to chloroplasts (Garcia 1997) since trehalose stabilize proteins and lipid bilayer integrity during stress conditions (Elbein 2003). This may allow plants to continue growing without loss of chlorophyll (Garcia 1997). In agreement with the above results, trehalase is downregulated at the transcription level in root nodules of Medicago truncatula under NaCl stress, allowing trehalose accumulation (López 2008b). Similarly, trehalose levels also increased in a range of wheat cultivars during NaCl stress, potentially due to increased TPS activity and decreased trehalase activity (El-Bashiti 2005). Likewise, there was a transient induction of OsTPP1 expression in roots and shoots when rice plants were treated with NaCl (Pramanik and Imai 2005). One should take into consideration the fact that although trehalose levels increased during salt stress, the levels were still too low to function as an osmoprotectant. Thus, it is likely that there is no direct protective effect of trehalose against salt stress. Maize metabolomic analyses revealed an increase in T6P levels in the leaves, kernels, and cobs at the silking stage when 75 mM NaCl was treated. Moreover, an accumulation of sucrose was also observed in the leaf, kernel, and cob tissue at silking, pollination, and 3 days after pollination under salt stress (Henry 2015). Together with these, the levels of many primary metabolites of sugar metabolism, glycolysis, and Krebs cycle were also affected upon NaCl stress. For instance, intermediates of sucrose synthesis, soluble sugars, and starch increased, whereas most of the other metabolites decreased. However, it still remains unclear whether a change in the T6P/sucrose ratios resulting from an increase in T6P at the very early developmental stage (the silking stage) represents a primary response to salt stress, and after- ward cellular metabolism is shaped in salt-stressed plants.

4.2 Drought Stress

Trehalose is massively present in some desiccation-tolerant resurrection plants such as S. lepidophylla, Myrothamnus flabellifolius, and Sporobolus spp. These plants can persist in metabolic stasis for several years until they are rewatered (Iturriaga 2000, 2006). In addition, sucrose is also present in very high levels in the resurrection plants. It was believed that trehalose and sucrose work together as osmoprotectants to stabilize membranes, proteins, and other cellular components during stasis (Drennan 1993). Nonetheless, it was reported that S. moellendorffii, a drought stress-sensitive relative, contains even higher trehalose levels than S. lepidophylla (Pampurova and Van Dijck 2014; Pampurova et al. 2014), which suggests that high trehalose levels possibly contribute to but are not sufficient for desiccation tolerance of S. lepidophylla. More recently, it was discovered that high levels of trehalose in S. lepidophylla could be originating from endophytes, which are endosymbionts living in this host (Pampurova and Van Dijck 2014; Pampurova et al. 2014). Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 187

Interestingly, in S. lepidophylla polyols, such as sorbitol and xylitol, are present much more abundantly in comparison with S. moellendorffii. Hence, the question of whether sorbitol or xylitol is linked to drought stress tolerance of S. lepidophylla needs to be addressed. In contrast to those desiccation-tolerant resurrection plants, trehalose levels are limited in crop plants. In drought-tolerant wheat and cotton varieties, dehydration induces a small rise in trehalose levels. This could be due to an induction of TPS expression or a reduction of trehalase activity (El-Bashiti 2005; Kosmas 2006). However, the question of how small changes in trehalose content potentially lead to protective effects remains to be established. It has been stated that water shortage can change seed chemical composition and reduce seed quality (Ali and Ashraf 2011). Obviously, maintenance of seed quality is an important aspect in agriculture for many crops. Seed oil extracted from maize is considered as one of the best oils in the world since it contains high levels of unsaturated fatty acids, such as oleic and linolenic acid, and antioxidants as flavonoids and phenolics. Nonetheless, dehydration reduces the yield of seed oil and the levels of unsaturated fatty acids (Ali and Ashraf 2011). Noticeably, negative impacts of drought stress on maize seeds were rescued when spraying a trehalose solution on the leaves. For instance, contents of oleic and linolenic acid were improved, and the oil antioxidant activity was increased when exogenous trehalose was applied (Ali 2012).

4.3 Temperature Stress

Cold or heat stress triggers many changes in physiological and biochemical processes in plants. Those changes include up- or downregulation of many genes and proteins, alteration in metabolite contents, and modification of membrane components and conformation (Sanghera 2011). A. thaliana metabolomic analyses revealed that levels of trehalose and other compatible solutes are elevated upon both cold or heat stresses. It suggests that trehalose may act in combination with other solutes to create synergistic effects during thermotolerant responses (Kaplan 2004). Nevertheless, temperature stress also induces sucrose; thus it is important to clarify if thermal stress directly drives changes in trehalose levels or trehalose induction is just a secondary response caused by changes in sucrose levels. Another evidence for a role of trehalose metabolism in high temperature stress was provided. A yeast two-hybrid screening demonstrated that AtTPS5 interacts with MBF1c (multiprotein bridging factor 1c), a key regulator of thermotolerance in A. thaliana. AtTPS5 is also heat-inducible, and the tps5 null mutants were hypersensitive to high temperature (Suzuki 2008). On the other hand, trehalose metabolism also plays a role in low temperature tolerance. Cold stress stimulated expression 188 L. C. H. B. T. Phan and P. Van Dijck of OsTPP1 and OsTPP2 genes in rice (Shima 2007; Pramanik and Imai 2005). Similarly, V. vinifera TPPA levels were induced upon chilling stress in grapevine (Fernandez 2012). Iordachescu and Imai (2008) demonstrated that an increase in trehalose and T6P levels results from an induction of AtTPPA during cold stress. Remarkably, T6P levels were positively correlated with sucrose contents in all plant tissues, which suggests that low temperature-induced sucrose accumulation might mediate an increase of T6P levels (Fernandez 2012).

4.4 Hypoxia

Hypoxia and anoxia which are caused by flooding are significant problems for many plants. Transcription profiling revealed that genes encoding TPP proteins (e.g., AtTPPA, AtTPPB, and AtTPPJ) and trehalase protein were upregulated, whereas expression of AtTPS11 gene was decreased in A. thaliana in response to low-oxygen stress (Liu 2005). Similarly, upregulation of TPP genes upon hypoxia stress was also observed in poplar (Populus x canescens) (Christianson 2010). The changes in expression of trehalose metabolism genes might lead to a reduction in T6P levels; consequently, it possibly lessens its inhibition on glycolysis and assists the sugar influx into anaerobic respiration (Liu2005 ). In agreement with this, a decrease in T6P contents was detected in wild-type A. thaliana plants under hypoxic stress (Thiel 2011).

4.5 Oxidative Stress

An accumulation of ROS (reactive oxygen species) resulting from abiotic and biotic stresses has both positive and negative impacts for the plants. On one hand, ROS act as signaling molecules which regulate different processes such as pathogen defense and programmed cell death (Grant and Loake 2000; Dangl and Jones 2001). On the other hand, when ROS mount up to toxic levels, they have to be scavenged to prevent oxidative damage. To deal with this kind of stress, plants develop multiple defense strategies, including accumulation of sugars. Studies both in vitro and in vivo provided evidences that trehalose protects against hydroxyl radicals (Roitsch 1999; Couee 2006). Tolerance to methyl viologen-induced oxidative stress was increased in transgenic tobacco and tomato plants expressing the yeast TPS1 gene (Romero 2002; Cortina and Culiáñez-Macià 2005). In addition, trehalose also scavenges free radicals generated by heat stress in wheat (Luo 2008). Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 189

5 Role of Trehalose-6-Phosphate/Trehalose in Biotic Stress Tolerance

5.1 Trehalose and Plant-Microorganism Symbiosis

Trehalose production is a common feature of many microbes, including microbial pathogens and beneficial microbes. Hence, the plant needs to discriminate various potential sources of extracellular trehalose. Under symbiotic and beneficial interactions, defense responses in plants have to be inhibited (Lunn 2014). Symbiotic plant-microorganism interactions promote growth and productivity in both the host and microorganism, as well as enhances plant tolerance to stress (Fernandez 2010). Trehalose accumulates in root nodules when nodules of several legumes form symbioses with rhizobia (Salminen and Streeter 1986; Müller 1992; Farias-Rodriguez 1998; Domínguez-Ferreras 2009; Brechenmacher 2010). Addition of exogenous trehalose to the growth medium increased sucrose synthase and alkaline invertase activities in soybean, potentially enhancing the source of hexose sugars to the symbionts (Müller 1998). Inoculation of common bean (Phaseolus vulgaris) with the symbiotic bacterium Rhizobium etli, which was engineered to overexpress the E. coli trehalose-6-phosphate synthase (otsA), resulted in more trehalose production and formation of more nodules with higher nitrogenase activity. The result was improved plant yield in comparison with plants infected with a wild-type strain (Suárez 2008). In agreement with this, deletion of the endogenous otsA gene in R. etli reduced the number of nodules, nitrogenase activity, and plant biomass. In addition, trehalose also plays an important role in mycorrhizal fungi-plant relationships. Trehalose represents a main carbon source in Amanita muscaria and Pisolithus microcarpus associated with poplar (Populus tremula x tremuloides) and Eucalyptus globulus roots, respectively (López 2007; Martin 1998). Upon formation of the ectomycorrhizal symbiosis, expression of trehalose metabolism-related genes in A. muscaria was induced (López 2008a, b), while an increase in carbon distribution to trehalose was seen in the mycelium when E. globulus interacts with P. tinctorius (Martin 1998).

5.2 Trehalose: An Elicitor of the Plant Defense Responses

Several studies have presented that trehalose might act as an elicitor of the plant defense responses. Transcriptional profiling revealed that exogenous trehalose feeding induced plant defense-related genes such as WRKY6 and β-1,3-glucanase genes, encoding a defense-related transcription factor and a PR (pathogenesis-related) protein, respectively (Schluepmann 2004). Moreover, trehalose treatment also enhanced partial resistance against wheat powdery mildew caused by the pathogen Blumeria graminis (Renard-Merlier 2007). Surprisingly, trehalose feeding changes neither 190 L. C. H. B. T. Phan and P. Van Dijck pathogen membrane lipid composition (Muchembled 2006) nor conidia germination (Renard-Merlier 2007). These findings support the elicitor hypothesis since trehalose does not seem to affect the pathogen in a direct way. Nonetheless, the concentration of trehalose used in those experiments was 15 g l−1, which is much higher than the physiological levels. Therefore the question of whether defenses were stimulated following osmotic stress or by true elicitation remains to be addressed.

5.3 Trehalose: A Signal of Pathogen Attack

Extracellular trehalose is a significant sign of danger to the plant, and this trehalose could be originating from fungi, bacteria (Lunn 2014), insects (Singh 2011), or nematodes (Hofmann 2010). For example, aphid honeydew contains high levels of trehalose which shows a potential signal of aphid attack. An increase in trehalose levels was seen in A. thaliana leaves, resulting from infestation of the peach potato aphid, Myzus persicae. Interestingly, high levels of trehalose were also detected in the phloem sap of plants attacked by aphids, indicating that aphids induce the plant to produce high amounts of trehalose. This trehalose is likely to move through the plant and into the aphids via the phloem sap. However, answers to the questions of whether the accumulation of trehalose in A. thaliana is modulated by the aphids in order to produce a carbohydrate storage substance favorable to themselves or whether trehalose acts as a signaling compound inducing resistance mechanisms in plants are required for further clarification (Hodge 2013). A second example to show the role of trehalose during virulence was obtained in the plant pathogenic fungus Magnaporthe oryzae. Deletion of TPS1 in M. oryzae resulted in low trehalose levels, and this resulted in a reduction of its pathogenicity (Wilson 2007). Similarly, a mutant strain of Pseudomonas aeruginosa which cannot synthesize trehalose is unable to infect the host plant A. thaliana, but this strain still has full capacity to infect non-host plants (Djonovic et al. 2013). These studies demonstrate that trehalose is a virulence factor of those pathogens in order to infect plants. An accumulation of trehalose in infected organs of A. thaliana was observed when plants were infected by P. brassicae, the clubroot pathogen. During the interaction of P. brassicae with A. thaliana, it was shown that the PbTPS1 gene is also upregulated, indicating that the accumulated trehalose was produced by the pathogen. The infection results in a defense response in the plants as AtTRE1 was induced in roots and hypocotyls probably to limit the accumulation of trehalose, which might have negative effects on the plant’s metabolism. Thus, AtTRE1 potentially acts as a sensor of extracellular, pathogen-produced trehalose and also as a defense against excessive accumulation of trehalose (Gravot 2011). In summary, microorganism or insect-derived trehalose represents a virulence or signal molecule during the interactions between plants and those organisms. These interactions interfere significantly with the plant’s own trehalose metabolism. Yet, the mechanisms and physiological changes upon these responses are still unknown. Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 191

Unraveling the precise mechanisms that take place during these interactions may offer potential ways to improve the resistance of crop plants against microbial pathogens and insect pests while also promoting beneficial interactions with bacterial and fungal symbionts.

6 Application of Trehalose-6-Phosphate/Trehalose Modifications in Plants

Due to the well-known stress-protecting properties of trehalose in microorganisms and some resurrection plants, trehalose metabolism has emerged as a valuable pathway to target in order to improve abiotic stress tolerance in crop plants. In early studies, tobacco (N. tabacum) and potato (Solanum tuberosum) were engineered by introducing bacterial or fungal genes encoding trehalose-synthesizing enzymes in order to improve the drought stress tolerance (Romero 1997; Goddijn and van Dun 1999). The heterologous TPS/TPP-expressing transgenic plants showed enhanced stress tolerance; however, unexpectedly abnormal phenotypes are exhibited such as stunted growth, lancet-shaped leaves, early flowering, and delayed senescence (Goddijn 1997; Pellny 2004; Goddijn and van Dun 1999; Schluepmann 2003; Paul 2008). The morphological defects were obviously due to changes in the level of T6P resulting from TPS versus TPP overexpression as simultaneous overexpression of both enzymes did not result in abnormal phenotypes (Schluepmann 2003; Salazar 2009; Garg 2002). Enzymes showing both enzymatic activities have been described for Cytophaga hutchinsonii, and such enzymes may be of interest to engineer plants (Avonce 2010). Over the past decades, several approaches in improving stress tolerance in plants without morphological defects have been attempted, apart from the abovementioned expression of both enzymes. An alternative for this approach would be to use enzymes that have both a synthesis and phosphatase activity, and such an enzyme has been described in the bacterium C. hutchinsonii (Avonce 2010). As far as we know, transgenic plants that express such a bifunctional enzyme have not been described yet. Another possibility is the use of conditional expression of the yeast TPS1 gene. This was achieved in potato and tobacco, where transgenic lines that expressed the yeast ScTPS1 gene under the control of a drought-inducible promoter were more tolerant to drought stress. Interestingly, these transgenic plants did not show major phenotypic growth defects (Stiller 2008; Kondrák 2012; Karim 2007). Interestingly, Arabidopsis plants engineered to overexpress ScTPS1 showed growth aberrations, whereas the plants engineered by introducing the AtRbcS1A promoter together with a chloroplast transit peptide in front of the coding sequence of ScTPS1 enhanced drought tolerance without undesired side effects (Karim 2007). This finding indicates that the cytosolic accumulation of T6P disturbs sugar signaling, generating the negative effects on plant growth and development. Another example is overexpression of the endogenous AtTPS1 in A. thaliana which also resulted in enhanced drought stress tolerance without visible effects on the plant’s morphology, 192 L. C. H. B. T. Phan and P. Van Dijck except for delay in flowering (Avonce2004 ). A similar study was performed in rice as overexpression of OsTPS1 increased the tolerance to drought, cold, and salinity stresses in this case without any severe morphological alterations (Li 2011). In addition, sugarcane (Saccharum officinarum L.) engineered to overexpress a Grifola frondosa trehalose synthase (TSase) gene exhibited an improved tolerance to drought stress. The transgenic sugarcane lines had no obvious aberrations in morphology and growth (Zhang 2006). The results obtained suggest that engineering by introducing trehalose biosynthesis genes to enhance abiotic stress tolerance in monocots seems to have less disturbance in morphology and growth when compared to dicots. However, more studies in monocots and dicots need to be investigated to support this statement. Although overexpression of endogenous trehalose-metabolizing genes improved abiotic stress tolerance, only a small increase in trehalose levels was observed (Avonce 2004; Li 2011). This could be due to the presence of the N-terminal autoinhibitory domain of the plant TPS proteins (Van Dijck 2002) as transgenic Arabidopsis lines overexpressing the N-terminal truncated version of AtTPS1 resulted in similar abnormalities as the expression of microbial TPS genes, and such alleles show much higher TPS activity (Van Dijck 2002). The N-terminal domain clearly results in lower enzymatic activity of the TPS enzymes. In addition, the enzymatic activity upon overexpressing of endogenous TPS genes might be controlled by endogenous regulatory mechanisms, hence potentially reducing the metabolic disturbance. This also could be a possible explanation for the minor phenotypic changes in plants engineered to constitutively express endogenous genes when compared to the serious abnormal phenotypes in plants overexpressing heterologous TPS proteins. An alternative approach to improve drought stress tolerance is suppressing the endogenous trehalase (AtTRE1) in A. thaliana (Van Houtte 2013). However, both the tre1-1 knockout mutant and tre1-2 knockdown mutant with higher concentrations of trehalose were surprisingly more sensitive to drought stress than wild-type plants. In line with this, AtTRE1 overexpressing lines such as tre1-3OE and 35S::AtTRE1 with reduced trehalose levels showed improved drought tolerance. Overexpression of AtTRE1 not only affects trehalose levels but also lowers T6P levels. These results indicate that drought tolerance observed in plants overexpressing TPS or TPP was not due to the small increase in trehalose levels but the results of a regulatory mechanism. This leads to further doubt on the role of trehalose as an osmoprotectant in plants, except for resurrection species. Even in some resurrection plants, the high levels of trehalose may come from endophytes (Pampurova and Van Dijck 2014; Pampurova et al. 2014). One of the regulatory mechanisms of trehalose metabolism on drought stress tolerance may be its effect on stomatal conductance and water-use efficiency. AtTPPG and AtTRE1 are strongly expressed in guard cells of A. thaliana leaves (Vandesteene 2012; Van Houtte 2013). Likewise, the AtTPS1 enzyme has hardly been detected in proteomic analyses of different types of cells (Tanz 2013), with the exception of guard cells, where it can be measured (Zhao 2008). This suggests that the AtTPS1 enzyme is probably present at higher contents in guard cells as compared to other cell types or tissues in A. thaliana. Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 193

Abscisic acid (ABA) is an important plant hormone that regulates stomatal closure during drought stress in order to minimize water loss from the leaves. The Attppg, Attre1-1, and Attre1-2 mutants were resistant to ABA as their stomata failed to close when exogenous ABA was added (Vandesteene 2012; Van Houtte 2013). These results indicate that AtTPPG and AtTRE1 are important for ABA-regulated stomatal closure. There is no doubt on the importance of trehalose metabolism in controlling ABA-mediated stomatal conductance. Nonetheless, the underlying signaling mechanisms remain unclear. Therefore, studies that give more insights about the link between ABA signaling and trehalose metabolism or sugar signaling, especially in guard cells, need to be further investigated.

7 Conclusions and Future Perspectives

There is no doubt that trehalose metabolism plays an important role in plant growth, development, metabolism, and stress tolerance. The exact mechanisms by which trehalose metabolism is affecting all these characteristics are still unclear. Also the regulation of trehalose metabolism at the cell, tissue, and development level remains to be determined at the molecular level. It is clear that there is a tight regulation between biosynthesis and hydrolysis of T6P at the cellular level as imbalance in the level of T6P results in aberrant phenotypes. The same is probably true for the level of trehalose itself where a tight balance between biosynthesis and hydrolysis of this metabolite is important. Currently, how trehalase, of which the catalytic domain is proposed to be apoplastic, can affect intracellular trehalose levels remains to be investigated. One approach to improve our understanding of the role of trehalose metabolism enzymes on plant growth or stress tolerance is an investigation of A. thaliana natural variations that affect trehalose biosynthesis and degradation. In addition, an extensive –omics approach at the cellular/tissue level would be a good approach for elucidating a holistic view of trehalose metabolism in plants during developmental stages and under stress conditions. Results obtained from such analysis could provide us with useful information for future engineering of trehalose metabolism in order to improve yield and tolerance against multiple stresses in crop plants.

References

Ali Q, Ashraf M (2011) Exogenously applied glycinebetaine enhances seed and seed oil quality of maize (Zea mays L.) under water deficit conditions. Environ Exp Bot 71:249–259 Ali Q, Ashraf M, Anwar F, Al-Qurainy F (2012) Trehalose-induced changes in seed oil composition and antioxidant potential of maize grown under drought stress. J Am Oil Chem Soc 89:1485–1493 Anselmino O, Gilg E (1913) Trehalose in Selaginella. Bericth der Deutschen pharmazeutischen Gesellschaft 23:326–327 Avonce N, Leyman B, Mascorro-Gallardo JO, Van Dijck P, Thevelein JM, Iturriaga G (2004) The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid and stress signaling. Plant Physiol 136:3649–3659 194 L. C. H. B. T. Phan and P. Van Dijck

Avonce N, Mendoza-Vargas A, Morett E, Iturriaga G (2006) Insights on the evolution of trehalose biosynthesis. BMC Evol Biol 6:109 Avonce N, Wuyts J, Verschooten K, Vandesteene L, Van Dijck P (2010) The Cytophaga hutchinsonii ChTPSP: first characterized Bifunctional TPS–TPP protein as putative ancestor of all eukaryotic Trehalose biosynthesis proteins. Mol Biol Evol 27(2):359–269 Baena-González E, Sheen J (2008) Convergent energy and stress signaling. Trends Plant Sci 13:474–482 Baena-González E, Rolland F, Thevelein JM, Sheen J (2007) A central integrator of transcription networks in plant stress and energy signaling. Nature 448:938–942 Barraza A, Contreras-Cubas C, Estrada-Navarrete G, Reyes JL, Juárez-Verdayes MA, Avonce N, Quinto C, Díaz-Camino C, Sanchez F (2016) The class II Trehalose 6-phosphate synthase gene PvTPS9 modulates Trehalose metabolism in Phaseolus vulgaris nodules. Front Plant Sci 7:1589 Brechenmacher L, Lei Z, Libault M, Findley S, Sugawara M, Sadowsky MJ, Lloyd W, Sumner LW, Stacey G (2010) Soybean metabolites regulated in root hairs in response to the symbiotic bacterium Bradyrhizobium japonicum. Plant Physiol 153:1808–1822 Brodmann D, Schuller A, Muller JL, Aeschbacher RA, Wiemken A, Boller T, Wingler A (2002) Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae. Mol Plant-Microbe Interact 15(7):693–700 Chary SN, Hicks GR, Choi YG, Carter D, Raikhel NV (2008) Trehalose-6-phosphate synthase/phosphatase regulates cell shape and plant architecture in Arabidopsis. Plant Physiol 146:97–107 Cho YH, Hong JW, Kim EC, Yoo SD (2012) Regulatory functions of SnRK1 in stress-responsive gene expression and in plant growth and development. Plant Physiol 158:1955–1964 Christianson JA, Llewellyn DJ, Dennis ES, Wilson IW (2010) Comparisons of early transcriptome responses to low-oxygen environments in three dicotyledonous plant species. Plant Signal Behav 5(8):1006–1009 Cortina C, Culiáñez-Macià FA (2005) Tomato abiotic stress enhanced tolerance by trehalose biosynthesis. Plant Sci 169:75–82 Couee I, Sulmon C, Gouesbet G, El-Amrani A (2006) Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. J Exp Bot 57:449–459 Dangl JL, Jones JDG (2001) Plant pathogens and integrated defence responses to infection. Nature 411:826–833 De Smet KAL, Weston A, Brown IN, Young DB, Robertson BD (2000) Three pathways for trehalose biosynthesis in mycobacteria. Microbiology 146:199–208 Delorge I, Figueroa CM, Feil R, Lunn JE, Van Dijck P (2015) Trehalose-6-phosphate synthase 1 is not the only active TPS in Arabidopsis thaliana. Biochem J 466(2):283–290 Djonović S, Urbach JM, Drenkard E et al (2013) Trehalose biosynthesis promotes Pseudomonas aeruginosa pathogenicity in plants. PLoS Pathog 9:e1003217 Domínguez-Ferreras A, Soto MJ, Pérez-Arnedo R, Olivares J, Sanjuán J (2009) Importance of trehalose biosynthesis for Sinorhizobium meliloti osmotolerance and nodulation of alfalfa roots. J Bacteriol 191:7490–7499 Drennan PM, Smith MT, Goldsworth D, Van Staden J (1993) The occurrence of trehalose in the leaves of the desiccation-tolerant angiosperm Myrothamnus flabellifolius Welw. J Plant Physiol 142:493–496 Eastmond PI, van Dijken AJ, Spielman M, Kerr A, Tissier AF, Dickinson HG, Jones JD, Smeekens SC, Graham IA (2002) Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation. Plant J 29:225–235 El-Bashiti T, Hamamci H, Oktem HA, Yucel M (2005) Biochemical analysis of trehalose and its metabolizing enzymes in wheat under abiotic stress conditions. Plant Sci 169:47–54 Elbein AD, Pan YT, Pastuszak I, Carroll D (2003) New insights on trehalose: a multifunctional molecule. Glycobiology 13(4):17–27 Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 195

Farias-Rodriguez R, Mellor RB, Arias C, Pena-Cabriales JJ (1998) The accumulation of trehalose in nodules of several cultivars of common bean (Phaseolus vulgaris) and its correlation with resistance to drought stress. Physiol Plant 102:353–359 Fernandez O, Béthencourt L, Quero A, Sangwan RS, Clément C (2010) Trehalose and plant stress responses: friend or foe? Trends Plant Sci 15:409–417 Fernandez O, Vandesteene L, Feil R, Baillieul F, Lunn JE, Clément C (2012) Trehalose metabolism is activated upon chilling in grapevine and might participate in Burkholderia phytofirmans induced chilling tolerance. Planta 236:355–369 Frison M, Parrou JL, Guillaumot D, Masquelier D, François J, Chaumont F, Batoko H (2007) The Arabidopsis thaliana trehalase is a plasma membrane-bound enzyme with extracellular activity. FEBS Lett 581:4010–4016 Garcia AB, Engler JD, Iyer S, Gerats T, Van Montagu M, Caplan AB (1997) Effects of osmoprotectants upon NaCl stress in rice. Plant Physiol 115:159–169 Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci U S A 99(25):15898–15903 Glinski M, Weckwerth W (2005) Differential multisite phosphorylation of the trehalose-6- phosphate synthase gene family in Arabidopsis thaliana - a mass spectrometry-based process for multiparallel peptide library phosphorylation analysis. Mol Cell Proteomics 4:1614–1625 Goddijn OJM, van Dun K (1999) Trehalose metabolism in plants. Trends Plant Sci 4(8):315–319 Goddijn OJ, Verwoerd TC, Voogd E, Krutwagen RWHH, de Graaf PTHM, Poels J, van Dun K, Ponstein AS, Damm B, Pen J (1997) Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants. Plant Physiol 113:181–190 Gómez LD, Gilday A, Feil R, Lunn JE, Graham IA (2010) AtTPS1-mediated trehalose 6-phosphate synthesis is essential for embryogenic and vegetative growth and responsiveness to ABA in germinating seeds and stomatal guard cells. Plant J 64:1–13 Grant JJ, Loake GJ (2000) Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol 124:21–29 Gravot A, Grillet L, Wagner G, Jubault M, Lariagon C, Baron C, De-leu C, Delourme R, Bouchereau A, Manzanares-Dauleux MJ (2011) Genetic and physiological analysis of the relationship between partial resistance to clubroot and tolerance to trehalose in Arabidopsis thaliana. New Phytol 191:1083–1094 Harthill JE, Meek SE, Morrice N, Peggie MW, Borch J, Wong BH, Mackintosh C (2006) Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose. Plant J 47:211–223 Henry C, Bledsoe SW, Griffiths CA, Kollman A, Paul MJ, Sakr S, Lagrimini LM (2015) Differential role for trehalose metabolism in salt-stressed maize. Plant Physiol 169:1072–1089 Hirt H, Shinozaki K (eds) (2004) Plant responses to abiotic stress. Springer, Berlin Hodge S, Ward JL, Beale MH, Bennett M, Mansfield JW, Powell G (2013) Aphid-induced accumulation of trehalose in Arabidopsis thaliana is systemic and dependent upon aphid density. Planta 237:1057–1064 Hofmann J, Ashry E, Ael N, Anwar S, Erban A, Kopka J, Grundler F (2010) Metabolic profiling reveals local and systemic responses of host plants to nematode parasitism. Plant J 62:1058–1071 Iordachescu M, Imai R (2008) Trehalose biosynthesis in response to abiotic stresses. Plant Biol 50:1223–1229 Iturriaga G, Gaff DF, Zentella R (2000) New desiccation-tolerant plants, including a grass, in the central high-lands of Mexico, accumulate trehalose. Aust J Bot 48:153–158 Iturriaga G, Cushman MAF, Cushman JC (2006) An EST catalogue from the resurrection plant Selaginella lepidophylla reveals abiotic stress-adaptive genes. Plant Sci 170:1173–1184 Kaplan F, Kopka J, Haskell DW, Zhao W, Schiller KC, Gatzke N, Sung DY, Guy CL (2004) Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiol 136:4159–4168 196 L. C. H. B. T. Phan and P. Van Dijck

Karim S, Aronsson H, Ericson H, Pirhonen M, Leyman B, Welin B, Mäntylä E, Palva ET, Van Dijck P, Holmström KO (2007) Improved drought tolerance without undesired side effects in transgenic plants producing trehalose. Plant Mol Biol 64:371–386 Kolbe A, Tiessen A, Schluepmann H, Paul M, Ulrich S, Geigenberger P (2005) Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase. Proc Natl Acad Sci U S A 102(31):11118–11123 Kondrák M, Marincs F, Antal F, Juhász Z, Bánfalvi Z (2012) Effects of yeast trehalose-6-phosphate synthase 1 on gene expression and carbohydrate contents of potato leaves under drought stress conditions. BMC Plant Biol 12:e74 Kosmas SA, Argyrokastritis A, Loukas MG, Eliopoulos E, Tsakas S, Kaltsikes PJ (2006) Isolation and characterization of drought-related trehalose 6-phosphate-synthase gene from cultivated cotton (Gossypium hirsutum L.). Planta 223:329–339 Leyman B, Van Dijck P, Thevelein JM (2001) An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana. Trends Plant Sci 6:510–513 Li HW, Zang BS, Deng XW, Wang XP (2011) Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta 234:1007–1018 Liu F, VanToai T, Moy LP, Bock G, Linford LD, Quackenbush J (2005) Global transcription profiling reveals comprehensive insights into hypoxic response inArabidopsis . Plant Physiol 137:1115–1129 López MF, Männer P, Willmann A, Hampp R, Nehls U (2007) Increased trehalose biosynthesis in Hartig net hyphae of ectomycorrhizas. New Phytol 174:389–398 López M, Herrera-Cervera JA, Iribarne C, Tejera NA, Lluch C (2008a) Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism. J Plant Physiol 165:641–650 López M, Tejera NA, Iribarne C, Lluch C, Herrera-Cervera JA (2008b) Trehalose and trehalase in root nodules of Medicago truncatula and Phaseolus vulgaris in response to salt stress. Physiol Plant 134:575–582 López M, Tejera NA, Lluch C (2009) Validamycin A improves the response of Medicago truncatula plants to salt stress by inducing trehalose accumulation in the root nodules. J Plant Physiol 166:1218–1222 Lunn JE, Feil R, Hendriks JHM, Gibon Y, Morcuende R, Osuna D, Scheible W-R, Carillo P, Hajirezaei M-R, Stitt M (2006) Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADP-glucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana. Biochem J 397:139–148 Lunn JE, Delorge I, Figueroa CM, Van Dijck P, Stitt M (2007) Gene families and evolution of trehalose metabolism in plants. Funct Plant Biol 34:550–563 Lunn JE, Delorge I, Figueroa CM, Van Dijck P, Stitt M (2014) Trehalose metabolisms in plants. Plant J 79:544–567 Luo Y, Li WM, Wang W (2008) Trehalose: protector of antioxidant enzymes or reactive oxygen species scavenger under heat stress? Environ Exp Bot 63:378–384 Martin F, Boiffin V, Pfeffer PE (1998) Carbohydrate and amino acid metabolism in the Eucalyptus globulus–Pisolithus tinctorius ectomycorrhiza during glucose utilization. Plant Physiol 118:627–635 Maruta K, Mitsuzumi H, Nakada T, Kubota M, Chaen H, Fukuda S, Sugimoto T, Kurimoto M (1996) Cloning and sequencing of a cluster of genes encoding novel enzymes of trehalose biosynthesis from thermophilic archaebacterium Sulfolobus acidocaldarius. Biochim Biophys Acta 1291:177–181 Muchembled J (2006) Changes in lipid composition of Blumeria graminis f. sp. tritici conidia produced on wheat leaves treated with heptanoyl salicylic acid. Phytochemistry 67:1104–1109 Müller J, Staehelin C, Mellor RB, Boller T, Wiemken A (1992) Partial purification and characterization of trehalase from soybean nodules. Plant Physiol 140:8–13 Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 197

Müller J, Boller T, Wiemken A (1995) Effects of validamycin A, a potent trehalase inhibitor, and phytohormones on trehalose metabolism in roots and root-nodules of soybean and cowpea. Planta 197:362–368 Müller J, Boller T, Wiemken A (1998) Trehalose affects sucrose synthase and invertase activities in soybean (Glycine max [L.] Merr.) roots. Plant Physiol 153:255–257 Müller J, Aeschbacher RA, Wingler A, Boller T, Wiemken A (2001a) Trehalose and trehalase in Arabidopsis. Plant Physiol 125:1086–1093 Müller J, Boller T, Wiemken A (2001b) Trehalose becomes the most abundant non-structural carbohydrate during senescence of soybean nodules. J Exp Bot 52:943–947 O’Hara LE, Paul MJ, Wingler A (2013) How do sugars regulate plant growth and development? New insight into the role of trehalose-6-phosphate. Mol Plant 6(2):261–274 Osuna D, Usadel B, Morcuende R, Gibon Y, Blasing OE (2007) Temporal responses of transcripts, enzyme activities and metabolites after adding sucrose to carbon-deprived Arabidopsis seedlings. Plant J 49:463–491 Pampurova S, Van Dijck P (2014) The desiccation tolerant secrets of Selaginella lepidophylla: what we have learned so far? Plant Physiol Biochem 80:285–290 Pampurova S, Verschooten K, Avonce N, Van Dijck P (2014) Functional screening of a cDNA library from the desiccation-tolerant plant Selaginella lepidophylla in yeast mutants identifies trehalose biosynthesis genes of plant and microbial origin. J Plant Res 127:803–813 Parrou JL, Jules M, Beltran G, François J (2005) Acid trehalase in yeasts and filamentous fungi: localization, regulation and physiological function. FEMS Yeast Res 5:503–511 Paul MJ, Primavesi LF, Jhurreea D, Zhang Y (2008) Trehalose metabolism and signaling. Annu Rev Plant Biol 59:417–441 Pellny TK, Ghannoum O, Conroy JP, Schluepmann H, Smeekens S, Andralojc J, Krause KP, Goddijn O, Paul MJ (2004) Genetic modification of photosynthesis with E. coli genes for trehalose synthesis. Plant Biotechnol J 2:71–82 Pramanik MHR, Imai R (2005) Functional identification of a trehalose 6-phosphate phosphatase gene that is involved in transient induction of trehalose biosynthesis during chilling stress in rice. Plant Mol Biol 58:751–762 Qu Q, Lee SJ, Boss W (2004) TreT, a novel trehalose glycosyltransferring synthase of the hyper- thermophilic archeon Thermococcus litoralis. J Biol Chem 279:47890–47897 Ramon M, Rolland F, Thevelein JM, Van Dijck P, Leyman B (2007) ABI4 mediates the effects of exogenous trehalose on Arabidopsis growth and starch breakdown. Plant Mol Biol 63:195–206 Ramon M, De Smet I, Vandesteene L, Naudts M, Leyman B, Van Dijck P, Rolland F, Beeckman T, Thevelein JM (2009) Extensive expression regulation and lack of heterologous enzymatic activity of the class II trehalose metabolism proteins from Arabidopsis thaliana. Plant Cell Environ 32:1015–1032 Renard-Merlier Dea (2007) Iodus 40, salicylic acid, heptanoyl salicylic acid and trehalose exhibit different efficacies and defence targets during a wheat/powdery mildew interaction. Phytochemistry 68:1156–1164 Richards A, Krakowka S, Dexter L, Schmid H, Wolterbeek A, Waalkens-Berendsen D, Shigoyuki A, Kurimoto M (2002) Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food Chem Toxicol 40(7):871–898 Roitsch T (1999) Source–sink regulation by sugar and stress. Curr Opin Plant Biol 2:198–206 Romero C, Bellés JM, Vayá JL, Serrano R, Culiáñez-Macià FA (1997) Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance. Planta 201:293–297 Romero C, Cruz Cutanda M, Cortina C, Primo J, Culiáñez-Macià FA (2002) Plant environmental stress response by trehalose biosynthesis. Curr Top Plant Biol 3:73–88 Salazar JR, Suárez R, Caballero-Mellado J, Iturriaga G (2009) Trehalose accumulation in Azospirillum brasilense improves drought tolerance and biomass in maize plants. FEMS Microbiol Lett 296:52–59 198 L. C. H. B. T. Phan and P. Van Dijck

Salminen SO, Streeter JG (1986) Enzymes of α, α-trehalose metabolism in soybean nodules. Plant Physiol 81:538–541 Sanghera GS, Wani SH, Hussain W, Singh NB (2011) Engineering cold stress tolerance in crop plants. Curr Genomics 12:30–43 Satoh-Nagasawa N, Nagasawa N, Malcomber S, Sakai H, Jackson D (2006) A trehalose metabolic enzyme controls inflorescence architecture in maize. Nature 441:227–230 Schluepmann H, Pellny T, Van Dijken A, Smeekens S, Paul M (2003) Trehalose-6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proc Natl Acad Sci U S A 100:6849–6854 Schluepmann H, van Dijken A, Aghdasi M, Wobbes B, Paul M, Smeekens S (2004) Trehalose mediated growth inhibition of Arabidopsis seedlings is due to Trehalose-6-phosphate accumulation. Plant Physiol 135:879–890 Schluepmann H, Berke L, Sanchez-Perez GF (2012) Metabolism control over growth: a case for trehalose-6-phosphate in plants. J Exp Bot 63:3379–3390 Schmid M, Davison TS, Henz SR, Pape UJ, Bemar M, Vingron M, Scholkopf B, Weigel D, Lohmann JU (2005) A gene expression map of Arabidopsis thaliana development. Nat Genet 37:501–506 Shima S, Matsui H, Tahara S, Imai R (2007) Biochemical characterization of rice trehalose- 6-phosphate phosphatases supports distinctive functions of these plant enzymes. FEBS J 274:1192–1201 Singh V, Louis J, Ayre BG, Reese JC, Shah J (2011) TREHALOSE PHOSPHATE SYNTHASE 11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae. Plant J 67:94–104 Stiller I, Dulai S, Kondrák M, Tarnai R, Szabo L, Toldi O, Bánfalvi Z (2008) Effects of drought on water content and photosynthetic parameters in potato plants expressing the trehalose-6- phosphate synthase gene of Saccharomyces cerevisiae. Planta 227:299–308 Suárez R, Wong A, Ramiréz M, Barraza A, Orozco MDC, Cevallos MA, Lara M, Hernandéz G, Iturriaga G (2008) Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia. Mol Plant-Microbe Interact 21:958–966 Suzuki N, Bajad S, Shuman J, Shulaev V, Mittler R (2008) The transcriptional co-activator MBF1c is a key regulator of thermotolerance in Arabidopsis thaliana. J Biol Chem 283:9269–9275 Tanz SK, Castleden I, Hooper CM, Vacher M, Small I, Millar AH (2013) SUBA3: a database for integrating experimentation and prediction to define the SUBcellular location of proteins in Arabidopsis. Nucleic Acids Res 41:1185–1191 Thiel J, Rolletschek H, Friedel S, Lunn JE, Nguyen TH, Feil R, Tschiersch H, Muller M, Borisjuk L (2011) Seed-specific elevation of non-symbiotic hemoglobinAtHb1 : beneficial effects and underlying molecular networks in Arabidopsis thaliana. BMC Plant Biol 11:48 Trevisol ETV, Panek AD, De Mesquita JF, Eleutherio ECA (2014) Regulation of the yeast trehalose-synthase complex by cyclic AMP-dependent phosphorylation. Biochim Biophys Acta 1840:1646–1650 Van Dijck P, Mascorro-Gallardo JO, De Bus M, Royackers K, Iturriagan G, Thevelein JM (2002) Truncation of Arabidopsis thaliana and Selaginella lepidophylla trehalose-6-phosphate synthase unlocks high catalytic activity and supports high trehalose levels on expression in yeast. Biochem J 366:63–71 van Dijken AJH, Schluepmann H, Smeekens SCM (2004) Arabidopsis trehalose-6-phosphate synthase 1 is essential for normal vegetative growthand transition to flowering. Plant Physiol 135:969–977 Van Houtte H, Vandesteene L, López-Galvis L, Lemmens L, Kissel E, Carpentier S et al (2013) Overexpression of the Trehalase gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in Abscisic acid-induced stomatal closure. Plant Physiol 161:1158–1171 Biosynthesis and Degradation of Trehalose and Its Potential to Control Plant Growth… 199

Vandesteene L, Ramon M, Le Roy K, Van Dijck P, Rolland F (2010) A single active trehalose-6-P synthase (TPS) and a family of putative regulatory TPS-like proteins in Arabidopsis. Mol Plant 3:406–419 Vandesteene L, López-Galvis L, Vanneste K, Feil R, Maere S, Lammens W, Rolland F, Lunn JE, Avonce N, Beeckman T, Van Dijck P (2012) Expansive evolution of the TREHALOSE-6- PHOSPHATE PHOSPHATASE gene family in Arabidopsis. Plant Physiol 160:884–896 Vauclare P, Bligny R, Gout E, De Meuron V, Widmer F (2010) Metabolic and structural rearrange- ment during dark-induced autophagy in soybean (Glycine max L.) nodules: an electron microscopy and 31P and 13C nuclear magnetic resonance study. Planta 231:1495–1504 Veluthambi K, Mahadevan S, Maheshwari R (1981) Trehalose toxicity in Cuscuta reflexa: correlation with low trehalase activity. Plant Physiol 68:1369–1374 Veluthambi K, Mahadevan S, Maheshwari R (1982a) Trehalose toxicity in Cuscuta reflexa: cell wall synthesis is inhibited upon trehalose feeding. Plant Physiol 70:686–688 Veluthambi K, Mahadevan S, Maheshwari R (1982b) Trehalose toxicity in Cuscuta reflexa: sucrose content decreases in shoot tips upon trehalose feeding. Plant Physiol 69:1247–1251 Wannet WJB, Op den Camp HJM, Wisselink HW, van der Drift C, Van Griensven LJLD, Vogels GD (1998) Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus. Biochim Biophys Acta 1425:177–188 Wilson RA, Jenkinson JM, Gibson RP, Littlechild JA, Wang ZY, Talbot NJ (2007) Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence. EMBO J 26:3673–3685 Wingler A, Fritzius T, Wiemken A, Boller T, Aeschbacher RA (2000) Trehalose induces the ADP- glucose pyrophosphorylase gene, ApL3, and starch synthesis in Arabidopsis. Plant Physiol 124:105–114 Wingler A, Delatte TL, O’Hara LE, Primavesi LF, Jhurreea D, Paul MJ, Schluepmann H (2012) Trehalose 6-phosphate is required for the onset of leaf senescence associated with high carbon availability. Plant Physiol 158:1241–1251 Zang B, Li H, Li W, Deng XW, Wang X (2011) Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice. Plant Mol Biol 76:507–522 Zhang SZ, Yang BP, Feng CL, Chen RK, Luo JP, Cai WW, Liu FH (2006) Expression of the Grifola frondosa Trehalose synthase gene and improvement of drought-tolerance in sugarcane (Saccharum officinarum L.). J Integr Plant Biol 48(4):453–459 Zhang Y, Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RAC, Powers SJ, Schluepmann H, Delatte T, Wingler A, Paul MJ (2009) Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiol 149:1860–1871 Zhao Z, Zhang W, Stanley BA, Assmann SA (2008) Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways. Plant Cell 20:3210–3226 Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants Under Stress

Suriyan Cha-um, Vandna Rai, and Teruhiro Takabe

1 Introduction

Compatible solutes, proline, glycinebetaine, and trehalose are upregulated in higher plants under abiotic stress, playing a key role in physiological responses and enabling the plants to better tolerate the adverse effects of abiotic stresses. Biosynthetic pathways of proline, glycinebetaine, and trehalose are well known. In addition, the metabolic flux analysis has been established. However, the information on the uptake and inter-organ transport in plants are largely unknown. Here, we review the update information on the transport of these osmoprotectants under abiotic stresses. Proline is mainly synthesized from glutamate in cytosol and/or chloroplast, which is reduced to glutamate-semialdehyde (GSA) by the pyrroline-5-carboxylate synthetase (P5CS), spontaneously converted to pyrroline-5-carboxylate (P5C) and reduced to proline by P5C reductase (P5CR). As an alternative pathway, proline can be synthesized from ornithine, which is transaminated first by ornithine-delta- aminotransferase (OAT) producing GSA and P5C, which is then converted to proline. Glycinebetaine (GB) is synthesized from choline by two-step oxidation of choline with enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). Glycinebetaine is mainly localized in cytosol and chloroplast. Biosynthesis of trehalose involves the enzyme trehalose-6-phosphate synthase (TPS), which produces trehalose-6-phosphate (T6P) from UDP-glucose and

S. Cha-um National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand V. Rai National Research Center on Plant Biotechnology, IARI, New Delhi, India T. Takabe (*) Research Institute, Meijo University, Nagoya, Japan e-mail: [email protected]

© Springer Nature Switzerland AG 2019 201 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_9 202 S. Cha-um et al. glucose-6-phosphate, and then glucose-6-phosphate is converted to trehalose and phosphate by the trehalose-6-phosphate phosphatase (TPP). Trehalose is synthesized in cytosol. We described the uptake and inter-organ transport of exogenous applied these osmoprotectants for cellular homeostasis, including redox balance and energy status, and signaling molecule.

2 Proline Uptake in Plants

Accumulation of proline occurs due to the increased synthesis of proline and the decreased degradation of proline under various stress conditions. Plant transporters mediating proline uptake across the plasma membrane have been identified both in the amino acid transporter (ATF)/amino acid/auxin permease (AAAP) family and in the amino acid–polyamine–choline (APC) family (Table 1) (Rentsch et al. 2007). In the ATF/AAAP family, transporters that recognize proline have been identified in several subfamilies, namely, in the amino acid permease (AAP) family, the lysine/ histidine transporter (LHT) family, and the proline transporter (ProT) family. AAPs mediate proton-coupled uptake of glutamate (aspartate) and neutral amino acids including proline (Frommer et al. 1993; Fischer et al. 1995, 2002; Okumoto et al. 2002; Lee et al. 2007; Schmidt et al. 2007). LHTs transport neutral amino acids (including proline) and acidic amino acids with high affinity (Chen and Bush 1997; Lee and Tegeder 2004; Hirner et al. 2006). In contrast to transporters of the AAP and LHT family, ProTs transport proline but no other proteinogenic amino acids (Rentsch et al. 1996). Moreover, ProTs from Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum L.), and gray mangrove [Avicennia marina (Forsk.) Vierh.] also transport glycinebetaine, though only the latter is a glycinebetaine- accumulating species (Breitkreuz et al. 1999; Schwacke et al. 1999; Waditee et al. 2002; Grallath et al. 2005). Three Arabidopsis ProTs and a ProT1 of tomato transport the stress-induced compound γ-aminobutyric acid (GABA), while GABA was not a substrate for the gray mangrove proline transporters (AmTs) (Breitkreuz et al. 1999; Schwacke et al. 1999; Waditee et al. 2002; Grallath et al. 2005). The affinity of the AtProTs for GABA was much lower than for proline or glycinebetaine (4.5 mM compared to 0.5 and 0.2 mM, respectively; Grallath et al. 2005). Barley (Hordeum vulgare L.) HvProT recognized only L-proline efficiently (Igarashi et al. 2000; Ueda et al. 2001). These data on substrate selectivity of AAPs, LHTs, and ProTs show that both transporters in plants with low and high selectivity for proline exist, indicating a role in general transfer of nitrogen and in proline-specific functions, respectively. In yeast, the general amino acid permease Gap1p and the proline transporter Put4p together mediate the major part of proline uptake (Lasko and Brandriss 1981). Gap1p transports all proteinogenic amino acids with low affinity, but Put4p transports only GABA, alanine, glycine, and proline. Other amino acid permeases are Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 203

Table 1 Plant transporters for proline

Target Accession Km for proline Plant species proteins number (μM) Expression organs ATF/AAAP (amino acid transporter family/amino acid/auxin permease) gene family ProT proline transporter (substrates: proline, glycinebetainea) Arabidopsis AtProT1 At2g39890 427b All organsa AtProT2 At3g55740 500b Root, leafq AtProT3 At2g36590 999b Leafb Tomato LeProT1 AF014808 1900c Pollenc Barley HvProT AB073084 25d Rootr HvProT2 AB545851 246e Root, leaf e Rice OsProT AB022783 –f All organ Gray AmT1 AB075902 430g Root, leaf g mangrove AmT2 AB075903 320g Root, leaf g Oil palm EgProT1 AB597035 –h Root, leaf h Sugar beet BvBet/ProT1 AB477096 2100i Root, leafi Salt bush AqBet/ProT1 AB597034 1860i Root, leafi Amaranthus AmtBet/ AB597033 555i Root, leafi ProT1 AAP amino acid permease (substrates: neutral amino acids and glutamate, aspartatea) Arabidopsis AtAAP1 At1g58360 601, 1900j Root, flower, endosperms,t,u,v AtAAP2 At5g09220 140l Root, stem, leafs,t AtAAP3 At1g77380 250m Rootw AtAAP4 At5g63850 134m Leaf, stems AtAAP5 At1g44100 500m Root, leaf, stem, flowers AtAAP6 At5g49630 67n All organsx LHT (lysine/histidine) transporter (substrates: neutral and acidic amino acidsa) Arabidopsis AtLHT1 At5g49780 10° Root, leaf, stemy AtLHT1 At1g24400 13n Flowerz APC (amino acid–polyamine–choline) family CAT cationic amino acid transporters Arabidopsis AtCAT1 At4g21120 3000p Root, flower, leafp aRentsch et al. 2007, bGrallath et al. 2005, cSchwacke et al. 1999, dUeda et al. 2001, eFujiwara et al. 2010, fIgarashi et al. 2000, gWaditee et al. 2002, hYamada et al. 2011a, iYamada et al. 2011b, jFrom- mer et al. 1993, kBoorer et al. 1996, lKwart et al. 1993, mFischer et al. 1998, nLee and Tegeder 2004, oHirner et al. 2006, pFrommer et al. 1995, qRentsch et al. 1996, rUeda et al. 2007, sFischer et al. 1995, tHirner et al. 1998, uLee et al. 2007, vSanders et al. 2009, wOkumoto et al. 2004, xOkumoto et al. 2002, yChen and Bush 1997, zFoster et al. 2008 also known but may contribute to residual low-affinity proline uptake (Andreasson et al. 2004). In bacteria, the accumulation of compatible solutes is controlled by synthesis, uptake, and export, though uptake is preferred over biosynthesis provided that proline or glycinebetaine is available (Kempf and Bremer 1998; Roeβler and Müller 204 S. Cha-um et al.

2001). A range of secondary active transporters (e.g., the E. coli H+/proline sym- porter ProP) and binding protein-dependent ABC transporters (e.g., E. coli ProU) mediate the uptake of proline and/or glycinebetaine and related substrates (Csonka 1989; Wood et al. 2001). Under osmotic stress, these transporters may be regulated via both increased gene expression and higher activity (Wood et al. 2001), and some were additionally shown to function as osmosensors (Morbach and Kramer 2002; Wood 2006). In addition to osmolyte uptake systems, E. coli uses the PutP transporter for uptake of proline as a nitrogen and carbon source (Csonka 1989). PutP expression is repressed by the trifunctional PutA protein (combining PDH, P5CDH, and regulatory functions in a single protein) in the absence of proline and becomes activated once the PutA protein is recruited to the membrane during proline degradation (Tanner 2008; Zhou et al. 2008).

3 Proline Uptake and Inter-Organ Transport During Stress

ProT1 was expressed in all organs of the plants. High-level expression of ProT1 was observed during flowering and seed set. In contrast, mRNA levels ofProT2 were observed throughout the plant, but their expression was strongly induced by water or salt stress. This suggests that ProT2 is involved in nitrogen distribution during drought stress unlike the members of amino acid permease gene family such as AtAAP1–6, the expressions of which are generally suppressed under similar conditions. High proline concentrations were reported in the phloem sap of drought-stressed alfalfa. Active expression of ProT1 and ProT2 was observed in roots. High expression of ProT2 in root tip of maize was reported during stress (Verslues and Sharp 1999). These facts suggest that proline synthesis occurs more in roots and transported to shoot tissues and root tip under salt-stress conditions, while transport of broad specificity amino acids is suppressed. It was reported that LeProT1 supplies proline to both mature and germinating pollen. LeProT1 transports proline and g-amino butyric acid with low affinity and glycinebetaine with high affinity. Igarashi et al. (2000) found that OsProT specifically transported L-proline in a transport assay. Andreasson et al. (2004) found that proline and the toxic proline analogue azetidine-2-carboxylic acid are efficiently imported into yeast cells by four amino acid permeases, including two nitrogen- regulated permeases. Four ProT-homologous genes (ClProT1–4) from Chrysanthemum lavandulifolium were shown to be expressed in different organs under various stresses (Zhang et al. 2014). The expression of ClProT2 was restricted to above-ground organs and induced by various stress conditions. Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 205

4 Physiological Role of Plant Proline Transporters

Since AAP and LHT families are not selective for proline, these transporters may play a role in general acquisition and allocation of nitrogen in plants. Indeed, Arabidopsis knockout mutants LHT1, AAP8, and AAP1 as well as overexpression of AAP1 revealed complex changes in amino acid levels (Rolletschek et al. 2005; Hirner et al. 2006; Schmidt et al. 2007; Weigelt et al. 2008; Sanders et al. 2009). By contrast, the selectivity of ProTs for proline and other compatible solutes indicates a specific role in proline homeostasis under stress and non-stress conditions. Under salt stress, proline accumulation is accompanied by an increased expression of Arabidopsis ProT2; mangrove AmT1, AmT2, and AmT3; as well as HvProT (Rentsch et al. 1996; Ueda et al. 2001; Waditee et al. 2002; Table 1). Likewise, the high transcript abundance of AtProT1 and LeProT1 in pollen and of AtProT3 in the epidermis correlated with an elevated proline content (Schwacke et al. 1999). In spite of these correlations, only few reports show the direct role of ProTs in proline transport in planta. The Arabidopsis knockout mutants AtProT1, AtProT2, or AtProT3 did not reveal phenotypic differences or altered proline content in the absence or presence of abiotic stress, indicating compensation by other transporters or altered proline metabolism (Lehmann and Rentsch, personal communication). However, the overexpression of HvProT in Arabidopsis resulted in reduced biomass and decreased proline levels in shoots, an effect that could be compensated by exogenous supply of low concentrations of proline (Ueda et al. 2008). On the other hand, root cap-specific expression ofHvProT in Arabidopsis resulted in higher proline levels in root tips and enhanced root elongation (Ueda et al. 2008), supporting a role of proline in organ development.

5 Intracellular Transport

Proline biosynthesis takes place in the cytosol and probably in chloroplasts under stress conditions (Szabados and Savoure 2010). Thus, at least in the absence of stress, proline import into plastids is necessary. Furthermore, transfer into mitochondria is essential for proline catabolism. Whereas information on proline transport into or out of plastids is lacking, proline uptake into mitochondria has been demonstrated to be mediated by two transport systems, i.e., a proline uniporter as well as a proline/glutamate antiport system (Elthon et al. 1984; di Martino et al. 2006), though a reversible switch of the transport mode as shown for other mitochondrial carriers cannot yet be excluded (Krämer 1998). 206 S. Cha-um et al.

6 Endogenous Proline Enrichment Using Proline Exogenous Application for Abiotic Stress-Tolerant Traits

Alternatively, proline accumulation in higher plants using exogenous application is a useful method to elevate the high level of proline in the cellular levels. Exogenous foliar proline can uptake and translocate to other organs, especially under stressful conditions to function as major osmolyts as abiotic tolerant strategies (Osman 2015; Zouari et al. 2016a, b; Kahlaoui et al. 2018). The exogenous proline application for abiotic stress-tolerant traits, i.e., salinity, heavy metals, drought, and oxidative constraints, is summarized in Table 2. Based on the literatures, proline can be applied in both root via culture media (Ozden et al. 2009; Nounjan et al. 2012; Zheng et al. 2015) and foliar spray in the aerial parts (Moustakas et al. 2011; Kahlaoui et al. 2018; Zouari et al. 2016b). Therefore, the level of proline in each plant species depends on the plant species, exogenous supply rates, growth conditions (in vitro culture, pot experiment, and field trials), plant developmental stages, and their interaction. In addition, proline biosynthesis key enzymes, i.e., P5CS and P5CR, are regulated by both stress and proline exogenous application, leading to play a role as antioxidant activities under abiotic stresses (Nounjan et al. 2012). However, real mechanism on gene(s) regulation relating to proline metabolism by final product application is still uncleared.

7 Trehalose Uptake in Plants

Trehalose is a nonreducing disaccharide formed by two glucose molecules. In plants, this disaccharide has diverse functions and plays an essential role in various stages of development (Griffiths et al. 2016). In Saccharomyces cerevisiae, it was shown that increasing intracellular trehalose is sufficient to confer desiccation tolerance to yeast (Tapia et al. 2015). Sugar transporters have essential roles in the appropriate distribution of carbohydrates throughout the plants (Mueckler 1994). They are typically categorized in two groups: (i) secondary active membrane transporters, which promote the uphill permeation of sugars driven by electrochemical gradients of Na+ or H+ ions across the cellular membranes, and (ii) facilitative sugar transporters, which enable sugars to flow across membranes down the concentration gradients (Wood and Trayhurn 2003). Trehalose transporters have been identified from yeasts and insects (Stambuk et al. 1998; Kikawada et al. 2007). Saccharomyces cerevisiae possesses the α-glucoside transporter AGT1, which promotes uptake of disaccharides, including trehalose, sucrose, and maltose, via an electrochemical proton gradient, which suggests that AGT1 belongs to the group of secondary active transporters (Han et al. 1995). AGT1 acts as an H+-dependent trehalose transporter for the uptake of low-level trehalose as a nutrient from culture medium under low pH conditions. Insects have a facilitative trehalose transporter, TRET1, which seems Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 207

Table 2 Proline accumulation using exogenous proline application for abiotic tolerant traits Exogenous Plant Plant species proline Endogenous proline Target traits organs References Rice cv. KDML105 10 mM 500 μmol g−1 FW Salt tolerance Leaf Nounjan et al. (2012) cvs. MR 5–20 mM 270 μmol g−1 Salt tolerance Leaf Teh et al. (2015) varieties cvs. BRRI 5 mM 6.5 μmol g−1 FW Salt tolerance Leaf Hasanuzzaman varieties et al. (2014) Maize cv. BHM-7 15 mM 6 μmol g−1 FW Salt tolerance Leaf Rohman et al. (2015) Tomato cv. Rio 10 mg L−1 85 mg g−1 DW Salt tolerance Leaf Kahlaoui et al. Grande 60 mg g−1 DW Root (2018) Eurya 10 mM 6 μg g−1 FW Salt tolerance Leaf Zheng et al. (2015) emarginata Olive tree cv. Chemlali 25–50 mM 4.99 μmol mg−1 FW Salt tolerance Leaf Ahmed et al. 3.92 μmol mg−1 FW Root (2010) Pea cv. Sprinter 60 mM 2.77 mg g−1 DW Nickle (Ni) Leaf Shahid et al. (2014) 2.70 mg g−1 DW Salt tolerance Olive tree cv. Chemlali 10–20 mM 140 μmol g−1 FW Cadmium Leaf Zouari et al. 200 μmol g−1 FW Root (2016a) Tobacco cv. BY-2 10 mM 132.5 mM Cadmium Cell Islam et al. (2009) culture Eggplant 25 μM 3.3 μg g−1 FW Arsenate Leaf Singh et al. (2015) Date palm cv. Deglet 20 mM 50 mg g−1 FW Cadmium Leaf Zouari et al. nour 4.2 mg g−1 FW Root (2016b) Rapeseed 5–20 mM 170 μmol g−1 Cold Leaf Jonytienė et al. tolerance (2012) Pea cv. Master B 4 mM 5.1 mg g−1 FW Drought Leaf Osman (2015) 30.2 mg g−1 FW tolerance Seed Arabidopsis 10 mM 900 μmol g−1 DW Drought Leaf Moustakas et al. tolerance (2011) Grapevine −1 cv. Öküzgözü 20 mM 0.45 μmol g FW H2O2 Leaf Ozden et al. (2009) oxidative stress Wild almond −1 8 species 10 mM 0.6 μmol g FW H2O2 Leaf Sorkheh et al. oxidative (2012) stress 208 S. Cha-um et al. to be responsible for the regulation of trehalose levels in the hemolymph (Kikawada et al. 2007; Kanamori et al. 2010). Secondary active transporters for trehalose in multicellular organisms, including insects, have not been reported. It is difficult to predict the characteristics of sugar transporters based solely on the amino acid sequences because only subtle difference exists between proton-dependent sugar transporters and facilitative sugar transporters in the major facilitator superfamily (MFS; Pao et al. 1998).

8 Endogenous Trehalose Using Exogenous Trehalose Application for Abiotic Stress-Tolerant Traits

In the natural habitat based on evaluation, high level of trehalose accumulation in higher plant is a rare report. Transgenic plants regulating on trehalose-metabolizing enzymes derived from microbial organisms are evidently discovered by several scientists (Garg et al. 2002; Paul et al. 2017; Kosar et al. 2018). On the other hand, it has been reported that non-stressed and stressed plants pretreated with trehalose increased the endogenous level of trehalose, which indicated that trehalose is readily absorbed by the roots and easily transported to the aerial parts to be functioned as major defensive responses to several abiotic stresses, i.e., salt tolerance, drought tolerance, heavy metal tolerance, heat tolerance, and stress-responsive regulation (Table 3). Therefore, there are large distributions in the exogenous concentration of trehalose and endogenous content depending on plant species, different providing methods, the stressors, and plant developmental stages (Yang et al. 2014; Ma et al. 2013; Abdallah et al. 2016).

9 Glycinebetaine Accumulation in Plants

Plants in taxonomically distant species can synthesize GB and accumulate larger amounts when they are exposed to abiotic stress conditions, such as drought (Wyn Jones and Storey 1981), salt, and cold stress (Rhodes and Hanson 1993). But many plants do not accumulate GB and accumulate other osmolytes. Sugar beet (Beta vulgaris L.), spinach (Spinacia oleracea L.), barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), and sorghum [Sorghum bicolor (L.) Monech] are known as GB accumulators. GB does not function only as “compatible solute” – a substance compatible with the cellular metabolism that accumulates in the cytoplasm to balance external osmotic pressure. It can protect proteins against thermodynamic perturbation caused by dehydration and heat denaturation. GB also protects the inhibition of several enzyme activity induced by NaCl. GB protects sugar beet root membranes against heat destabilization and spinach thylakoids against freezing stress. The concentra- Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 209

Table 3 Endogenous trehalose using exogenous trehalose application for abiotic tolerant traits Exogenous Endogenous Plant Plant species trehalose trehalose Target traits organs References Arabidopsis 30 mM 0.29 mg g−1 FW Stress Cell Bae et al. responses culture (2005) 5 mM 5.5 μmol g−1 Salt tolerance Seedling Yang et al. DW (2014) Rice cv. Nakdong 25 mM 0.242 mg g−1 Drought Seedling Redillas et al. FW tolerance (2012) cv. BRRI 10 mM 1.5 μmol g−1 FW Copper Seedling Mostafa et al. dhan29 (2015) cvs. Giza 25 mM 210 nmol g−1 Salt tolerance Seedling Abdallah et al. varieties FW (2016) Wheat cv. Zhoumai 50 mM 380 μg g−1 FW Water Callus Ma et al. (2013) 18 tolerance culture 1.5 mM 1658 μg g−1 DW Heat Seedling Luo et al. tolerance (2010) Duckweed 0.5–2.0 mM 11.8 μg g−1 DW Cadmium Frond Duman et al. (2011) tions of GB required to produce these protective effects are often high (>0.5 M) (Rhodes and Hanson 1993). GB is thought to be distributed exclusively in cytoplasm and chloroplasts in plant cells; however, clear experimental data have not been reported. The amount of GB in the chloroplasts of spinach leaves was estimated to be close to 50% of the total GB in plant cells (Rhodes and Hanson 1993). Concentrations of GB of up to 0.3 M (20 times as high as the concentration calculated if GB distributes uniformly in leaf tissue) have been reported for chloroplasts isolated from salinized spinach plants. Such concentrations could contribute to chloroplast osmotic adjustment, facilitating maintenance of chloroplast volume and photosynthetic activity at low leaf-water potentials. The GB concentration was below detection limit in vacuoles of Sea Blite [Suaeda maritima (L.) Dumort.] leaf cells. Major solutes in vacuole are considered to be inorganic ions in leaves and sucrose in roots (Bell et al. 1996).

10 Glycinebetaine Biosynthesis in Monocot and Dicot Plants

In plant, GB is synthesized by two-step oxidations of choline. Choline is first oxidized to betaine aldehyde by choline monooxygenase (CMO). Betaine aldehyde is further oxidized by betaine aldehyde dehydrogenase (BADH) to GB. The CMO enzyme in GB-accumulating dicotyledonous Amaranthaceae (Chenopodiaceae, Beta vulgaris L., spinach, Atriplex, etc.) is localized in chloroplast, whereas the localization of CMO in GB-accumulating monocot such as barley and wheat seems 210 S. Cha-um et al. to be different. Peroxysome localization of barley CMO and BADH has been reported (Fujiwara et al. 2008; Mitsuya et al. 2011). In addition to the localization of CMO and BADH, the sequence of CMO should be carefully analyzed. Sequence homology did not give us the information on the gene of CMO. For example, CMO homolog gene(s) can be found in Arabidopsis genome which is a well-known GB non-accumulator. The Arabidopsis CMO homolog did not show the choline oxidation activity (Hibino et al. 2002). Therefore, the real function of CMO homolog genes in GB non-accumulator must be clarified in the future. The experimental design is straightforward and the obtained results are clear.

11 Regulation of Gene Expression

It has been known that the gene expression of CMO and BADH is induced by salt and drought stress. Production of GB involves expression of not only the abovementioned genes but also many genes that encode enzymes such as AdoMet synthetase, AdoHcy hydrolase, and methionine synthase. Tabuchi et al. (2006) examined transcriptional regulation of the GB-related genes in leaf beet. The expression of GB-related genes in leaf tissue showed induction under salt stress. Their induction showed diurnal rhythms were reduced in a dark condition. Salinization of spinach plants increases PEAMT mRNA abundance and enzyme activity in leaves by about tenfold, consistent with the high demand in stressed plants for choline to support GB synthesis (Nuccio et al. 2000).

12 Translocation of GB in Sugar Beet

Halotolerant plants often accumulate GB under non-stress condition. GB of 420 μmol g−1 FW accumulated in young leaves of B. vulgaris L. even under normal growth conditions, whereas levels in mature leaves, cotyledons, hypocotyls, and roots were low (Yamada et al. 2009). Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. Incubation of segments of mature and young leaves with deuterium-labeled choline revealed that the rate of deuterium-labeled GB synthesis in mature leaves was about ninefold higher than that in young leaves. The levels of CMO mRNA were higher in mature leaves than in young leaves. Thus, the differential accumulation of CMO was essentially controlled by transcription level. When deuterium-substituted GB was foliar- applied to one of the attached mature leaf, labeled GB were preferentially detected in young leaves and root, but not in other expanded leaves. These data indicate that GB is primarily synthesized in mature leaves and translocated into young leaves. In response to salt stress, GB levels increased in all tissues, but most significantly increased in young leaves with only small increase in the levels of CMO (Yamada Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 211 et al. 2009). GB functions as a chemical chaperone to protect the inactivation of proteins under high salinity and high temperature. In actively developing cells, the levels of protein synthesis and translocation would be high. GB might facilitate folding and translocation, serving as a chemical chaperone. Another possibility is that GB might be utilized to maintain high cellular pressure. It is known that high cellular pressure is required for cell growth (Bourot et al. 2000). Higher osmolarity in young leaves than in mature leaves is compatible with this idea. In animal cells, it has been shown that GB plays a role in cell volume homeostasis. The high concentration of GB in young leaves with a little de novo synthesis of GB suggests the importance of GB transporters in GB accumulation. To date, many amino acid transporter genes have been identified in the plant genomes (Yamada et al. 2009; Lehmann et al. 2010). Proline transporters (ProTs) were first isolated from A. thaliana as highly selective transporters for proline. However, ProTs from tomato (LeProTs) were subsequently shown to transport GB as well as proline, although tomato is a GB non-accumulating plant. Hitherto, functional properties of ProTs have been reported on GB non-accumulating plants, A. thaliana (AtProT1–3), tomato (LeProT1–3), oil palm (EgProT1), and rice (OsProT1), and GB-accumulating plants, mangrove (AmBet/ProT1–2), sugar beet (BvBet/ProT1), Atriplex (AqBet/ ProT1), Amaranthus (AmtBet/ProT1), and barley (HvProT1–2) (Ueda et al. 2001; Waditee et al. 2002; Grallath et al. 2005; Yamada et al. 2009, 2011a, b; Fujiwara et al. 2010). Among them, the selectivity of rice ProT for GB remains uninvesti- gated, and the barley HvProT1 was reported to recognize proline, but not GB. All other transporters mediate transport of both GB and proline. The selectivity of ProTs for proline suggests their specific role in proline homeostasis under stressed and non-stressed conditions (Lehmann et al. 2010). Oil production from oil palm is adversely affected by drought and salt. Under drought and salt stress, proline content increases in oil palm. The proline transporter gene from oil palm (Elaeis guineensis Jacq.) showed high similarity to Bet/ProT genes from several plants, but the highest homology to rice ProT1 (Yamada et al. 2011a). Expression of EgProT1 in Escherichia coli mutant exhibited uptake activities for GB and choline as well as proline. Under salt-stressed conditions, exogenous applied GB was taken up into the root more rapidly than the control. Two betaine/proline transporters (AmT1, AmT2) were isolated from GB-accumulating mangrove Avicennia marina (Waditee et al. 2002). Am1 and Am2 could efficiently take up GB and proline with similar affinities (Km, 0.32–0.43 mM). The uptakes of GB and proline were significantly inhibited by mono- and dimethylglycine but only partially inhibited by betaine aldehyde, choline, and 4-aminobutyrate. Betaine/proline transporter genes were isolated from GB-accumulating plants, sugar beet, Amaranthus, and Atriplex, as well as GB non- accumulating (Arabidopsis) plant (Yamada et al. 2011b). Using a yeast mutant deficient for uptake of proline and GB, it was shown that all these transporters exhibited higher affinity for GB than proline. The uptake of GB and proline was pH-dependent and inhibited by the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). These transporters exhibited a higher affinity for choline uptake rather than GB uptake. Uptake of choline by sugar beet BvBet/ProT1 was indepen- 212 S. Cha-um et al. dent of the proton gradient, and the inhibition by CCCP was reduced compared with that for uptake of GB, suggesting different proton-binding properties between the transport of choline and GB. The physiological role of GB uptake by Bet/ProTs still remains obscure. A homologous gene BvBet/ProT1 was isolated from sugar beet. The fusion protein of green fluorescent protein and BvBet/ProT1 showed that BvBet/ProT1 was localized at the plasma membrane. Levels of mRNA for BvBet/ProT1 were much higher in mature leaves than in young leaves under normal and salt-stress conditions. In situ hybridization experiments revealed the localization of BvBet/ProT1 in phloem and xylem parenchyma cells (Yamada et al. 2011b). Since GB is transported from the mature leaves to young leaves and the accumulated levels of BvBet/ProT1 gene transcript were higher in mature leaves than in young leaves, BvBet/ProT1 might play a role in the efflux of GB from CMO-expressing cells.

13 Exogenous Application for Crop Production

In many crop plants, the natural accumulation of GB is lower than sufficient to ameliorate the adverse effects of dehydration caused by various environmental stresses. Exogenous application of GB to low-accumulating or non-accumulating plants may help reduce adverse effects of environmental stresses (Mäkelä 2004; Ashraf and Foolad 2007). Exogenous applied GB is, at least, partially immediately taken up by plant tissues and is readily translocated to roots, meristems, and expanding leaves (Mäkelä et al. 2000). Because GB is metabolically quite inert in plant, it remains in the plant tissue for several weeks. There is now strong evidence that GB plays an important role in tolerance to abiotic stress. Exogenous application of GB to non-accumulator plants may be a possible alternative approach for tolerance against multiple abiotic stresses (Table 4). Foliar spray of GB improved salt and drought tolerance in tomato. Application of tomato plants in field condition with 3.36 Kg ha−1 GB during mid flowering period increased fruit yield 36% in salt stress, as compared to control (Mäkelä 2004). Salt stress decreased net photosynthetic rate of tomato plant, and application of GB increased the rate of photosynthesis of the salt-stressed plants to the same level as control plants, which was not applied with GB. The increase of net photosynthetic rate by GB application was also observed in control plants. Stomatal conductance was increased by GB treatment, resulting in efficient gas exchange and reduced photorespiration. GB application protected chloroplast ultrastructure and prevented the decrease of chlorophyll and RuBisCO activity under salt and drought stress conditions (Mäkelä et al. 2000). Relative water content of total shoot of tomato decreased under drought stress, and GB had increasing effect (Rezaei et al. 2012). The positive effect of GB on drought stress tolerance was more efficient atvegetative growth stage (Hussain et al. 2008). Exogenous application of GB increases chilling tolerance in tomato plants (Park et al. 2006). GB levels in tomato plants which took up exogenous GB were high in meristematic tissues such as shoot apices and flower Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 213

Table 4 Endogenous glycinebetaine using exogenous glycinebetaine supplementation for abiotic tolerant traits Exogenous Plant Plant species GB Endogenous GB Target traits organs References Rice cv. KDML105 2–6 mM 90 μmol g−1 DW Salt Leaf Cha-um et al. cv. Super-basmati 50–150 mM 17 μmol g−1 DW tolerance Leaf (2006) Drought Farooq et al. tolerance (2008) Cotton cv. MNH886 5.0 mM 70 μmol g−1 Cadmium Leaf Farooq et al. cv. CIM-496 100 mg L−1 140 μmol g−1 Drought Root (2016) 18 μmol g−1 FW tolerance Leaf Ahmad et al. (2014) Maize cv. Black Mexican 2–5 mM 240 μmol g−1 Chilling Cell Chen et al. sweet 30 mM DW tolerance culture (2000) cv. Agaiti-2002, 50 mg L−1 45 μmol g−1 DW Drought Leaf Ali and Ashraf EV-1098 51.02 g plant−1 tolerance Leaf (2011) cv. S9, S911 Drought LiXin et al. tolerance (2009) Tomato cv. Moneymaker 10 mM 10.88 μmol g−1 Chilling Leaf Park et al. (2006) FW tolerance Sunflower cv. Hysun −33 100 mM 16.38 μmol g−1 Drought Leaf Hussain et al. DW tolerance (2008) Cvs. Glushan-98, 50–100 mM 800 μg g−1 FW Drought Leaf Iqbal et al. (2011) Suncross tolerance Tobacco cv. DHJ5210, 80 mM 900 μmol g−1 Drought Leaf Ma et al. (2007) ZY100 DW tolerance Wheat 19 genotypes 100 mM 7.5 μmol g−1 Drought Leaf Gupta et al. DW tolerance (2014) cv. HF9703, 56.8 μg g−1 DW Drought Leaf Zhao et al. (2007) SN215953 tolerance Bean cv. Tendergreen 10 mM 1 μmol g−1 DW Drought Leaf Xing and tolerance Rajashekar (1999) buds. In leaves, GB existed mainly in cytosol with at most 22% in chloroplasts. The transport of GB from cytosol to chloroplasts might be inefficient. However, GB-treated plants exhibited increased levels of photosystem II (PSII) activity compared with control plants. GB-treated plants had significantly greater catalase (CAT) activity and CAT1 gene expression, although their H2O2 levels remained unchanged. During chilling treatment, H2O2 level was lower, and catalase activity was higher in 214 S. Cha-um et al.

GB-treated plants than those in control plants. These results suggest that GB may enhance the induction of antioxidant mechanisms under chilling stress condition (Park et al. 2006). When 28-day-old rice seedlings were exposed to NaCl, relative water content in the leaves drastically decreased. This adverse effect was largely prevented when the seedlings were treated with 15 mM GB before exposure to NaCl stress (Harinasut et al. 1996). GB added in culture medium was taken up by the roots and accumulated in the leaves to reach a concentration of 5.0 μmol g−1 FW−1 before transfer to NaCl stress. This level of GB is comparable with those of barley, GB accumulator. The level of GB in rice seedlings did not changed significantly after transfer to NaCl stress. No apparent difference in relative water content was found between control and GB-treated rice seedling before transfer to salt stress condition, and changes in relative water content after transfer to salt stress was significantly different. Control rice seedlings showed large decrease in relative water content (RWC) after transfer to salt stress; the extent of the decrease in RWC was small in GB-treated seedlings. Photosynthetic capacity of control seedlings also drastically decreased whereas that of GB-treated seedlings was maintained near the value of non-stressed seedlings. Higher water use efficiency in GB-treated rice plants than that in control plants under salt stress condition was reported (Cha-um et al. 2006). Foliar spray of 50 mM GB to a salt-sensitive cultivar of rice enhanced proline accumulation under salt stress condition and resulted in maintaining transpiration efficiency and high photosynthetic performance. The photosynthetic abilities in plants were positively related to seed fertility percentage and total seed grain weight after recovery from the stress. This performance was rather inhibited with the high dose of 200 mM GB. Under salt stress, GB-treated rice plants had significantly lower Na+ and higher K+ concentrations in the shoots, compared with untreated plants (Cha-um et al. 2006). Moreover, salt-induced ultrastructural damages in chloroplasts and mitochondria were prevented by GB pretreatment. Although the inhibition of root growth by NaCl was not alleviated, formation of large vacuoles was observed in root cells by GB pretreatment. It was proposed that the mitigation of the NaCl- induced damages in the shoots is due to the enhanced sequestration of Na+ into the vacuoles in root cells by GB pretreatment. The foliar GB application (50–100 mM) in cv. PT1 rice demonstrated the increase in water use efficiency, photosynthetic abilities, and seed grain yield under salt stress (150 mM NaCl) (Cha-um and Kirdmanee 2010) as well as under drought stress (25% soil water content) (Cha-um et al. 2013). Studies with wheat showed contradictory results. There were reports that exogenous GB delayed the canopy senescence of wheat, but this was not associated with its grain yields after the exposure to drought stress. On the other hand, there are reports that GB had effect to mitigate adverse effect of drought stress in wheat. There are also reports for the protective effects of GB against NaCl stress and freezing stress. Drought stress reduced plant biomass, grain yield, and phosphate (P) and K+ contents in shoot and root, but foliar-applied GB mitigated the adverse effects of drought stress (Ashraf and Foolad 2007). It was reported that mitigation of decrease in photosynthetic rates was not prominent but transpiration rate was apparently Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 215 decreased by GB application. The extent of the effect of GB was varied among wheat cultivars. Drought stress caused an increase in osmotic pressure in flag leaves of wheat. The drought-resistant cultivar had higher values of osmotic pressure than the sensitive one. Drought stress induced marked decrease in grain yield, but spray of 10 mM GB mitigated the effect of drought stress. The application of GB increased the osmotic pressure, the osmolyte concentrations, as well as the grain yield (Ma et al. 2006). Among the organic osmolytes, proline and keto acid concentrations were positively correlated with the grain yield. The drought-resistant cultivar had a higher concentration of K+ than the sensitive one, and the application of GB caused an accumulation in K+ in both wheat cultivars. Because K+ has some role in stabilization of native proteins, the increase in K+ level may play a role in adaptation of wheat plants to drought stress conditions (Raza et al. 2014).

14 Genetic Engineering of GB in Plants

Various genes have been used to generate transgenic plants that accumulate GB and exhibit enhanced tolerance to abiotic stress (Chen and Murata 2011). Most of the transgenic plants of GB non-accumulators accumulate only low concentration of GB compared with that in GB accumulators. However, these transgenic plants often exhibited increases in abiotic stress tolerance. Two known factors have been reported to limit the accumulation of GB in transgenic plants: the availability of endogenous choline and the transport of choline across the chloroplast envelope (Nuccio et al. 2000). Exogenous supply of choline showed significant increase in the level of GB accumulation in transgenic plants. Indeed, the activity of phosphoethanolamine N-methyltransferase (PEAMT) was 30 to 100 times lower in tobacco, a GB non- accumulator, compared with spinach, a GB accumulator. Furthermore, it was revealed that the import of choline, a precursor of GB, into chloroplasts limits GB synthesis. A halotolerant cyanobacterium Aphanothece halophytica synthesizes GB by a three-step methylation of glycine with two genes: ApGSMT encodes the methylation of glycine and sarcosine and ApDMT encodes the methylation of dimethylglycine to GB. The Arabidopsis plants expressing the glycine methylation genes (ApGSMT+ApDMT) showed improved tolerances for various abiotic stresses more than the CMO-expressing plants (Waditee et al. 2005). The reason for the accumulation of GB in various tissues of the glycine methylation genes-expressing plants might be the availability of substrates. Glycine and serine are readily inter- convertible via the action of serine hydroxymethyltransferase (SHMT) and glycine decarboxylase enzymes. The GB level in the transformed plants expressing the glycine methylation genes increased at high salinity. The high accumulation of serine at high salinity is reported (Ho and Saito 2001). Supply of exogenous glycine to Arabidopsis transformants enhanced the accumulation levels of GB and stress tolerance. Furthermore, a transgenic plant expressing ApGSMT and ApDMT together with 3-phosphoglycerate dehydrogenase (PGDH) encoding serine biosynthesis 216 S. Cha-um et al. gene could accumulate more GB than plants transformed with ApGSMT and ApDMT (Waditee et al. 2007). This demonstrates the importance of precursor supply for biosynthesis of GB.

15 Summary and Future Prospects

As described above, uptake and inter-organ transport of proline, glycinebetaine, and trehalose play important roles for plant adaptation under various abiotic stresses. While many studies have indicated a positive relationship between accumulation of these compatible solutes and plant stress tolerance, the accumulation levels are still low. Further efforts are required to increase the information on the transport mechanisms of these solutes, especially on the localization of solutes and transporters as well as the regulation mechanisms of these compounds. The future research using the important plants both in laboratory and field would contribute to understand the mechanisms of transport and their effective utilization in crop production under stress environments.

References

Abdallah MMS, Abdelgawad ZA, El-Bassiouny HMS (2016) Alleviation of the adverse effects of salinity stress using trehalose in two rice varieties. South Afri J Bot 103:275–282. https://doi. org/10.1016/j.sajb.2015.09.019 Ahmad S, Raza I, Ali H, Shahzad AN, Rehman A, Sarwar N (2014) Response of cotton crop to exogenous application of glycinebetaine under sufficient and scare water conditions. Braz J Bot 37:407–415. https://doi.org/10.1007/s40415-014-0092-z Ahmed CB, Rouina BB, Sensoy S, Boukhriss M, Abdullah FB (2010) Exogenous proline effects on photosynthetic performance and antioxidant defense system of young olive tree. J Agric Food Chem 58:4216–4222. https://doi.org/10.1021/jf9041479 Ali Q, Ashraf M (2011) Exogenously applied glycinebetaine enhances seed and seed oil quality of maize (Zea mays L.) under water deficit conditions. Environ Exp Bot 71:249–259.https://doi. org/10.1016/j.envexpbot.2010.12.009 Andreasson C, Neve EPA, Ljungdahl PO (2004) Four permeases import proline and the toxic proline analogue azetidine-2-carboxylate into yeast. Yeast 21:193–199. https://doi.org/10.1002/ yea.1052 Ashraf MFMR, Foolad M (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot 59:206–216. https://doi.org/10.1016/j. envexpbot.2005.12.006 Bell CI, Milford GFJ, Leigh RA (1996) Photoassimilate distribution in plants and crops: source- sink relationships. In: Zamski E, Schaffer AA (eds) Sugar beet. Marcel Dekker, Inc., New York, pp 691–707 Boorer KJ, Frommer WB, Bush DR, Kreman M, Loo DDF, Wright EM (1996) Kinetics and specificity of a +H /amino acid transporter from Arabidopsis thaliana. J BioI Chem 271:2213–2220. https://doi.org/10.1074/jbc.271.4.2213 Bourot S, Sire O, Trautwetter A, Touze T, Wu LF, Blanco C, Bernard T (2000) Glycine betaine- assisted protein folding in a lysA mutant of Escherichia coli. J Biol Chem 275:1050–1056. https://doi.org/10.1074/jbc.275.2.1050 Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 217

Breitkreuz KE, Shelp BJ, Fischer WN, Schwacke R, Rentsch D (1999) Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana. FEBS Lett 450:280–284. https://doi.org/10.1016/S0014-5793(99)00516-5 Bae H, Herman E, Bailey B, Bae H-J, Sicher R (2005) Exogenous trehalose alters Arabidopsis transcripts involved in cell wall modification, abiotic stress, nitrogen metabolism, and plant defense. Physiol Plant 125(1):114–126 Cha-um S, Kirdmanee C (2010) Effect of glycinebetaine on proline, water use, and photosynthetic efficiencies, and growth of rice seedlings under salt stress. Turk J Agric Forest 34:517–527. https://doi.org/10.3906/tar-0906-34 Cha-um S, Supaibulwatana K, Kirdmanee C (2006) Water relation, photosynthetic ability and growth of Thai jasmine rice (Oryza sativa L. ssp. indica cv. KDMK105) to salt stress by application of exogenous glycinebetaine and choline. J Agron Crop Sci 192:25–36. https://doi. org/10.1111/j.1439-037X.2006.00186.x Cha-um S, Samphumphuang T, Kidmanee C (2013) Glycinebetaine alleviates water deficit stress in indica rice using proline accumulation, photosynthetic efficiencies, growth performances and yield traits. Aust J Crop Sci 7:213–218 Chen L, Bush DR (1997) LHTl, a lysine- and histidine-specific amino acid transporter in Arabidopsis. Plant Physiol 115:1127–1134. https://doi.org/10.1104/pp.115.3.1127 Chen TH, Murata N (2011) Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 34:1–20. https://doi. org/10.1111/j.1365-3040.2010.02232.x Chen WP, Li PH, Chen THH (2000) Glycinebetaine increase chilling-induced lipid peroxidation in Zea mays L. Plant Cell Environ 23:609–618. https://doi.org/10.1046/j.1365-3040.2000.00570.x Csonka LN (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiol Mol Biol Rev 53:121–147. doi:0146-0749/89/010121-27 Di Martino C, Pizzuto R, Pallotta M, De Santis A, Passarella S (2006) Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings. Planta 223:1123–1133. https://doi.org/10.1007/ s00425-005-0166-z Duman F, Aksoy A, Aydin Z, Temizgul R (2011) Effects of exogenous glycinebetaine and trehalose on cadmium accumulation and biological responses of an aquatic plant (Lemna gibba L.). Water Air Pollut 217:545–556. https://doi.org/10.1007/s11270-010-0608-5 Elthon TE, Stewart CR, Bonner WD (1984) Energetics of proline transport in corn mitochondria. Plant Physiol 7:951–955. https://doi.org/10.1104/pp.75.4.951 Farooq M, Basra SMA, Wahid A, Cheema ZA, Cheema MA, Khaliq A (2008) Physiological role of exogenously applied glycinebetaine to improve drought tolerance in fine grain aromatic rice (Oryza sativa L.). J Agron Crop Sci 194:325–333. https://doi.org/ 10.1111/j.1439-037X.2008.00323.x Farooq MA, Ali S, Hameed A, Bharwana SA, Rizwan M, Ishaque W, Farid M, Mahmood K, Iqbal Z (2016) Cadmium stress in cotton seedlings: physiological, photosynthesis and oxidative damages alleviated by glycinebetaine. South Afri J Bot 104:61–68. https://doi.org/10.1016/j. sajb.2015.11.006 Fischer WN, Kwart M, Hummel S, Frommer WB (1995) Substrate specificity and expression profile of amino acid transporters (AAPs) inArabidopsis . J Biol Chem 270:16315–16320. https:// doi.org/10.1074/jbc.270.27.16315 Fischer WN, André B, Rentsch D, Krolkiewicz S, Tegeder M, Breitkreuz K, Frommer WB (1998) Amino acid transport in plants. Trends Plant Sci 3:188–195. https://doi.org/10.1016/ S1360-1385(98)01231-X Fischer WN, Loo DDF, Koch W, Ludewig U, Boorer KJ, Tegeder M, Rentsch D, Wright EM, Frommer WB (2002) Low and high affinity amino acid +H -co transporters for cellular import of neutral and charged amino acids. Plant J 29:717–731. https://doi. org/10.1046/j.1365-313X.2002.01248.x 218 S. Cha-um et al.

Foster J, Lee YH, Tegeder M (2008) Distinct expression of members of the LHT amino acid transporter family in flowers indicates specific roles in plant reproduction. Sex Plant Reprod 21:143–152. https://doi.org/10.1007/s00497-008-0074-z Frommer WB, Hummel S, Riesmeier JW (1993) Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease fromArabidopsis thaliana. Proc Natl Acad Sci 90:5944–5948. https://doi.org/10.1073/pnas.90.13.5944 Frommer WB, Hummel S, Unseld M, Ninnemann O (1995) Seed and vascular expression of a high-affinity transporter for cationic amino acids in Arabidopsis. Proc Natl Acad Sci 92:12036– 12040. https://doi.org/10.1073/pnas.92.26.12036 Fujiwara T, Hori K, Ozaki K, Yokota Y, Mitsuya S, Ichiyanagi T, Hattori T, Takabe T (2008) Enzymatic characterization of peroxisomal and cytosolic betaine aldehyde dehydrogenases in barley. Physiol Plant 134:22–30. https://doi.org/10.1111/j.1399-3054.2008.01122.x Fujiwara T, Mitsuya S, Miyake H, Hattori T, Takabe T (2010) Characterization of a novel glycinebetaine/proline transporter gene expressed in the mestome sheath and lateral root cap cells in barley. Planta 232:133–143. https://doi.org/10.1007/s00425-010-1155-4 Garg AK, Kim JK, Owens TG, Ranwala AP, Do Choi Y, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Nat Acad Sci 99:15898–15903. https://doi.org/10.1073/pnas.252637799 Grallath S, Weimar T, Meyer A, Gumy C, Suter-Grotemeyer M, Neuhaus JM, Rentsch D (2005) The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns. Plant Physiol 137:117–126. https://doi.org/10.1104/pp.104.055079 Griffiths CA, Paul MJ, Foyer CH (2016) Metabolite transport and associated sugar signalling systems underpinning source/sink interactions. Biochim Biophys Acta 1857:1715–1725. https:// doi.org/10.1016/j.bbabio.2016.07.007 Gupta N, Thind SK, Bains NS (2014) Glycine betaine application modifies biochemical attributes of osmotic adjustment in drought stressed wheat. Plant Growth Regul 72:221–228. https://doi. org/10.1007/s10725-013-9853-0 Han EK, Cotty F, Sottas C, Jiang H, Michels CA (1995) Characterization of AGT1 encoding a general α-glucoside transporter from Saccharomyces. Mol Microbiol 17:1093–1107. https:// doi.org/10.1111/j.1365-2958.1995.mmi_17061093.x Harinasut P, Tsutsui K, Takabe T, Nomura M, Takabe T, Kishitani S (1996) Exogenous glycinebetaine accumulation and increased salt-tolerance in rice seedlings. Biosci Biotechnol Biochem 60:366–368. https://doi.org/10.1271/bbb.60.366 Hasanuzzaman M, Alam MM, Rehman A, Hasanuzzaman M, Nahar K, Fujita M (2014, 2014) Exogenous proline and glycine betaine upregulation of antioxidant defense and glyoxalase systems provides better protection against salt-induced oxidative stress in two rice (Oryza sativa L.) varieties. BioMed Res Inter:17. https://doi.org/10.1155/2014/757219 Hibino T, Waditee R, Araki E, Ishikawa H, Aoki K, Tanaka Y, Takabe T (2002) Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants. J Biol Chem 277:41352–41360. https://doi.org/10.1074/jbc.M205965200 Hirner B, Fischer WN, Rentsch D, Kwart M, Frommer WB (1998) Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis. Plant J 14:535–544. https://doi.org/10.1046/j.1365-313X.1998.00151.x Hirner A, Ladwig F, Stransky H, Okumoto S, Keinath M, Harms A, Frommer WB, Koch W (2006) Arabidopsis LHTI is a high affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll. Plant Cell 18:1931–1946. https://doi.org/10.1105/tpc.106.041012 Ho CL, Saito K (2001) Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana. Amino Acids 20:243–259. https://doi.org/10.1007/ s007260170042 Hussain M, Malik MA, Farooq M, Ashraf MY, Cheema MA (2008) Improving drought tolerance by exogenous application of glycinebetaine and salicylic acid in sunflower. J Agron Crop Sci 194:193–199. https://doi.org/10.1111/j.1439-037X.2008.00305.x Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 219

Igarashi Y, Yoshiba Y, Takeshita T, Nomura S, Otomo J, Yamaguchi-Shinozaki K, Shinozaki K (2000) Molecular cloning and characterization of a cDNA encoding proline transporter in rice. Plant Cell Physiol 41:750–756. https://doi.org/10.1093/pcp/41.6.750 Iqbal N, Ashraf Y, Ashraf M (2011) Modulation of endogenous levels of some key organic metabolites by exogenous application of glycine betaine in drought stressed plants of sunflower Helianthus( annuus L.). Plant Growth Regul 63:7–12. https://doi.org/10.1007/ s10725-010-9506-5 Islam MM, Hoque MA, Okuma E, Banu MNA, Shimoishi Y, Nakamura Y, Murata Y (2009) Exogenous proline and glycinebetaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells. J Plant Physiol 166:1587–1597. https://doi. org/10.1016/j.jplph.2009.04.002 Jonytienė V, Burbulis N, Kuprienė R, Blinstrubienė A (2012) Effect of exogenous proline and de- acclimation treatment on cold tolerance in Brassica napus shoots culture in vitro. J Food Agric Environ 10:327–330 Kahlaoui B, Hachicha M, Misle E, Fidalgo F, Teixeira J (2018) Physiological and biochemical responses to the exogenous application of proline of tomato plants irrigated with saline water. J Saudi Soc Agric Sci 17:17–23. https://doi.org/10.1016/j.jssas.2015.12.002 Kanamori Y, Saito A, Hagiwara-Komoda Y, Tanaka D, Mitsumasu K, Kikuta S, Watanabe M, Cornette R, Kikawada T, Okuda T (2010) The trehalose transporter 1 gene sequence is conserved in insects and encodes proteins with different kinetic properties involved in trehalose import into peripheral tissues. Insect Biochem Mol Biol 40:30e37. https://doi.org/10.1016/j. ibmb.2009.12.006 Kempf B, Bremer E (1998) Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch Microbiol 170:319–330. https://doi. org/10.1007/s002030050649 Kikawada T, Saito A, Kanamori Y, Nakahara Y, Iwata KI, Tanaka D, Watanabe M, Okuda T (2007) Trehalose transporter 1, a facilitated and high-capacity trehalose transporter, allows exogenous trehalose uptake into cells. Proc Nat Acad Sci 104:11585–11590. https://doi.org/10.1073/ pnas.0702538104 Kosar F, Akram NA, Sadiq M, Al-Qurainy F, Ashraf M (2018) Trehalose: a key organic osmolyte effectively involved in plant abiotic stress tolerance. J Plant Growth Regul:1–13. https://doi. org/10.1007/s00344-018-9876-x Krämer R (1998) Mitochondrial carrier proteins can reversibly change their transport mode: the cases of the aspartate/glutamate and the phosphate carrier. Exp Physiol 83:259–265. https:// doi.org/10.1113/expphysiol.1998.sp004111 Kwart M, Hirner B, Hummel S, Frommer WB (1993) Differential expression of two related amino acid transporters with differing substrate specificity in Arabidopsis thaliana. Plant J 4:993– 1002. https://doi.org/10.1046/j.1365-313X.1993.04060993.x Lasko PF, Brandriss MC (1981) Proline transport in Saccharomyces cerevisiae. J Bacteriol 148:241–247 Lee YH, Tegeder M (2004) Selective expression of a novel high affinity transport system for acidic and neutral amino acids in the tapetum cells of Arabidopsis flowers. Plant J 40:60–74. https:// doi.org/10.1111/j.1365-313X.2004.02186.x Lee YH, Foster J, Chen J, Voll LM, Weber APM, Tegeder M (2007) AAP1 transports uncharged amino acids into roots of Arabidopsis. Plant J 50:305–319. https://doi.org/10.1111/j.1365- 313X.2007.03045.x Lehmann S, Funck D, Szabados L, Rentsch D (2010) Proline metabolism and transport in plant development. Amino Acids 39:949–962. https://doi.org/10.1111/j.1365-313X.2004.02186.x LiXin Z, ShengXiu L, ZongSuo L (2009) Differential plant growth and osmotic effects of two maize (Zea mays L.) cultivars to exogenous glycinebetaine application under drought stress. Plant Growth Regul 58:297–305. https://doi.org/10.1007/s10725-009-9379-7 220 S. Cha-um et al.

Luo Y, Li F, Wang GP, Yang XH, Wang W (2010) Exogenously-supplied trehalose protects thylakoid membranes of winter wheat from heat-induced damage. Biol Plant 54:495–501. https:// doi.org/10.1007/s10535-010-0087-y Ma QQ, Wang W, Li YH, Li DQ, Zou Q (2006) Alleviation of photoinhibition in drought-stressed wheat (Triticum aestivum) by foliar-applied glycinebetaine. J Plant Physiol 163:165–175. https://doi.org/10.1016/j.jplph.2005.04.023 Ma XL, Wang YJ, Xie SL, Wang C, Wang W (2007) Glycinebetaine application ameliorates negative effects of drought stress in tobacco. Russ J Plant Physiol 54:472–479. https://doi. org/10.1134/S1021443707040061 Ma C, Wang Z, Kong B, Lin T (2013) Exogenous trehalose differentially modulate antioxidant defense system in wheat callus during water deficit and subsequent recovery. Plant Growth Regul 70:275–285. https://doi.org/10.1007/s10725-013-9799-2 Mäkelä P (2004) Agro -industrial uses of glycinebetaine. Sugar Technol 6:207–212. https://doi. org/10.1007/BF02942500 Mäkelä P, Kärkkäinen J, Somersalo S (2000) Effect of glycinebetaine on chloroplast ultrastructure, chlorophyll and protein content, and RuBPCO activities in tomato grown under drought or salinity. Biol Plant 43:471–475. https://doi.org/10.1023/A:1026712426180 Mitsuya S, Kuwahara J, Ozaki K, Saeki E, Fujiwara T, Takabe T (2011) Isolation and characterization of a novel peroxisomal choline monooxygenase in barley. Planta 234:1215–1226. https:// doi.org/10.1007/s00425-011-1478-9 Morbach S, Kramer R (2002) Body shaping under water stress: osmosensing and osmoregulation of solute transport in bacteria. Chem Biol Chem 3:384–397. https://doi. org/10.1002/1439-7633(20020503)3:5<384::AID-CBIC384>3.0.CO;2-H Mostafa MG, Hossain MA, Fujita M, Tran LSP (2015) Physiological and biochemical mechanisms associated with trehalose-induced copper-stress tolerance in rice. Sci Rep 5:11433. https://doi. org/10.1038/srep11433 Moustakas M, Sperdouli I, Kouna T, Antonopoulou CI, Therios I (2011) Exogenous proline induces soluble sugar accumulation and alleviates drought stress effects on photosystem II functioning of Arabidopsis thaliana leaves. Plant Growth Regul 65:315. https://doi.org/10.1007/ s10725-011-9604-z Mueckler M (1994) Facilitative glucose transporters. Eur J Biochem 219:713e725. https://doi. org/10.1111/j.1432-1033.1994.tb18550.x Nounjan N, Nghia PT, Theerakulpisut P (2012) Exogenous proline and trehalose promote recovery of rice seedlings from salt-stress and differentially modulate antioxidant enzymes and expression of related genes. J Plant Physiol 169:596–604. https://doi.org/10.1016/j.jplph.2012.01.004 Nuccio ML, McNeil SD, Ziemak MJ, Hanson AD, Jain RK, Selvaraj G (2000) Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway. Met Eng 2:300–311. https://doi.org/10.1006/ mben.2000.0158 Okumoto S, Schmidt R, Tegeder M, Fischer WN, Rentsch D, Frommer WB, Koch W (2002) High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis. J Biol Chem 277:45338–45346. https://doi.org/10.1074/jbc.M207730200 Okumoto S, Koch W, Tegeder M, Fischer WN, Biehl A, Leister D, Stierhof YD, Frommer WB (2004) Root phloem-specific expression of the plasma membrane amino acid proton cotrans- porter AAP3. J Exp Bot 55:2155–2168. https://doi.org/10.1093/jxb/erh233 Osman HS (2015) Enhancing antioxidant-yield relationship of pea plant under drought at different growth stages by exogenously applied glycine betaine and proline. Ann Agric Sci 60:389–402. https://doi.org/10.1016/j.aoas.2015.10.004 Ozden M, Demirel U, Karaman A (2009) Effects of proline on antioxidant system in leaves of

grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2. Sci Hortic 119:163–168. https://doi.org/10.1016/j.scienta.2008.07.031 Pao SS, Paulsen IT, Saier MH Jr (1998) Major facilitator superfamily. Microbiol Mol Biol Rev 62:1–34 Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 221

Park EJ, Jeknić Z, Chen HH (2006) Exogenous application of glycinebetaine increases chilling tolerance in tomato plants. Plant Cell Physiol 47:706–714. https://doi.org/10.1093/pcp/pcj041 Paul MJ, Oszvald M, Jesus C, Rajulu C, Griffiths CA (2017) Increasing crop yield and resilience with trehalose 6-phosphate: targeting a feast–famine mechanism in cereals for better source– sink optimization. J Exp Bot 68:4455–4462. https://doi.org/10.1093/jxb/erx083 Raza MAS, Saleem MF, Shah GM, Khan IH, Raza A (2014) Exogenous application of glycinebetaine and potassium for improving water relations and grain yield of wheat under drought. J Soil Sci Plant Nutr 14:348–364. https://doi.org/10.4067/S0718-95162014005000028 Rentsch D, Hirner B, Schmelzer E, Frommer WB (1996) Salt stress induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant. Plant Cell 8:1437–1446. https://doi.org/10.1105/ tpc.8.8.1437 Rentsch D, Schmidt S, Tegeder M (2007) Transporters for uptake and allocation of organic nitrogen compounds in plants. FEBS Lett 581:2281–2289. https://doi.org/10.1016/j.febslet.2007.04.013 Rezaei MA, Jokar I, Ghorbanli M, Kaviani B, Kharabian-Masouleh A (2012) Morpho-physiological improving effects of exogenous glycine betaine on tomato (Lycopersicum esculentum Mill.) cv. PS under drought stress conditions. Plants Omics J 5:79–86 Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Ann Rev Plant Physiol Plant Mol Biol 44:357–384. https://doi.org/10.1146/annurev. pp.44.060193.002041 Roeβler M, Müller V (2001) Osmoadaptation in bacteria and archaea: common principles and differences. Environ Microbiol 3:743–754. https://doi.org/10.1046/j.1462-2920.2001.00252.x Rohman MM, Begum S, Akhi AH, Ahsan AFMS, Uddin MS, Amiruzzaman M, Banik BR (2015) Protective role of antioxidants in maize seedlings under saline stress: exogenous proline provided better tolerance than betaine. Bothalia J 45:17–35 Rolletschek H, Hosein F, Miranda M, Heim U, Gotz KP, Schlereth A, Borisjuk L, Saalbach I, Wobus U, Weber H (2005) Ectopic expression of an amino acid transporter (VfAAPI) in seeds of Vicia narbonensis and pea increases storage proteins. Plant Physiol 137:1236–1249. https:// doi.org/10.1104/pp.104.056523 Redillas MCFR, Jeong JS, Kim YS, Jung H, Bang SW, Choi YD, Ha S-H, Reuzeau C, Kim J-K (2012) The overexpression of alters the root architecture of rice plants enhancing drought resistance and grain yield under field conditions. Plant Biotechnol J 10(7):792–805 Sanders A, Collier R, Trethewy A, Gould G, Sieker R, Tegeder M (2009) AAP1 regulates import of amino acids into developing Arabidopsis embryos. Plant J 59:540–552. https://doi. org/10.1111/j.1365-313X.2009.03890.x Schmidt R, Stransky H, Koch W (2007) The amino acid permease AAP8 is important for early seed development in Arabidopsis thaliana. Planta 226:805–813. https://doi.org/10.1007/ s00425-007-0527-x Schwacke R, Grallath S, Breitkreuz KE, Stransky E, Stransky H, Frommer WB, Rentsch D (1999) LeProT1, a transporter for proline, glycine betaine, and γ-amino butyric acid in tomato pollen. Plant Cell 11:377–392. https://doi.org/10.1105/tpc.11.3.377 Shahid MA, Balal RM, Pervez MA, Abbas T, Aqeel MJ, Javaid MM, Gacia-Sanchez F (2014) Exogenous proline and proline-enriched Lolium perenne leaf extract protects against phytotoxic effects of nickel and salinity in Pisum sativum by altering polyamine metabolism in leaves. Tuk J Bot 38:914–926. https://doi.org/10.3906/bot-1312-13 Singh M, Singh VP, Dubey G, Prasad SM (2015) Exogenous proline application ameliorates toxic effects of arsenate in Solanum melongena L. seedlings. Ecotoxicol Environ Safe 117:164–173. https://doi.org/10.1016/j.ecoenv.2015.03.021 Sorkheh K, Shiran B, Khodambashi M, Rouhi V, Mosavei S, Sofo A (2012) Exogenous proline

alleviates the effects of H2O2-induced oxidative stress in wild almond species. Russ J Plant Physiol 59:788–798. https://doi.org/10.1134/S1021443712060167 222 S. Cha-um et al.

Stambuk BU, Panek AD, Crowe JH, Crowe LM, de Araujo PS (1998) Expression of high-affinity trehalose–H+ symport in Saccharomyces cerevisiae. Biochim et Biophys Acta -Gen Sub 1379:118–128. https://doi.org/10.1016/S0304-4165(97)00087-1 Szabados L, Savoure A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97. https://doi.org/10.1016/j.tplants.2009.11.009 Tabuchi T, Okada T, Takashima Y, Azuma T, Nanmori T, Yasuda T (2006) Transcriptional response of glycinebetaine-related genes to salt stress and light in leaf beet. Plant Biotechnol 23:317– 320. https://doi.org/10.5511/plantbiotechnology.23.317 Tanner J (2008) Structural biology of proline catabolism. Amino Acids 35:719–730. https://doi. org/10.1007/s00726-008-0062-5 Tapia H, Young L, Fox D, Bertozzi CR, Koshland D (2015) Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 112:6122–6127. https://doi.org/10.1073/pnas.1506415112 Teh CH, Mahmood M, Shaharuddin NA, Ho CL (2015) In vitro shoot apices as simple model to study the effect of NaCl and the potential of exogenous proline and glutathione in mitigating salinity stress. Plant Growth Regul 75:771–781. https://doi.org/10.1007/s10725-014-9980-2 Ueda A, Shi W, Sanmiya K, Shono M, Takabe T (2001) Functional analysis of salt-inducible proline transporter of barley roots. Plant Cell Physiol 42:1282–1289. https://doi.org/10.1093/pcp/ pce166 Ueda A, Yamamoto-Yamane Y, Takabe T (2007) Salt stress enhances proline utilization in the apical region of barley roots. Biochem Biophys Res Commun 355:61–66. https://doi.org/10.1016/j. bbrc.2007.01.098 Ueda A, Shi W, Shimada T, Miyake H, Takabe T (2008) Altered expression of barley proline transporter causes different growth responses in Arabidopsis. Planta 227:277–286. https://doi. org/10.1007/s00425-007-0615-y Verslues PE, Sharp RE (1999) Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. II. Metabolic source of increased proline deposition in the elongation zone. Plant Physiol 119:1349–1360. https://doi.org/10.1104/pp.119.4.1349 Waditee R, Hibino T, Tanaka Y, Nakamura T, Incharoensakdi A, Hayakawa S, Suzuki S, Futsuhara Y, Kawamitsu Y, Takabe T, Takabe T (2002) Functional characterization of betaine/proline transporters in betaine-accumulating mangrove. J Biol Chem 277:18373–18382. https://doi. org/10.1074/jbc.M112012200 Waditee R, Bhuiyan MNH, Rai V, Aoki K, Tanaka Y, Hibino T, Suzuki S, Takano J, Jagendorf AT, Takabe T, Takabe T (2005) Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc Nat Acad Sci 102:1318–1323. https://doi.org/10.1073/pnas.0409017102 Waditee R, Bhuiyan NH, Hirata E, Hibino T, Tanaka Y, Shikata M, Takabe T (2007) Metabolic engineering for betaine accumulation in microbes and plants. J Biol Chem 282:34185–34193. https://doi.org/10.1074/jbc.M704939200 Weigelt K, Kiister H, Radchuk R, Miiller M, Weichert H, Fait A, Fernie AR, Saalbach I, Weber H (2008) Increasing amino acid supply in pea embryos reveals specific interactions of N and C metabolism, and highlights the importance of mitochondrial metabolism. Plant J 55:909–926. https://doi.org/10.1111/j.1365-313X.2008.03560.x Wood JM (2006) Osmosensing by bacteria. Sci STKE 357:pe43. https://doi.org/10.1126/ stke.3572006pe43 Wood IS, Trayhurn P (2003) Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. British J Nutr 89:3e9. https://doi.org/10.1079/BJN2002763 Wood JM, Bremer E, Csonka LN, Kraemer R, Poolman B, van der Heide T, Smith LT (2001) Osmosensing and osmoregulatory compatible solute accumulation by bacteria. Comp Biochem Physiol A Mol Integr Physiol 130:437–460. https://doi.org/10.1016/S1095-6433(01)00442-1 Wyn Jones RG, Storey R (1981) Betaines. In: Paleg LG, Aspinall D (eds) The physiology and biochemistry of drought resistance in plants, Academic Press, Sydney, pp 171–204 Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants… 223

Xing W, Rajashekar CB (1999) Alleviation of water stress in beans by exogenous glycine betaine. Plant Sci 48:185–195. https://doi.org/10.1016/S0168-9452(99)00137-5 Yamada N, Promden W, Yamane K, Tamagake H, Hibino T, Tanaka Y, Takabe T (2009) Preferential accumulation of betaine uncoupled to choline monooxygenase in young leaves of sugar beet– importance of long-distance translocation of betaine under normal and salt-stressed conditions. J Plant Physiol 166:2058–2070. https://doi.org/10.1016/j.jplph.2009.06.016 Yamada N, Cha-um S, Kageyama H, Promden W, Tanaka Y, Kirdmanee C, Takabe T (2011a) Isolation and characterization of proline/betaine transporter gene from oil palm. Tree Physiol 31:462–468. https://doi.org/10.1093/treephys/tpr017 Yamada N, Sakakibara S, Tsutsumi K, Waditee R, Tanaka Y, Takabe T (2011b) Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet. J Plant Physiol 168:1609–1616. https://doi.org/10.1016/j.jplph.2011.03.007 Yang L, Zhao X, Zhu H, Paul M, Zu Y, Tang Z (2014) Exogenous trehalose largely alleviates ionic unbalance, ROS bust, and PCD occurrence induced by high salinity in Arabidopsis seedlings. Front Plant Sci 5:570. https://doi.org/10.3389/fpls.2014.00570 Zhang M, Huang H, Dai S (2014) Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium. Gene 537:203–213. https://doi.org/10.1016/j. gene.2014.01.002 Zhao XX, Ma QQ, Liang C, Fang Y, Wang YQ, Wang W (2007) Effect of glycinebetaine on function of thylakoid membranes in wheat flag leaves under drought stress. Biol Plant 51:584–588. https://doi.org/10.1007/s10535-007-0128-3 Zheng JL, Zhao LY, Wu CW, Shen B, Zhu AY (2015) Exogenous proline reduces NaCl-induced damage by mediating ionic and osmotic adjustment and enhancing antioxidant defense in Eurya emarginata. Acta Physiol Plant 37:181. https://doi.org/10.1007/s11738-015-1921-9 Zhou Y, Zhu W, Bellur PS, Rewinkel D, Becker DF (2008) Direct linking of metabolism and gene expression in the proline utilization a protein from Escherichia coli. Amino Acids 35:711–718. https://doi.org/10.1007/s00726-008-0053-6 Zouari M, Ahmed CB, Elloumi N, Bellassoued K, Delmail D, Labrousse P, Abdallah FB, Rouina BB (2016a) Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress. Ecotoxicol Environ Safe 128:195–205. https://doi.org/10.1016/j.ecoenv.2016.02.024 Zouari M, Ahmed CB, Zorrig W, Elloumi N, Delmail D, Rouina BB, Labrousse P, Abdallah FB (2016b) Exogenous proline mediates alleviation of cadmium stress by promoting photosynthetic activity, water status and antioxidative enzymes activities of young date palm (Phoenix dactylifera L.). Ecotoxicol Environ Safe 128:100–108. https://doi.org/10.1016/j.ecoenv.2016.02.015 Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress Tolerance in Plants

Zsófia Bánfalvi

1 Introduction

A common response to water deficit is the accumulation of osmoprotectants such as sugars and amino acids. High concentrations of disaccharide trehalose occur in many anhydrobiotic organisms that survive complete dehydration and can be found in a relatively large amount in some resurrection plants. Accumulation of sucrose and trehalose during desiccation is proposed to prevent protein denaturation and membrane fusion (Suprasanna et al. 2016). In the yeast Saccharomyces cerevisiae, stress or starvation can lead to variations in trehalose content to over 20% of the cell dry weight. The most common pathway to produce trehalose involves two enzymes: trehalose-6-phosphate synthase (TPS), which catalyses the synthesis of trehalose-6- phosphate (T6P) from glucose-6-phosphate (G6P) and UDP-glucose (UDPG), and trehalose-6-phosphate phosphatase (TPP), which dephosphorylates T6P to trehalose. This pathway is found in a great variety of species, like insects, Escherichia coli and Mycobacterium tuberculosis and some species of nematodes and plants (Eleutherio et al. 2015). In higher plants, however, trehalose is generally present in a low abundance. To improve abiotic stress tolerance of plants, several different approaches were used to increase the trehalose content of plants.

2 Abiotic Stress Tolerance with Phenotypic Alterations

To increase the trehalose content of tobacco, as a model plant, Holmström et al. (1996) transformed and expressed the TPS1 gene of yeast by the Rubisco promoter in tobacco (Nicotiana tabacum). Leaves of transgenic plants contained 0.8–3.2 mg

Z. Bánfalvi (*) NARIC, Agricultural Biotechnology Institute, Gödöllő, Hungary e-mail: [email protected]

© Springer Nature Switzerland AG 2019 225 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_10 226 Z. Bánfalvi trehalose per g dry weight in contrast to 0.06 mg/g in non-transformed plants. T6P was not found in plants, suggesting that a tobacco enzyme dephosphorylated T6P to trehalose. When detached leaves were air-dried, those from trehalose-producing plants lost water more slowly than control leaves. When seedlings were drought- stressed, the trehalose-producing plants recovered turgor and recommenced growth, while the controls died. Nevertheless, TPS1 expression decreased the growth rate of the plants by 30–50% under optimal growth conditions. Constitutive expression of yeast TPS1 from the CaMV35S promoter also improved the drought tolerance of tobacco and, however, resulted in the same phenotypical changes as its expression by the Rubisco promoter (Romero et al. 1997; Lee et al. 2003; Karim et al. 2007). In prokaryotes, trehalose is frequently used as a compatible solute to contend with osmotic stress and can be used as an external carbon source. In Escherichia coli, trehalose biosynthesis reactions are catalysed also by TPS and TPP, encoded by the otsA and otsB gene, respectively (Giaever et al. 1988). Constitutive expression of otsA or otsA and otsB, like that of yeast TPS1, resulted in drought-tolerant tobacco plants. Nevertheless, these transgenic plants were similar also in phenotype to those with constitutive expression of yeast TPS1 because they were characterised by stunted growth and lancet-shaped leaves (Pilon-Smits et al. 1998; Jun et al. 2005). The situation was similar in Chinese cabbage (Brassica rapa subsp. pekinensis) expressing otsA. Although the transgenic Chinese cabbage plants were drought, salt and heat tolerant, they showed stunted growth and aberrant root development (Park et al. 2003). Constitutive expression of TPS1 with plant, namely, Arabidopsis origin in tobacco, had a similar effect on growth of the plants. However, it was demonstrated that these plants were not even drought but also salt and temperature tolerant and showed better acclimation to cadmium and excess copper compared to nontransformed control (Almeida et al. 2005, 2007a; Martins et al. 2014). Constitutive expression of yeast TPS1 by either Rubisco (RbcS1) or CaMV35S promoter in Arabidopsis also increased the tolerance of plants to different stresses, like drought, salt, freezing and heat, but also had negative effect on growth and root development (Karim et al. 2007; Miranda et al. 2007). The situation was the same in potato (Solanum tuberosum) and tomato (Lycopersicon esculentum), in which expression of yeast TPS1 by the CaMV35S promoter improved drought, salt and oxidative stress tolerance but the tomato plants had thick shoots, rigid dark-green leaves and aberrant root development (Yeo et al. 2000; Cortina and Culianez-Macia 2005). It should be noted, however, that potato plants showing altered phenotype in vitro recovered when planted in soil (Yeo et al. 2000). To avoid the negative effects of constitutive yeast TPS1 expression, Zhao et al. (2000) and Stiller et al. (2008) expressed TPS1 from a drought-inducible promoter in tobacco and potato, respectively. Both transgenic tobaccos and potatoes demonstrated increased drought tolerance under drought stress. Despite the drought- inducible nature of the promoters, a few transgenic tobacco plants had some obvious morphological changes including dwarf and fine shoot, lancet-shaped leaves and vigorous auxiliary buds, while the potato plants were smaller than the nontransformed control plants. These results indicated that tobacco and potato are so Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress… 227 sensitive to expression of yeast TPS1 that even a low, basic activity of an inducible promoter can lead to phenotypical changes. It was known that in the basidiomycete Grifola frondosa, trehalose is synthesised directly from D-glucose and α-glucose 1-phosphate catalysed by trehalose synthase (TSase). To avoid potential regulatory problems with the introduction of multiple genes and expecting a higher catalytic efficiency for trehalose synthesis, Zhang et al. (2005) expressed the G. frondosa TSase gene by the CaMV35S promoter for manipulating abiotic stress tolerance in tobacco. These transgenic plants, however, also had obvious morphological changes, including thick and deep-coloured leaves, but showed no growth inhibition. However, the morphological changes disappeared in T2 progenies. Results are summarised in Table 1.

3 Abiotic Stress Tolerance Without Phenotypic Alterations

Unlike expression of yeast TPS1 from the drought-inducible promoters Prd29A and StDS2 (Zhao et al. 2000; Stiller et al. 2008), fusion of the gene to the drought- inducible Arabidopsis promoter RAB18 clearly retained its drought stress responsiveness in tobacco and led to drought-tolerant transgenic plants with normal growth phenotype (Karim et al. 2007). Expression of yeast TPS1 together with the yeast phosphatase TPP or in fusion with TPP not only improved the drought tolerance of plants but raised the morphological aberrations even when expressed by constitutive promoters both in tobacco and Arabidopsis (Karim et al. 2007; Miranda et al. 2007). Fusion of otsA with otsB had the same effect in rice (Oryza sativa subsp. indica) and tomato (Garg et al. 2002; Jang et al. 2003; Lyu et al. 2013). By fusing the TPS1 and TPP genes of Zygosaccharomyces rouxii, the drought tolerance of potato could be improved when expressing the fused genes by the CaMV35S promoter (Kwon et al. 2004). An alternative solution was the transformation of chloroplast by yeast TPS1 or targeting it into the chloroplast with a transit peptide in front of the TPS1 coding sequence. In this way, osmotic and drought stress-tolerant tobacco and Arabidopsis lines were isolated with constitutive expression of TPS1 in chloroplasts (Lee et al. 2003; Karim et al. 2007). A similar result was obtained when otsA-otsB was targeted into rice chloroplasts (Garg et al. 2002). Overexpression of trehalose biosynthetic genes from plants may also be a good solution to overcome the problem of phenotypic alterations elicited by the E. coli or yeast TPS genes. For example, transgenic Arabidopsis plants overexpressing their own TPS1 gene displayed a dehydration tolerance phenotype without any visible morphological alterations, except for delayed flowering (Avonce et al. 2004). Another good example is the expression of TPS gene of Zostera marina in rice. Z. marina is a kind of seed plant growing in seawater during its whole life history. Expressing its TPS gene by the CaMV35S promoter in rice resulted in salt-tolerant transgenic lines (Zhao et al. 2013). Another example is the constitutive expression 228 Z. Bánfalvi

Table 1 Stress-tolerant transgenic plants overexpressing trehalose biosynthetic genes with phenotypical changes Species Promoter Gene(s) Tolerance Changes in phenotype Reference Nicotiana RbcS1 TPS1 Yeast Drought 30–50% decreased Holmström tabacum growth rate et al. (1996) CaMV35S TPS1 Yeast Drought Stunted growth, Romero et al. lancet-shaped leaves (1997) CaMV35S otsA, otsB Drought Stunted growth, more Pilon-Smits E. coli leaves and larger leaf et al. (1998) area Prd29A TPS1 Yeast Drought Dwarf and fine shoot, Zhao et al. lancet-shaped leaves (2000) CaMV35S TPS1 Yeast Osmotic stress Stunted growth, Lee et al. sterility (2003) CaMV35S otsA E. coli Drought, salt Stunted growth, Jun et al. lancet-shaped leaves (2005) CaMV35S TSase Drought, salt Thick and deep- Zhang et al. Grifola coloured leaves (2005) CaMV35S TPS1 Yeast Drought Delayed growth Karim et al. (2007) CaMV35S TPS1 Drought, salt, Stunted growth Almeida et al. Arabidopsis temperature (2005, 2007a) CaMV35S TPS1 Cu, Cd Stunted growth Martins et al. Arabidopsis (2014) Arabidopsis RbcS1A TPS1 Yeast Drought Retarded growth and Karim et al. thaliana root development (2007) CaMV35S TPS1 Yeast Drought, salt, Aberrant growth, Miranda et al. freezing, heat colour and shape (2007) Solanum CaMV35S TPS1 Yeast Drought Dwarfish growth, Yeo et al. tuberosum lancet-shaped leaves, (2000) aberrant root (in vitro); recovered in soil StDS2 TPS1 Yeast Drought Smaller plants Stiller et al. (2008) Lycopersicon CaMV35S TPS1 Yeast Drought, salt, Thick shoots, rigid Cortina and esculentum oxidative dark-green leaves, Culianez- stress aberrant root Macia (2005) development Brassica CaMV35S otsA E. coli Drought, salt, Stunted growth and Park et al. rapa heat aberrant root (2003) development of TPS1 gene of cassava (Manihot esculenta Crantz) in tobacco leading to improved drought stress tolerance (Han et al. 2016). Nevertheless, overexpression of a plant TPS is not a guarantee of success. For example, TPS11 gene of cotton (Gossypium hirsutum), which is heat, drought and salt inducible, increased not the chilling stress tolerance but the chilling stress sensitivity of transgenic Arabidopsis seeds probably via the expression changes of some chilling-related genes (Wang et al. 2016). Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress… 229

In various microorganisms including green algae and various fungi, trehalose phosphorylase (TP) produces trehalose by glucose-1-phosphate and glucose. When the TP gene of Pleurotus sajor-caju (oyster mushroom, one of the most commonly cultivated edible mushrooms in the world) was introduced and constitutively expressed in tobacco, it became drought tolerant (Han et al. 2005). In some bacteria, the biosynthesis of trehalose is mediated by maltooligosyl trehalose synthase (MTS) and maltooligosyl trehalose trehalohydrolase (MTH). In this pathway, T6P is not generated as an intermediate. One nonpathogenic bacterium, Brevibacterium helvolum, is known to contain these two enzymes. Joo et al. (2014) generated transgenic rice plants that overexpressed the bifunctional in-frame fusion gene of MTS and MTH and found that transgenic rice overexpressing the fused genes is drought stress tolerant without growth aberrations. Cassava (Manihot esculenta) is a perennial dicotyledonous crop belonging to the family of Euphorbiaceae. Its starchy tuberous roots have global importance as food and feed. Cassava is highly tolerant to abiotic stress and can be grown on arid and marginal land. Recently, unexpected high levels of trehalose in all tested cassava varieties have been detected may be due to the constant expression of a key active TPS gene. Transgenic tobacco lines constitutively expressing this gene accumulated significant level of trehalose and possessed improved drought stress tolerance (Han et al. 2016). Rice possesses several TPS genes divided in two classes: class I genes encoding enzymes with TPS activity and class II genes encoding proteins with no TPS activity. TPS1 in rice belongs to class I. N-terminal truncated rice TPS1 lacking the region just before the TPS domain has increased TPS activity compared to the full length protein. When it was overexpressed in transgenic rice, the truncated protein enhanced the tolerance to drought, salt and cold. To investigate the function of class II TPS genes, TPS2, 4, 5, 8 and 9 were overexpressed in rice. All of these class II TPSs improved the tolerance of rice to cold and salinity stress, and some of them also improved the drought tolerance of transgenic rice seedlings (Li et al. 2011). Vishal et al. (2019) supported Li and co-workers’ (2011) finding because it showed that overexpression of rice TPS8 was adequate to confer enhanced salinity tolerance of rice without any yield penalty, suggesting its usefulness in rice genetic improvement. Trehalose is hydrolysed to glucose by trehalase. TRE1 encodes the Arabidopsis trehalase. TRE1-overexpressing Arabidopsis plants had decreased trehalose levels and interestingly recovered better after drought stress. Leaf detachment assays showed that TRE1-overexpressing plants have a better water-retaining capacity (Van Houtte et al. 2013). Transgenic rice plants overexpressing the trehalase gene showed remarkable increases in trehalase activity and dramatic decreases in trehalose abundance compared with the wild type. The TRE1 overexpressors did not have notable morphological alterations or growth defects but interestingly exhibited enhanced salt tolerance, suggesting the involvement of TRE1 in salt stress tolerance in rice (Islam et al. 2019). Results are summarised in Table 2. 230 Z. Bánfalvi

Table 2 Stress-tolerant transgenic plants overexpressing trehalose biosynthetic genes without phenotypical changes Species Promoter Gene(s) Tolerance Reference Nicotiana CaMV35S TPS1 Osmotic stress Lee et al. tabacum Yeast (2003) Chloroplast transformation CaMV35S TP Drought Han et al. Pleurotus (2005) RbcS1A TPS1, TPP Drought Karim et al. Yeast (2007) RAB18 TPS1 Drought Yeast CaMV35S TPS1 Drought Han et al. Cassava (2016) Arabidopsis CaMV35S TPS1 Drought Avonce et al. thaliana Arabidopsis (2004) RbcS1A TPS1 Drought, fast recovery of Karim et al. Yeast dehydrated leaves (2007) Chloroplast targeting CaMV35S TPS1-TPP Drought, salt, freezing, Miranda et al. Yeast heat (2007) rd29A TPS1-TPP Drought, salt, freezing, Yeast heat CaMV35S TRE1 Drought Van Houtte Arabidopsis et al. (2013) CaMV35S TPS11 Chilling stress sensitivity Wang et al. Cotton (2016) Solanum CaMV35S TPS1-TPP Drought Kwon et al. tuberosum Zygosaccharomyces (2004) Lycopersicon CaMV35S otsA-otsB Drought, salt, heat Lyu et al. esculentum E. coli (2013, 2018) Oryza ABA- otsA-otsB Drought, salt, cold Garg et al. sativa inducible E. coli (2002) RbcS otsA-otsB Drought, salt, cold E. coli Chloroplast targeting Ubi1 otsA-otsB Drought, salt, cold Jang et al. E. coli (2003) CaMV35S TPP1 Salt, cold Ge et al. (2008) Rice CaMV35S TPS1 (class I) Drought, salt, cold Li et al. (2011) Rice CaMV35S TPS (class II) Salt, cold Rice 101MTSH MTS-MTH Drought Joo et al. (2014) Brevibacterium (continued) Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress… 231

Table 2 (continued) Species Promoter Gene(s) Tolerance Reference 105MTSH MTS-MTH Drought Brevibacterium CaMV35S TPS Salt Zhao et al. Zostera (2013) Ubi1 TRE1 Salt Islam et al. Rice (2019) OsTPS8 TPS8 Salt Vishal et al. Rice (2019) Zea rd29A TPS1 Drought Liu et al. (2015) mays Yeast MADS6 TPP Drought (field) Nuccio et al. Rice (2015)

4 The Role of Stomata in Drought Tolerance

The water status of a plant is mainly regulated by the opening and closing of stomata. To avoid desiccation and ultimate death, stomata typically close during periods of water stress. However, stomata are the main gateways not only for water transpiration but also for photosynthetic CO2 exchange. Drought stress is known to depress gas exchange characteristics to a varying extent thereby affecting overall photosynthetic capacity of most plants. As drought continues, the stomata closure occurs for longer periods during the day. This, in turn, leads to the reduced carbon assimilation rate and water loss, resulting in maintenance of the carbon assimilation at the cost of low water availability with a final outcome in reduction of growth, biomass and yield (Ashraf and Harris 2013). Just a few months after publication by Holmström et al. (1996) on drought- tolerant, but growth-retarded tobacco plants expressing the yeast TPS1 gene, Gaff (1996) raised the possibility of relating the phenotype of transgenic plants to altered stomatal movement. According to Gaff’s hypothesis, the transgenic plants might pass into the slow water-loss phase at higher fresh weights than non-transgenic plants, and thus transgenic stomata commenced closing at milder drought stress. As a result, water was retained for a longer time. During plant cultivation then, CO2 supply for photosynthesis would be restricted more frequently by stomatal closure, causing slower growth in the transgenic plants. Although no publication on the stomatal movement of TPS1 transgenic tobacco plants generated by Holmström et al. (1996) appeared in the literature to support or disprove Gaff’s theory, the results obtained by Stiller et al. (2008) in potato and Liu et al. (2015) in maize demonstrate a correlation between the number of stomata, drought tolerance and stunted growth of plants. In the case of TPS1 transgenic potatoes, detached leaves showed an 8-hour delay in wilting and a 30–40% reduction in stomatal densities compared to the non-transformed control, while under optimal growth conditions, lower CO2 fixation was detected in the smaller transgenic than in the larger control plants. The stomata density on the leaf surface of the TPS1 232 Z. Bánfalvi transgenic maize was 20% lower than that of the wild-type plants (Liu et al. 2015). Stomatal development in the Arabidopsis model includes a signalling pathway with TMM, MAPK3, MAPK6 and SDD1 that negatively regulates the basal pathway of stomata lineage (Casson and Hetherington 2010). The expression of TMM, MAPK3, MAPK6 and SDD1 was increased in TPS1 transgenic maize (Zea mays) plants, indicating that TPS1 can negatively regulate stomata development and reduce the number of stomata to improve drought tolerance (Liu et al. 2015). In vitro studies revealed that trehalase TRE1-overexpressing, drought-tolerant Arabidopsis plants are more sensitive towards abscisic acid (ABA)-dependent stomatal closure. This observation is further supported by the altered leaf temperatures seen in trehalase-modified plantlets during in vivo drought stress studies (Van Houtte et al. 2013).

5 Transcriptional Changes Associated with Abiotic Stress Tolerance

Detection of transcriptional changes in abiotic stress-tolerant plants based on alteration in a trehalose biosynthetic pathway goes back to 2004, when the findings by Avonce et al. suggested that TPS1 in Arabidopsis has a pivotal role in the regulation of glucose and ABA signalling during vegetative development. The transgenic Arabidopsis plants overexpressing the Arabidopsis TPS1 gene showed up-regulation of the ABI4 and CAB1 genes. In the presence of glucose, CAB1 expression remained high, whereas ABI4, HXK1 and ApL3 levels were downregulated in the TPS1 overexpressing lines. Analysis of TPS1 expression in HXK1-antisense or HXK1-sense transgenic lines suggested the possible involvement of TPS1 in the hexokinase-dependent glucose signalling pathway in Arabidopsis. To obtain a general picture on gene expression influenced by the trehalose pathway genes, microarray analyses of transgenic Arabidopsis seedlings constitutively expressing otsA to elevate T6P level and otsB to decrease T6P level were performed. Analysis of microarray data showed up-regulation by T6P of genes involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle and mitochondrial electron transport, which are normally downregulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were downregulated by T6P (Zhang et al. 2009). SnRKs are evolutionarily conserved SNF1- related kinase complexes. SnRK1 is a key regulator in adjusting cellular metabolism during starvation, stress conditions and growth-promoting conditions. A number of different transcription factors have been identified recently as direct SnRK1 targets in plants (Wurzinger et al. 2018). Thus, we can conclude that T6P inhibits SnRK1 to activate biosynthetic processes, at least in part, via transcription factors. Another interesting finding highlighting the intimate connection between TPS1 and SnRK1 was obtained by Antal et al. (2013) demonstrating that repression of the regulatory Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress… 233 subunit of the SnRK1 complex attenuates growth aberrations caused by the yeast TPS1 expression in potato. These plants, however, behaved like wild type in detached leaf assays. It therefore seems likely that the yeast TPS1-triggered morphological changes are indispensable for increased drought tolerance in potato. Kondrák et al. (2011) analysed the transcriptome of potato leaves with a low level of yeast TPS1 expression, which improved drought tolerance, but slower growth and less stomata than in the wild type (Stiller et al. 2008). Even under optimal growth conditions, 74 and 25 genes were up- and downregulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild type. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes were involved in transcriptional and post-transcriptional regulation. By comparing the effect of otsA in Arabidopsis (Zhang et al. 2009) with that of yeast TPS1 in potato, expression of 22 genes was changed in both plants. However, 10 of these displayed an inverse regulation (Kondrák et al. 2011). Under drought stress conditions, wild-type potato leaves had twice as many genes with altered expression than TPS1 transgenic leaves, but 112 genes were differentially expressed in both strains (Kondrák et al. 2012). In transgenic rice lines overexpressing their own TPS1, some stress-related genes were up-regulated, including WSI18 (water stress-inducible protein), RAB16C (responsive to ABA), HSP70 (heat shock protein), DHN6 (dehydrin), LEA14A (late embryogenesis abundant protein) and ELIP (early light-inducible protein). These results demonstrate that rice TPS1 may enhance the abiotic stress tolerance of plants by regulating the expression of stress-related genes (Li et al. 2011). The salt-tolerant rice TPS8 expressing rice lines is ABA sensitive. TPS8 affects the gene expression level of ABA-responsive genes (RAB21, RAB16C, Xd422) and SnRK2s (Vishal et al. 2019), another family of SnRKs involved mainly in maintaining osmotic homeostasis (Yang and Guo 2018). It was demonstrated that TPP1 also plays a role in the activation of stress response genes in rice, which might be the main reason for the enhanced tolerance to abiotic stress of TPP1 overexpression lines (Ge et al. 2008).

6 Metabolic Changes Associated with Abiotic Stress Tolerance

Expression of TPS genes with various origins increased the trehalose level to different extent in different plant species from 1 to 2 μg/g fresh weight (FW) to approximately 1 mg/g FW or 3.2 mg/g dry weight (DW) (Iordachescu and Imai 2008). In contrast, some resurrection plants are able to accumulate trehalose in concentrations as high as 36 mg/g DW under dehydration condition (Drennan et al. 1993). In the organisms naturally accumulating osmolyte, the osmolyte concentration is 13–23 mg/g FW (Chen and Murata 2002). Thus, it was concluded that the amount of trehalose found in tissues of TPS expressing plants is too low for it to act merely 234 Z. Bánfalvi as an osmoprotectant. It may be true even for tobacco plants expressing the TSase gene of Grifola, which accumulated 2.1–2.6 mg/g FW trehalose that was 400-fold higher than that of transgenic tobacco plants co-transformed with otsA and otsB (Zhang et al. 2005). According to a recent theory, trehalose may promote the autophagy that is associated with drought tolerance (Williams et al. 2015). It is interesting to note that in the drought-tolerant crop cassava, high amounts of trehalose (0.23–1.29 mg/g FW) were detected and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. This is, however, not a general feature of plants. The biosynthesis of trehalose in cassava may have been enhanced in the evolutionary history by growing in arid and marginal areas (Han et al. 2016). Enhanced TPS expression unavoidably coincides with enhanced T6P levels, which might be the cause of phenotypic alterations. Although T6P measurements were rarely performed in abiotic stress-tolerant transgenic plants, the lack of morphological aberrations of tobacco plants co-expressing TPS1 and TPP or Arabidopsis plants expressing a fusion protein of TPS1 and TPP indicated that growth aberrations and improved drought tolerance can be uncoupled (Karim et al. 2007; Miranda et al. 2007). This was demonstrated also for potato and tomato (Kwon et al. 2004; Lyu et al. 2013). A possible explanation was that overproduction of both TPS1 and TPP enzymes should lead to more rapid removal of T6P and thus results in normal plants. The role of T6P in regulation of whole-plant carbohydrate allocation and utilisation is recently reviewed and showed that decreasing T6P promotes resource mobilisation enabling better performance under abiotic stress (Paul et al. 2018). As early as in 1998, differences in levels of carbohydrates were detected in tobacco expressing the otsA and otsB. Leaves of one of the stressed transgenic plants contained about threefold higher levels of each of the four non-structural carbohydrates starch, sucrose, glucose and fructose, than leaves of stressed wild- type plants (Pilon-Smits et al. 1998). In contrast, starch concentration of the stressed potato leaves was very low, and the contents of fructose, galactose and glucose were increased and decreased in the wild-type and TPS1 transgenic potato leaves, respectively, while the amounts of proline, inositol and raffinose were highly increased in both the wild-type and TPS1 transgenic leaves under drought conditions (Kondrák et al. 2012). Concerning the proline content, a similar result was obtained in TPS1-expressing rice plants in which cold stress treatment increased proline levels four to five times than those without the treatment both in wild-type and transgenic plants (Li et al. 2011). Thus it is fairly likely that alterations in the trehalose synthesis pathway do not influence the proline synthesis. The general accumulation of proline in stressed plants is elicited in a trehalose independent pathway (Fichman et al. 2015). Salt-tolerant transgenic rice plants overexpressing the rice trehalase TRE1 showed remarkable increases in trehalase activity and dramatic decreases in trehalose abundance compared with the wild type, with little change in the levels of other soluble sugars, such as glucose, fructose and sucrose (Islam et al. 2019). Together with the previous finding that trehalase is involved in drought stress Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress… 235 tolerance in Arabidopsis (Van Houtte et al. 2013), these data suggest that activation of the entire trehalose metabolic pathway is one of the strategies by which plants can adapt to abiotic stresses in their environment. Similarly, the improvement of stress tolerance without trehalose accumulation was previously reported for TPP1- overexpressing plants (Ge et al. 2008).

7 Conclusions

The expression of both trehalose biosynthesis and degradation genes are induced in plants in response to abiotic stresses (Iordachescu and Imai 2008) and overexpression of both trehalose biosynthesis and degradation genes improves abiotic stress tolerance (Tables 1 and 2). Thus accumulation of trehalose is not a prerequisite of better adaptation to stress conditions. In dicotyledonous plant species, however, constitutive overexpression of trehalose biosynthetic genes resulted in stunted growth and lancet-shaped leaves. In contrast, no phenotypic alterations were detected in rice plants, and while alterations in carbohydrate metabolism were demonstrated in dicotyledonous plants both at transcript at metabolite levels, the rice plants altered in trehalose biosynthesis were characterised by induction the transcription of ABA-regulated stress-related genes. One of the explanations relates this difference to the so-called T6P:sucrose nexus, which forms part of a homeostatic mechanism to maintain sucrose levels within a range and that is related to the role of T6P in regulation of SnRK1 activity (Yadav et al. 2014; Paul et al. 2018). It is possible that while, for example, in Arabidopsis, T6P level appears to consistently correlate with sucrose level, this correlation, like in maize kernels (Bledsoe et al. 2017), is much weaker in rice than in Arabidopsis. Therefore, unlike in Arabidopsis, the alteration of T6P:sucrose ratio by TPS expression does not influence the growth rate in rice. An alternative explanation for different influence of constitutiveTPS expression on dicotyledonous and rice plants can be based on a new finding related to the function of TPS1 in yeast. It was demonstrated that TPS1 protein itself, not trehalose, was the main determinant for stress resistance and protection of cells from commitment to apoptosis during growth in yeast (Petitjean et al. 2015, 2016). Immunocytochemical staining, followed by electron microscopy, showed that the yeast TPS1 protein in transgenic tobacco with growth alteration was markedly present in the vacuoles and the cell wall (Almeida et al. 2007b). Chloroplast transformation or targeting of yeast TPS1 into the chloroplast raised the aberrant phenotype of plants (Lee et al. 2003; Karim et al. 2007). A similar result was obtained by fusing TPS1 with TPP (Kwon et al. 2004; Miranda et al. 2007) or otsA with otsB (Lyu et al. 2013). These results indicate that growth aberrations and abiotic stress tolerance of plants can be uncoupled and suggest that it is probably due to the prevention of TPS1 admission into the vacuoles or cell wall. Nevertheless, further experiments are needed to clarify the role of TPS1 as a protein per se in 236 Z. Bánfalvi abiotic stress tolerance of plants. Overexpression of an inactive variant of yeast TPS1 in Arabidopsis or tobacco would be a possible approach to clarify this point. Despite the fact that using inducible promoters or gene fusions the abiotic stress tolerance of different crops could be improved without phenotypic alterations (Table 2), there is no sign that these crops have been transferred to field cultivation. The only exception is transgenic maize that expresses rice TPP1 from the rice MADS6 promoter, which is active over the flowering period, and produces higher yields than wild type with or without drought conditions (Nuccio et al. 2015). Studying this maize line, it was found that decreasing the level of T6P in reproductive tissues downregulates primary metabolism and up-regulates secondary metabolism (Oszvald et al. 2018). Thus, we can conclude that modification of the trehalose pathway provides multiple possibilities in crop improvement as a tool to increase abiotic stress tolerance and cereal yields as well as to alter the metabolite composition of seeds and tubers.

References

Antal F, Kondrák M, Kovács G, Bánfalvi Z (2013) Influence of the StubSNF1 kinase complex and the expression of the yeast TPS1 gene on growth and tuber yield in potato. Plant Growth Regul 69:51–61 Almeida AM, Santos M, Villalobos E, Araujo SS, van Dijck P, Leyman B, Cardoso LA, Santos D, Fevereiro PS, Torne JM (2007a) Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana. Protoplasma 230:41–49 Almeida AM, Silva AB, Araujo SS, Cardoso LA, Santos DM, Torne JM, Silva JM, Paul MJ, Fevereiro PS (2007b) Responses to water withdrawal of tobacco plants genetically engineered with the AtTPS1 gene: a special reference to photosynthetic parameters. Euphytica 154:113–126 Almeida AM, Villalobos E, Araujo SS, Leyman B, Van Dijck P, Alfaro-Cardoso L, Fevereiro PS, Torne JM, Santos DM (2005) Transformation of tobacco with an Arabidopsis thaliana gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses. Euphytica 146:165–176 Ashraf M, Harris PJC (2013) Photosynthesis under stressful environments: an overview. Photosynthetica 51:163–190 Avonce N, Leyman B, Mascorro-Gallardo JO, Van Dijck P, Thevelein JM, Iturriaga G (2004) The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signaling. Plant Physiol 136:3649–3659 Bledsoe SW, Henry C, Griffiths CA, Paul MJ, Feil R, Lunn JE, Stitt M, Lagrimini LM (2017) The role of Tre6P and SnRK1 in maize early kernel development and events leading to stress induced kernel abortion. BMC Plant Biol 17:74 Casson SA, Hetherington AM (2010) Environmental regulation of stomatal development. Curr Opin Plant Biol 13:90–95 Chen THH, Murata N (2002) Enhancement of tolerance of abioticstress by metabolic engineering of betaines and other compatiblesolutes. Curr Opin Plant Biol 5:250–257 Cortina C, Culianez-Macia FA (2005) Tomato abiotic stress enhanced tolerance by trehalose biosynthesis. Plant Sci 169:75–82 Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress… 237

Drennan PM, Smith MT, Goldsworth D, van Staden J (1993) The occurrence of trehalose in the leaves of the desiccation tolerant angiosperm Myrothamnus flabellifolia welw. J Plant Physiol 142:493–496 Eleutherio E, Panek A, De Mesquita JF, Trevisol E, Magalhães R (2015) Revisiting yeast trehalose metabolism. Curr Genet 61:263–274 Fichman Y, Gerdes SY, Kovács H, Szabados L, Zilberstein A, Csonka LN (2015) Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation. Biol Rev Camb Philos Soc 90:1065–1099 Gaff D (1996) Tobacco-plant desiccation tolerance. Nature 382:502 Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA 99:15898–15903 Ge LF, Chao DY, Shi M, Zhu MZ, Gao JP, Lin HX (2008) Overexpression of thetrehalose-6- phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes. Planta 228:191–201 Giaever HM, Styrvold OB, Kaasen I, Strom AR (1988) Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli. J Bacteriol 170:2841–2849 Han B, Fu L, Zhang D, He X, Chen Q, Peng M, Zhang J (2016) Interspecies and intraspecies analysis of trehalose contents and the biosynthesis pathway gene family reveals crucial roles of trehalose in osmotic-stress tolerance in cassava. Int J Mol Sci 17:E1077 Han SE, Park SR, Kwon HB, Yi BY, Lee GB, Byun MO (2005) Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phosphorylase (TP) gene from Pleurotussajor caju. Plant Cell Tissue Organ Cult 82:151–158 Holmström KO, Mantyla E, Welin B, Mandal A, Palva ET, Tunnela OE, Londesborough J (1996) Drought tolerance in tobacco. Nature 379:683–684 Iordachescu M, Imai R (2008) Trehalose biosynthesis in response to abiotic stresses. J Integr Plant Biol 50:1223–1229 Islam MO, Kato H, Shima S, Tezuka D, Matsui H, Imai R (2019) Functional identification of a rice trehalase gene involved in salt stress tolerance. Gene 685:42–49 Jang IC, Oh SJ, Seo JS, Choi WB, Song SI, Kim CH, Kim YS, SeoHS CYD, Nahm BH, Kim JK (2003) Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6- phosphate synthase and trehalose-6-phosphate phosphatase intransgenic rice plants increases trehalose accumulation andabiotic stress tolerance without stunting growth. Plant Physiol 131:516–524 Joo J, Choi HJ, Lee YH, Lee S, Lee CH, Kim CH, Cheong JJ, Do Choi Y, Song SI (2014) Over- expression of BvMTSH, a fusion gene for maltooligosyltrehalose synthase and maltooligosyl- trehalosetrehalohydrolase, enhances drought tolerance in transgenic rice. BMB Rep 47:27–32 Jun SS, Yang JY, Choi HY, Kim NR, Park MC, Hong YN (2005) Altered physiology in trehalose- producing transgenic tobacco plants: enhanced tolerance to drought and salinity stresses. J Plant Biol 48:456–466 Karim S, Aronsson H, Ericson H, Pirhonen M, Leyman B, Welin B, Mäntylä E, Palva ET, Van Dijck P, Holmström KO (2007) Improved drought tolerance without undesired side effects in transgenic plants producing trehalose. Plant Mol Biol 64:371–386 Kondrák M, Marincs F, Antal F, Juhász Z, Bánfalvi Z (2012) Effects of yeast trehalose-6-phosphate synthase 1 on gene expression and carbohydrate contents of potato leaves under drought stress conditions. BMC Plant Biol 12:74 Kondrák M, Marincs F, Kalapos B, Juhász Z, Bánfalvi Z (2011) Transcriptome analysis of potato leaves expressing the trehalose-6-phosphate synthase 1 gene of yeast. PLoS One 6:e23466 Kwon SJ, Hwang EW, Kwon HB (2004) Genetic engineering of drought resistant potato plants by co-introduction of genes encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Zygosaccharomyces rouxii. Korean J Genetic 26:199–206 Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun M-O, Daniell H (2003) Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol Breed 11:1–13 238 Z. Bánfalvi

Li HW, Zang BS, Deng XW, Wang XP (2011) Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta 234:1007–1018 Liu YB, Han LZ, Qin LJ, Zhao DG (2015) Saccharomyces cerevisiae gene TPS1 improves drought tolerance in Zea mays L. by increasing the expression of SDD1 and reducing stomatal density. Plant Cell Tissue Organ Cult 120:779–789 Lyu JI, Min SR, Lee JH, Lim YH, Kim J-K, Bae C-H, Liu JR (2013) Overexpression of a trehalose- 6-phosphate synthase/phosphatase fusion gene enhances tolerance and photosynthesis during drought and salt stress without growth aberrations in tomato. Plant Cell Tissue Organ Cult 112:257–262 Lyu JI, Park JH, Kim JK, Bae CH, Jeong WJ, Min SR, Liu JR (2018) Enhanced tolerance to heat stress in transgenic tomato seeds and seedlings overexpressing a trehalose-6-phosphate synthase/phosphatase fusion gene. Plant Biotechnol Rep 12:399–408 Martins LL, Mourato MP, Baptista S, Reis R, Carvalheiro F, Almeida AM, Fevereiro P, Cuypers A (2014) Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1). Acta Physiol Plant 36:755–765 Miranda JA, Avonce N, Suarez R, Thevelein JM, Van Dijck P, Iturriaga G (2007) A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis. Planta 226:1411–1421 Nuccio ML, Wu J, Mowers R, Zhou HP, Meghji M, Primavesi LF, Paul MJ, Chen X, Gao Y, Haque E, Basu SS, Lagrimini LM (2015) Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions. Nature Biotechnol 33:862–869 Oszvald M, Primavesi LF, Griffiths CA, Cohn J, Basu SS, Nucio ML, Paul MJ (2018) Trehalose 6-phosphate in maize reproductive tissue regulates assimilate partitioning and photosynthesis. Plant Physiol 176:2623–2638 Park SH, Jun SS, An G, Hong YN, Park MC (2003) A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (Brassica campestris) overexpressing CaLEA or OtsA. J Plant Biol 46:277–286 Paul MJ, Gonzalez-Uriarte A, Griffiths CA, Hassani-Pak K (2018) The role of trehalose 6-phosphate in crop yield and resilience. Plant Physiol 177:12–23 Petitjean M, Teste M-A, François JM, Parrou J-L (2015) Yeast trehalose-6P tolerance to various stresses relies on the synthase (Tps1) protein, not on trehalose. J Biol Chem 290:16177–16190 Petitjean M, Teste MA, Leger-Silvestre FJM, Parrou JL (2016) Anew function for the yeast trehalose-6P synthase (Tps1) protein as key pro-survival factor during growth, chronological ageing and apoptotic stress. Mech Ageing Dev 161:234–246 Pilon-Smits EAH, Terry N, Sears T, Kim H, Zayed A, Hwang S, Dun K, Voogd E, Verwoerd TC, Krutwagen RWHH, Goddijn OJM (1998) Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress. J Plant Physiol 152:525–532 Romero C, Belles JM, Vaya JL, Serrano R, Culianez-Macia FA (1997) Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance. Planta 201:293–297 Stiller I, Dulai S, Kondrák M, Tarnai R, Szabó L, Toldi O, Bánfalvi Z (2008) Effects of drought on water content and photosynthetic parameters in potato plants expressing the trehalose-6- phosphate synthase gene of Saccharomyces cerevisiae. Planta 227:299–308 Suprasanna P, Nikalje G, Rai AN (2016) Osmolyte accumulation and implications in plant abiotic stress tolerance. In: Iqbal N, Khan NA, Nazar R (eds) Osmolytes and plants acclimation to changing environment: emerging omics technologies. Springer, New Delhi, pp 1–12 Yadav UP, Ivakov A, Feil R, Duan GY, Walther D, Giavalisco P, Piques M, Carillo P, Hubberten HM, Stitt M et al (2014) The sucrose-trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P. J Exp Bot 65:1051–1068 Yang Y, Guo Y (2018) Unraveling salt stress signaling in plants. J Integr Plant Biol 60:796–804 Yeo ET, Kwon HB, Han SE, Lee JT, Ryu JC, Byun MO (2000) Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae. Mol Cells 10:263–268 Transgenic Plants Overexpressing Trehalose Biosynthetic Genes and Abiotic Stress… 239

Van Houtte H, Vandesteene L, Lopez-Galvis L, Lemmens L, Kissel E, Carpentier SC, Feil R, Avonce N, Beeckman T, Lunn JE, Van Dijck P (2013) Overexpression of the trehalase gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in ABA- induced stomatal closure. Plant Physiol 161:1158–1171 Vishal B, Krishnamurthy P, Ramamoorthy R, Kumar PP (2019) OsTPS8 controls yield-related traits and confers salt stress tolerance in rice by enhancing suberin deposition. New Phytol 221:1369–1386 Wang CL, Zhang SC, Qi SD, Zheng CC, Wu CA (2016) Delayed germination of Arabidopsis seeds under chilling stress by overexpressing an abiotic stress inducible GhTPS11. Gene 575:206–212 Williams B, Njaci I, MoghaddamL LH, Dickman MB, Zhang X, Mundree S (2015) Trehalose accumulation triggers autophagy during plant desiccation. PLoS Genet 11:e1005705 Wurzinger B, Nukarinen E, Nägele T, Weckwerth W, Teige M (2018) The SnRK1 kinase as central mediator of energy signaling between different organelles. Plant Physiol 176:1085–1094 Zhang Y, Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RAC, Powers SJ, Schluepmann H, Delatte T, Wingler A, Paul MJ (2009) Inhibition of Snf1-related protein kinase (SnRK1) activity and regulation of metabolic pathways by trehalose 6-phosphate. Plant Physiol 149:1860–1871 Zhang SZ, Yang BP, Feng CL, Tang HL (2005) Genetic transformation of tobacco with the trehalose synthase gene from Grifola frondosa Fr. enhances the resistance to drought and salt in tobacco. J Integr Plant Biol 47:579–587 Zhao HW, Chen YJ, Hu YL, Gao Y, Lin ZP (2000) Construction of a trehalose-6-phosphate synthase gene driven by drought-responsive promoter and expression of drought-resistance in transgenic tobacco. Acta Bot Sin 42:616–619 Zhao F, Li QY, Weng ML, Wang XL, Guo BT, Wang L, Wang W, Duan DL, Wang B (2013) Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice. Gene 531:205–211 The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression

Merve Kahraman, Gulcin Sevim, and Melike Bor

1 Introduction

Biosynthesis of osmoprotectant molecules such as proline, glycinebetaine, trehalose, polyols, poliamines, and sugars are among the most common protective mechanisms against stresses which affect the osmotic potential of the cells. Introducing or increasing the expression of genes related to the biosynthesis of osmoprotectant molecules was reported to be promising for accelerating stress tolerance in plants. There has been a huge amount of information regarding the contribution of these solutes to tolerance against drought and any other types of stress that cause osmotic effect; however, we still lack the knowledge on their exact mode of action. For instance, since the first resurrection plant was discovered, high concentration and rapid accumulation of trehalose were demonstrated to be a unique feature of these plants. However, drought-sensitive Selaginella sp. accumulated more trehalose than drought-tolerant Selaginella sp. (Pampurova and Van Dijck 2014; Bledsoe et al. 2017) which indicated that even in the drought-tolerant plants, the role and contribution of trehalose or other osmoprotectant molecules to stress tolerance was not completely deciphered. Protective function of proline, glycinebetaine, and trehalose has been known since the 1990s with confirmation from transgenic studies inA. thaliana, tobacco, rice, and wheat (Liu and Zhu 1997; Sakamoto and Murata 1998, Bor and Ozdemir 2018). Several crop plants have been genetically engineered for proline-, glycinebetaine-, and trehalose-related genes which were reported to have improved tolerance to several environmental constraints. Among the pioneer investigations, overexpression of proline biosynthetic geneΔ-pyrroline-5-carboxylate synthase in A. thaliana and tobacco plants (Liu and Zhu 1997) and overexpression of choline oxidase

M. Kahraman · G. Sevim · M. Bor (*) University of Ege, Faculty of Science, Department of Biology, Bornova-Izmir, Turkey e-mail: [email protected]

(glycinebetaine biosynthesis-related gene) in rice (Sakamoto and Murata 1998) might be given as examples of which resulted in increased salinity tolerance in relation to proline and glycinebetaine accumulation, respectively. A more recent transgenic approach was reported by Nuccio et al. (2015). Maize plants overexpressing a rice trehalose-6-phosphate phosphatase gene had better yield performance under drought stress conditions at field trials (Nuccio et al. 2015). The amino acid proline, is reported to be accumulated to high levels when plants encounter different type of stress conditions. Besides its function in growth and development, it acts as an osmoprotectant and redox-buffering agent with an antioxidant characteristic under abiotic stresses (Kishor and Sreenivasulu 2014). On the other hand, high levels of proline have detrimental effects in plant cells leading to cell death; therefore, keeping cellular proline content in balance was reported to be critical for plant survival (Szabados and Savoure 2010; Kishor and Sreenivasulu 2014). The second well-known osmoprotectant molecule, glycinebetaine (GB), is an N-methyl-substituted glycine derivative found in microorganisms, animals, and plants such as sugar beet, wheat, and spinach (Sakamoto and Murata 2002; Ahmad et al. 2013). Besides osmotic adjustment capacity, stabilization of macromolecules, protection of membrane integrity, and contribution to regulating reactive oxygen species (ROS) are among the major roles for GB under stress conditions (Chen and Murata 2011; Ahmad et al. 2013). The third and most studied osmoprotectant molecule, trehalose, is a non-reducing sugar which was reported to be responsible for osmoregulation and protection against environmental stresses in different organisms including plants (Houtte et al. 2013). Unlike other osmotic solutes, trehalose concentrations in wild-type and genetically engineered plants were reported to be low, and cellular compartmentalization was important. Therefore, trehalose-mediated improvement in abiotic stress responses was suggested to be related to the activation of stress-responsive genes and transcription factors rather than being as an osmoprotectant molecule (Lunn et al. 2006; Zhang et al. 2009; Houtte et al. 2013). Contribution of proline, glycinebetaine, and trehalose to stress-responsive gene expression for increased tolerance has been investigated extensively (Table 1). Understanding which genes and especially which transcription factors are up- or downregulated by these molecules would be important not only for a better understanding of stress-coping mechanisms in plants but also for maintenance of better crop performance and yield through manipulation of these genes in cultivated plants. In this chapter, we summarized the latest information regarding the effects of proline, glycinebetaine, and trehalose on the expression of stress-responsive genes in plants. Among these three molecules, only glycinebetaine was reported to be compatible which had no toxic effects even at high levels. Keeping the balance in proline and trehalose contents of the cells need to be tightly regulated. Therefore, for these two molecules, we have given information both for the genes that they regulate and the genes which are related to their biosynthesis and hydrolysis. The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 243

Table 1 Proline-, glycinebetaine-, and trehalose-induced transcription factors and their roles in plant development and stress responses Transcription Factors Function in Plants Proline bZIP11 ProDH-related sugar signaling (Verslues and Sharma bZIP53 2010) bZIP44 Proline catabolism (Satoh et al. 2004) bZIP2 Upregulation of P5CS1 and P5CS2 (Su et al. 2011) MYBCORE Regulation of P5CS (Gao et al.2008; Zhang et al. WRKY 2013; Yang et al. 2016) MYC2 ABA and proline signaling (Li et al. 2018) AP2/ERF TSRF1 JERF1 JERF3 SpERF1 DREB21 ERF71 Glycinebetaine DREB2A CMO gene expression (Khattab et al. 2014) NAC5 BADH gene expression (Liang et al. 2017). WRKY Chilling tolerance (Einset et al. 2007) bZIP53 Fruit development (Zhang et al. 2019) IAA9 Dehydration response (Ahmad et al. 2013) bHLH-FRO2 NDPK2 Trehalose bZIP11 Fine-tuning of carbon and nitrogen metabolism bZIP12 (Garapati et al. 2015; Chen et al. 2016; Laser and bZIP53 Weiste 2018) bZIP44 Development and growth responses (Garapati et al. bZIP2 2015; Tsai and Gazzattini 2014; Chen et al. 2016; WRINKL1 Laser and Weiste 2018) HY5 Low energy signaling (Garapati et al. 2015; Laser and ABI5 Weiste 2018) EEL Sugar signaling (Sun et al. 2003; Bae et al. 2005; KNOTTED1 Kretzshmer et al. 2015; Zhai et al. 2018) LEAFY Fatty acid signaling (Zhai et al. 2018) WUSCHEL ABA signaling (Bae et al. 2005; Tsai and Gazzattini ATAF1 2014) MYBS1 Meristem identity function (Tsai and Gazzattini 2014; CIPK15 Coelho et al. 2018) SUSIBA2 Autophagy (Garapati et al. 2015) WRYK6 Anaerobic germination tolerance (Kretzschmer et al. AGL4 2015) RNA polymerase σ Starch mobilization (Kretzschmer et al. 2015) 70-type initiation Leaf senescence (Bae et al. 2005) factor Floral morphogenesis (Bae et al. 2005; Coelho et al. JUMANJI 2018) Plastid genome transcription, chromatin modification, transcriptional repression (Kondrak et al. 2012) Floral transition and shoot development (Coelho et al. 2018) 244 M. Kahraman et al.

2 Proline

Proline is an amino acid which serves as an osmoprotectant and protective molecule at drought, salt, and other stress conditions. Although the accumulation of proline is a well-known response in stress-tolerant plants, the mode of action is still unclear (Ghars et al. 2012). Different roles have been attributed to proline such as scavenging of the hydroxyl radical, interacting with enzymes responsible for stress tolerance, protecting protein structure and enzyme activity, maintaining pH and redox balance, and supplementation of carbon, nitrogen, and energy (Hare et al. 1999; Szabados and Savoure 2010; Ghars et al. 2012). Biosynthesis of proline occurs in two different pathways which include glutamate and ornithine. Glutamate pathway is the predominant route with two steps: first, glutamate is phosphorylated and reduced to Δ-pyrroline-5-carboxylate (P5C) by PC5 synthase enzyme (P5CS), and then it is reduced to proline by P5C reductase (P5CR) enzyme (Kim and Nam 2013). The second pathway is related to the activity of ornithine δ-aminotransferase (OAT) which also produces P5C that contributes to proline (Szabados and Savoure 2010; Liang et al. 2013). Recent findings have proved that proline has a significant role in osmotic adjustment, stabilization of cellular structures, and protection of photosynthetic apparatus. The translation start site of proline metabolism-related genes has putative cis-regulatory elements (CREs) site which interacts with several general transcription factors such as HD-HOX, AP2/EREBP, MYB, WRKY, and bZIP (Fichman et al. 2015). Therefore, regulation of proline content might be important not only for proline biosynthesis and catabolism but also for the control of the expression of different stress-responsive transcription factors and genes (Table 1). Accordingly, proline inhibited stomatal closure while promoted Ca+2 uptake in contrast to other amino acids such as histidine, methionine, aspartic acid, glutamic acid, and alanine (Rai and Sharma.1991; Rana and Rai 1996; Hayat et al. 2012). However, high levels of proline lead to impairment in the destabilization of DNA helix and susceptibility to S1 nuclease activity (Rajendrakumar et al. 1997; Szabados and Savoure 2010). To date, all of the defined stress response and tolerance processes in plants are regulated by complex signaling networks and have multigenic characteristics. Since proline is a common stress-responsive and adaptive molecule, it would be a good candidate for manipulating stress responses and tolerance mechanisms. Understanding which genes and especially which transcription factors are induced by proline will be beneficial for providing solutions to agricultural practices under changing environmental conditions. Enhanced proline accumulation at stress conditions was reported to be parallel to increased transcriptional activation of P5CS and P5CR genes while ornithine route seemed to have a less impact (Fig. 1). There are two P5CS enzymes in A. thaliana; one is chloroplastic and the other is cytosolic (Liang et al. 2013). P5CS1 is reported to be responsible for stress-induced proline biosynthesis, while the second one is required for developmental processes (Strizhov et al. 1997; Mattioli et al. 2009). P5CS1 transcription and proline accumulation are induced by cooperation of Ca+2-dependent calmodulin with MYB2 The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 245

ABIOTIC & BIOTIC STRESS HR

+2 Ca Level Salicylic acid related-pathway

mETC NADPH oxidase pH NADPH NADP FADH2 FAD maintanence P5CS1 P5CDH ProDH P5CR P5CS2

L-GLUTAMATE GSA P5C L-PROLINE P5C GSA L-GLUTAMATE

Carbon & Nitrogen Sources OAT ORNITHINE CATABOLiSM BIOSYNTHESIS

Membrane traﬃcking Toxicity Lipid seconder messenger

Phospholipase D &C Anoxidant-related Ion channels ABA signalling & genes H O pathway Proline transporters 2 2

Fig. 1 Model of proline-related and proline-regulated processes in the plant metabolism. Proline biosynthesis and catabolism-related enzymes contribute to different physiological processes in development and stress responses. P5CS pyrroline-5-carboxylate synthase, P5CDH pyrroline-5- carboxylate dehydrogenase, P5CR pyrroline-5-carboxylate reductase, ProDH proline dehydrogenase, HR hypersensitive response transcription factor (Yoo et al. 2005). P5CS2 affected development of reproductive organs, and this was proposed to be related to flowering regulator CONSTANS genes (Samach et al. 2000). Expression level of P5CS, which encodes the enzyme that catalyzes the rate- limiting step in proline biosynthesis, was increased in response to salinity and drought. In addition, transcript level of P5CR encoding gene was also found out to be upregulated in the leaves of A. thaliana and in the roots of soybean and pea under osmotic stress (Delauney and Verma 1990; Williamson and Slocum 1992; Verbruggen et al. 1993; Liang et al. 2013). Transcription of P5CS is tightly regulated by proline levels by feedback inhibition (Zhang et al. 1995; Liang et al. 2013). On the other hand, proline levels are determined by the activities of proline dehydrogenase (proDH), P5CR, and pyrroline-5-carboxylate dehydrogenase (P5CDH) which are transcriptionally regulated and alter ROS-mediated signaling processes (Liang et al. 2013). The analyses of the promoter regions of several stress marker genes by bioinformatics tools have revealed that many of them had at least one proline-responsive element (PRE) in their promoter regions although their expressions were not affected by proline (Sharma and Verslues 2010). For example, a bZIP transcription 246 M. Kahraman et al. factor which has a proline binding element was related to the induction of proDH by exogenous proline treatment. However, at stress conditions, the presence of neither proline nor ABA did not alter proDH expression (Sharma and Verslues 2010). Accordingly, bHLH-related two G-BOX motifs were found at Oryza sativa P5CS promoters. Overexpression of bHLH leads to enhanced osmotic and cold stress tolerance with increased proline levels (Liu et al. 2014, 2015; Jin et al. 2016). Similarly, OsP5CS2 and OsP5CR promoters had CACG NAC-core motif in their promoter regions, and overexpression of NAC genes increased drought and salt tolerance in relation to proline accumulation (Liu et al. 2013; Hong et al. 2016). Moreover, P5CS expression can be negatively regulated by different proteins such as annexins. These proteins are light-dependent Ca+2 and phospholipid binding proteins, and annexin mutants have increased P5CS expression which leads to drought and salt tolerance (Huh et al. 2010). Exogenous treatment of plants with proline or proline precursors affected the expression of different stress-related genes which resulted in tolerance against not only to abiotic stresses but to biotic stress. A recent confirmation was reported by Wang et al. (2017). Amino acid permease 1 (AAP1)-mediated proline uptake has improved salt stress tolerance in A. thaliana (Wang et al. 2017). When treated with the precursor of proline, P5C increased HR-like responses against pathogens by the activation of AvrB and AvrRpt2 genes (Funck et al. 2008). Chen et al. (2011) reported that proline affected calcium-mediated production of H2O2 and increased NDR1 expression-activated SA signaling pathway which lead to pathogenesis- related (PR) gene expression. In abiotic stress responses, exogenous proline was reported to be also responsible for protection of plants; however, there are contro- versial results which indicated the negative impact of proline on growth and metabolic processes. A. thaliana plants treated with proline at salt stress conditions had growth inhibition and accelerated senescence (Yamada et al. 2005). Antioxidant enzymes Cu/ZnSOD and MnSOD encoding genes were upregulated in rice plants when treated with proline under salinity; however, in the absence of NaCl, the expression of these genes was suppressed (Nounjan et al. 2012). In the light of these findings, regulation of biosynthesis and catabolism of proline within the plant cells seemed to be more effective than exogenous proline treatment.

3 Glycinebetaine

Glycinebetaine (GB) is the most common and best-known compatible solute that is found in several organisms including bacteria and plants (Castiglioni et al. 2018). GB is biosynthesized by two pathways; the most common route is via the oxidation of choline, while the other one is a bacteria-specific glycine methylation pathway (Fig. 2). In plants, choline is oxidized to betaine aldehyde by a ferredoxin-dependent choline monooxygenase (CMO) which is then converted to GB by the activity of betaine aldehyde dehydrogenase (BADH) (Nuccio et al. 1998; Ahmad et al. 2013). Plants are divided into two classes: GB accumulators and non-accumulators The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 247

Plants

CMO

BADH

CHOLINE Escherichia coli

CDH

BADH GLYCINE BETAINE

Artrobacter globiformis

COD/COX

2O2 + H2 2O2

GLYCINE Acnopolispora halophilia

GSMT

SDMT

Fig. 2 Biosynthesis of glycinebetaine from different precursor molecules in different organisms. CMO choline monooxygenase, COD choline oxidase, BADH betaine aldehyde dehydrogenase, CDH choline dehydrogenase, GSMT glycine sarcosine methyltransferase, SDMT sarcosine dimethylglycine methyltransferase according to their ability for GB biosynthesis. Accumulator plants such as sugar beet, spinach, and mangrove have the ability to well-adapt to drought and salinity conditions (Bor et al. 2003; Ahmad et al. 2013). Under osmotic stress-imposing conditions, even exogenous GB treatment was found out to have a protective role in plants; therefore, engineering non-accumulator plants for genes related to GB biosynthesis was proposed to be important for increasing yield of crop plants (Castiglioni et al. 2018; Bor and Ozdemir 2018). Crop plants such as rice, carrot, tomato, and potato are non-accumulators of GB, and in the recent years, transgenic studies for GB were accelerated for increasing crop biomass and yield (Ahmad et al. 2013). In GB-synthesizing transgenic rice plants, more than 165 genes were upregulated and 76 genes were downregulated (Kathuria et al. 2009; Ahmad et al. 2013). Within the upregulated genes, 50 of them were related to the alleviation of various stress effects, and 115 of them were involved in regulation of gene expression, membrane transport, growth and development, signal transduction, and metabolism (Kathuria et al. 2009). GB functions at 248 M. Kahraman et al. important processes such as osmoprotection, destabilization of DNA, refolding and thermal stabilization of proteins, maintenance of membrane integrity, and protection of enzymes (rubisco, rubisco activase, malate dehydrogenase, etc.) which are all remarkable components of plant tolerance to abiotic stresses (Chen and Murata 2011; Ahmad et al. 2013). Wei et al. (2017) reported that the activity of ion channels and transporters was regulated by GB which provided high potassium and low sodium levels conferring to salt tolerance in transgenic tomato plants. On the other hand, codA-and BADH-transgenic tomato plants had differential regulation of cell wall invertase, protein kinase, sucrose transporter, cyclin-dependent kinase, auxin transcription factor, and miniature zinc-finger protein (IMA) encoding genes which might be responsible for flower and fruit development (Wei et al. 2017). A generalized scheme for the processes and contribution of these genes to overall plant metabolism and stress responses was given in Fig. 3. In the case of stress-coping mechanisms, the possibility of different interactions between GB and stress-related metabolites was proposed (Fig. 3). For instance, maize plants treated with a nitric oxide (NO) inhibitor (Nω-nitro-L-arginine methyl ester; L-NAME) had reduced BADH gene expression which leads to low GB levels (Phillips et al. 2018). NO is known to contribute to ROS detoxification, regulation of antioxidant enzymes, and compatible solutes during abiotic and biotic stresses

OSMOPROTECTION DEFENSE MECHANISM Proline Choline availibility Synthesis of Trehalose antioxidant Carotenoids IMPROVING enzymes APX PLANT GROWTH CAT SOD *Biomass ROS *Yield *Growth of reproductive Transgenic plants CHOLINE organs e- O2 2O2 CMO PQ COD LIGHT CYT H2O2 BADH COMPLEX PC GLYCINE BETAIN PSII PSI D1 H2 O2 TRANSCRIPTIONAL CHANGES Synergistic Effect Repairing process Stress-related of TFs PSII *Ethylene *ABA *Salicylic Acid Inhibition CO2 of LOX and PLD levels RUBİSCO Calvin Chaperon-mediate Cycle protein disaggregation Protecting of CO Assimilation 2 Interaction with chaperon-like ASR1

Fig. 3 The direct and indirect contribution of glycinebetaine metabolism to stress-coping processes in plants. SOD superoxide dismutase, CAT catalase, APX ascorbate peroxidase, CMO choline monooxygenase, COD choline oxidase, BADH betaine aldehyde dehydrogenase, ABA abscisic acid, PLD phospholipase D, LOX lipoxygenase The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 249

(Uchida et al. 2002; Zhang et al. 2006; Guo et al. 2009; Phillips et al. 2018). Several metabolic routes are affected by GB accumulation and/or exogenous GB treatment. As indicated before, GB served not only by protecting proteins and enzymes but also by triggering transcription of stress-responsive genes or their transcription factors. Antioxidant enzymes, fatty acid metabolism-related enzymes such as lipoxygenase (LOX) and phospholipase-D (PLD) are among the most important enzymes which are regulated by GB levels.

4 Trehalose

Trehalose is synthesized from uridine diphosphate glucose (UDP-Glc) and glucose- 6-phosphate (G6P) via trehalose-6-phosphate synthase (TPS) enzyme which dephosphorylated to a more effective form, trehalose-6-phosphate (T6P) by the activity of trehalose-6-phosphate phosphatase (Figueroa et al. 2016). In A. thaliana, T6P proposed to act as a signaling molecule in the regulation of sucrose level in order to provide optimal level of sucrose within the cell (Fig. 4). Oryza sativa TPS overexpressing lines, trehalose, and proline levels were highly induced with or without stress treatment. Expression of stress-related genes such as ELIP, HSP70, CRP, DHN6, LEA14A, and WS118 were increased up to twofold in these plants as compared to wild-type plants (Li et al. 2011). Increased level of T6P was related to the activation of nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPC) through posttranslational modifications (Figueroa et al.2016 ). Protein kinases, protein phosphatases, and other enzymes involved in these modifications were proposed to be the potential targets of T6P (Fig. 4). Trehalose was reported to serve as a compatible solute for the stabilization of membranes and biomolecules (Fernandez et al. 2010). In plant cells, trehalose is synthesized at low levels as compared to other compatible solutes such as proline, glycinebetaine, mannitol, etc. Hence, its being a common compatible solute is still under debate. High levels of trehalose were detected only in resurrection plants and in specific organs upon stress exposure (Avonce et al. 2004; Schluepmann et al. 2003; Grennan 2007; El-Bashiti et al. 2005; Garg et al. 2002; Fernandez et al. 2010). Since trehalose and T6P levels are usually very low in plants, they were proposed to have regulatory or sensing roles for source-sink relationship. Trehalose pathway might be a facilitator between the cellular compartments via regulation of different transcription factors under different environmental stresses (Table 1 and Fig. 5). T6P was thought to be a negative-feedback regulator for the adjustment of sucrose levels by interaction with SnRK1 (Bledsoe et al. 2017). T6P-sucrose interaction is adjusted according to developmental stage, tissue and cell type, and various environmental factors such as low temperature stress (Figueroa et al. 2016). Various studies indicated the importance of trehalose metabolism at transcriptional, translational, and posttranslational levels for controlling and regulating stress responses in plants (Table 1). In plant cells, sucrose:T6P ratio affects important metabolic processes in multiple ways via induction or repression of several 250 M. Kahraman et al.

TRE1

UDP-

HEXOSE-P PHOTOASSIMILATES SnRK1 AMINO ACID TFs SUCROSE ORGANIC ACID

TCATCA Cycle

PEPCPEPC STARCH HEXOSE-P COO2 Glycolysis AGPase NR ADP-glc CARBON MMETABOLISETA M & NITROGEN FIXATION

TRANSPORT ABA related OF stomatal funcon POST-TRANSLATIONAL SUGAR MODIFICATIONMODIFICATION *PHOSPHORILATIONPHOSPHORILATION SUC1 *MONOUBIQUITINATION SWEET1

Fig. 4 The interaction of trehalose pathway with different metabolic processes. TPS trehalose phosphate synthase, TPP trehalose phosphate phosphatase, TRE trehalose, T6P trehalose-6- phosphate, ABI4 ABA insensitive 4; Glc-6P glucose-6-phosphate, AGPase ADP-glucose- pyrophosphorylase, PEPC phosphoenolpyruvate carboxylase, ABA abscisic acid, SnRK sucrose non-fermenting receptor kinase, FUS3 mitogen-activated kinase, bZIP11 basic leucine zipper transcription factor 11

stress-responsive transcription factors (Fig. 4). For instance, increased T6P levels resulted in the repression of SnRK1 which is a key transcriptional regulator that responds to carbon and energy supply (Nuccio et al. 2015). Therefore, T6P influences SnRK1-upregulated genes negatively and SnRK1-downregulated genes positively. Another transcription factor bZIP11 which affects the regulation of carbohydrate metabolism is also regulated by T6P. The developmental phase transi- tions, carbohydrate, and amino acid metabolisms are regulated by bZIPs (Tsai and Gazzarini 2014). Accordingly, it has been suggested that OsTPP7 contributes to anaerobic germination tolerance by modulating local T6P:sucrose ratios in germinating tissues which lead to upregulation of MYBS1 and CIPK15 genes for regulating amylase activation for increased starch mobilization (Kretzshmer et al. 2015). Trehalase catalyzes the hydrolysis of trehalose into two glucose monomers which was reported to be important for osmotic regulation and stress responses (Lunn 2007; Avonce et al. 2010; Houtte et al. 2013). A. thaliana had one trehalase The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 251

SUCROSE LEVELS SnRK1

FUS3

BZIP11 ABI4 SUCROSE:T6P NUCLEUS

DEVELOPMENTAL PHASE TRANSITION L

O TPS TPP TRE ABA S O T

Y UDP-Glc + Glc6P TREHALOSE 2GLU

C Tre6P

STOMATAL AGPase PEPC OPENING Tre6P STARCH MALATE CHLOROPLAST PHOTOSYNTHETIC ACTIVITY

Fig. 5 Impact of T6P-mediated photoassimilates partitioning on the plant metabolism. TPS trehalose phosphate synthase, TPP trehalose phosphate phosphatase, TRE trehalose, T6P trehalose-6- phosphate, SnRK sucrose non-fermenting receptor kinase, ABA abscisic acid, PEPC phosphoenolpyruvate carboxylase, NR nitrate reductase, SUC1 sucrose transporter 1 encoding gene, TRE1, that has a MYB4 binding site in its promoter region (Lunn 2007; Avonce et al. 2010; Houtte et al. 2013). Besides this, a W-box promoter motif was identified in the AtTRE1 promoter for MYB102 and WRKY transcription factors which are known to be involved in ABA signaling at dehydration and osmotic stress conditions (Houtte et al. 2013). Since both MYB4 and MYB102 are members of the R2R3-type MYB family, these transcription factors can induce AtTRE1 expression during developmental processes such as guard cell differentiation (Houtte et al. 2013). Genetic control of trehalase would be a good tool for adjusting endogenous trehalose levels; therefore, drought tolerance might be manipulated by regulation of AtTRE1 (Houtte et al. 2013). Increased trehalase activity affected the sensitivity of guard cells to exogenous ABA treatments; thus, AtTRE1 may be essential for the ABA-induced stoma closure. One confirmation was reported from a study with Attre1-1 and Attre1-2 mutants which were unable to close their stomata in response to the ABA treatments (Houtte et al. 2013). On the other hand, hydrolysis of trehalose would be essential for different developmental processes. AtTRE1 was strongly upregulated during senescence in A. thaliana which indicated the contribution of trehalose degradation during programmed cell death (Yamada et al. 2005). 252 M. Kahraman et al.

Although upregulation of trehalose biosynthesis and exogenous trehalose treatments both have protective and regulatory functions in various plants such as tomato, tobacco, and rice under drought, salt, and cold stresses, we are still far from explaining the exact mode of action of trehalose in plants. Despite increasing stress tolerance in plants, overexpression of trehalose pathway-related genes has frequently resulted in dwarfism, delay in flowering, and abnormalities in leaf and root morphologies (Li et al. 2011). Exogenous trehalose treatment in rice resulted in reduced damage under salinity which was proposed to be related to preservation of root integrity, decreased Na+ accumulation, and regulation of the genes responsible for osmotic adjustment (Garcia et al. 1997; Bae et al. 2005; Fernandez et al. 2010).

5 Conclusion

Understanding stress-coping mechanisms is among the hot topics of plant science not only for basic scientific curiosity but also for improving agricultural yield and performance. Plants have evolved sophisticated stress tolerance mechanisms against abiotic and biotic stresses of which can be introduced to crop plants by transgenic approaches. Stress tolerance is a complex network of gene activation and signaling transduction routes; therefore, manipulation of one metabolic process may lead to undesired or unsufficient effects. Among these mechanisms, accumulation of osmoprotectant solutes was found out to be the most effective and compatible one since most of the crop plants have at least one type of these molecules or their precursors. Studies presented in this chapter might give an idea for how the biosynthetic and catabolic routes of these three molecules might be manipulated by genetic approach for improvement of stress responses in plants. Different characteristics and features of these molecules and how they affected transcription of stress-responsive and stress-related genes were discussed in detail. All of these molecules have an impact and ameliorative effect on stress tolerance in plants, and one might consider carefully for choosing the best candidate. Proline, for being a component of free amino acid; glycinebetaine, for being the most compatible solute among these molecules; and trehalose, for being an unusual sugar molecule with ability to preserve water, are all promising for regulating and controlling stress tolerance processes in plants.

References

Ahmad R, Lim CJ, Kwon SY (2013) Glycine betaine: a versatile compound with great potential for gene pyramiding to improve crop plant performance against environmental stresses. Plant Biotechnol Rep 7:49–57 Avonce N, Leyman B, Mascorro-Gallardo JO, Van Dijck P, Thevelein JM, Iturriaga G (2004) The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signaling. Plant Physiol 136:3649–3659 The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 253

Avonce N, Wuyts J, Verschooten K, Vandesteene L, Van Dijck P (2010) The Cytophaga hutchinsonii ChTPSP: first characterized bifunctional TPS–TPP protein as putative ancestor of all eukaryotic trehalose biosynthesis proteins. Mol Biol Evol 27(2):359–369 Bae H, Herman E, Bailey B, Bae HJ, Sicher R (2005) Exogenous trehalose alters Arabidopsis transcripts involved in cell wall modification, abiotic stress, nitrogen metabolism, and plant defense. Physiol Plant 125:114–126 Bledsoe SW, Henry C, Griffiths CA, Paul MJ, Feil R, Lunn JE, Lagrimini LM (2017) The role of Tre6P and SnRK1 in maize early kernel development and events leading to stress-induced kernel abortion. BMC Plant Biol 17:74 Bor M, Ozdemir F (2018) Manipulating metabolic pathways for development of salt-tolerant crops. In: Kumar V, Wani S, Suprasanna P, Tran LS (eds) Salinity responses and tolerance in plants, vol 1. Springer, Cham Bor M, Ozdemir F, Turkan I (2003) The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. Plant Sci 164:77–84 Castiglioni P, Bell E, Lund A, Rosenberg AF, Meghan GM, Hinchey BS, Bauer S, Nelson DE, Robert J, Bensen RJ (2018) Identification of GB1, a gene whose constitutive overexpression increases glycinebetaine content in maize and soybean. Plant Direct 2(2):1–7 Chen TH, Murata N (2011) Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ 34:1–20 Chen J, Zhang Y, Wang C, Lu W, Jin JB, Hua X (2011) Proline induces calcium-mediated oxidative burst and salicylic acid signaling. Amino Acids 40:1473–1484 Chen X, Yao Q, Gao X, Jiang C, Harberd NP, Fu X (2016) Shoot-to-root mobile transcription factor HY5 coordinates plant carbon and nitrogen acquisition. Curr Biol 26:640–646 Coelho CP, Huang P, Lee DY, Brutnell TP (2018) Making roots, shoots, and seeds: IDD gene family diversification in plants. Trends Plant Sci 23:66–78 Delauney AJ, Verma DPS (1990) A soybean gene encoding delta-1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia-coli and is foundtobeosmo- regulated. Mol Gen Genet 221:299–305 Dröge-Laser W, Weiste C (2018) The C/S1 bZIP network: a regulatory hub orchestrating plant energy homeostasis. Trends Plant Sci 23:422–433 Einset J, Nielsen E, Connolly EL, Bones A, Sparstad T, Winge P (2007) Membrane trafficking RabA4c involved in the effect of glycine betaine on recovery from chilling stress in Arabidopsis. Physiol Plant 130:511–518 El-Bashiti T, Hamamcı H, Oktem H, Yucel M (2005) Biochemical analysis of trehalose and its metabolizing enzymes in wheat under abiotic stress conditions. Plant Sci 169:47–54 Fernandez O, Béthencourt L, Quero A, Sangwan RS, Clément C (2010) Trehalose and plant stress responses: friend or foe? Trends Plant Sci 15:409–417 Fichman Y, Gerdes SY, Kovács H, Szabados L, Zilberstein A, Csonka LN (2015) Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation. Biol Rev 90:1065–1099 Figueroa CM, Feil R, Ishihara H, Watanabe M, Kölling K, Krause U, Höhne M, Encke B, Plaxton WC, Zeeman SC, Li Z, Schulze WX, Hoefgen R, Stitt M, Lunn JE (2016) Trehalose 6-phosphate coordinates organic and amino acid metabolism with carbon availability. Plant J 85:410–423 Funck D et al (2008) Ornithine-delta-aminotransferase is essential for arginine catabolism but not for proline biosynthesis. BMC Plant Biol 8:40 Gao S, Ouyang C, Wang S, Xu Y, Tang L, Chen F (2008) Effects of salt stress on growth, antioxidant enzyme and phenyalanine ammonia-lyase activities in Jatropha curcas L seedlings. Plant Soil Environ 54:374–381 Garapati P, Feil R, Lunn JE, Van Dijck P, Balazadeh S, Mueller-Roeber B (2015) Transcription factor Arabidopsis activating factor1 integrates carbon starvation responses with trehalose metabolism. Plant Physiol 169:379–390 Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci U S A 99:15898–15903 254 M. Kahraman et al.

Ghars MA, Richard L, Lefebvre-De Vos D, Leprince AS, Parre E, Bordenave M, Abdelly C, Savoure A (2012) Phospholipases C and D modulate Proline accumulation in Thellungiellahalophila/ salsuginea differently according to the severity of salt or hyperosmotic stress. Plant Cell Physiol 53(1):183–192 Grennan AK (2007) The role of trehalose biosynthesis in plants. Plant Physiol 144:3–5 Guo Y, Tian Z, Yan D, Zhang J, Qin P (2009) Effects of nitric oxide on salt stress tolerance in Kosteletzkya virginica. Life Sci J 6:67–75 Garcia AB, Engler JA, Iyer S, Gerats T, Montagu MV, Caplan AB (1997) Effects of Osmoprotectants upon NaCl stress in rice. Plant Physiol 115(1):159–169 Hare P, Cress W, van Staden J (1999) Proline synthesis and degradation: a model system for elucidating stress-related signal transduction. J Exp Bot 50:413–434 Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A (2012) Role of proline under changing environments. Plant Signal Behav 7(11):1456–1466 Hong Y, Zhang H, Huang L, Li D, Song F (2016) Overexpression of a stress-responsive NAC transcription factor gene ONAC022 improves drought and salt tolerance in rice. Front Plant Sci 7:4 Houtte H, Vandesteene L, Lopes-Galvis L, Lemmens L, Kissel E, Carpentier S, Feil R, Avonce N, Beeckman T, Lunn JE, Van Dijck P (2013) Overexpression of the trehalose gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in absisic acid-induced stomatal closure. Plant Physiol 161:1158–1171 Huh SM, Noh EK, Kim HG, Jeon BW, Bae K, Hu HC, Kwak JM, Park OK (2010) Arabidopsis annexins AnnAt1 and AnnAt4 interactwith each other and regulate drought and salt stress responses. Plant Cell Physiol 51:1499–1514 Jin C, Huang X-S, Li K-Q, Yin H, Li L-T, Yao Z-H (2016) Overexpression of a bHLH1 transcription factor of Pyrus ussuriensis confers enhanced cold tolerance and increases expression of stress-responsive genes. Front Plant Sci 7:441 Kathuria H, Giri J, Nataraja KN, Murata N, Udayakumar M, Tyagi AK (2009) Glycinebetaine induced water-stress tolerance in codA-expressing transgenic indica rice is associated with upregulation of several stress responsive genes. Plant Biotechnol J 7:512–526 Khattab HI, Emam MA, Emam MM, Helal NM, Mohamed MR (2014) Effect of selenium and silicon on transcription factors NAC5 and DREB2A involved in drought-responsive gene expression in rice. Biol Plant 58:265 Kim GB, Nam YW (2013) A novel 1-pyrroline-5-carboxylate synthetase gene of Medicagotruncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation. J Plant Physiol 170:291–302 Kishor PBK, Sreenivasulu N (2014) Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant Cell Environ 37:300–311 Kondrák M, Marincs F, Antal F, Juhász Z, Bánfalvi Z (2012) Effects of yeast trehalose-6-phosphate synthase 1 on gene expression and carbohydrate contents of potato leaves under drought stress conditions. BMC Plant Biol 12:74 Kretzschmar T, Pelayo MAF, Trijatmiko KR, Gabunada LFM, Alam R, Jimenez R, Ismail AM (2015) A trehalose-6-phosphate phosphatase enhances anaerobic germination tolerance in rice. Nat Plants 1:15124 Li HW, Zang BS, Deng XW, Wang XP (2011) Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta 234(5):1007–1018 Liang X, Zhang L, Natarajan SK, Becker DF (2013) Proline mechanisms of stress survival. Antioxid Redox Signal 19(9):998–1011 Liang W, Xiaoli M, Peng W, Lianyin L (2017) Plant salt-tolerance mechanism: a review. Biochem Biophys Res Comm 495(1):286–291 Liu JP, Zhu JK (1997) Proline accumulation and salt-stress-induced gene expression in a salt- hypersensitive mutant of Arabidopsis. Plant Physiol 114:591–596 Liu X, Liu S, Wu J, Zhang B, Li X, Yan Y (2013) Overexpression of Arachis hypogaea NAC3 in tobacco enhances dehydration and drought tolerance by increasing superoxide scavenging. Plant Physiol Biochem 70:354–359 The Role of Proline, Glycinebetaine, and Trehalose in Stress-Responsive Gene Expression 255

Liu W, Tai H, Li S, Gao W, Zhao M, Xie C (2014) bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism. New Phytol 201:1192–1204 Liu Y, Ji X, Nie X, Qu M, Zheng L, Tan Z et al (2015) Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs. New Phytol 207:692–709 Lunn JE, Feil R, Hendriks JH, Gibon Y, Morcuende R, Osuna D (2006) Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana. Biochem J 397:139–148 Lunn JE (2007) Gene families and evolution of trehalose metabolism in plants. Funct Plant Biol 34(6):550–563 Li J, Guo X, Zhang M, Wang X, Zhao Y, Yin Z, Zhang Z, Wang Y, Xiong H, Zhang H, Todorovska E, Li Z (2018) OsERF71 confers drought tolerance via modulating ABA signaling and proline biosynthesis. Plant Sci 270:131–139 Mattioli R, Falasca G, Sabatini S, Altamura MM, Costantino P, Trovato M (2009) The proline biosynthetic genes P5CS1 and P5CS2 play overlapping roles in Arabidopsis flower transition but not in embryo development. Physiol Plant 137:72–85 Nounjana N, Nghiab PT, Theerakulpisut P (2012) Exogenous proline and trehalose promote recovery of rice seedlings from salt-stress and differentially modulate antioxidant enzymes and expression of related genes. J Plant Physiol 169(6):596–604 Nuccio ML, Russel BL, Nolte KD, Rathinasabapathi B, Gage DA, Hanson AD (1998) The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J 16:487–496 Nuccio ML, Wu J, Mowers R, Zhou H, Meghji M, Primavesi LF, Basu SS (2015) Expression of trehalose-6-phosphate in maize ears improves yield in well-watered and drought conditions. Nat Biotech 33:862–869 Pampurova S, Van Dijck P (2014) The desiccation tolerant secrets of Selaginella lepidophylla: what we have learned so far? Plant Physiol Biochem 80:285–290 Phillips K, Majola A, Gokul A, Keyster M, Ludidi N, Egbichi I (2018) Inhibition of NOS- like activity in maize alters the expression of genes involved in H2O2 scavenging and glycine betaine biosynthesis. Sci Rep 8(1):12628 Rai VK, Sharma UD (1991) Amino acids can modulate ABA induced stomatal closure, stomatal resistance and K+ fluxes in Vicia faba leaves. Beitr Biol Pflanzenphysiol 66:393–405 Rana U, Rai VK (1996) Modulation of calcium uptake by exogenous amino acids in Phaseolus vulgaris seedlings. Acta Physiol Plant 18:117–120 Rajendrakumar CSV, Suryanarayana T, Reddy AR (1997) DNA helix destabilization by proline and betaine: possible role in the salinity tolerance process. FEBS Letters 410 (2-3):201–205 Sakamoto A, Murata A (1998) Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold. Plant Mol Biol 38:1011–1019 Sakamoto A, Murata N (2002) The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant Cell Environ 25:163–171 Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky MF, Coupland G (2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288:1613–1616 Satoh R, Fujita Y, Nakashima K, Shinozaki K, Yamaguchi- Shinozaki K. (2004) A novel subgroup of bZIP proteins functions as transcriptional activators in hypoosmolarity responsive expression of the ProDH gene in Arabidopsis. Plant Cell Physiol 45:309–317 Schluepmann H, Pellny T, van Dijken A, Smeekens S, Paul M (2003) Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proc Natl Acad Sci U S A 100:6849–6854 Sharma S, Verslues PE (2010) Mechanisms independent of abscisic acid (ABA) or proline feedback have a predominant role in transcriptional regulation of proline metabolism during low water potential and stress recovery. Plant Cell Environ 33:1838–1851 256 M. Kahraman et al.

Strizhov N, Abraham E, Okresz L, Blickling S, Zilberstein A, Schell J, Koncz C, Szabados L (1997) Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. Plant J 12:557–569 Su M, Li XF, Ma XY, Peng XJ, Zhao AG, Cheng LQ, Chen SY, Liu GS (2011) Cloning two P5CS genes from bioenergy sorghum and their expression profiles under abiotic stresses and MeJA treatment. Plant Sci 181:652–659 Sun C, Palmqvist S, Olsson H, Borén M, Ahlandsberg S, Jansson C (2003) A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar- responsive elements of the iso1 promoter. Plant Cell 15:2076–2092 Szabados L, Savoure A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15(2):89–97 Tsai AY, Gazzarrini S (2014) Trehalose-6-phosphate and SnRK1 kinases in plant development and signaling: the emerging picture. Front Plant Sci 5:119 Uchida A, Jagendorf AT, Hibino T, Takabe T, Takabe T (2002) Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice. Plant Sci 63:515–523 Verbruggen N, Villarroel R, Van Montagu M (1993) Osmoregulation of a pyrroline-5-carboxylate reductase gene in Arabidopsis thaliana. Plant Physiol 103:771–781 Verslues PE, Sharma S (2010) Proline metabolism and its implications for plant-environment interaction. Arabidopsis Book, American Society of Plant Biologists. USA 8:e0140 Wang T, Chen Y, Zhang M, Chen J, Liu J, Han H, Hua X (2017) Arabidopsis amino acid Permease1 contributes to salt stress-induced proline uptake from exegenous sources. Front Plant Sci 8:2182 Wei DD, Zhang W, Wang CC, Meng QW, Li G, Chen THH, Yang XH (2017) Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants. Plant Sci 257:74–83 Williamson CL, Slocum RD (1992) Molecular cloning and evidence for osmoregulation of the delta 1-pyrroline-5-carboxylate reductase (proC) gene in pea (Pisumsativum L.). Plant Physiol 100:1464–1470 Yamada M, Morishita H, Urano K, Shiozaki N, Yamaguchi-Shinozaki K, Shinozaki K et al (2005) Effects of free proline accumulation in petunias under drought stress. J Exp Bot 56:1975–1981 Yang Y, Dong C, Li X, Du J, Qian M, Sun X, Yang Y (2016) A novel Ap2/ERF transcription factor from Stipapurpurea leads to enhanced drought tolerance in Arabidopsis thaliana. Plant Cell Rep 35:2227 Yoo JH, Park CY, Kim JC, Heo WD, Cheong MS, Park HC (2005) Direct interaction of a divergent CaM isoform and the transcription factor, MYB2, enhances salt tolerance in Arabidopsis. J Biol Chem 280:3697–3706 Zhai Z, Keereetaweep J, Liu H, Feil R, Lunn JE, Shanklin J (2018) Trehalose 6-phosphate positively regulates fatty acid synthesis by stabilizing WRINKLED1. Plant Cell 30:2616–2627 Zhang CS, Lu Q, Verma DP (1995) Removal of feedback inhibition of delta 1-pyrroline-5- carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270:20491–20496 Zhang Y, Wang L, Liu Y, Zhang Q, Wei Q, Zhang W (2006) Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast. Planta 224:545–555 Zhang Y, Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RA, Powers SJ, Schluepmann H, Delatte T, Wingler A, Paul MJ (2009) Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiol 149:1860–1871 Zhang XX, Tang YJ, Ma QB, Yang CY, Mu YH, Suo HC, Luo LH, Nian H (2013) OsDREB2A, a rice transcription factor, significantly affects salt tolerance in transgenic soybean. PLoS One 8:e83011 Zhang T, Liang J, Wang M, Li D, Liu Y, Chen THH, Yang X (2019) Genetic engineering of the biosynthesis of glycinebetaine enhances the fruit development and size of tomato. Plant Sci 280:355–366 Seed Osmolyte Priming and Abiotic Stress Tolerance

Danny Ginzburg and Joshua D. Klein

1 Introduction

Seed priming is any technique in which seeds are imbibed in a solution prior to sowing in order to improve germination rates and uniformity and/or to confer abiotic and biotic stress tolerance. Priming can be performed with water alone (hydropriming) or with chemical or bioactive compounds (Paparella et al. 2015). The controlled seed rehydration induced by priming triggers metabolic processes associated with early states of germination such as restoration of cellular integrity, initiation of respiration and DNA repair functions, and increased activity of antioxidant enzymes and reactive oxygen species (ROS) scavenging. Priming with water, chemical, or biological solutions can enhance seedling or mature-plant tolerance to abiotic stresses. The method of introducing a priming compound into a seed (soaking, solid matrix priming, seed coating) can influence the efficacy of the treatment (Klein et al.2017 ; Wang et al. 2016). Priming conditions – chemical concentrations/osmotic potentials, durations, and temperatures – can in turn affect the amount of material taken up by the seed (El-Araby and Hegazi 2004; Posmyk et al. 2008). With the correct priming conditions, a seed has a high enough water content to initiate germination-related processes, but low enough to prevent germination (radicle emergence) and to retain desiccation tolerance upon drying. The specific processes induced by priming and the degree of their expression may also vary depending on plant species, seed structure, and quality (Paparella et al. 2015). Embryo location (external as in monocots or internal as in dicots) may influence the ability of a priming compound to affect seed germination and seedling growth, as can amount of endosperm (low in onions or tomatoes versus high in wheat or beans). The amount of mucilage generated on the seed coat (as in basil or rocket seeds) may also affect uptake and effectiveness of the priming compounds (Western 2012, and references therein).

D. Ginzburg · J. D. Klein (*) Institute for Plant Science, ARO-Volcani Center, Rishon LeZion, Israel e-mail: [email protected]

© Springer Nature Switzerland AG 2019 257 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_12 258 D. Ginzburg and J. D. Klein

We review here recent and foundational literature on methods and compounds used in seed priming to achieve tolerance to abiotic stress in emerging and growing plants, as well as proposed mechanisms of action and potential for future research and development.

2 Abiotic Stress in Plants: Phenomenon and Induced Tolerance

Abiotic stress can affect all major aspects of plant development, from seed germination to growth, flowering, and seed development. The degree to which a given stress affects a crop depends both on the severity and on the crop’s tolerance or resistance to the stress. The effects of abiotic stress, and the plant’s responses, are specific to the type of stress (i.e., limited photosynthesis and nutrient availability under drought and salinity, suboptimal cellular respiration under heavy metal stress, and slowed metabolic activity in cold temperatures). Under all abiotic stresses, though, plants produce toxic levels of ROS capable of lipid peroxidation, protein denaturation, and DNA mutation (Jaspers and Kangasjärvi 2010). Although necessary for proper development at low concentrations, ROS concentrations accumulate to toxic levels under abiotic stress and become particularly damaging to organelles such as mitochondria and chloroplasts, which facilitate a high rate of electron flow (Gill and Tuteja 2010). A common response to abiotic stress is therefore the induction of antioxidative enzymes and metabolites to neutralize the damaging effects of ROS (Bernstein et al. 2010). The specific antioxidant compounds (enzymatic and nonenzymatic) upregulated during stress conditions and the degree of their expression are influenced by the plant developmental state, duration of stress, and subcellular localization of ROS accumulation (Reddy et al. 2004; Gill and Tuteja 2010). Another general characteristic of stress tolerance is maintaining osmotic homeostasis, which is critical for protecting membranes from desiccation and facilitating continued nutrient uptake from a potentially high-ionic root zone, such as during salinity or heavy metal stress. Maintaining osmotic homeostasis is achieved in general by increasing the concentration of osmolytic compounds, for example, total soluble sugars (Jisha and Puthur 2016b), and/or amino acids such as proline (Hayat et al. 2012). Abiotic stress tolerance is a complex biological trait expressed both physiologically and morphologically through altered gene expression, modified hormone levels, metabolite biosynthesis, and antioxidant activity (Zhang et al. 2014; Nguyen et al. 2018). Examples of stress tolerance responses include increasing root growth, modifying cellular relative water content via osmotic adjustment, and abscisic acid-induced stomatal closure to improve water use efficiency upon drought and salinity stress (Bartels and Sunkar 2005; Taiz and Zeiger 2006), and faster and more efficient utilization of storage compounds during chilling stress (Hussain et al. 2017). Seed Osmolyte Priming and Abiotic Stress Tolerance 259

3 Priming Compounds Conferring Abiotic Stress Tolerance in Plants

Plant growth regulators (PGRs) are naturally occurring or synthetic compounds which modify developmental and/or metabolic processes by specifically affecting a plant’s natural hormone system (Rademacher 2015). Often bioactive at very small concentrations, PGRs are widely used to modify plant morphology, confer stress tolerance, improve yield, and increase harvesting efficiencies. In addition to the bioactive compound used, the method of PGR application and plant developmental stage is critical to achieve the desired effects. Under abiotic stress conditions, exogenous application of PGRs induces common stress tolerance responses such as increased antioxidant enzyme activity, leaf proline content, and relative water content. Examples include the brassinosteroid 28-homobrassinolide on Indian mustard (Brassica juncea) (Zhang et al. 2014), melatonin on fava beans (Vicia faba) (Dawood and El-Awadi 2015), salicylic acid on rice (Oryza sativa) (Wang et al. 2016), and gamma aminobutyric acid (GABA) on ryegrass (Lolium perenne) (Krishnan et al. 2013). While PGRs are usually applied as a foliar spray, they have proven to be valuable seed priming agents as well. Wheat seeds (Triticum aestivum) primed with benzyl aminopurine had increased α-amylase activity and soluble sugar concentrations during salt stress (Bajwa et al. 2018). Under drought conditions, rice grown from seeds primed with salicylic acid had increased antioxidant activity, osmolyte concentration, and cellular water potential (Farooq et al. 2009). Red cabbage seedlings (Brassica oleracea rubrum) grown from melatonin-primed seeds had higher germination rates, decreased levels of lipid peroxidation, and higher fresh weight when grown under high concentrations of copper (Posmyk et al. 2008). In addition to traditional PGRs, essential oils (EOs) and botanical extracts are increasingly used in place of synthetic compounds to protect against biotic and abiotic stresses. Thymol and carvacrol, monoterpenes derived from the essential oils of thyme and oregano, have antioxidant properties which support membrane integrity during abiotic or biotic stresses (De Azeredo et al. 2011; Ye et al. 2016). Seedlings grown from radish seeds (Raphanus raphanistrum) imbibed in carvacrol had increased pigment (carotenoid and anthocyanin) concentrations, antioxidant activity, and survival rate under drought conditions (Klein et al. 2017). Thymol-priming reduced the effects of salinity stress on pea seedlings (Pisum sativum) by increasing superoxide dismutase (SOD) activity (Kazemi 2013). Rice seeds primed with sunflower extract had increased root and shoot length and increased fresh weight when grown under high salinity (Farooq et al. 2011). Allelopathic sorghum extract priming increased total phenolics and soluble sugar concentrations and decreased Na+ content in wheat grown under high salinity (Bajwa et al. 2018). Given their bioactivity at small concentrations, optimal priming concentrations for PGRs, EOs, and botanical extracts need to be determined to avoid negatively affecting germination and seedling development (Fariduddin et al. 2003; Posmyk et al. 2008; Martino et al. 2010; Arteca 2013). Recently, priming with osmolytic compounds has received considerable interest by the scientific community. Some of 260 D. Ginzburg and J. D. Klein the beneficial effects of priming with osmolytic compounds in various plant species against different abiotic stresses and at various stages of plant growth are shown in Table 1.

Table 1 Selected references for seed priming with osmolytes and resulting abiotic stress tolerance Concentration Priming Plant and duration of Positive effects of priming compound species priming regulating stress tolerance Reference Proline Vigna 5 mM and Increased germination %, Posmyk and radiata 150 mM proline; proline content, and hypocotyl Janas (2007) 6 hours growth, decreased lipid peroxidation under chilling stress Oryza 1, 5, and 10 mM Increased root and shoot Deivanai sativa L. proline; 12 hours length, chlorophyll et al. (2011) concentration, and proline biosynthesis under salinity stress Mannitol Cakile 2% mannitol Increased RWC, GSH and Ellouzi et al. maritima solution; proline content, and SOD (2017) L. 12 hours activity, decreased MDA levels under drought and salinity stresses Medicago 4% mannitol; Increased root and shoot Amooaghaie sativa L. 12 hours length, proline content, and (2011) antioxidant activity, decreased ion leakage under salinity stress Cicer 1–10% mannitol; Increased root and root fresh Kaur et al. arietinum 24, 48, 72 hours and dry weight, amylase and (2005) L. sucrose synthase activities, and total leaf sugar content under osmotic stress Glycinebetaine Four 50, 100, 150, Increased germination rate, Zhang and (GB) turfgrass and seedling fresh weight, and Rue (2012) species 200 mM GB; water content under both 24 hours osmotic and salinity stresses Oryza 50, 100, and Increased leaf GB content, Farooq et al. sativa L. 150 mg/L GB; soluble sugar and antioxidant (2008) 48 hours concentrations, increased RWC, and decreased ion leakage under drought stress Trehalose Zea mays 10 mM Decreased ion leakage and Zeid (2009) L. trehalose; lipid peroxidation, increased 8 hours leaf K/Na ratio under salinity stress Raphanus 25 and 50 mM Increased root fresh weight, Shafiq et al. sativus L. trehalose; GB content, antioxidant (2015) 14 hours content and activity, decreased lipid peroxidation under drought stress (continued) Seed Osmolyte Priming and Abiotic Stress Tolerance 261

Table 1 (continued) Concentration Priming Plant and duration of Positive effects of priming compound species priming regulating stress tolerance Reference Spermidine Oryza 5 mM Increased phenolic, GB, Sheteiwy sativa L. spermidine; soluble sugar and protein et al. (2017) 24 hours contents, α-amylase, and antioxidant activities under chilling stress Triticum 5 mM Increased stomatal Iqbal and aestivum spermidine; conductance and grain yield, Ashraf (2005) L. 12 hours decreased shoot [Na+] and [Cl−] under salinity stress Trifolium 30 μM Increased α-amylase activity, Li et al. repens spermidine; fructose and glucose (2014) 3 hours concentrations, increased antioxidant activity, and decreased lipid peroxidation under osmotic stress GABA/BABA Trifolium 1 μM GABA; Increased root and shoot Cheng et al. repens 2 hours length, fresh and dry weight, (2018) and dehydrin concentrations, decreased peroxidation levels under salinity stress Oryza 0–2.5 mM Increased pigment Jisha and sativa L. BABA; 12 hours concentration, PS I and II Puthur activities, antioxidant enzyme (2016b) activity, and proline content, decreased MDA concentration under salinity and osmotic stresses

4 Possible Mechanisms of Osmolyte Priming: Induced Abiotic Stress Tolerance

The currently understood mechanisms of priming for abiotic stress tolerance are twofold. Seed imbibition and drying promote the seed to an advanced germinative state such that primed seeds germinate faster and at higher rates than non-primed seeds under adverse conditions, resulting in increased competitiveness for limited resources and overall yield increases (Chen and Arora 2013; Paparella et al. 2015). This advanced germinative state is the result of multiple induced processes such as repair and increased synthesis of metabolic machinery, cell-cycle components, aquaporin activity, and ion transporters. Seed imbibition additionally promotes increased gibberellic acid (GA) biosynthesis and abscisic acid (ABA) degradation, thus furthering the germinative state via reserve mobilization and endosperm weak- ening (Bewley et al. 2012; Chen and Arora 2013; Zhang et al. 2014). Seed imbibition with water alone also imparts a stress imprint or “memory” to the seed which remains after drying. Even under optimal priming conditions, and regardless of the protocol used, seed hydration and drying are inevitably damaging 262 D. Ginzburg and J. D. Klein to a seed due to ROS production and membrane disruption upon imbibition and the likely decreased desiccation tolerance caused by drying (Chen and Arora 2013). Improper priming conditions, such as rapid water uptake, priming at low temperatures, and/or rapid drying, would therefore only exacerbate such damage (Bewley et al. 2012). This stress imprint is often connected to increased antioxidant activity and protective compounds in the seedling. Antioxidant enzyme activity has been correlated to stress tolerance in many plant species (Munns 2002; Ashraf and Ali 2008; Posmyk et al. 2008; Zhang et al. 2014; Hussain et al. 2017). Increased antioxidant activity can be attributed both to seed rehydration in general and to specific priming agents and conditions to induce stress (osmotic, salinity, heavy metal, etc.) such as seed microencapsulation (Murungu et al. 2004) or priming temperature and duration (Bujalski and Nienow 1991). Based on their bioactivity and concentration, the specific priming compounds used may further influence seed germination and seedling growth beyond that of water alone (hydropriming) (Posmyk et al. 2008; Baier et al. 2019; Bajwa et al. 2018). The protective effects of seed priming against oxidative stress have been extensively reported. Primed seedlings exposed to abiotic stress had increased activities of ROS scavenging enzymes such as superoxide dismutase, catalase, and ascorbate peroxidase (Bailly et al. 2000; El-Araby and Hegazi 2004; Lei et al. 2005; Hussain et al. 2017; Latif et al. 2016). In all cases, seedlings from primed seeds had higher germination rates and decreased levels of lipid peroxidation. Priming also combats oxidative stress by increasing polyphenol and pigment biosynthesis (Bailly 2004; Nouman et al. 2012; Latif et al. 2016; Klein et al. 2017). In addition to increasing ROS scavenging, seed priming with osmolytes directly supports membrane integrity and protein structure during abiotic stress. This has been achieved by increasing the concentration of late embryogenesis abundant (LEA) proteins in polyethylene-glycol (PEG)-primed pepper (Cortez-Baheza et al. 2007), heat shock proteins in hydroprimed sugar beet (Catusse et al. 2011), and protein folding and stabilization proteins in PEG-primed rapeseed (Kubala et al. 2015). The ability of plants to break down starch into an adequate supply of soluble sugars for metabolism is crucial for survival and tolerance to abiotic stresses (Rosa et al. 2009; Zheng et al. 2016). Rice seedlings from KCl and CaCl2 primed seeds demonstrated increased starch hydrolysis and resulted in greater yield (Farooq et al. 2006). Under salinity stress, BABA-primed mung bean seedlings had increased soluble carbohydrate levels, contributing to increased photosynthetic pigment concentration and seedling fresh weight (Jisha and Puthur 2016a). An additional benefit of increased soluble sugar concentrations is to support cellular osmotic homeostasis (Jisha and Puthur 2016a), which, as previously noted, supports nutrient uptake and membrane integrity. Priming-induced mechanisms to increase osmolyte concentrations also include increasing proline, as noted in cauliflower (Latif et al. 2016), and total soluble protein concentrations in wheat (Bajwa et al. 2018). Seed Osmolyte Priming and Abiotic Stress Tolerance 263

5 Conclusion and Future Perspectives

The beneficial effects of seed priming have not been observed consistently. Most studies to date do not report beneficial residual effects of seed priming beyond the seedling stage or without early stress exposure. Protection induced by priming is often more pronounced if the stress is present at sowing, upon germination or shortly after emergence. Certain priming treatments are species-specific, perhaps based on the structure of the seeds and the amount of endosperm they contain that can “store” the priming compound (Dawood 2018; Zheng et al. 2016). The protective effects of a priming treatment, if expressed at all, do not always persist as the plant matures or if it grows in nonstressed conditions (Cayuela et al. 1996; Posmyk et al. 2008; Poonam et al. 2013; Jisha and Puthur 2016a; Savvides et al. 2016; Ye et al. 2016; Ellouzi et al. 2017). Some studies have reported a beneficial residual effect of seed priming beyond the seedling stage and/or without the immediate induction of stress such as maize seeds soaked in water (Murungu et al. 2004), or microencapsulated with the fungicide tebuconazole (Yang et al. 2014), and melatonin-primed fava beans (Dawood and El-Awadi 2015). Although epigenetics have been invoked as a possible mode of action for the protection provided by seed priming (Savvides et al. 2016), there is not yet evidence that plants that grow from primed seeds can themselves produce seeds with endogenous protection. There is no evidence that any of the osmolytes or other compounds thus far studied as priming materials are injurious to human health or to the environment at the concentrations used with seed treatments. However, fungal and bacterial populations on seeds can increase during extended treatment with osmolytes (Wright et al. 2003), even if the seeds are subsequently coated with antimicrobial compounds, but this is unlikely to affect human health in seeds that germinate. In extreme cases, ingesting plants with an induced overproduction of natural antioxidants can cause internal injury (Bast and Haenen 2002), although this is unlikely to occur as a result of seed priming. However, there is evidence that antioxidant activity measured in vitro may not correlate with the actual activity in vivo (Ndhlala et al. 2010). Noninvasive methods of measuring both plant structures (Tardieu et al. 2017) and physiological compounds or mechanisms (Boughton et al. 2016) which protect against abiotic stress must be further developed to allow in vivo measurement of the effects of priming treatments. This will provide both more accurate measurements of the true influence of seed treatments and may well provide an impetus to the development/discovery of more and better-targeted osmolytes and other compounds. The resulting improved plant resistance to abiotic stress will in turn enhance food security in an era of global uncertainty. We suggest three fronts for further investigations of seed priming for abiotic stress resistance: 1. Growing plants from treated seeds to maturity, so as to ensure that there are no negative effects of treatments on quality or yield, despite the fact that the seedling can withstand stress. 264 D. Ginzburg and J. D. Klein

2. Developing treatments with effects that persist if treated seeds are stored for one or more seasons after treatment. 3. Always testing seeds from at least two cultivars of the plant being investigated, to ensure that the treatment being developed will be suitable for as broad a genetic background as possible.

References

Amooaghaie R (2011) The effect of hydro and osmopriming on alfalfa seed germination and antioxidant defenses under salt stress. Afr J Biotechnol 10:6269–6275 Arteca RN (2013) Plant growth substances: principles and applications. Springer Science & Business Media Ashraf M, Ali Q (2008) Relative membrane permeability and activities of some antioxidant enzymes as the key determinants of salt tolerance in canola (Brassica napus L.). Environ Exp Bot 63:266–273 Bailly C, Benamar A, Corbineau F, Côme D (2000) Antioxidant systems in sunflower (Helianthus annuus L.) seeds as affected by priming. Seed Sci Res 10:35–42 Bailly C (2004) Active oxygen species and antioxidants in seed biology. Seed Sci Res 14:93–107 Baier M, Bittner A, Prescher A, van Buer J (2019) Preparing plants for improved cold tolerance by priming. Plant Cell Environ 42:782–800 Bajwa AA, Farooq M, Nawaz A (2018) Seed priming with sorghum extracts and benzyl aminopurine improves the tolerance against salt stress in wheat (Triticum aestivum L.). Physiol Mol Biol Plants 24:239–249 Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24:23–58 Bast A, Haenen GR (2002) The toxicity of antioxidants and their metabolites. Environ Toxicol Pharmacol 1:251–258 Bernstein N, Shoresh M, Xu Y, Huang B (2010) Involvement of the plant antioxidative response in the differential growth sensitivity to salinity of leaves vs roots during cell development. Free Radic Biol Med 49:1161–1171 Bewley JD, Bradford K, Hilhorst H (2012) Seeds: physiology of development, germination and dormancy. Springer Science & Business Media Boughton BA, Thinagaran D, Sarabia D, Bacic A, Roessner U (2016) Mass spectrometry imaging for plant biology: a review. Phytochem Rev 15:445–488 Bujalski W, Nienow AW (1991) Large-scale osmotic priming of onion seeds: a comparison of different strategies for oxygenation. Sci Hortic 46:13–24 Catusse J, Meinhard J, Job C, Strub JM, Fischer U, Pestsova E, Job D (2011) Proteomics reveals potential biomarkers of seed vigor in sugarbeet. Proteomics 11:1569–1580 Cayuela E, Pérez-Alfocea F, Caro M, Bolarin MC (1996) Priming of seeds with NaCl induces physiological changes in tomato plants grown under salt stress. Physiol Plant 96:231–236 Chen K, Arora R (2013) Priming memory invokes seed stress tolerance. Environ Exp Bot 94:33–45 Cheng B, Li Z, Liang L, Cao Y, Zeng W, Zhang X, Peng Y (2018) The γ-aminobutyric acid (GABA) alleviates salt stress damage during seeds germination of white clover associated with Na+/K+ transportation, Dehydrins accumulation, and stress-related genes expression in white clover. Int J Mol Sci 19:2520 Cortez-Baheza E, Peraza-Luna F, Hernandez-Alvarez MI, Aguado-Santacruz GA, Torres-Pacheco I, González-Chavira MM, Guevara-Gonzalez RG (2007) Profiling the transcriptome in Capsicum annuum L. seeds during osmopriming. Am J Plant Physiol 2:99–106 Dawood MG (2018) Stimulating plant tolerance against abiotic stress through seed priming. In: Advances in seed priming. Springer, Singapore, pp 147–183 Seed Osmolyte Priming and Abiotic Stress Tolerance 265

Dawood MG, El-Awadi ME (2015) Alleviation of salinity stress on Vicia faba L. plants via seed priming with melatonin. Acta Biológica Colombiana 20:223–235 Deivanai S, Xavier R, Vinod V, Timalata K, Lim OF (2011) Role of exogenous proline in ameliorating salt stress at early stage in two rice cultivars. J Stress Physiol Biochem 7:157–174 De Azeredo GA, Stamford TLM, Nunes PC, Neto NJG, De Oliveira MEG, De Souza EL (2011) Combined application of essential oils from Origanum vulgare L. and Rosmarinus officinalis L. to inhibit bacteria and autochthonous microflora associated with minimally processed vegetables. Food Res Int 44:1541–1548

Ellouzi H, Sghayar S, Abdelly C (2017) H2O2 seed priming improves tolerance to salinity; drought and their combined effect more than mannitol in Cakile maritima when compared to Eutrema salsugineum. J Plant Physiol 210:38–50 El-Araby MM, Hegazi AZ (2004) Responses of tomato seeds to hydro-and osmo-priming, and possible relations of some antioxidant enzymes and endogenous polyamine fractions. Egypt J Biol 6:81–93 Fariduddin Q, Hayat S, Ahmad A (2003) Salicylic acid influences net photosynthetic rate, carboxylation efficiency, nitrate reductase activity, and seed yield in Brassica juncea. Photosynthetica 41:281–284 Farooq M, Basra SMA, Hafeez K (2006) Seed invigoration by osmohardening in fine and course rice. Seed Sci Technol 34:181–186 Farooq M, Basra SMA, Wahid A, Ahmad N, Saleem BA (2009) Improving the drought tolerance in rice (Oryza sativa L.) by exogenous application of salicylic acid. J Agron Crop Sci 195:237–246 Farooq M, Basra SMA, Wahid A, Cheema ZA, Cheema MA, Khaliq A (2008) Physiological role of exogenously applied glycinebetaine to improve drought tolerance in fine grain aromatic rice (Oryza sativa L.). J Agron Crop Sci 194:325–333 Farooq M, Habib M, Rehman A, Wahid A, Munir R (2011) Employing aqueous allelopathic extracts of sunflower in improving salinity tolerance of rice. J Agric Soc Sci 7:75–80 Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930 Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A (2012) Role of proline under changing environments: a review. Plant Signal Behav 7:1456–1466 Hussain M, Farooq M, Lee DJ (2017) Evaluating the role of seed priming in improving drought tolerance of pigmented and non-pigmented rice. J Agron Crop Sci 203:269–276 Iqbal M, Ashraf M (2005) Changes in growth, photosynthetic capacity and ionic relations in spring wheat (Triticum aestivum L.) due to pre-sowing seed treatment with polyamines. Plant Growth Regul 46:19–30 Jaspers P, Kangasjärvi J (2010) Reactive oxygen species in abiotic stress signaling. Physiol Plant 138:405–413 Jisha KC, Puthur JT (2016a) Seed priming with BABA (β-amino butyric acid): a cost-effective method of abiotic stress tolerance in Vigna radiata (L.) Wilczek. Protoplasma 253:277–289 Jisha KC, Puthur JT (2016b) Seed priming with beta-amino butyric acid improves abiotic stress tolerance in rice seedlings. Rice Sci 23:242–254 Kaur S, Gupta AK, Kaur N (2005) Seed priming increases crop yield possibly by modulating enzymes of sucrose metabolism in chickpea. J Agron Crop Sci 191:81–87 Kazemi M (2013) Priming with 5-SSA, glutamine and thyme oil improves the emergence and early seedling growth in pea (Pisum sativum L.). bulletin of environment, pharmacology. Life Sci 3:21–27 Klein JD, Firmansyah A, Panga N, Abu-Aklin W, Dekalo-Keren M, Gefen T, Kohen R, Raz Shalev Y, Dudai N, Mazor L (2017) Seed treatments with essential oils protect radish seedlings against drought. AIMS Agriculture and Food 2:345–353 Krishnan S, Laskowski K, Shukla V, Merewitz EB (2013) Mitigation of drought stress damage by exogenous application of a non-protein amino acid γ–aminobutyric acid on perennial ryegrass. J Am Soc Hortic Sci 138:358–366 266 D. Ginzburg and J. D. Klein

Kubala S, Garnczarska M, Wojtyla Ł, Clippe A, Kosmala A, Żmieńko A, Quinet M (2015) Deciphering priming-induced improvement of rapeseed (Brassica napus L.) germination through an integrated transcriptomic and proteomic approach. Plant Sci 231:94–113 Latif M, Akram NA, Ashraf M (2016) Regulation of some biochemical attributes in drought- stressed cauliflower Brassica( oleracea L.) by seed pre-treatment with ascorbic acid. J Hortic Sci Biotechnol 91:129–137 Lei YB, Song SQ, Fu JR (2005) Possible involvement of anti-oxidant enzymes in the cross- tolerance of the germination/growth of wheat seeds to salinity and heat stress. J Integr Plant Biol 47:1211–1219 Li Z, Peng Y, Zhang XQ, Ma X, Huang LK, Yan YH (2014) Exogenous spermidine improves seed germination of white clover under water stress via involvement in starch metabolism, antioxidant defenses and relevant gene expression. Molecules 19:18003–18024 Martino LD, Mancini E, Almeida LFRD, Feo VD (2010) The antigerminative activity of twenty- seven monoterpenes. Molecules 15:6630–6637 Munns R (2002) Comparative physiology of salt and water stress. Plant Cell Environ 25:239–250 Murungu FS, Chiduza C, Nyamugafata P, Clark LJ, Whalley WR, Finch-Savage WE (2004) Effects of ‘on-farm seed priming’on consecutive daily sowing occasions on the emergence and growth of maize in semi-arid Zimbabwe. Field Crop Res 89:49–57 Ndhlala A, Moyo M, Van Staden J (2010) Natural antioxidants: fascinating or mythical biomolecules? Molecules 15:6905–6930 Nouman W, Siddiqui MT, Basra SMA, Afzal I, Rehman HU (2012) Enhancement of emergence potential and stand establishment of Moringa oleifera Lam. by seed priming. Turk J Agric For 36:227–235 Nguyen HC, Lin KH, Ho SL, Chiang CM, Yang CM (2018) Enhancing the abiotic stress tolerance of plants: from chemical treatment to biotechnological approaches. Physiol Plant 164:452–466 Paparella S, Araújo SS, Rossi G, Wijayasinghe M, Carbonera D, Balestrazzi A (2015) Seed priming: state of the art and new perspectives. Plant Cell Rep 34:1281–1293 Poonam S, Kaur H, Geetika S (2013) Effect of jasmonic acid on photosynthetic pigments and stress markers in Cajanus cajan (L.) Millsp. seedlings under copper stress. Am J Plant Sci 4:817 Posmyk MM, Janas KM (2007) Effects of seed hydropriming in presence of exogenous proline on chilling injury limitation in Vigna radiata L. seedlings. Acta Physiol Plant 29:509–517 Posmyk MM, Kuran H, Marciniak K, Janas KM (2008) Presowing seed treatment with melatonin protects red cabbage seedlings against toxic copper ion concentrations. J Pineal Res 45:24–31 Rademacher W (2015) Plant growth regulators: backgrounds and uses in plant production. J Plant Growth Regul 34:845–872 Reddy AR, Chaitanya KV, Vivekanandan M (2004) Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. J Plant Physiol 161:1189–1202 Rosa M, Prado C, Podazza G, Interdonato R, González JA, Hilal M, Prado FE (2009) Soluble sugars: metabolism, sensing and abiotic stress: a complex network in the life of plants. Plant Signal Behav 4:388–393 Taiz L, Zeiger E (2006) Plant physiology, vol 25, 5th edn. Sinauer Associates, Sunderland, pp 591–623 Savvides A, Ali S, Tester M, Fotopoulos V (2016) Chemical priming of plants against multiple abiotic stresses: mission possible? Trends Plant Sci 21:329–340 Shafiq S, Akram NA, Ashraf M (2015) Does exogenously-applied trehalose alter oxidative defense system in the edible part of radish (Raphanus sativus L.) under water-deficit conditions? Sci Hortic 185:68–75 Sheteiwy M, Shen H, Xu J, Guan Y, Song W, Hu J (2017) Seed polyamines metabolism induced by seed priming with spermidine and 5-aminolevulinic acid for chilling tolerance improvement in rice (Oryza sativa L.) seedlings. Environ Exp Bot 137:58–72 Tardieu F, Cabrera-Bosquet L, Pridmore T, Bennett M (2017) Plant phenomics, from sensors to knowledge. Curr Biol 27:R770–R783 Seed Osmolyte Priming and Abiotic Stress Tolerance 267

Wang WQ, Chen Q, Hussain S, Mei JH, Dong HL, Peng SB, Nie LX (2016) Pre-sowing seed treatments in direct-seeded early rice: consequences for emergence, seedling growth and associated metabolic events under chilling stress. Sci Rep 6:19637 Wright B, Rowse H, Whipps JM (2003) Microbial population dynamics on seeds during drum and steeping priming. Plant Soil 255:631–640 Western TL (2012) The sticky tale of seed coat mucilages: production, genetics, and role in seed germination and dispersal. Seed Sci Res 22:1–25 Yang D, Wang N, Yan X, Shi J, Zhang M, Wang Z, Yuan H (2014) Microencapsulation of seed- coating tebuconazole and its effects on physiology and biochemistry of maize seedlings. Colloids Surf B: Biointerfaces 114:241–246 Ye X, Ling T, Xue Y, Xu C, Zhou W, Hu L, Chen J, Shi Z (2016) Thymol mitigates cadmium stress by regulating glutathione levels and reactive oxygen species homeostasis in tobacco seedlings. Molecules 21:1339 Zeid IM (2009) Trehalose as osmoprotectant for maize under salinity-induced stress. Res J Agric Biol Sci 5:613–622 Zhang X, Lu G, Long W, Zou X, Li F, Nishio T (2014) Recent progress in drought and salt tolerance studies in Brassica crops. Breed Sci 64:60–73 Zhang Q, Rue K (2012) Glycinebetaine seed priming improved osmotic and salinity tolerance in turfgrasses. HortScience 47:1171–1174 Zheng M, Tao Y, Hussain S, Jiang Q, Peng S, Huang J, Nie L (2016) Seed priming in dry direct- seeded rice: consequences for emergence, seedling growth and associated metabolic events under drought stress. Plant Growth Regul 78:167–178 Relationship Between Polyamines and Osmoprotectants in the Response to Salinity of the Legume-Rhizobia Symbiosis

Miguel López-Gómez, Javier Hidalgo-Castellanos, Agustín J. Marín-Peña, and J. Antonio Herrera-Cervera

1 Salinity

Salinity is an environmental factor produced by the accumulation of mineral salts in soils and waters. The dissolved salts form cationic electrolytes such as Na+, 2+ 2+ + − 2− − 2− Ca , Mg , and K and anionic electrolytes such as Cl , SO4 , HCO3 , CO3 , and − NO3 (Manchanda and Garg 2008). Soil salinity negatively affects the normal development of plants, being one of the main environmental factors that limit agricultural productivity in arid and semiarid regions of the planet. It is estimated that about 45 million hectares of arable land, approximately 20% of the total, are affected by salinity (Munns 2009). This is a serious problem to reach the objective of increasing agricultural production by 70% by the year 2050 (FAO 2009), which is what would be needed to meet the demand of an expected world population close to 10 billion people. The causes of salinity are varied: primary salinity is produced by the erosion of rocks with a high content of soluble salts and also by the deposition of marine salts carried by the wind, rain, or tides, with sodium chloride being the main salt present (Rengasamy 2006). Secondary salinity is caused by anthropogenic activities, such as the misuse of fertilizers, excessive irrigation with low-quality water, drainage system deficient in crops, deforestation, monocultures, or excess urbanization of the fields, which alters the water balance of the soil causing salinization (Abiala et al. 2018). As a result, salinity affects the physicochemical properties of the soil, which leads to an adverse effect on the ecological balance of the affected area, which at the agrarian level results in a decrease in crop production as well as a degradation of the arable land.

M. López-Gómez (*) · J. Hidalgo-Castellanos · A. J. Marín-Peña · J. A. Herrera-Cervera Dpto. Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Granada, Spain e-mail: [email protected]

© Springer Nature Switzerland AG 2019 269 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_13 270 M. López-Gómez et al.

2 Effects of Salinity on Plants

Soil salinity results in plant growth limitation, development, and survival due to the saline stress induction with alterations in physiological and metabolic processes of plants depending on the severity of stress, duration, and tolerance of the plant (Hasanuzzaman et al. 2013). The main physiological effect of saline stress on vegetables is related with a reduction in photosynthesis and restricted water and nutrient uptake by the plant with a concomitant growth inhibition produced, initially, by an osmotic effect and then followed by an ionic toxicity and a nutritional imbalance due to the interference of saline ions with nutrients. Saline stress triggers oxidative damage (Huang et al. 2017), which causes alterations in proteins and membranes structure.

2.1 Osmotic Stress

Elevated salt concentration in the rhizosphere provokes a reduction in the capacity of water absorption by the roots due to a reduction of the soil water potential, generating a hyperosmotic stress that entails several changes at the physiological level. Among these changes are a reduction in cell division and elongation, decrease in leaf area and photosynthetic capacity caused by alterations in electronic transport and inhibition of key enzymes of the Calvin cycle (Manchanda and Garg 2008), a disruption of the membranes, reduction of the ability to eliminate reactive oxygen species and stomatal closure due to the reduction of the turgor pressure of the stomatal cells (Munns and Tester 2008).

2.2 Ionic Stress

Another harmful effect of salt stress is the excessive accumulation of ions from the dissolved salts, especially Na+ and Cl-, which causes an ionic imbalance in the plant. Ionic toxicity produces a nutritional imbalance when Na+ ions compete with essential ions such as Ca2+ and K+ (Tejera et al. 2006), which causes severe physiological and structural disorders, since K+ is essential for growth and development.

2.3 Oxidative Stress

Saline stress induces the formation of reactive oxygen species (ROS) in the cell due to imbalances and breaks in the electron transport chain in chloroplasts and mitochondria. Among the ROS that accumulate are hydrogen peroxide (H2O2), hydroxyl Relationship Between Polyamines and Osmoprotectants in the Response to Salinity… 271

⋅ − 1 radical (OH ), superoxide anion radical (O2 ), and singlet oxygen ( O2). ROS produce oxidative damage in different cellular components such as DNA, proteins, and lipids, producing alterations in cellular functions (Foyer and Noctor 2005). At the protein level, these radicals are able to act on the side chains of amino acid residues, which completely change the structure and protein function. Fatty acids, especially membrane phospholipids, are very susceptible to oxidation, generating lipid perox- ides. This causes the alteration of the fluidity and produces changes in the transporters and receptors of the membrane (Koca et al. 2007). In addition, ROS are able to break and alter the DNA chains, originating mutations (Møller et al. 2007).

3 Agrarian Importance and Legume-Rhizobia Symbiosis

Legumes are plants belonging to the Fabaceae family with approximately 20,000 species distributed in about 700 genera of cosmopolitan distribution, with capacity to adapt to all kinds of ecological conditions thanks to a great variety of strategies (Smýkal et al. 2015). This family contains three subfamilies – Mimosoideae, Caesalpinoidae (tropical subtropical trees and shrubs), and Papilionoideae – formed by shrub and herbaceous species that include most of the cultivable species (Doyle and Luckow 2003). Within this subfamily are the legumes with greater agronomic interest such as groundnut (Arachis hypogaea), soybean (Glycine max), common bean (Phaseolus vulgaris), pea (Pisum sativum), and beans (Vicia faba), among others (Le et al. 2007). The legumes are the second family in agronomic importance after the grasses (Gepts et al. 2005) due to their ability to establish symbiosis with soil diazotrophic bacteria known as rhizobia and fix atmospheric nitrogen. This important characteristic makes legumes fundamental pieces in crop rotations, since they allow the addition of organic nitrogen to the soil (Mantri et al. 2013), reducing the need of chemical fertilizers, with a concomitant reduction in the use of energy and CO2 emissions associated with their production (Jensen et al. 2012). In addition, legumes can improve saline soil fertility and help to reintroduce agriculture to these lands due to their capacity to grow on nitrogen-poor soils (Crespi and Gálvez 2000; Coba de la Peña and Pueyo 2012). The symbiosis legume-rhizobia results in the formation of nodules in the roots of legumes where the rhizobia differentiate into bacteroides, and the proper conditions for reduction of atmospheric nitrogen to ammonium are provided by the plant (Burris 1984). Under such conditions, bacteroides experiment a genetic reprogram- ing that allow the synthesis of the nitrogenase complex, where the nitrogen fixation takes place, providing the plant with ammonia in exchange of carbon and energy supply (Udvardi and Poole 2013). 272 M. López-Gómez et al.

4 Effect of Salt Stress on Legume-Rhizobia Symbiosis

One of the most successful strategies of plants to cope with environmental changes is to establish symbiotic relationships with soil microorganisms. However, in spite of the capacity to establish symbiotic interactions with rhizobia, legumes are considered salinity sensitive species, since the establishment of the symbiosis and its efficiency in fixing nitrogen are very sensitive to this stress. Nevertheless, considerable variability in salinity tolerance among crop legumes has been reported (Bruning and Rozema 2013) showing grain legumes such as Phaseolus vulgaris, Cicer arietinum, and Vigna radiata high sensitivity to salinity (Abdel-Wahab et al. 2002), meanwhile Pisum sativum, Vicia faba, and Medicago sativa are moderately salt sensitive. Under these conditions is reduced the ability of rhizobia to infect the root and nitrogenase activity (Aranjuelo et al. 2014). This decrease in nitrogen fixation under saline stress may be related to limitation in the energetic substrate shortage to bacteroides following photosynthetic activity decline and nodule sucrose breakdown reduction (Lopez et al. 2008). Sucrose is the main carbon source required by the nodule; therefore, sucrose synthase (SS) is a key enzyme for the functioning of nitrogenase (Gordon 1995) and has been shown to be sensitive to different stresses, among them salinity (López et al. 2008). In addition, Na+ toxicity reduces homeostasis and uptake of essential nutrients, including Ca, K, and P, which reduces the growth of the nodule and its proper functioning (Ben Salah et al. 2010).

5 Tolerance to Salinity in the Legume-Rhizobia Symbiosis

Legumes in symbiosis have adopted physiological, morphological, and molecular changes to tolerate salt stress in the nodule and in the rest of the plant. These changes include the accumulation of osmoprotectants (such as proline and sugars) to counteract osmotic stress (López-Gómez et al. 2011). These molecules are small and electrically neutral that are nontoxic at molar concentrations and stabilize proteins and membranes against the denaturing effect of high concentrations of salts. In dry or saline environments, osmoprotectants can therefore serve both to raise cellular osmotic pressure and to protect cell constituents (McNeil et al. 1999). Exclusion of toxic ions to balance nutrient acquisition (Tejera et al. 2006) and increment of antioxidant metabolites and enzymes to prevent accumulation of ROS and protect membranes, proteins, and DNA in the nodular tissues are also mechanisms involved in nodule salt stress responses (Rubio et al. 2009). Previous studies have highlighted the increment in the concentrations of disaccharide trehalose by the inhibition of its degrading enzyme, trehalase, as one of the strategies to cope with salt stress in root nodules of Medicago truncatula (Lopez et al. 2008), a fact that corroborates the high concentrations of trehalose found in nodules of Lotus japonicus during a saline stress (Lopez et al. 2006). Relationship Between Polyamines and Osmoprotectants in the Response to Salinity… 273

Within the amino acids, proline shows the largest increase in the nodular tissue in plants of M. truncatula and L. japonicus (López-Gómez et al. 2011). An increase in proline has also been observed in nodules of P. vulgaris and M. sativa related to osmotic adjustment (Tejera García et al. 2007; Lopez-Gomez et al. 2014). Biosynthesis of antioxidants (Becana et al. 2010) and differential gene expression (Molina et al. 2011) are other responses described in root nodules under salt stress conditions. All these responses are orchestrated by specific hormones such as abscisic acid (ABA) (Palma et al. 2014) and salicylic acid (SA) (Palma et al. 2013). These endogenous molecules regulate, via synergistic and antagonistic actions, the expression of different but overlapping suites of genes, which is referred to as signaling crosstalk (Nishi et al. 2015).

6 Polyamines

Polyamines (PAs) are hormonal compounds and growth regulators, with low molecular weight, aliphatic nature, and polycationic character at physiological pH, present in different types of organisms and particularly in plants, where they are involved in the regulation of various physiological processes related to growth and development as well as in responses to abiotic and biotic stresses (Pal et al. 2015). Polyamines also participate in the responses to drought, high and low temperatures, wounds, ozone, heavy metals and oxidative stress (Wimalasekera et al. 2011). During the last years, a special attention to the involvement of PAs in the response to salinity has been paid, since a relationship between the accumulation of this type of compounds and tolerance to salinity has been described (Minocha et al. 2014). The most common PAs in nature are putrescine (Put), spermidine (Spd), and spermine (Spm), as well as other diamines such as 1–3 diaminopropane (DAP) and cadaverine (Cad). There are also rare PAs that have a very limited distribution in nature, found mainly in prokaryotes (Terui et al. 2005). These consist of molecules derived or related to Spd and Spm, among which are homospermidine (HomSpd), homospermine (HomSpm), norspermidine (NorSpd), and other pentamines and hexamines (Sagor et al. 2013).

6.1 Polyamines Metabolism

Common PAs, Put, Spd, and Spm are synthesized by the decarboxylation of arginine and ornithine by arginine and ornithine decarboxylases (ADC and ODC), respectively. The triamine and tetraamine Spd and Spm are derivatives from the diamine Put by the addition of aminopropyl groups donated by S-adenosylmethionine in reactions catalyzed by Spd synthase (SPSD) and Spm synthase (SPMS), respectively (Handa et al. 2018) (Fig. 1). However, the content of PAs can also be regulated by their degradation rates by the catabolic enzymes diamine oxidase (DAO) 274 M. López-Gómez et al.

OAT N Arginine Urea Cycle Ornithine Glu 2 GS/GOGAT

ADC + Proline NH4 Agmatine α -KG AIH CPA DAO Put 4-Aminobutanal GABA Krebs SAM Cycle H2O2 SPDS PAO

SAMDC

dcSAM Spd 4-Aminobutanal ∆¹-Pirroline Asp PAO

PAO + Dap H2O2 Lys

3-(Aminopropil) LDC Spm 4-Aminobutanal Cad PAO

Fig. 1 Polyamines (PAs) metabolism and interaction with the synthesis of osmoprotectants proline and γ-aminobutiric acid (GABA) and Krebs and glutamate cycles. Put (putrescine), Spd (spermidine), Spm (spermine), Cad (cadaverine), Glu (glutamate), Asp (aspartic acid), Lys (lysine), α-KG (α-ketoglutarate), DAO (diamine oxidase), PAO (polyamine oxidase), H2O2 (hydrogen peroxide), ADC (arginine decarboxylase), ODC (ornithine decarboxylase), SPDS (spermidine synthase), SPMS (spermine synthase), SAMDC (S-adenosyl methionine decarboxylase), LDC (lysine decarboxylase), OAT (ornithine aminotransferase), AIH (agmatine iminohydrolase), CPA (carbamoil putrescine aminohydrolase) and polyamine oxidase (PAO). Spermidine and Spm are preferably oxidized by PAO activity, classified depending on whether they terminally oxidize PAs or catalyze their back conversion producing Put and Spd from Spd and Spm, respectively. In addition to contribute to the homeostasis of PAs, plant amine oxidases contribute to other physiological processes through the production of H2O2, which has a versatile role in plants as a signal molecule during abiotic and biotic stresses (Gupta et al. 2016), and γ-aminobutyric acid (GABA), also produced by cytosolic glutamate decarboxylase, and with important functions in response to biotic and abiotic stresses (Podlešáková et al. 2019).

6.2 Polyamines Against Salt Stress

Numerous studies have shown the participation of PAs in salt stress tolerance in plants (Tang and Newton 2005; Jiménez-Bremont et al. 2007; Li et al. 2016; López-Gómez et al. 2017). The accumulation of PAs in response to salt stress is one of the strategies of plants to acquire tolerance to this stress; however, the levels of PAs change depending on several factors such as the species, the organ Relationship Between Polyamines and Osmoprotectants in the Response to Salinity… 275 analyzed, their tolerance or sensitivity to the salinity stress, or the duration of it (Groppa and Benavides 2008). In plant cells, PAs occur as free bases but may also be covalently linked to small molecules such as hydroxycinnamic acids to form soluble conjugated PAs. The conjugation of PAs has a role in the regulation of free PAs levels, which may be related to the variations of PA levels in the responses to salt stress. The application of exogenous PAs has been shown to alleviate the effects of NaCl stress on various plants (Verma and Mishra 2005). Especially Spm and Spd resulted in increased reactive oxygen metabolism and photosynthesis, which improved plant growth and reduced the inhibitory effects of salt stress (Baniasadi et al. 2018). Implication of PAs in the protection against salinity is due to their capacity to stabilize macromolecules such as DNA, RNA, proteins and phospholipids, as well as their free radical scavenging activity (Hussain et al. 2011). Some of these properties are shared with other compatible solutes such as proline; however, the concentration of stress-induced PAs is lower than proline and other compatible solutes such as sucrose and trehalose, which suggests that PAs do not behave as compatible solutes.

6.3 Polyamines Interaction with Other Abiotic Stress Regulators

Polyamines play critical roles in the adaptation of plants to stress conditions through an intricate crosstalk with other growth regulators and signaling molecules, which is confirmed by the fact that metabolic pathways that regulate the levels of PAs share common substrates with other molecules that also participate in stress responses, such as nitric oxide (NO), H2O2, GABA, ethylene, or proline; so it is difficult to discern the relationships between PAs and the involvement of other molecules in the abiotic stress responses (Shi et al. 2013). The alteration of PA metabolism has been used as a strategy to uncover its relationships with other molecules involved in defense against abiotic stresses (Duque et al. 2016). For instance, the inhibition of PA catabolism has been shown to reduce the level of osmoprotectants such as proline and GABA (Su and Bai 2008; Xing et al. 2007), suggesting that PAs might act as stress alleviators through the modulation of the levels of these amino acids (Fig. 2). However, the inhibition of PAO activity in Arabidopsis thaliana increased the tolerance to salinity and drought (Sagor et al. 2016) by reducing ROS production and inducing stress responsive genes under stress conditions. Glutamate is a common precursor for the biosynthesis of proline and PAs (Fig. 1), and therefore considerable changes in the pool of PAs can be caused by a shift between syntheses of both compounds. All together suggests that PAs would be in the center of a complex regulation network in which the levels of osmoprotectants, such as proline and GABA, would depend on PAs metabolic alterations (Fig. 2).

Additionally, production of signaling molecules such as H2O2 by PAs catabolism has been related with the induction of defensive responses of hypersensitivity 276 M. López-Gómez et al.

Salt Stress

BRs ABABA

PAs SA Ethylene

Proline H2O2

GABA NO

Fig. 2 Schematic representation of polyamines interaction with hormones in the response to salt stress. PAs (polyamines), BRs (brasinosteroids), ABA (abscisic acid), SA (salicylic acid), GABA (γ-aminobutiric acid), NO (nitric oxide)

(Jasso-Robles et al. 2016), including the activation and signaling of abiotic stress responses (Moschou et al. 2008). Nitric oxide (NO) also participates in the PA signaling as it is produced in the course of PA metabolism and is a key signaling molecule that mediates a variety of physiological functions, including defense responses against abiotic stresses in plants (Diao et al. 2017). Polyamines play a positive role in NO production with an inverse correlation between NO and ethylene presence in abscission tissue (Parra-Lobato and Gomez-Jimenez 2011). The reduction of the expression level of de NCED2 gene, involved in the abscisic acid (ABA) synthesis, by a DAO inhibitor and osmotic stress, indicates a relationship between PA oxidation and ABA synthesis under osmotic stress conditions (Hatmi et al. 2018), which highlights the role of PAs in controlling abiotic stress responses through ABA responses (Fig. 2). Indeed, ABA pretreatment alleviates the negative effect of salinity in plants of M. sativa by increasing antioxidant responses and the level of Put derived PAs such as Spd and Spm (Palma et al. 2014). Exogenous ABA also increases the Put contents in chickpea, while ABA has been reported to trigger PAs synthesis through transcriptional regulation of genes encoding SPDS (Jiménez-Bremont et al. 2007). All these results suggest that ABA would be involved in the modulation of PAs metabolism at transcriptional level. By contrary, salicylic acid (SA), which participates in the signaling of salt stress in legumes (Palma et al. 2009), prevents the accumulation of PAs under salt stress conditions (Palma et al. 2013). This negative relationship has been related with the linkage between PAs and ethylene synthesis that is favored by SA through the common precursor S-adenosylmethionine (SAM). Another example of interaction is in Relationship Between Polyamines and Osmoprotectants in the Response to Salinity… 277 mutants impaired on the biosynthetic pathways of both ethylene and PAs, which prevented premature cell death of xylem elements (Milhinhos and Miguel 2013). Brassinosteroids (BRs) are steroid hormones that regulate salt stress responses in plants (Bajguz 2011) in crosstalk with PAs (Zheng et al. 2016). Indeed, foliar treatment with epibrassinolide induces a protective effect in M. truncatula against salt stress by an increment in the Spm levels (López-Gómez et al. 2016). In addition, regulation of the nodule number in legumes has been shown to occur through an interrelation between PAs and BRs (Terakado-Tonooka and Fujihara 2008), which indicates a complex network in which different hormones and plant growth regulators, including PAs, would interact during the abiotic stress responses and the biotic interactions of plants (Fig. 2).

6.4 PAs in the Legume-Rhizobia Symbiosis

The levels of PAs in plants depend on the species and the stage of development, being more abundant in growing tissues or in plants exposed to different stress conditions, including biotic interactions (Jiménez Bremont et al. 2014). In general, the levels of Put are higher than the rest of the PAs; however, in legumes, higher concentrations of Cad can be found (Tomar et al. 2013). Polyamines are also necessary for growth and division of microorganisms, including rhizobia (Becerra-Rivera et al. 2018), in which among the usual PAs (Put, Spd, and Spm), rare PAs considered structural analogues of the main PAs have also been identified. Therefore, PAs composition in the root nodules of legumes is the result of the mixture and cooperation of the plant and rhizobia metabolisms. In general, PAs in root nodules of legumes accumulate in values 5–10 times higher than any other organ of the plant, and the composition of these PAs can vary depending on the species of legume. Most of these PAs are specific to the nodule, since they are synthesized by the bacteroides (Fujihara 2009), including unusual PAs such as HomSpd, which is the most abundant in nodules of M. sativa and P. vulgaris (López-Gómez et al. 2014a, b). In nodules of P. vulgaris has also been found 4-ami- nobutilcadaverine (4-Abcad), a polyamine exclusive of the bacteroides, whose synthesis depends on the enzyme HomSpd synthase in the bacteroides (Lopez-Gomez et al. 2016a). The presence of both uncommon PAs in nodules is possible by the supply of Cad from the plant cytosol to the bacteroides, since Cad is neither produced by the free-living bacteria nor the bacteroides (Fujihara 2009) (Fig. 3). During the establishment of the legume-rhizobia symbiosis, alterations in PAs levels have been described (Jimenez-Bremont et al. 2014; Terakado-Tonooka and Fujihara 2008), suggesting an active role of these molecules in the symbiotic interaction. The production of H2O2 by PAs catabolism has proved to be a requirement for the infection of roots in M. truncatula during the establishment of the symbiosis (Jamet et al. 2007) and the nodule functioning (Andrio et al. 2013). In this regard, the oxidation of PAs has been concluded to be involved in the establishment of the symbiosis between M. truncatula and S. meliloti, as shown by the reduction of the nodulation by the inhibitor of DAO, aminoguanidine (AG) (Hidalgo-Castellanos et al. Data not published). 278 M. López-Gómez et al.

ArgOrn

ADC ODC

Put Spd Spm SPDS SPMS Cad Cad + Put

LDC HSS dcSAM

SAMDC Lys Put HomSpd SAM

Plantcytosol Bacteroid

Fig. 3 Schematic representation of polyamines biosynthesis in root nodules of legumes. ADC, (arginine decarboxylase), Arg (arginine), ABCad (aminobutylcadaverine), Cad (cadaverine), Homspd (homospermidine), HSS (homospermidine synthase), LDC (lysine decarboxylase), Lys (lysine), ODC (ornithine decarboxylase), Orn (ornithine), Put (putrescine), SAM (S-adenosylmethionine), SAMDC (S-adenosylmethionine decarboxylase), Spd (spermidine), SPDS (spermidine synthase), Spm (spermine), SPMS (spermine synthase). (Adapted from Plant and Soil (2016) 404: 413–425)

H2O2 PAs

NO Infection

Fig. 4 Schematic representation for the role of signal molecules induced by polyamines (PAs) in the legume–rhizobia interaction. NO (nitric oxide), H2O2 (hydrogen peroxide)

NO has been shown to be required for an optimal establishment of the M. truncatula-S. meliloti symbiosis and has been suggested that it could have functions in bacterial infection as well as in nodule development (del Giudice et al. 2011). Interaction between PAs metabolism and NO has been shown by the exogenous application of Spd and Spm that induced the generation of NO by the nitrate reductase pathway (Diao et al. 2017). Therefore, PAs metabolism has an active role in the legume-rhizobia symbiosis providing signal molecules necessary for the infection threads formation and nodule organogenesis (Fig. 4). In addition, PA concentrations participate in the control of root nodule number and biomass in crosstalk with BRs, as demonstrated by the inhibition of Spd synthesis in a supernodulating genotype of soybean, that restored Relationship Between Polyamines and Osmoprotectants in the Response to Salinity… 279 the wild-type nodule number and the fact that exogenous application of BRs restored the Spd levels and reduced the nodule number in this supernodulating mutant (Terakado et al. 2006). The involvement of PAs in nodule organogenesis has also been shown in nodules of L. japonicus, where the expression of genes involved in the synthesis of PAs is induced during nodule development and declines with aging (Efrose et al. 2008). Indeed, significant linear correlations between the total concentrations of free PAs in nodules and nitrogenase activity and leghaemoglobin content have been reported in field-grown bean (Lahiri et al.2004 ), suggesting their implication in other functions related to nitrogen fixation.

6.5 Polyamines in the Salt Stress Response in the Legume-Rhizobia Symbiosis

Nodule-specific PAs such as HomSpd, Cad, or AbCad are involved in mechanisms of tolerance to salinity in the legume-rhizobia symbiosis (López-Gómez et al. 2014a, 2016). Specifically, the concentration of the bacteroidal-produced polyamine AbCad augmented under salinity stress in nodules of P. vulgaris, suggesting the modification of the bacteroidal metabolism toward the synthesis of this compound as a strategy to cope with salt stress. HomSpd synthase seems to be the enzyme responsible for this nodular metabolic response to salinity, based on the reduction of the nodulation observed in plants of P. vulgaris inoculated with a mutant strain of Rhizobium tropici impaired in the synthesis of HomSpd (Rt hss::Ω, Spr) (Lopez-Gomez et al. 2016). In addition, this enzyme is involved in the adaptation to salt stress conditions in the free living bacteria, since the reduction of the growth by the salinity observed in the mutant strain (Rt hss::Ω, Spr) was restored by the exogenous addition of HomSpd to the growth medium. The involvement of PAs in the tolerance to salinity in the symbiosis M. truncatula-Sinorhizobium meliloti has been shown by the exogenous addition of Spd and Spm to the growth medium (López-Gómez et al. 2017). In this work, a reduction of the oxidative stress was detected, and in addition, evidence of the crosstalk between PAs and BRs was reported based on the induction of the expression of genes involved in BRs biosynthesis by exogenous PAs. Indeed, a similar result was induced by foliar treatment with BRs which induced an increment in the Spd levels in leaves and restored growth under salt stress conditions (López- Gómez et al. 2016b).

7 Conclusion

The involvement of PAs in the response to salinity has acquired a special attention since a relationship between the accumulation of this type of compounds and tolerance to salinity has been described. Additionally, PAs share common metabolic 280 M. López-Gómez et al. pathways with other molecules that also participate in salt stress responses, such as GABA or proline, which suggest that PAs are in the center of a complex regulation network in which the levels of these osmoprotectants depend on PAs metabolic alterations. Additionally, the production of signaling molecules such as H2O2 by PAs catabolism are also involved in salt stress responses. The symbiotic interaction between legumes and soil nitrogen-fixing bacteria reduces the need to use chemical fertilizers but is a salt-sensitive process, which make of special interest the improvement of the tolerance to salinity of this symbiotic interaction. PAs metabolism has an active role in the legume-rhizobia symbiosis, providing signal molecules necessary for the infection threads formation and nodule organogenesis. In addition, PAs composition in the root nodules of legumes is the result of the cooperation of the plant and rhizobia metabolism with some of the nodule-specific PAs synthesized by the bacteroides. Nodule-specific PAs are involved in mechanisms of tolerance to salinity in the legume-rhizobia symbiosis, suggesting the modification of the bacteroidal metabolism toward the synthesis of this compound as a strategy to cope with salt stress. Therefore, the gain of knowledge in the alterations of the metabolism of PAs in the legume-rhizobia symbiosis and its interaction with other molecules involved in salt stress tolerance is of great interest to improve the ability to fix atmospheric nitrogen of legumes and to reduce the need to use chemical fertilizers.

References

Abdel-Wahab AM, Shabeb MSA, Younis MAM (2002) Studies on the effect of salinity, drought stress and soil type on nodule activities of Lablab purpureus (L.) sweet (Kashrangeeg). J Arid Environ 51(4):587–602. https://doi.org/10.1006/jare.2002.0974 Abiala MA, Abdelrahman M, Burritt DJ, Tran L-SP (2018) Salt stress tolerance mechanisms and potential applications of legumes for sustainable reclamation of salt-degraded soils. Land Degrad Dev 29(10):3812–3822. https://doi.org/10.1002/ldr.3095 Andrio E, Marino D, Marmeys A, de Segonzac MD, Damiani I, Genre A, Huguet S, Frendo P, Puppo A, Pauly N (2013) Hydrogen peroxide-regulated genes in the Medicago truncatula- Sinorhizobium meliloti symbiosis. New Phytol 198(1):190–202. https://doi.org/10.1111/ nph.12120 Aranjuelo I, Arrese-Igor C, Molero G (2014) Nodule performance within a changing environmental context. J Plant Physiol 171(12):1076–1090. https://doi.org/10.1016/j.jplph.2014.04.002 Bajguz A (2011) Brassinosteroids - Occurence and chemical structures in plants. In: Brassinosteroids: a class of plant hormone, pp 1–27. https://doi.org/10.1007/978-94-007-0189-2_1 Baniasadi F, Saffari VR, Maghsoudi Moud AA (2018) Physiological and growth responses of Calendula officinalis L. plants to the interaction effects of polyamines and salt stress. Sci Hortic 234:312–317. https://doi.org/10.1016/j.scienta.2018.02.069 Becana M, Matamoros MA, Udvardi M, Dalton DA (2010) Recent insights into antioxidant defenses of legume root nodules. New Phytol 188(4):960–976. https://doi. org/10.1111/j.1469-8137.2010.03512.x Becerra-Rivera VA, Bergström E, Thomas-Oates J, Dunn MF (2018) Polyamines are required for normal growth in Sinorhizobium meliloti. Microbiology 164(4):600–613. https://doi. org/10.1099/mic.0.000615 Relationship Between Polyamines and Osmoprotectants in the Response to Salinity… 281

Ben Salah I, Slatni T, Albacete A, Gandour M, Martínez Andújar C, Houmani H, Ben Hamed K, Martinez V, Pérez-Alfocea F, Abdelly C (2010) Salt tolerance of nitrogen fixation in Medicago ciliaris is related to nodule sucrose metabolism performance rather than antioxidant system. Symbiosis 51(2):187–195. https://doi.org/10.1007/s13199-010-0073-3 Bruning B, Rozema J (2013) Symbiotic nitrogen fixation in legumes: perspectives for saline agriculture. Environ Exp Bot 92:134–143 Burris RH (1984) The fundamentals of nitrogen fixation- Posgate, JR. Am Sci 72(5):517 Coba de la Peña T, Pueyo JJ (2012) Legumes in the reclamation of marginal soils, from cultivar and inoculant selection to transgenic approaches. Agron Sustain Dev 32(1):65–91. https://doi. org/10.1007/s13593-011-0024-2 Crespi M, Gálvez S (2000) Molecular mechanisms in root nodule development. J Plant Growth Regul 19(2):155–166. https://doi.org/10.1007/s003440000023 del Giudice J, Cam Y, Damiani I, Fung-Chat F, Meilhoc E, Bruand C, Brouquisse R, Puppo A, Boscari A (2011) Nitric oxide is required for an optimal establishment of the Medicago truncatula–Sinorhizobium meliloti symbiosis. New Phytol 191(2):405–417. https://doi. org/10.1111/j.1469-8137.2011.03693.x Diao QN, Song YJ, Shi DM, Qi HY (2017) Interaction of polyamines, abscisic acid, nitric oxide, and hydrogen peroxide under chilling stress in tomato (Lycopersicon esculentum Mill.) seedlings. Front Plant Sci 8. https://doi.org/10.3389/fpls.2017.00203 Doyle JJ, Luckow MA (2003) The rest of the iceberg. Legume diversity and evolution in a phylogenetic context. Plant Physiol 131(3):900–910. https://doi.org/10.1104/pp.102.018150 Duque AS, López-Gómez M, Kráčmarová J, Gomes CN, Araújo SS, Lluch C, Fevereiro P (2016) Genetic engineering of polyamine metabolism changes Medicago truncatula responses to water deficit. Plant Cell Tissue Organ Cult 127(3):681–690.https://doi.org/10.1007/ s11240-016-1107-1 Efrose RC, Flemetakis E, Sfichi L, Stedel C, Kouri ED, Udvardi MK, Kotzabasis K, Katinakis P (2008) Characterization of spermidine and spermine synthases in Lotus japonicus: induction and spatial organization of polyamine biosynthesis in nitrogen fixing nodules. Planta 228(1):37–49. https://doi.org/10.1007/s00425-008-0717-1 FAO (2009). El estado mundial de la agricultura y la alimentación Foyer CH, Noctor G (2005) Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28(8):1056–1071. https://doi.org/10.1111/j.1365-3040.2005.01327.x Fujihara S (2009) Biogenic amines in rhizobia and legume root nodules. Microbes Environ 24(1):1–13. https://doi.org/10.1264/jsme2.ME08557 Gepts P, Beavis WD, Brummer EC, Shoemaker RC, Stalker HT, Weeden NF, Young ND (2005) Legumes as a model plant family. Genomics for food and feed report of the cross-legume advances through genomics conference. Plant Physiol 137(4):1228–1235. https://doi. org/10.1104/pp.105.060871 Gordon AJ (1995) Sucrose metabolism to support N2 fixation in legume root nodules. In: Tikhonovich I, Provorov N, Romanov V, Newton W (eds) Nitrogen fixation: fundamentals and applications, vol 27. Current plant science and biotechnology in agriculture. Springer Netherlands, pp 533–538. https://doi.org/10.1007/978-94-011-0379-4_62 Groppa MD, Benavides MP (2008) Polyamines and abiotic stress: recent advances. Amino Acids 34(1):35–45. https://doi.org/10.1007/s00726-007-0501-8 Gupta K, Sengupta A, Chakraborty M, Gupta B (2016) Hydrogen peroxide and polyamines act as double edged swords in plant abiotic stress responses. Front Plant Sci 7(1343). https://doi. org/10.3389/fpls.2016.01343 Handa AK, Fatima T, Mattoo AK (2018) Polyamines: bio-molecules with diverse functions in plant and human health and disease. Front Chem 6:10–10. https://doi.org/10.3389/fchem.2018.00010 Hasanuzzaman M, Nahar K, Fujita M (2013) Plant response to salt stress and role of exogenous protectants to mitigate salt-induced damages. In: Ahmad P, Azooz MM, Prasad MNV (eds) Ecophysiology and responses of plants under salt stress. Springer New York, New York, pp 25–87. https://doi.org/10.1007/978-1-4614-4747-4_2 282 M. López-Gómez et al.

Hatmi S, Villaume S, Trotel-Aziz P, Barka EA, Clément C, Aziz A (2018) Osmotic stress and ABA affect immune response and susceptibility of Grapevine Berries to Gray Mold by priming polyamine accumulatioN. Front Plant Sci 9(1010). https://doi.org/10.3389/fpls.2018.01010 Huang X, He J, Yan X, Hong Q, Chen K, He Q, Zhang L, Liu X, Chuang S, Li S, Jiang J (2017) Microbial catabolism of chemical herbicides: microbial resources, metabolic pathways and catabolic genes. Pestic Biochem Physiol 143:272–297. https://doi.org/10.1016/j. pestbp.2016.11.010 Hussain SS, Ali M, Ahmad M, Siddique KHM (2011) Polyamines: natural and engineered abiotic and biotic stress tolerance in plants. Biotechnol Adv 29(3):300–311. https://doi.org/10.1016/j. biotechadv.2011.01.003 Jamet A, Mandon K, Puppo A, Herouart D (2007) H2O2 is required for optimal establishment of the Medicago sativa/Sinorhizobium meliloti symbiosis. J Bacteriol 189(23):8741–8745. https://doi.org/10.1128/jb.01130-07 Jasso-Robles FI, Jimenez-Bremont JF, Becerra-Flora A, Juarez-Montiel M, Gonzalez ME, Pieckenstain FL, de la Cruz RFG, Rodriguez-Kessler M (2016) Inhibition of polyamine oxidase activity affects tumor development during the maize-Ustilago maydis interaction. Plant Physiol Biochem 102:115–124. https://doi.org/10.1016/j.plaphy.2016.02.019 Jensen E, Peoples M, Boddey R, Gresshoff P, Hauggaard-Nielsen H, Alves BJR, Morrison M (2012) Legumes for mitigation of climate change and the provision of feedstock for biofu- els and biorefineries: a review. Agron Sustain Dev 32(2):329–364.https://doi.org/10.1007/ s13593-011-0056-7 Jiménez Bremont J, Marina M, de la Luz Guerrero-González M, Rossi F, Sánchez-Rangel D, Rodríguez-Kessler M, Ruiz O, Gárriz A (2014) Physiological and molecular implications of plant polyamine metabolism during biotic interactions. Front Plant Sci 5(95). https://doi. org/10.3389/fpls.2014.00095 Jiménez-Bremont JF, Ruiz OA, Rodríguez-Kessler M (2007) Modulation of spermidine and spermine levels in maize seedlings subjected to long-term salt stress. Plant Physiol Biochem 45(10):812–821 Jimenez-Bremont JF, Marina M, Guerrero-Gonzalez MD, Rossi FR, Sanchez-Rangel D, Rodriguez-Kessler M, Ruiz O, Garriz A (2014) Physiological and molecular implications of plant polyamine metabolism during biotic interactions. Front Plant Sci 5. https://doi. org/10.3389/fpls.2014.00095 Koca H, Bor M, Ozdemir F, Turkan I (2007) The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environ Exp Bot 60. https://doi. org/10.1016/j.envexpbot.2006.12.005 Lahiri K, Chattopadhyay S, Ghosh B (2004) Correlation of endogenous free polyamine levels with root nodule senescence in different genotypes in Vigna mungo L. J Plant Physiol 161(5):563– 571. https://doi.org/10.1078/0176-1617-01057 Le BH, Wagmaister JA, Kawashima T, Bui AQ, Harada JJ, Goldberg RB (2007) Using genomics to study legume seed development. Plant Physiol 144(2):562–574. https://doi.org/10.1104/ pp.107.100362 Li S, Jin H, Zhang Q (2016) The effect of exogenous spermidine concentration on polyamine metabolism and salt tolerance in zoysiagrass (Zoysia japonica steud) subjected to short-term salinity stress. Front Plant Sci 7. https://doi.org/10.3389/fpls.2016.01221 Lopez M, Herrera-Cervera JA, Lluch C, Tejera NA (2006) Trehalose metabolism in root nodules of the model legume Lotus japonicus in response to salt stress. Physiol Plant 128(4):701–709. https://doi.org/10.1111/j.1399-3054.2006.00802.x Lopez M, Herrera-Cervera JA, Iribarne C, Tejera NA, Lluch C (2008) Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism. J Plant Physiol 165(6):641–650. https://doi.org/10.1016/j.jplph.2007.05.009 López M, Herrera-Cervera JA, Iribarne C, Tejera NA, Lluch C (2008) Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism. J Plant Physiol 165(6):641–650 Relationship Between Polyamines and Osmoprotectants in the Response to Salinity… 283

López-Gómez M, Tejera NA, Iribarne C, Herrera-Cervera JA, Lluch C (2011) Different strategies for salt tolerance in determined and indeterminate nodules of Lotus japonicus and Medicago truncatula. Arch Agron Soil Sci 58(9):1061–1073. https://doi.org/10.1080/03650340.2011.56 1836 López-Gómez M, Cobos-Porras L, Hidalgo-Castellanos J, Lluch C (2014a) Occurrence of polyamines in root nodules of Phaseolus vulgaris in symbiosis with Rhizobium tropici in response to salt stress. Phytochemistry 107:32–41. https://doi.org/10.1016/j.phytochem.2014.08.017 López-Gómez M, Hidalgo-Castellanos J, Iribarne C, Lluch C (2014b) Proline accumulation has prevalence over polyamines in nodules of Medicago sativa in symbiosis with Sinorhizobium meliloti during the initial response to salinity. Plant Soil 374(1-2):149–159 Lopez-Gomez M, Cobos-Porras L, Prell J, Lluch C (2016a) Homospermidine synthase contributes to salt tolerance in free-living Rhizobium tropici and in symbiosis with Phaseolus vulgaris. Plant Soil 404(1-2):413–425. https://doi.org/10.1007/s11104-016-2848-7 López-Gómez M, Hidalgo-Castellanos J, Lluch C, Herrera-Cervera JA (2016b) 24-Epibrassinolide ameliorates salt stress effects in the symbiosis Medicago truncatula-Sinorhizobium meliloti and regulates the nodulation in cross-talk with polyamines. Plant Physiol Biochem 108:212– 221. https://doi.org/10.1016/j.plaphy.2016.07.017 López-Gómez M, Hidalgo-Castellanos J, Muñoz-Sánchez JR, Marín-Peña AJ, Lluch C, Herrera- Cervera JA (2017) Polyamines contribute to salinity tolerance in the symbiosis Medicago truncatula-Sinorhizobium meliloti by preventing oxidative damage. Plant Physiol Biochem 116:9–17. https://doi.org/10.1016/j.plaphy.2017.04.024 Manchanda G, Garg N (2008) Salinity and its effects on the functional biology of legumes. Acta Physiol Plant 30(5):595–618. https://doi.org/10.1007/s11738-008-0173-3 Mantri N, Basker N, Ford R, Pang E, Pardeshi V (2013) The role of micro-ribonucleic acids in legumes with a focus on abiotic stress response. Plant Genome 6. https://doi.org/10.3835/ plantgenome2013.05.0013 McNeil SD, Nuccio ML, Hanson AD (1999) Betaines and related osmoprotectants. Targets for metabolic engineering of stress resistance. Plant Physiol 120(4):945. https://doi.org/10.1104/ pp.120.4.945 Milhinhos A, Miguel CM (2013) Hormone interactions in xylem development: a matter of signals. Plant Cell Rep 32(6):867–883. https://doi.org/10.1007/s00299-013-1420-7 Minocha R, Majumdar R, Minocha SC (2014) Polyamines and abiotic stress in plants: a complex relationship. Front Plant Sci 5. https://doi.org/10.3389/fpls.2014.00175 Molina C, Zaman-Allah M, Khan F, Fatnassi N, Horres R, Rotter B, Steinhauer D, Amenc L, Drevon J-J, Winter P, Kahl G (2011) The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE. BMC Plant Biol 11(1):31. https://doi.org/10.1186/1471-2229-11-31 Møller I, Jensen P, Hansson A (2007) Oxidative modifications to cellular components in plants. Annu Rev Plant Biol 58. https://doi.org/10.1146/annurev.arplant.58.032806.103946 Moschou PN, Paschalidis KA, Delis ID, Andriopoulou AH, Lagiotis GD, Yakoumakis DI, Roubelakis-Angelakis KA (2008) Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco. Plant Cell 20(6):1708–1724. https://doi.org/10.1105/tpc.108.059733 Munns R (2009) Strategies for crop improvement in saline soils. In: Ashraf M, Ozturk M, Athar HR (eds) Salinity and water stress, vol 44. Tasks for vegetation sciences. Springer Netherlands, pp 99–110. https://doi.org/10.1007/978-1-4020-9065-3_11 Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59(1):651–681. https://doi.org/10.1146/annurev.arplant.59.032607.092911 Nishi H, Demir E, Panchenko AR (2015) Crosstalk between signaling pathways provided by single and multiple protein phosphorylation sites. J Mol Biol 427(2):511–520. https://doi. org/10.1016/j.jmb.2014.11.001 Pal M, Szalai G, Janda T (2015) Speculation: polyamines are important in abiotic stress signaling. Plant Sci 237:16–23. https://doi.org/10.1016/j.plantsci.2015.05.003 284 M. López-Gómez et al.

Palma F, Lluch C, Iribarne C, Garcia-Garrido JM, Tejera Garcia NA (2009) Combined effect of salicylic acid and salinity on some antioxidant activities, oxidative stress and metabolite accumulation in Phaseolus vulgaris. Plant Growth Regul 58(3):307–316. https://doi.org/10.1007/ s10725-009-9380-1 Palma F, López-Gómez M, Tejera NA, Lluch C (2013) Salicylic acid improves the salinity tolerance of Medicago sativa in symbiosis with Sinorhizobium meliloti by preventing nitrogen fixation inhibition. Plant Sci 208(0):75–82. https://doi.org/10.1016/j.plantsci.2013.03.015 Palma F, López-Gómez M, Tejera NA, Lluch C (2014) Involvement of abscisic acid in the response of Medicago sativa plants in symbiosis with Sinorhizobium meliloti to salinity. Plant Sci 223:16–24. https://doi.org/10.1016/j.plantsci.2014.02.005 Parra-Lobato MC, Gomez-Jimenez MC (2011) Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission. J Exp Bot 62(13):4447–4465. https://doi.org/10.1093/jxb/err124 Podlešáková K, Ugena L, Spíchal L, Doležal K, De Diego N (2019) Phytohormones and polyamines regulate plant stress responses by altering GABA pathway. New Biotechnol 48:53–65. https://doi.org/10.1016/j.nbt.2018.07.003 Rengasamy P (2006) World salinization with emphasis on Australia. J Exp Bot 57(5):1017–1023. https://doi.org/10.1093/jxb/erj108 Rubio MC, Bustos-Sanmamed P, Clemente MR, Becana M (2009) Effects of salt stress on the expression of antioxidant genes and proteins in the model legume Lotus japonicus. New Phytol 181(4):851–859. https://doi.org/10.1111/j.1469-8137.2008.02718.x Sagor GHM, Berberich T, Takahashi Y, Niitsu M, Kusano T (2013) The polyamine spermine protects Arabidopsis from heat stress-induced damage by increasing expression of heat shock- related genes. Transgenic Res 22(3):595–605. https://doi.org/10.1007/s11248-012-9666-3 Sagor GHM, Zhang SY, Kojima S, Simm S, Berberich T, Kusano T (2016) Reducing cytoplasmic polyamine oxidase activity in arabidopsis increases salt and drought tolerance by reducing reactive oxygen species production and increasing defense gene expression. Front Plant Sci 7. https://doi.org/10.3389/fpls.2016.00214 Shi H, Ye T, Chan Z (2013) Comparative proteomic and physiological analyses reveal the protective effect of exogenous polyamines in the Bermudagrass (Cynodon dactylon) response to salt and drought stresses. J Proteome Res 12(11):4951–4964. https://doi.org/10.1021/pr400479k Smýkal P, Coyne CJ, Ambrose MJ, Maxted N, Schaefer H, Blair MW, Berger J, Greene SL, Nelson MN, Besharat N, Vymyslický T, Toker C, Saxena RK, Roorkiwal M, Pandey MK, Hu J, Li YH, Wang LX, Guo Y, Qiu LJ, Redden RJ, Varshney RK (2015) Legume crops phylogeny and genetic diversity for science and breeding. Crit Rev Plant Sci 34(1-3):43–104. https://doi.org/ 10.1080/07352689.2014.897904 Su GX, Bai X (2008) Contribution of putrescine degradation to proline accumulation in soybean leaves under salinity. Biol Plantarum 52(4):796–799. https://doi.org/10.1007/ s10535-008-0156-7 Tang W, Newton RJ (2005) Polyamines reduce salt-induced oxidative damage by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation in Virginia pine. Plant Growth Regul 46(1):31–43. https://doi.org/10.1007/s10725-005-6395-0 Tejera García NA, Iribarne C, Palma F, Lluch C (2007) Inhibition of the catalase activity from Phaseolus vulgaris and Medicago sativa by sodium chloride. Plant Physiol Biochem 45(8):535– 541. https://doi.org/10.1016/j.plaphy.2007.04.008 Tejera NA, Soussi M, Lluch C (2006) Physiological and nutritional indicators of tolerance to salinity in chickpea plants growing under symbiotic conditions. Environ Exp Bot 58(1-3):17–24. https://doi.org/10.1016/j.envexpbot.2005.06.007 Terakado J, Yoneyama T, Fujihara S (2006) Shoot-applied polyamines suppress nodule formation in soybean (Glycine max). J Plant Physiol 163(5):497–505. https://doi.org/10.1016/j. jplph.2005.05.007 Terakado-Tonooka J, Fujihara S (2008) Involvement of polyamines in the root nodule regulation of soybeans (Glycine max). Plant Root 2:46–53. https://doi.org/10.3117/plantroot.2.46 Relationship Between Polyamines and Osmoprotectants in the Response to Salinity… 285

Terui Y, Ohnuma M, Hiraga K, Kawashima E, Oshima T (2005) Stabilization of nucleic acids by unusual polyamines produced by an extreme thermophile, Thermus thermophilus. Biochem J 388:427–433 Tomar PC, Lakra N, Mishra SN (2013) Cadaverine: a lysine catabolite involved in plant growth and development. Plant Signal Behav 8(10):e25850. https://doi.org/10.4161/psb.25850 Udvardi M, Poole PS (2013) Transport and metabolism in legume-rhizobia symbioses. Annu Rev Plant Biol 64:781–805 Verma S, Mishra SN (2005) Putrescine alleviation of growth in salt stressed Brassica juncea by inducing antioxidative defense system. J Plant Physiol 162(6):669–677. https://doi. org/10.1016/j.jplph.2004.08.008 Wimalasekera R, Tebartz F, Scherer GFE (2011) Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses. Plant Sci 181(5):593–603. https://doi.org/10.1016/j. plantsci.2011.04.002 Xing SG, Jun YB, Hau ZW, Liang LY (2007) Higher accumulation of γ-aminobutyric acid induced by salt stress through stimulating the activity of diamine oxidases in Glycine max (L.) Merr. roots. Plant Physiol Biochem 45(8):560–566. https://doi.org/10.1016/j.plaphy.2007.05.007 Zheng Q, Liu J, Liu R, Wu H, Jiang C, Wang C, Guan Y (2016) Temporal and spatial distributions of sodium and polyamines regulated by brassinosteroids in enhancing tomato salt resistance. Plant Soil 400(1-2):147–164. https://doi.org/10.1007/s11104-015-2712-1 Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants

Susana de Sousa Araújo, André Luis Wendt dos Santos, and Ana Sofia Duque

1 Introduction

Polyamines (PAs) are low-molecular-weight polycationic nitrogenous compounds that are ubiquitously distributed in eukaryotic and prokaryotic cells (Liu et al. 2015a) and may even be found in plant RNA viruses and plant tumors (Chen et al. 2019). In plants, the most abundant PAs include putrescine (Put, 1,4-diaminobutane), spermidine (Spd, N-(3-Aminopropyl)-1,4-diaminobutane), spermine (Spm, N,N’-Bis(3-aminopropyl)-1,4-diaminobutane), and its structural isomer thermospermine (tSpm) (Saha et al. 2015). Cadaverine (Cad, 1,5-Diaminopentane), a diamine less known compared to major PAs, is commonly found in plants belonging to the families Gramineae, Leguminosae, and Solanaceae (Kakkar and Sawhney 2002; Lutts et al. 2013; Saha et al. 2015). They are synthesized from decarboxylation of amino acid precursors including arginine, ornithine, methionine, and lysine (Falahi et al. 2018). PAs are a class of plant biomolecules that have been implicated in several plant growth and development processes, which include the promotion of cell division, responses to biotic and abiotic stresses, rhizogenesis, senescence, flower development, N:C balance, fruit ripening, and embryogenesis (Baron and Stasolla 2008; Duque et al. 2016; de Oliveira et al. 2017, 2018). The polycationic structure of PAs, at physiological pH, mediates their biological activity, since they are able to electrostatically bind negatively charged macromolecules such as DNA, proteins, membrane phospholipids, and pectic polysaccharides via amine and imine groups

S. de Sousa Araújo · A. S. Duque (*) Laboratory of Plant Cell Biotechnology (BCV), Instituto de Tecnologia Química e Biológica António Xavier (Green-it Unit), Universidade Nova de Lisboa, Oeiras, Portugal e-mail: [email protected] A. L. W. dos Santos Laboratory of Plant Cellular Biology (BIOCEL), Instituto de Biociências (IB), Universidade de São Paulo (USP), São Paulo, Brazil

© Springer Nature Switzerland AG 2019 287 M. A. Hossain et al. (eds.), Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, https://doi.org/10.1007/978-3-030-27423-8_14 288 S. de Sousa Araújo et al.

(Martin-Tanguy 2001; Liu et al. 2015a). Consequently, these electrostatic interactions can affect DNA replication and gene transcription, protein conformational structure, cellular structures integrity, and membrane permeability and therefore ion homeostasis (Liu et al. 2015a; Mattoo et al. 2014). Furthermore, the presence of cationic amine and imine groups also suggests that PAs may act as free radical scavengers and/or as signaling molecules that trigger enzymatic and nonenzymatic antioxidative components during exposition to biotic and abiotic stresses in plants (Liu et al. 2015a; Saha et al. 2015). PAs can exist as free amines present either inside or outside the plant cells (free cations or involved in ionic interactions), as soluble bound or soluble conjugated in association with low molecular mass compounds (coumaroyl, ferulic, phenolics, caffeic, or hydrocinnamic acids), and insoluble bound or insoluble conjugated amines associated with high molecular mass compounds (lipids, nucleic acids, and proteins) and cell wall (Yadav and Rajam 1997; Bouchereau et al. 2000; Wuddineh et al. 2018). PA homeostasis, which refers to the adjustments made on cellular levels of Put, Spd, and Spm, is tightly regulated in plants. Intracellular PA levels are regulated not only by fine-tuning of their biosynthesis and catabolism or turnover but also through their interconversion, conversion into secondary metabolites, and allocation to other tissues/organs (Wuddineh et al. 2018). Unlike mammals, plants can withstand the accumulation of large amounts of PAs (up to mmolar concentration) as they can buffer this excess by binding PAs to TCA-soluble conjugates or by storing them in the vacuole (Serafini-Fracassini and Del Duca2008 ; Wuddineh et al. 2018). However, high cellular accumulation of PAs as well as high rate of PAs catabolism may be prejudicial to plant cells (Wuddineh et al. 2018). In plants, PA endogenous levels are influenced by different factors such as species, stress tolerance capacity, stress types and conditions, and the physiological status of the examined tissue/organs (Liu et al. 2015a). In this context, numerous review articles have described the effects of PA modulation in plants from an agricultural and biotechnology point of view (Tiburcio and Alcázar 2018; Anwar et al. 2018; He et al. 2018; Seifi and Shelp2019 ). This is well reflected on the growing number of research studies and articles addressing these aspects. A non-exhaustive survey conducted in the PubMed repository of the National Center for Biotechnology Information NCBI (http://www.ncbi.nlm.nih.gov/ pubmed) retrieved 947 research articles dedicated to PA research in the context of plant sciences (6th March 2019). Of those, 799 articles were published during the period of 1998–2018. The evolution in the number of polyamine articles scored in the two last decades is depicted in Fig. 1a. One of the most interesting features is that the number of articles on plant PAs almost duplicated since 2008. Potentially, this aspect was supported by the development of new high-throughput methodologies to characterize the global gene expression or metabolite accumulation (omics) with reduced cost for user and the release of several plant genome sequences that supports the development of new research efforts on the topic. Another interesting outcome of the survey made is that the majority of the PAs studies have been conducted in non-model species (Fig. 1b), which may likely reflect a translational application of the early results obtained with models as Arabidopsis thaliana L. and Nicotiana Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 289

Fig. 1 Query of research scientific papers on plant polyamines found at NCBI between 1998 and 2018. The search for best match results was conducted using the following advanced search settings: (Polyamines[Title/Abstract]) AND (plant[Title/Abstract]) NOT “review” (Publication Type). Review manuscripts were not included. The search was conducted on the 6th of March 2018. (a) Evolution on the total number of scientific research papers published per year. b( ) Evolution on the number of paper published in main model species (Arabidopsis and tobacco) compared to the ones published on other plant species. This query was made by searching the name of the species on the title tabacum L. As one example, among the 76 articles on plants PAs published during the year of 2018, 69 describes research conducted in other species beside the refereed models. This reflects the interest of the scientific community to elucidate the different roles of these important biomolecules in plants. Indeed, based on the overall analysis of Fig. 1, it is tempting to claim that the number of scientific PA articles is expected to increase in forthcoming years. This book chapter focuses on the contributions that the modulation of the PAs content has brought to improve our knowledge on the role that these interesting biomolecules have on plant physiology, development, and response to environment. The recent development of plants with altered PAs contents will be discussed in a context of crop improvement and food and feed safety, under the current scenario of climate changes.

2 Polyamine Metabolism and Regulation

2.1 Biosynthetic Pathways

In animals and fungi, Put is synthesized primarily through l-ornithine decarboxylation via the catalytic action of ornithine decarboxylase (ODC, EC 4.1.1.17). In plants, Put synthesis involves the ODC pathway and the l-arginine decarboxylation via arginine decarboxylase (ADC, EC 4.1.1.19) (Flemetakis et al. 2004), except in 290 S. de Sousa Araújo et al. the model plant A. thaliana, in whose genome the ODC gene was found to be missing (Mattoo et al. 2014). The ODC pathway is a single step reaction, in which l-ornithine is directly converted to Put. In plants, Put biosynthesis via ADC pathway involves the production of the intermediate agmatine (Agm), followed by two successive steps catalyzed by agmatine iminohydrolase (AIH, EC 3.5.3.12) and N-carbamoylputrescine amidohydrolase (CPA, EC 3.5.1.53) (Fig. 2). In addition, plants can use ADC and arginase/agmatinase (ARGAH) as a third route for Put synthesis (Patel et al. 2017). Put is then converted into Spd by spermidine synthase (SPDS; EC 2.5.1.16), after addition of an aminopropyl group donated by decarboxylated S-adenosylmethionine (dcSAM). DcSAM is produced from l-methionine via two sequential reactions that are catalyzed by methionine adenosyltransferase (MAT, EC 2.5.1.6) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), respectively. Spd is then converted into Spm or thermospermine (tSpm), using dcSAM as an aminopropyl donor, in a reaction catalyzed by Spm synthase (SPMS, EC 2.5.1.22) and thermospermine synthase (ACL5, EC 2.5.1.79), respectively (Liu et al. 2015a). Cadaverine (Cad), a diamine less known as compared to major PAs (Put, Spd, and Spm), is formed directly from l-lysine decarboxylation via lysine decarboxylase (LDC, EC 4.1.1.18) (Kakkar and Sawhney 2002) (Fig. 2).

> Glutamate > α-Ketoglutarate LDC GlutamGlu ate CADAVERINE Lysine L-Arginine Arginino- succinate Citruline ADC Agmane

L-Ornithine Succinic Methionine N-Carbamoyl- y-AminobutyricAcid(GABA) semialdehyde putrescine ODC DAO ∆1-Pyrroline SAMDC S-Adenosylmethionine PUTRESCINE H O + NH Succinate (SAM) Decarboxylated 2 2 3 S-Adenosylmethionine 1- TCA (dcSAM) SPDS PAO ∆ Pyrroline cycle Aminocyclopropane ACL5 SPERMIDINE PAO CaboxylicAcid (ACC) SPMS PAO 1,3-Diaminopropane

THERMOSPERMINE SPERMINE Ethylene PAO 1,3-Diaminopropane

Fig. 2 Schematic representations of polyamines metabolism and ethylene biosynthesis in plants. Blue arrows represent PA anabolic and back conversion pathways, green arrows denote PA catabolic pathways, and black arrows indicate PA interaction with other metabolic routes. Abbreviations: arginine decarboxylase (ADC), ornithine decarboxylase (ODC), spermidine synthase (SPDS), spermine synthase (SPMS), S-adenosylmethionine decarboxylase (SAMDC), lysine decarboxylase (LDC), flavin-containing polyamine oxidase (PAO), diamine oxidase (DAO), thermospermine synthase (ACL5). (Adapted from Alcázar et al. 2010a) Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 291

2.2 Degradation Pathways

PAs catabolic pathways occur either through direct terminal oxidative deamination/ acetylation or indirectly through PAs back-conversion pathway and subsequent oxidation. In plants, PAs catabolism is catalyzed by two classes of amine oxidases (AOs), classified according to their cofactor: diamine oxidases (DAO), copper- containing AOs (CuAOs, also known as primary AOs, EC 1.4.3.6), and FAD- dependent AOs (FAD-AOs), also known as polyamine oxidase (PAOs, EC 1.5.3.3) (Podlešáková et al. 2018; Fig. 2). Both DAO and PAO are localized in the cytoplasm and cell wall and are involved in production of hydrogen peroxide (H2O2) required for cell wall stiffening (Saha et al. 2015). CuAOs catalyze the oxidation of the diamines Put and Cad at the primary amino groups. In this reaction, Put is converted to 4-aminobutanal (ABAL, that spontaneously cyclizes to Δ1-pyrroline, PYRR), H2O2, and ammonia (NH3) (Tiburcio et al. 1997; Podlešáková et al. 2018; Fig. 2). Following the oxidation of Put, PYRR is catabolyzed into γ-aminobutyric acid (GABA) by pyrroline dehydrogenase (PDH), which is ultimately converted into succinate, a component of the Krebs cycle (Gill and Tuteja 2010; Michaeli and Fromm 2015). CuAOs can also catalyze the oxidation of Spd and Spm, although with a lower affinity (Wuddineh et al.2018 ). Plant PAOs catabolize primarily Spd, Spm/tSpm, and their derivatives (Alcázar et al. 2010a). Furthermore, PAOs are able to back-convert Spm to Spd and Spd to Put (Fig. 2), in a two-step reaction with an acetylation (spermidine/spermine N-1 acetyl transferase (SSATs)) followed by an oxidation (PAOs) with the production of 3-acetamidopropanal and H2O2 (Moschou et al. 2008).

2.3 PA Signaling Pathways and Interconnection with Other Metabolic Pathways

Although early studies claimed a just protective role for PAs, nowadays PAs are known to be involved in a complex signaling system and have a key role in the regulation of biotic and abiotic stress tolerance (Pál et al. 2015; Romero et al. 2018). Oxygen and nitrogen reactive species (ROS and RNS, respectively) are considered one of the major links between polyamines and other metabolic pathways in the response to stresses. Indeed, H2O2 and nitric oxide (NO) that are produced during polyamine metabolism may be implicated in the transmission of signals that influence gene expression via an increase in the cytoplasmic calcium (Ca2+) level (Pál et al. 2015). Other links of PAs metabolism with other metabolic pathways are found on the amino acids (proline and GABA), alkaloids, and ethylene metabolisms. All together, these pathways represent an important way of assimilation and partitioning of carbon and nitrogen (producing other amino acids and signaling molecules) that play critical functions in responses to stress and developmental process in plants (Page et al. 2012; Minocha et al. 2014; Majumdar et al. 2016). 292 S. de Sousa Araújo et al.

PAs are biochemically related to NO through arginine, a common precursor in their biosynthetic routes, suggesting that alteration in NO homeostasis can affect PAs bioavailability and vice versa (Filippou et al. 2013; Tanou et al. 2014). Moreover, NO has been shown to be produced from PAs through a still uncharacterized mechanism (Tun et al. 2006; Silveira et al. 2006). It has been demonstrated that the addition of Spd and Spm to the culture medium may induce the formation of NO in A. thaliana roots (Tun et al. 2006) and in embryogenic cultures of Ocotea catharinensis Mez (Santa-Catarina et al. 2007) and Araucaria angustifolia (Bertol.) Kuntze (Silveira et al. 2006). These pioneering studies showed the first evidence of the relationship between PAs and NO in plants. Furthermore, the relationship between PAs and NO has provided new perspectives for the study of the biosynthesis and catabolism of PAs and production of ROS mediated by the enzymes copper amine oxidase1 (CuAO1) and polyamine oxidases (PAO) (Wimalasekera et al. 2011). The overlapping roles between PAs and NO raise the question of how both molecules may act in coordination during plant development and stress resistance (Tun et al. 2006; Silveira et al. 2006). In Cucumis sativus L. (cucumber), it was demonstrated that PAs have a regulatory effect on NO biosynthesis related to the drought stress response (Arasimowicz-Jelonek et al. 2009). More recently, Diao et al. (2016) investigated the effects of Put and Spd on NO generation and the potential role of Spd-induced NO in the tolerance of tomato (Solanum lycopersicum L.) seedlings to chilling stress. Spd increased NO release via the nitric oxide synthase (NOS)-like and nitrate reductase (NR) enzymatic pathways in the tomato seedlings, whereas Put had no such effect (Diao et al. 2016). One of the most interesting findings of these studies is that H2O2 might also act as an upstream signal to stimulate NO production, highlighting again the important crosstalk between PAs, ROS, and NRS in the modulation of abiotic stress responses in plants. Many potential links between PAs and hormones exist in processes related to plant growth and environmental stress (Wimalasekera et al. 2011). PA biosynthetic genes, such as those encoding ADC, SAMDC, and SPDS, have been shown to be induced under drought stress or following ABA treatment, and this is accompanied by an increase in the endogenous PAs (Liu et al. 2015a). Moreover, to reinforce the fact that PAs biosynthesis may be regulated by ABA, several stress-responsive elements, such as drought-responsive (DRE), low temperature-responsive (LTR), and ABA-responsive elements (ABRE and/or ABRE-related motifs), are present in the promoters of the polyamine biosynthetic genes (Alcázar et al. 2006). Plants with overexpression or downregulation of PAs implicated genes have been essential tools to provide new clarifications on the existing links between PAs and plant hormones. Plants with altered PA levels often presented abnormal phenotypic alterations as stem elongation or branching, root growth, leaf morphology, and flowering delay (Kumar et al. 1996; Masgrau et al. 1997; Hanzawa et al. 2000; Alcázar et al. 2005). Some of these phenotypic alterations were found very similar to the ones observed in mutant plants with defective hormone biosynthetic pathways (Hanzawa et al. 2000), being this a driving force for investigating the crosstalk between PAs and hormones. Alcázar et al. (2005) demonstrated that transgenic Arabidopsis plants with increased levels of Adc2 transcript and elevated Put content showed dwarfism and late flowering, a Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 293 phenotype that was rescued by gibberellin A3 (GA3) application. The data obtained by these authors in these transgenic plants showed that Put accumulation downregulated the expression of several GA oxidases as AtGA20ox1, AtGA3ox1, and AtGA3ox3 implicated in GA biosynthesis. Nevertheless, it remains to be fully understood the molecular mechanism by which Put might act as an endogenous signal regulating the expression of the dioxygenase genes in GA metabolism in a way similar to GAs. The metabolic pathway of PAs is also linked to ethylene, another important plant hormone, since both share a common precursor, the S-adenosylmethionine (SAM) (Fig. 2). A recent report on grain filling in wheat (Triticum aestivum L.) under severe water stress showed that an increase in Spd levels may affect the rates of ethylene biosynthesis (Yang et al. 2017). Indeed, severe stress decreased Spd levels but increased the 1-aminocylopropane-1-carboxylic acid (ACC) concentration which is an enzyme implicated in ethylene biosynthesis. This indicated that Spd and ACC exhibited an antagonistic relationship. Interestingly, the competition for the SAM pool between PAs and ethylene biosynthesis might explain its antagonistic effects during fruit ripening and plant senescence (Tiburcio et al. 1997). While ethylene is described as a ripening and senescence promoter, PAs are described as promoters of growth and act as anti-senescence regulators (Mattoo et al. 2014).

3 Involvement of Polyamines in Stress Responses

In the early 1990s, a possible role for polyamines in plant responses to abiotic stress was proposed by Flores (1991) based on studies indicating that plants subjected to osmotic stress showed a rapid increase in putrescine levels due to the activation of the arginine decarboxylase (ADC) enzyme and by the significant increase of its transcript level (Flores and Galston 1982). In the model A. thaliana, the Adc2 expression was correlated with free Put accumulation under salinity and dehydration (Urano et al. 2003, 2004). Several studies demonstrated that PA levels increased under a number of environmental stress conditions, including drought, high salinity, and low and high temperatures (Borrell et al. 1996; Kasinathan and Wingler 2002; Do et al. 2014; Liu et al. 2007; Ikbal et al. 2014; Zapata et al. 2017; Falahi et al. 2018). It was also reported that stress-tolerant plants accumulate higher levels of polyamines in response to several stresses, comparatively to sensitive plants (e.g., Chattopadhyay et al. 1997, 2002). There are also numerous references regarding the advantage of exogenous PAs application, at different concentrations, in an attempt to study stress effects and improve stress tolerance in several species. A recent review by Khare et al. (2018) considered the exogenous polyamine application has a convenient and effective approach to alleviate salt stress and eventually improve crop productivity under high salinity and presented several examples for exogenous PAs effect in various plant species. Others of many examples are further described. Exogenous Put application was shown to prevent abiotic stress damage and increase stress tolerance in Conyza bonariensis L. and wheat (Ye et al. 1998), in Glycine max 294 S. de Sousa Araújo et al.

(L.) Merr. (Nayyar et al. 2005), in Oryza sativa L. (Ndayiragije and Lutts 2006), in Medicago sativa L. (Zeid and Shedeed 2006), and in cucumber (Duan et al. 2008). Wheat showed enhanced thermotolerance by the application of Put at pre-anthesis stage (Kumar et al. 2014), better growth and productivity under lead stress (Rady et al. 2016), and the beneficial of Put pretreatment when subject to cadmium stress (Tajti et al. 2018). In field-grown plants ofThymus vulgaris L., the foliar application of Put could acts as elicitor to trigger physiological processes and induce valuable metabolites biosynthesis, which may compensate the negative impacts of drought stress (Mohammadi et al. 2018). The supplementation of Spd can alleviate salt stress effects in sorghum (Sorghum bicolor L.) (Yin et al. 2016); mitigate saline– alkaline stress in tomato (Solanum lycopersicum L.) at physiological and proteomic levels (Zhang et al. 2015); reduce grape (Vitis vinifera L.) berries’ chilling injury during storage at low temperature, increasing postharvest life (Champa et al. 2015); and also alleviate oxidative damage in rice caused by submergence stress (Liu et al. 2015b). Also in rice, exogenously applied PAs (Put, Spd, and Spm) as seed priming and foliar spray increased drought tolerance, being foliar application more effective and Spm the most efficient PA in improving leaf water status, photosynthesis, and membrane properties (Farooq et al. 2009). Additionally, Spm application in rice worked as antioxidant by inhibition of ROS and malondialdehyde (MDA) accumulation and enhanced plant growth (Farooq et al. 2009; Radhakrishnan and Lee 2013). The foliar application of Spm alleviated the negative effects of water stress in maize (Zea mays L.) plants by increasing the activity of antioxidant enzymes (Talaat et al. 2015). In a more recent study, application of Spm alleviated water stress-induced oxidative stress of Rosa damascene Mill. by improving the growth characters, relative water content (RWC), chlorophyll content, and stomatal conductance (Hassan et al. 2018). As a conclusion, the application of exogenous PAs, mainly Put, Spd, and Spm, benefits several plant species by enhanced drought, salt, flooding, heat and cold tolerance, heavy metal, ozone, and copper, among others. Without ceasing to attribute meaning to these studies, there are also other important works encompassing plant genetic engineering strategies. Transgenic approaches have been used with the advantage of endogenous manipulation of PAs levels, focused toward the agricultural use of PAs capacity for enhancing abiotic stress tolerance and also to further study the regulatory mechanisms controlling plant cellular polyamine levels.

4 Changes in Polyamine Metabolism by Genetic Engineering

4.1 A Brief Historical Overview

With the cloning of several genes coding for enzymes of the polyamine biosynthetic pathway, it has become possible to manipulate polyamine biosynthesis using transgenic approaches (for cloned genes available, see Kumar et al. 1997, Liu et al. Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 295

2007, Pathak et al. 2014). Studies using genes or coding sequences (cDNAs) for enzymes of the PA biosynthetic pathways have become frequent. A first discovery using transgenic approaches was that Put levels were increased in tobacco roots as a result of expressing yeast Odc (Hamill et al. 1990). The expression of a mammalian Odc in transgenic carrot (Daucus carota L.) and tobacco also resulted in a significant increase in cellular Put levels (DeScenso and Minocha 1993; Bastola and Minocha 1995). Burtin and Michael (1997) and Capell et al. (1998) reported that the ectopic expression of the Avena sativa L. Adc in tobacco and rice, respectively, resulted an increase in Agm and Put. Regarding the plant performance, Roy and Wu (2001) presented first evidences that transgenic rice plants, with enhancedAdc expression, showed increased biomass under saline conditions, compared to control plants. By the same time Hanfrey et al. (2001) reported that in A. thaliana, the gene coding for ODC enzyme was not present, and the corresponding enzyme activity could not be detected. In this way, Arabidopsis depended only in the ADC pathway for Put biosynthesis. Based on the relative frequencies of Adc and Odc sequences in large EST collections in many plant species, including Glycine max, Medicago truncatula Gaertn., and Lotus japonicus (Regel) K. Larsen, the ADC pathway was considered the primary source of putrescine in plants (Flemetakis et al. 2004); and consequently, the focus of the transgenic approaches favored the use of the Adc gene. Other genes additionally used for transgenic approaches included the ones involved in Spd and Spm synthesis (Spds and Spms); in the decarboxylation of S-adenosylmethionine (Samdc), related with PAs catabolic pathways (coding for DAO and PAO); and lately transcription factors positively involved with PAs biosynthesis (see Table 1). In addition to the choice of genes to be used in the transformation, it is also necessary to take into account the choice of the promoter used to regulate their expression. The promoters that have been most commonly employed include the cauliflower mosaic virus (CaMV) 35S promoter (used for dicot crops) and the actin 1 promoter (Act-1) (used for monocot crops) (Grover et al. 2003). The use of inducible promoters, that allow the expression of a transgene only when it is required, was also a strategy used for PAs studies (an example is the use of ABA-inducible promoter in rice (Roy and Wu 2001, 2002)). The methodologies used to change PA levels have also take into account if the purpose is to have the gene expression upregulated, by sense overexpression of the transgene, or downregulated, by the antisense or RNA interference (RNAi) techniques (review in Duque et al. 2013). Early studies were accomplished in model species like Arabidopsis, tobacco, or plants with agricultural value as rice. Some examples include the heterologous expression of mouse Odc in tobacco, driven by CaMV 35S promoter, which resulted in a significant increase in Put and Spd (two- to threefold) and conferred increased tolerance to salt stress (Kumria and Rajam 2002). Nevertheless, transgenic plants showed altered in vitro growth and development, an aspect that was correlated with the supraoptimal Put levels. Transgenic tobacco expressing human Samdc (driven by CaMv 35S) showed increased Put and Spd levels; tolerance to multiple abiotic and biotic stresses, including salinity and drought; as well as resistance against Fusarium and Verticillium wilts (Waie and Rajam 2003). The introduction of Datura stramonium L. spermidine synthase (SPDS) cDNA into tobacco has led to the 296 S. de Sousa Araújo et al.

Table 1 Examples of genetic engineering of plants toward the accumulation or reduction of polyamines levels and the subsequent responses to environmental stresses Transformed Effects on PA metabolism and plant Gene/origin/promoter responses to environmental stresses References Nicotiana Odc, mouse, CaMV Increased Put and Spd, increased Kumria and tabacum 35S tolerance to salt stress; altered in vitro Rajam (2002) plant growth correlated with Put levels Nicotiana Samdc, human, Increased Put and Spd, tolerance to Waie and tabacum CaMV 35S salinity and drought; resistance against Rajam (2003) Fusarium and Verticillium wilts Solanum Samdc, yeast, E8 Increased Spd and Spm, enhanced Mehta et al. lycopersicum promoter phytonutrient content and fruit quality (2002); Mattoo et al. (2006) Oryza sativa Adc, Datura Higher Put, Spd, and Spm levels; Capell et al. stramonium, increased drought tolerance (2004) Ubi-1promoter Arabidopsis Spds, Cucurbita Increased SPDS activity, increased Kasukabe et al. thaliana ficifolia, CaMV 35S Spd; enhanced tolerance to various (2004) stresses including chilling, freezing, salinity, hyperosmosis, drought, and paraquat toxicity Ipomoea Spds, C. ficifolia, Increased Spd; improved number of Kasukabe et al. batatas CaMV 35S storage roots; increased tolerance to (2006) chilling- and heat-mediated damage to photosynthesis and enhanced tolerance to paraquat Solanum Adc, Avena sativa, Increased Put, Spd, and Spm levels; Prabhavathi melongena CaMV 35S multiple abiotic stress resistance and Rajam (salinity, drought, low and high (2007) temperature, and cadmium), and fungal resistance Oryza sativa Samdc, Datura Increased Spd and Spm levels, normal Peremarti et al. stramonium, CaMV levels of Put. Plants showed drought (2009) 35S symptoms, however, demonstrate a more robust recovery from stress Arabidopsis Adc1, Adc2, Increased Put; improved freezing, and Altabella et al. thaliana A. thaliana, CaMV drought tolerance (2009); 35S Alcázar et al. (2010b) Pyrus Spds, Malus Higher Spd accumulation, strong Wen et al. communis sylvestris, CaMV35S tolerance to salt, osmotic stress, and (2008, 2009,

heavy metals (CuSO4, CdCl2, PbCl2, 2010, 2011) and ZnCl2). Additional tolerance to AlCl3 Arabidopsis Adc, A. sativa, Increment in endogenous Put levels; Alet et al. thaliana pRD29A stress- transgenic lines more resistant to both (2011) inducible promoter cold and dehydration stresses (continued) Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 297

Table 1 (continued) Transformed Effects on PA metabolism and plant Gene/origin/promoter responses to environmental stresses References Solanum Sams, Transgenic plants showed lower Gong et al. lycopersicum S. lycopersicum, concentrations of Put, but enhanced (2014) CaMV35S Spd and Spm. Improved tomato fruit setting and yield and increased tolerance to alkali stress. Higher photosynthetic capacity and lower oxidative stress Lotus tenuis Adc, A. sativa, Increased Put and improved cellular Espasandin pRD29A hydration and increased root growth. et al. (2014) Put also controls the level of ABA by modulating ABA synthesis at the transcriptional level Nicotiana Myb, Poncirus Higher mRNA levels of two ADC Sun et al. tabacum trifoliate, CaMV35S genes, higher levels of PAs (Put, Spd, (2014) and Spm); enhanced dehydration tolerance, and lower levels of ROS and MDA Gossypium Myb, Gossypium Decreased tolerance to drought stress; Chen et al. hirsutum barbadense, decrease in proline and antioxidant (2015) virus-induced gene enzyme, and increase in MDA silencing (VIGS) Nicotiana Myb, G. barbadense, Improved survival and reduced water Chen et al. tabacum CaMV35S loss in plants under drought stress; (2015) enhanced proline and antioxidant enzymes, increased transcript levels of transcript levels of ADC1 and SAMDC Solanum Odc, mouse, 2A11 Transgenic plants with enhanced levels Pandey et al. lycopersicum fruit-specific of Put, Spd, and Spm; concomitant (2015) promoter reduction in ethylene levels, respiration rate, and physiological loss of water. Tomato plants with enhanced fruit quality Medicago Adc, A. sativa, Transgenic plants with elevated Put and Duque et al. truncatula 2XCaMV35S Spd, higher leaf RWC, and increased (2016) photosynthetic parameters during drought stress; higher seed yield upon stress recovery Citrus Pao, C. sinensis, Transgenic plants with increased PAO Wang and Liu sinensis CaMV35S activity, concomitant with a marked (2016) decrease of Spm and Spd, and elevation

of H2O2; transgenic seeds germinate better under salt stress, but by contrast vegetative growth and root elongation are inhibited (continued) 298 S. de Sousa Araújo et al.

Table 1 (continued) Transformed Effects on PA metabolism and plant Gene/origin/promoter responses to environmental stresses References Arabidopsis Pao, G. hirsutum, Decreased Spm and increased Put in Cheng et al. thaliana CaMV35S transgenic lines; discrepancies on salt (2017) resistance during germination and seedling stage Eremochloa Samdc, Cynodon Transgenic plants with higher levels of Luo et al. ophiuroides dactylon, CaMV35S PAO activity; improved cold tolerance (2017)

through the involvement of H2O2 and NO signaling Nicotiana Myb21, Pyrus Overexpression of MYB21 plays a Li et al. (2017) tabacum betulaefolia, positive role in drought tolerance; CaMV35S possible modulation of polyamine biosynthesis by regulating the ADC expression Lotus tenuis Adc, A. sativa, Improved tolerance to salt stress in Espasandin pRD29A transgenic lines, smaller reduction in et al. (2018) shoot biomass, and a slight increase in root growth in response to stress; increased osmotic adjustment via proline production increased tolerance against multiple abiotic stresses and also provided evidences that spermidine synthase was not a limiting step in the biosynthesis of polyamines (Franceschetti et al. 2004). Transgenic rice expressing heterologous Samdc, driven by an ABA-inducible promoter, have showed increased Put and Spm levels (three- to fourfold), and transgenic seedlings showed increased growth under salinity conditions (Roy and Wu 2002). One the other side, the expression of Datura stramonium Adc gene, driven by the strong maize polyubiquitin-1 (Ubi-1) promoter, increased Put, Spd, and Spm levels and conferred drought tolerance to rice (Capell et al. 2004). The spermidine synthase cDNA from Cucurbita ficifolia Bouché was inserted into Arabidopsis (35S promoter), and this approach resulted in plants with increased tolerance against multiple abiotic stresses, including chilling, freezing, salinity, hyperosmosis, drought, and paraquat toxicity (Kasukabe et al. 2004). The constitutive expression of homologous Adc1 and Adc2 genes in Arabidopsis resulted in freezing and drought tolerance, respectively (Altabella et al. 2009; Alcázar et al. 2010b). As previously referred, from 2004 to 2006 onward, the majority of the PAs studies have been conducted in non-model species (see Fig. 1b), reflecting the interest of the scientific community on the possible use of these biomolecules on plant crop improvement toward abiotic stress. Several studies based on the engineering of PA biosynthesis for the production of stress-tolerant plants are summarized in Table 1 and will be further discussed on the following sections. A common feature observed in drought (here in the sense of water deficit), salinity, and cold/heat stress is the primary consequence of loss of water by the cells, resulting in a decrease in the osmotic potential. However, cell water loss in drought is due to water shortage in soil and/or in atmosphere; in salinity, stress is Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 299 related to the decreases caused by the increased ion concentration; in cold stress, the cell water content diminished due to the difficulty of the soil available water to be transported to the living cells; and finally, with increased temperatures, the root moisture is lost to harmful levels (reviewed in Duque et al. 2013; Lamaoui et al. 2018). For the sake of structuring this chapter, we decided to focus each subsection in a specific stress response; it is however important to take into consideration that stresses are related and that in nature they do not occur normally as isolated phenomena.

4.2 Polyamines and Drought Stress

Among abiotic stresses, drought affects foremost arable area and, in the current scenario of climate change and increased world population, is a major challenge for agriculture (reviewed in Duque et al. 2013; Araújo et al. 2015; Tardieu et al. 2018). Plant growth and productivity are greatly affected by drought, which causes a primary loss of cell water and, therefore, a decrease of cell osmotic potential (Duque et al. 2013). Plant reactions to water deficit can be divided in short-term and long-term responses. Physiological mechanisms primarily triggered act as short-term feedback: for example, an increase in transpiration rate tends to cause partial stomatal closure, thereby stabilizing transpiration, and a decrease in osmotic potential is rapidly compensated by rapid buildup and/or uptake of solutes to maintain turgor homeostasis (reviewed in Tardieu et al. 2018). Changes in photosynthetic parameters and in carbon metabolism are also promptly observed, with subsequent changes in the pool of sugars used for signaling cellular processes (Chaves et al. 2009; Liu et al. 2013). Long-term water shortage negatively affects photosynthesis, leaf and root growth, and reproductive development, resulting in lower biomass accumulation, lower yield, and poor quality of the harvested plant parts (e.g., grains, biomass, and stalks) (reviewed in Araújo et al. 2015). Additionally, it is well documented that root growth is usually less affected by water potential changes than shoot growth; thus, an increased root/shoot ratio is commonly observed under water deficit (e.g., Xu et al. 2015; Purushothaman et al. 2017). In Arabidopsis, drought stress induces Adc2 expression (Alcázar et al. 2005), and since most of the key genes involved in polyamine biosynthesis are duplicated and a functional ODC pathway is missing (Hanfrey et al. 2001), the constitutive expression of homologous Adc2 gene was the choice for plant transformation (Alcázar et al. 2010b). In this study, 4-week-old plant lines grown in soil were exposed to water deprivation by 2 weeks water withholding, followed by 7 days of recovery. Survival rates in transgenic lines were 18–75%, higher than the 12% wild- type (WT) survival rate. Measurements of leaf relative water content (RWC), stomata conductance (gs), and stomata aperture showed reduced water loss, reduced stomata conductance, and stomata closure in 35S::ADC2 drought-tolerant lines compared to WT (Alcázar et al. 2010b). Arabidopsis Adc2 overexpressing transgenic 300 S. de Sousa Araújo et al. lines presented Put accumulation but unaltered Spd or Spm. Moreover, a linear correlation between total Put content and drought resistance was observed (Alcázar et al. 2005, 2010b). Espasandin et al. (2014) studied the drought response of a transgenic Lotus tenuis cv INTA PAMPA line that expresses the oat Adc gene, driven by the stress- inducible Arabidopsis pRD29A promoter. The pRD29A promoter presents DRE (drought-responsive element) and ABRE (ABA responding element) cis acting elements, enabling induction under ABA and osmotic stress (Wu et al. 2008). Plants were grown for 6–8 weeks and subjected to continuous soil drying by water withholding until reaching Ψsoil = −2 MPa. Interestingly, the RWC of pRD29A::oatADC line under stress conditions did not change significantly with the decrease in soil water potential, when comparing to the WT line. In drought conditions, the transgenic line also presented higher stomatal conductance (gs) and transpiration (E) values (Espasandin et al. 2014). Put accumulation was considered sufficient to promote drought tolerance since Spm and Spd levels did not increase during the dehydration period. Additionally, the highest Put content in pRD29A::oatADC plants improved the cell’s water balance by adjusting the osmotic potential through the production of proline and by adapting the plant growth pattern through the redistribution of photo-assimilates in favor of root growth at the expense of leaf area (Espasandin et al. 2014). Nunes et al. (2008) evaluated the two main mechanisms of drought resistance, drought avoidance, and drought tolerance, in the model legume Medicago truncatula cv. Jemalong. Under mild stress conditions, when the soil water content (SWC) decreased to 1/2 of its maximum, M. truncatula plants maintained identical leaf

RWC, net CO2 fixation rate, and photochemical and biochemical photosynthetic processes, suggesting that plants are able to avoid leaf dehydration. However, under severe water deficit (SWD), ribulose-1,5-bisphosphate (RuBP) regeneration and Rubisco carboxylation efficiency were both decreased, suggesting that nonstomatal limitations also occur in addition to mechanisms involving osmotic adjustment (reviewed in Araújo et al. 2015). Based on the previous study, 11-week-old M. truncatula plants constructively expressing the oat Adc gene (35S::oatADC) were subjected to severe water deficit (SWD), by water withholding, until SWC reached values below 1/5, followed by 4 days of recovery (Duque et al. 2016). In the 35S::oatADC transformed line, higher leaf RWC was observed under SWD, compared to WT plants. Additionally, and independently of the light intensity, under SWD, the transgenic line stood out with increased photosynthetic parameters, namely, leaf internal CO2 concentration (Ci), net CO2 assimilation rate (A), transpiration (E), and stomatal conductance (gs). Elevated Put and Spd observed in the transgenic line could be responsible for preserving the guard cells turgor pressure (Duque et al. 2016), which ultimately regulate stomatal aperture (Damour et al. 2010). Moreover, 35S::oatADC transgenic plants that recovered from SWD had higher seed yield compared to control WT, suggesting a possible benefit of PA metabolism manipulation in preserving harvested grain yield in legumes exposed to drought stress. Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 301

Works focusing on the overexpression of the SAMDC and SPDS enzymes were also conducted toward the possibility of enhancing plants’ drought tolerance. A heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium was used to generate transgenic rice plants with altered Spd and Spm levels, although maintaining Put titers (Peremarti et al. 2009). SAM is a common precursor for both the synthesis of PAs and ethylene; SAMDC is essential for the production of decarboxylated S-adenosylmethionine (dcSAM) and to provide aminopropyl groups to convert Put into Spd and Spm (see Fig. 2). Two-month-old WT and Ubi:DsSAMDC-transformed rice plants (Oryza sativa L. subsp. Japonica cv. EYI105) were watered during 60 days and after were subjected to 6 days treatment with 20% polyethylene glycol. The plants were allowed to recover for 20 days by replacing the PEG solution with water. Symptoms of dehydration (wilting and curling of the leaves) were clear after 3 days and became more severe after 6 days of PEG treatment, with WT and transgenic plants showing similar drought stress phenotypes. Peremarti et al. (2009) demonstrated that the transgenic plants expressing SAMDC produced normal levels of Put and presented similar drought syndromes when compared to WT. However, transgenic plants demonstrated a more robust recovery upon return to normal conditions. They suggested that, while plants with elevated Put are able to tolerate stress because of Put direct protective role in preventing the symptoms of dehydration, the higher PAs (Spd and Spm) could play an important in role in stress recovery. Moreover, Spd and Spm can also feedback to regulate the Put pool, with the result that normal metabolic and morphological phenotypes could be restored under stress (Peremarti et al. 2009). In a previous study, sweet potato (Ipomoea batatas cv. Kokei) was constructively transformed with Spds from Cucurbita ficifolia, and resulting transgenic plants showed twofold Spd content compared to the WT counterpart, both in leaves and storage roots (Kasukabe et al. 2006). Potted plants were exposed to drought stress and revealed a marked growth reduction in both vines and storage roots. However, transgenic plants were less affected than WT in terms of the number of storage roots and their fresh weights. Drought stress also decreased starch content in WT storage roots, but not in the transgenic plants, which showed a 1.5-fold higher starch yield. Authors concluded that transgenic FSPD1 sweet potatoes were more tolerant to drought stresses in the root zone than the WT plants (Kasukabe et al. 2006). Interestingly, MYB transcription factor genes from Gossypium barbadense L. (GbMyb5) and from Poncirus trifoliata L. Raf. (PtsrMyb) were found to confer drought tolerance in cotton and transgenic tobacco, Chen et al. (2015) and Sun et al. (2014), respectively. In the first work, GbMyb5 virus-induced gene silencing compromised the tolerance of cotton plantlets to drought stress by decreasing the recovery survival rate after the re-watering of stressed plants (a reduction of 90% to 50% was observed, when comparing transgenic and WT plants). A decrease in proline and antioxidant enzyme activities, and the opposite increase in MDA, was observed as a consequence of GbMyb5 silencing in stressed cotton (Chen et al. 2015). Oppositely, the overexpression of GbMyb5 in tobacco revealed hypersensitivity to ABA, improved survival, and reduced water loss rates in plants under drought stress. Proline and antioxidant enzymes showed enhanced 302 S. de Sousa Araújo et al. accumulation, while MDA was reduced in transgenic tobacco under drought stress. The transcript levels of the antioxidant genes SOD, CAT, and GST and of polyamine biosynthesis genes Adc1 and Samdc were also generally higher in GbMyb5 transgenic tobacco plants (Chen et al. 2015). The overexpression of PtsrMyb in tobacco conferred enhanced dehydration tolerance (by diminishing water loss) and showed lower levels of ROS and MDA production. Transgenic tobacco lines presented higher mRNA levels of two Adc genes, before and after dehydration treatments, when compared to WT, and consequently higher levels of PAs (Sun et al. 2014). Additionally, several MYB-recognizing cis-acting elements are present on PtAdc gene promoters, and yeast-hybrid assays demonstrated that PtsrMYB interacts predominantly with two regions of the Adc promoter, indicating the PtAdc may be a target gene of PtsrMYB (Sun et al. 2014). Both works revealed that MYB transcription factors play a positive role in dehydration tolerance through the modulation of polyamine biosynthesis by the means of regulating the Adc gene expression (Sun et al. 2014; Chen et al. 2015). In this way, the identification of important transcriptional factors, capable to trigger the expression of the multiple genes encoding for enzymes of PA metabolic pathways, and their further use for PA modulation is a promising approach for future studies in this field.

4.3 Polyamines and Salinity Stress

A recent Springer book entitled Salinity Responses and Tolerance in Plant provided an excellent update to the state of the art of the complex machinery of plant responses and adaptive mechanisms to cope with hyper soil salinity (Kumar et al. 2018). This book also includes transgenic approaches to develop salt-tolerant crops in this challenge era of climate changes. Salt stress is a major environmental limitation affecting crop performance and yield due to its osmotic and ionic effects (Negrão et al. 2017; Bor and Özdemir 2018). Manipulating metabolic pathways for higher osmotic and ionic tolerance would be more realistic to mitigate the negative impact of salt stress on crop plants, in opposition of targeting signaling or regulatory networks (Bor and Özdemir 2018). Among possible metabolic pathways to be engineered is the overexpression of polyamine biosynthesis-related genes, consensually accepted to contribute for salt stress tolerance in several species (Bor and Özdemir 2018; Khare et al. 2018). Khare et al. (2018) presented an overview of transgenic plants that, by the overexpression of PA biosynthesis-related genes, are able to perform better under salt stress (see Table 13.1 in Khare et al. 2018 and references therein). Salinity effect on plants is similar to drought effect in an initial stage; however, in the long term the consequences of Na+ and Cl− leaf accumulation and hyperosmotic and hyper-ionic stresses enhance the severity and impact of this stress (Chaves et al.

2009; Bor and Özdemir 2018). Salinity directly affects stomata and mesophyll (gm) conductance as well as gene expression, resulting in alteration of photosynthetic metabolism (Chaves et al. 2009). The effects can be direct, with plants losing water Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 303 from their tissues, with a rapid effect on cell expansion and cell divisions (Negrão et al. 2017), suffering a decrease in CO2 availability caused by diffusion limitations through the stomata and the mesophyll, being damaged by photosynthetic alterations, and leading to abscisic acid (ABA) accumulation, or can be related to secondary effects, specifically oxidative stress (Chaves et al.2009 ). In experimental settings, one of the first observable responses after salinity imposition is a reduction in shoot growth (Negrão et al. 2017). Here we present some non-exhaustive examples of PA content modification toward salinity resistance given the actuality of the previously mentioned up-to-date revisions. Prabhavathi and Rajam (2007) reported an increase in Put but also in higher PAs, Spd, and Spm in eggplant (Solanum melongena L. cv. Pusa Purple Long) transformed with oat Adc (under the control of CaMV 35S promoter). Regarding the salinity tolerance assay, plant seeds were inoculated on MS basal medium containing 150 and 200 mM NaCl, and tolerance was accessed based on the percentage of seed germination according to Prabhavathi et al. (2002). Transgenic seeds germinated in 200 mM NaCl after 15 days of inoculation, and the seedlings grew well on salt-amended medium, while WT seeds failed to germinate. Seed survival was also tested with a sublethal 150 mM salt concentration; in this condition, transgenic seedling performed better, and their growth in terms of shoot length and fresh and dry weight was greater than in the WT seedlings. Additionally, in this study, authors also found that PA accumulation in transgenic eggplants resulted in increased tolerance to other abiotic stresses (drought, low and high temperature, and heavy metal) and also fungal resistance. Moreover, in addition to the ADC enzyme activity increase, the DAO activity was also augmented in transgenic plant lines (Prabhavathi and Rajam 2007). Espasandin et al. (2018) overexpressed the oat Adc gene in Lotus tenuis cv INTA PAMPA, using the stress-inducible pRD29A promoter (pRD29A::oatADC construct; previously used in Espasandin et al. 2014). In addition to the previous work regarding the contribution of ADC overexpression in drought tolerance (Espasandin et al. 2014) the authors also tested the improved tolerance to salt stress in 6-month-old WT and transgenic plants at vegetative stage, by gradually increasing salinity, that was applied by means of sodium chloride irrigation (NaCl from 0 to 0.3 mol.L−1) (Espasandin et al. 2018). Transgenic lines appeared healthier than the WT plants and showed a smaller reduction in shoot biomass and a slightly increase in root growth in response to the applied stress. ADC overexpression also increased osmotic adjustment by 5.8-fold, via proline production. Moreover, salinity treatment doubled the potassium uptake by transgenic ADC roots in stressed plants, with a concomitant decrease in the sodium accumulation, balancing the Na+/K+ ratio. The shoot/root ratio increased at the expense of roots in WT; contrastingly, the stressed transgenic plants showed lesser reduction in shoot biomass and a minor promotion in root growth. A detailed analysis of gene expression, of enzymatic activities, and of hormone metabolism suggested an important crosstalk between PAs and ABA in response to salinity, via the modulation of the ABA biosynthesis-related enzyme 9-cis-epoxycarotenoid dioxygenase (EC 1.13.11.51) at the transcriptional level (Espasandin et al. 2018). 304 S. de Sousa Araújo et al.

In Kasukabe et al. (2006) work, concerning sweet potato constructively transformed with Spds (previously discussed in Sect. 4.1 in respect to the drought tolerance), the response to high salinity was also evaluated. The establishment of a saline soil for plant growth was accomplished by the addition of 8 g NaCl, before planting, and by an extra addition of 4 g NaCl 45 days after planting (for 20 L of soil). Salt stress suppressed storage root growth; however, transgenic plants were less affected and produced significantly larger mass of storage roots and starches (1.2-fold higher fresh mass) than WT plants. Transgenic plants also showed increased tolerance to chilling- and heat-mediated damage to photosynthesis compared to the WT plants (Kasukabe et al. 2006). Overexpression of an apple (Malus sylvestris (L.) Mill.) spermidine synthase (SPDS) gene substantially increased the tolerance to multiple stresses by altering the PA levels in pear (Pyrus communis L.). A specific transgenic line, which revealed highest Spd accumulation, showed the strongest tolerance to salt (150 mM NaCl), osmotic stress (300 mM mannitol), and heavy metal (500 μM CuSO4). Additionally, this line showed the lowest growth inhibition and the least increased in the electrolyte leakage (EL) and in the production of thiobarbituric acid reactive substances (TBARS) under stress conditions (Wen et al. 2008). Wen et al. (2009) further studied the possibility of this MdSPDS1 pear transgenic lines to tolerate aluminum (Al) stress, a major cause of poor crop yields, particularly in countries with acidic soil prevalence. Overexpressing apple spermidine synthase (MdSPDS1) plants and WT plants was subjected to long-term stress with 30 μM AlCl3. The performance of transgenic line was superior in terms of shoot height increment (SHI), fresh weight increment (FWI), and observed morphological features, when compared to WT counterparts (Wen et al. 2009). Favorable to the better performance and survival of MdSPDS1 transgenic line was the observation that, upon this stress, there was an increase of the activities of superoxide dismutase (SOD), of glutathione reductase (GR), and of proline and MDA accumulation. Moreover, Ca2+ concentration and some co-factor metals of SOD were higher in transgenic than in WT, after the imposed stress. These antioxidant parameters were closely related to the Spd levels, evidencing that Spd is implicated in Al stress tolerance via ameliorating oxidative status as well as by affecting mineral element balance (Wen et al. 2009). Wen et al. (2010) also found that the transgenic MdSPDS1 line had better performance than

WT line when subjected to heavy metals stress (using CdCl2, PbCl2, ZnCl2, or a combination of those). Results were based on either shoot height increment or fresh weight and morphological changes upon heavy metal stress (Wen et al. 2010). The importance of PAs in stress responses can also be evaluated by using approaches of antisense inhibition of polyamine biosynthesis-related genes. An example was the transformation of P. communis with a construct containing an apple Spds gene (MdSPDS1) in antisense orientation (Wen et al. 2011). In this work, the antioxidant system was not effectively induced under both salt and cadmium stress in the antisense transgenic line, when compared to WT, as could be perceived by the determination of glutathione (GSH) content, glutathione reductase (GR) activity, SOD, and evaluating the proline accumulation. Moreover, under stress the antisense line showed greater accumulation of MDA; an important Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 305 indicator of lipid peroxidation. These results provide evidences for the important role of PAs in both salt and cadmium stress tolerance, in which the PAs act, to some extent, by influencing the antioxidant system (Wen et al. 2011). Attempts to change the PA concentrations by modulation of its catabolic pathway were also accomplished, and here we present an example for genetic engineering with the model Arabidopsis. Cotton (Gossypium hirsutum L.) PAO3 cDNA (genome-wide identified from a released genome database) was selected for

Arabidopsis transformation (35S::GhPAO3 construct) (Cheng et al. 2017). To com- pare the stress tolerance of WT and GhPAO3 transgenic line, 2-week-old Arabidopsis seedlings were treated with 300 mM NaCl. Overall, and during seed development, the WT plants performed better than the transgenic lines under NaCl treatment. Interestingly, WT and GhPAO3 Arabidopsis plants showed an opposite performance in soil under 300 mM NaCl and 100 mM NaCl treatment (Cheng et al. 2017). In the transgenic 35S::GhPAO3 line, the Spm content was significantly decreased, whereas the Put content was enhanced, which indicates a potential role of GhPAO3 in the back-conversion of Spd and Spm to Put. Hydrogen peroxide analysis in transgenic and WT indicated that H2O2 was mainly produced from polyamine oxidation in this process. The discrepancies in salt stress tolerance that were observed in germination and during seedling development (which might be controlled by the H2O2 concentration threshold) suggested an enhance resistance to NaCl stress in GhPAO3 plants at a certain level; whereas unusually higher H2O2 concentrations may become harmful for Arabidopsis development and be implicated in programed cell death (Cheng et al. 2017).

4.4 Polyamines and Tolerance to Extreme Temperatures

In the current scenario of climate change, plants are being challenged with frequent episodes of extreme weather events that include, among other aspects, spells of very high temperatures which are strongly connected to drought episodes (Rosenzweig et al. 2001). But not only high temperatures compromise serious plant growth, and suboptimal temperatures have been considered also a major threat to agricultural production in temperate regions. Despite most of the temperate plants acquiring chilling and freezing tolerance upon prior exposure to sublethal cold stress commonly called cold acclimation, still many important crops with agronomic relevance are incapable of cold acclimation (Yadav 2010). Many plant crops are sensitive to supraoptimal temperatures that lead to disruption of cellular and physiological homeostasis, a phenomena also known as heat stress (Xu et al. 2017). The impacts of heat stress on plant performance are well described and associated with alterations on photosynthesis, assimilating partitioning, growth, and development (Bokszczanin et al. 2013). Importantly, heat stress strongly negatively affects reproductive success on the majority of the plants species, which commonly translates into yield losses in agricultural settings (Asseng et al. 2011). 306 S. de Sousa Araújo et al.

Approaches to improve tolerance to extreme temperatures through the PA content modification embrace some case-stories of success. Tomato is one of the most important horticultural crops worldwide, and the damaging effects of heat stress in tomato production are very well documented (Sato et al. 2000, 2001; Xu et al. 2017). Thus, the identification and development of tomato cultivars better adapted to growth, and set fruits under supraoptimal temperature conditions are still an important need of the agricultural sector. In this context, Cheng and collaborators (2009) have generated transgenic plants of tomato variety “zhongshu No.6” constitutively overexpressing the Saccharomyces cerevisiae Samdc. In average, 1.7- to 2.4-fold higher levels of Spd and Spm were found in transgenic lines compared to WT under high temperature stress (38 °C/30 °C). Associated with this PA accumulation, a markedly increased antioxidant enzyme activity and decreased MDA contents were also noticed in transgenic plants. Heat stress increased lipid peroxidation in the nontransformed line, an aspect that was not observed in transgenic lines. The authors concluded that PA accumulation generated a heat tolerance mechanism, supported the PA antioxidant activity, and alleviated the membrane damage caused by ROS during heat stress (Cheng et al. 2009). Another interesting aspect, likely beyond the scope of this aspect, is that the overexpression of Samdc in tomato modulated the nutrient contents of the fruits (Kolotilin et al. 2011), which can have added value for the consumers or impact in product shelf-life. The physiological impact of low temperatures is also well described in plants. Low temperature reduces water availability for the plant, decreases membrane fluidity, and causes an imbalance between the light energy absorbed by photosystems and the energy consumed by metabolic reactions, compromising plant growth and survival (Ruelland et al. 2009). Not all plants or crops are well adapted to low temperatures or chilling, being this related mainly to their tropical or subtropical origin (Lyons 1973). One of those examples is the centipede grass (Eremochloa ophiuroides (Munro) Hack.), a warm-season (C4) perennial grass native to South and Central China (Hanna 1995). Centipede grass is widely used as turf and has gained growing interest as a low-input plant for soil conservation in many countries prone to erosion. In environments where frosts are not severe, centipede grass is green throughout the winter, but its growing period is from spring through late autumn, becoming brown or dormant in winter (Islam and Hirata 2005). Among the different research efforts conducted to improve cold tolerance in this grass, we highlight here one that included the overexpression of PA biosynthetic genes. Recently, Luo and collaborators (2017) overexpressed in centipede grass a Samdc gene from Bermuda grass (Cynodon dactylon (L.) Pers.) and investigated their effects in response to cold. In resulting SAMDC transgenic plants, Spd levels were in average 2.3- to 2.9-fold higher, while Spm levels were 1.6- to 1.8-fold higher, in comparison with the nontransformed plants. Freezing tolerance of transgenic and nontransgenic lines was evaluated by determination of the temperature that resulted in 50% lethality (LT50) and assessment of survival rate. Under non-acclimated conditions, transgenic lines showed a lower level of LT50 (−5.2 °C), when compared to the −3.2 °C observed in nontransformed plants, suggesting a better tolerance to freezing. After a progressive freezing treatment, the Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 307 survival rate of transgenic lines was higher (59–61%) comparing to those observed in nontransformed ones (40%) under non-acclimated conditions. Biochemical studies provided evidences about the mechanisms triggered by PA accumulation under cold. As one example, transgenic plants showed higher catalase and SOD activities and higher PAO activity and H2O2 levels upon cold. These results supported the assumption that the elevated cold tolerance was associated with PAO-catalyzed production of H2O2, a key signaling molecule that led to nitrate reductase (NR)- derived NO production (Lu et al. 2014) and triggered antioxidant enzyme activities in transgenic plants (Luo et al. 2017). Importantly, this work provided new evidences of the prevailing crosstalk between PAs, ROS, and NRS in the modulation of cold stress responses in plants. The knowledge about the crosstalk between PAs and hormones as ethylene was essential to derive or test new strategies to increase plants’ tolerance to cold stress. One of these approaches has as key player a cold-induced transcription factor, the ethylene responsive factor (ERF), which has been isolated from Medicago falcata L. (Pang et al. 2009). M. falcata is a forage legume well known by its enhanced cold tolerance, when compared to other species of the same genera (Lesins and Lesins 1979). The role of MfERF1 on cold acclimation has been further assessed in tobacco (Zhuo et al. 2018). These authors found that the constitutive overexpression of MfErf1 resulted in an increased tolerance to freezing and chilling in transgenic tobacco plants. Indeed, a freezing treatment at −3 °C for 6 h. was lethal for most of nontransformed plants, whereas 47–50% of transgenic plants were able to survive. One of the most interesting aspects of this work is that the overexpression of MfErf1 upregulated, among others, the expression of PA genes implicated in Spd and Spm synthesis (SAMDC1, SAMDC2, SPDS1, SPDS2, and SPMS) and PA catabolism (PAO) in comparison with WT plants, which is an indirect approach to modulate PA metabolism. Nevertheless, no significant accumulation of Spd and Spm was noticed in transgenic plants, likely as result of the increased PAO activity, which contributed for maintaining PAs at nonlethal levels. Among other findings, these results suggested that MfERF1 conferred cold tolerance by promoting polyamine turnover, antioxidant protection, and proline accumulation (Zhuo et al. 2018). The use of transcription factors, such as MfERF1, that trigger the expression of several protective genes like the ones responsible for PA biosynthesis constitutes a very promissory and still unexploited approach to enhance cold tolerance in plants. In this section, we have reviewed several successful approaches to improve heat and cold tolerance in plants through the modulation of the PA metabolism. Interestingly, most of the approaches targeted the constitutive overexpression of SAMDC, an enzyme essential for the addition of aminopropyl groups, needed for the conversion of Put to Spd and Spd to Spm. Most of the studies provided clear evidences of the link between PAs, ROS, NRS, and hormones to enhance plants adaptation to extreme temperatures. Still, there is an open window for the development of new strategies based on the modulation of PA contents and turnover. One promissory could be related to the identification of transcription factors capable to trigger the expression of the multiple genes encoding for enzymes PA metabolism. 308 S. de Sousa Araújo et al.

4.5 Oxidative Stress: A Common Mechanism in Response to Stress

All kinds of abiotic stresses previously referred trigger a generalized stress response denominated oxidative stress, due to the accumulation of ROS, and can seriously affect leaf photosynthetic machinery (Chaves and Oliveira 2004). Oxidative stress can be generally defined as a physiological state in which oxidation is superior to its opposite mechanism, the reduction (Harshavardhan et al. 2018). In respect to the antioxidant activity of PAs, the research data is contradictory; on the one hand, PAs have been suggested to protect cells against ROS, and on the other hand, their catabolism generates ROS. PA catabolism produces H2O2, a signaling molecule that not only acts by promoting an antioxidative defense response upon stress but can also act as a peroxidation agent (Groppa and Benavides 2008; Gupta et al. 2016) (Fig. 2). In a study using poplar cells in culture (isogenic cell lines of Populus nigra × Populus maximowiczii), the effect of increased Put accumulation was found to negatively impact their oxidative state owing to the enhanced turnover of Put (Mohapatra et al. 2009). Gill and Tuteja (2010) suggested that while increase Put accumulation could have a protective role against ROS in plants, an enhanced Put turnover can in reality make them more vulnerable to increased oxidative damage. Contrastingly, the higher polyamines, Spd, and Spm are believed to be most efficient antioxidants and considered as scavengers of oxyradicals (He et al. 2008; Channarayappa and Biradar 2018). The work by Cheng et al. (2017), previously described in Sect. 4.2, concerning GhPAO3 Arabidopsis transgenic plants, is a clear example of the duality of the balance process between consumption and production of H2O2. Another interesting work is the transformation of Citrus sinensis (L.) Osbeck with the homologous CsPAO4 by Wang and Liu (2016). These transgenic plants showed prominent increase in PAO activity, concomitantly with a marked decrease of Spm and Spd and elevation of H2O2. While transgenic seeds germinate better when compared with WT under salt stress, vegetative growth and root elongation are further inhibited (with the maintenance of stressed conditions), accompanied with higher accumulation of H2O2 and a more noticeable programmed cell death (PCD).

5 Conclusions

Polyamines are a class of plant biomolecules that have been implicated in plant growth and development processes and are also very important in plant responses to biotic and abiotic stresses. PAs’ endogenous levels are influenced by different factors such as plant species, stress tolerance capacity, stress types and conditions, and the physiological status of the studied tissue or organ. The major forms of PAs Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 309 are Put, Spd, and Spm; although plants also synthesized a variety of other related compounds. PA intracellular levels are regulated by anabolic and catabolic processes as well as by polyamine conjugation with hydroxycinnamic acids, fatty acids, and macromolecules. Moreover, polyamines metabolic pathways are closely interconnected with other metabolic pathways, such as the glutamate cycle, proline, urea, γ-aminobutiric acid (GABA), and TCA cycle. PA metabolic pathway is also closely related to ethylene by the means of sharing a common precursor, the S-adenosylmethionine (SAM). Several studies also provided evidences of the link between PAs, ROS, NRS, and hormones (besides ethylene), being a driving force for further studies on this subject. One interesting fact is that the number of articles on plant PAs almost duplicated since 2008. This may be due to the development of new high-throughput methodologies to characterize the global gene expression or metabolite accumulation (omics) with reduced cost for user, together with the release of several plant genome sequences. Another interesting fact is that the majority of the PA studies have been conducted into nonmodel species, which may likely reflect a translational application of the early results obtained with models for further use in important agricultural crops. In this chapter, we presented non- exhaustive examples of endogenous PA content modification toward the tolerance/ resistance to abiotic stresses, by the means of plant genetic engineering. We focus each subsection in a specific stress, drought, salinity, and tolerance to extreme temperatures; however, PA manipulation usually confers tolerance to multiple abiotic stresses. Moreover, stress phenomena are related, and in nature they typically do occur as isolated events. PA biosynthetic genes usually used for transgenic approaches included the ones involved in Put, Spd, and Spm synthesis (Adc, Spds, and Spms); in the decarboxylation of SAM (Samdc), related with PA catabolic pathways (coding for DAO and PAO); and lately, in transcription factors positively involved with PA biosynthesis. The identification and further study of transcription factors capable to trigger the expression of multiple genes encoding for enzymes of PA metabolism constitutes a promising path for future studies. Despite the complexity and intricate network of the mechanisms involved in the PA synthesis and regulation, the increased number of recent works, as well as the application of patents related to PAs and stress tolerance, corroborates the continued interest of the scientific community in elucidating the different roles of these important biomolecules in plants.

Acknowledgments Financial support from FCT (Fundação para a Ciência e Tecnologia, Lisbon, Portugal) is acknowledged through the research unit “GREEN-it: Bioresources for Sustainability” (UID/Multi/04551/2013) and through ASD and SSA PhD holders DL57 research contracts. ALWS is supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and Young Investigators Grants 15/21075-4 and 17/01284-3. ALWS thanks Dra. Eny IS Floh (Department of Botany, University of São Paulo) for her valuable collaboration and pioneering studies with polyamines in Brazil. 310 S. de Sousa Araújo et al.

References

Alcázar R, García-Martínez JL, Cuevas JC, Tiburcio AF, Altabella T (2005) Overexpression of ADC2 in Arabidopsis induces dwarfism and late-flowering through GA deficiency. Plant J 43:425–436. https://doi.org/10.1111/j.1365-313X.2005.02465.x Alcázar R, Cuevas JC, Patron M, Altabella T, Tiburcio AF (2006) Abscisic acid modulates polyamine metabolism under water stress in Arabidopsis thaliana. Physiol Plant 128:448–455 Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, Tiburcio AF (2010a) Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta 231:1237–1249 Alcázar R, Planas J, Saxena T, Zarza X, Bortolotti C, Cuevas JC, Bitrián M, Tiburcio AF, Altabella T (2010b) Putrescine accumulation confers drought tolerance in transgenic Arabidopsis plants overexpressing the homologous Arginine decarboxylase 2 gene. Plant Physiol Biochem 48(7):547–552 Alet AI, Sanchez DH, Cuevas JC, del Valle S, Altabella T, Tiburcio AF, Marco F, Ferrando A, Espasandín FD, González ME, Carrasco P, Ruiz OA (2011) Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress. Plant Signal Behav 6:278–286. https://doi.org/10.4161/psb.6.2.14702 Altabella T, Tiburcio AF, Ferrando A (2009) Plant with resistance to low temperature and method of production thereof. Spanish patent application; WO2010/004070; US patent application; No:2011/0126,322 Anwar A, She M, Wang K, Riaz B, Ye X (2018) Biological roles of ornithine aminotransferase (OAT) in plant stress tolerance: present progress and future perspectives. Int J Mol Sci 19:3681. https://doi.org/10.3390/ijms19113681 Arasimowicz-Jelonek M, Floryszak-Wieczorek J, Kubiś J (2009) Interaction between polyamine and nitric oxide signaling in adaptive responses to drought in cucumber. J Plant Growth Regul 28:177–186. https://doi.org/10.1007/s00344-009-9086-7 Araújo SS, Beebe S, Crespi M, Delbreil B, González EM, Gruber V, Lejeune-Henaut I, Link W, Monteros MJ, Prats E, Rao I, Vadez V, Vaz Patto MC (2015) Abiotic stress responses in Legumes: strategies used to cope with environmental challenges. Crit Rev Plant Sci 34:237– 280. https://doi.org/10.1080/07352689.2014.898450 Asseng S, Foster I, Turner NC (2011) The impact of temperature variability on wheat yields. Glob Chang Biol 17:997–1012. https://doi.org/10.1111/j.1365-2486.2010.02262.x Baron K, Stasolla C (2008) The role of polyamines during in vivo and in vitro development. In Vitro Cell Dev Biol Plant 44:384–395. https://doi.org/10.1007/s11627-008-9176-4 Bastola DR, Minocha SC (1995) Increased putrescine biosynthesis through transfer of mouse ornithine decarboxylase cDNA in carrot promotes somatic embryogenesis. Plant Physiol 109:63–71 Bokszczanin K, Fragkostefanakis S, Bostan H, Bovy A, Chaturvedi P, Chiusano M, Firon N, Iannacone R, Jegadeesan S, Klaczynskid K, Li H, Mariani C, Müller F, Paul P, Paupiere M, Pressman E, Rieu I, Scharf K, Schleiff E, Van Heusden A, Vriezen W, Weckwerth W, Winter P (2013) Perspectives on deciphering mechanisms underlying plant heat stress response and thermotolerance. Front Plant Sci 4:315. https://doi.org/10.3389/fpls.2013.00315 Bor M, Özdemir F (2018) Manipulating metabolic pathways for development of salt-tolerant crops. In: Kumar V, Wani S, Suprasanna P, Tran LS (eds) Salinity responses and tolerance in plants, vol 1. Springer International Publishing, Cham, pp 235–256 Borrell A, Besford RT, Altabella T, Masgrau C, Tiburcio AF (1996) Regulation of arginine decarboxylase by spermine in osmotically-stressed oat leaves. Physiol Plant 98:105–110 Bouchereau A, Guénot P, Larher F (2000) Analysis of amines in plant materials. J Chromatogr B 747:49–67 Burtin D, Michael AJ (1997) Over-expression of arginine decarboxylase in transgenic plants. Biochem J 325:331–337 Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 311

Capell T, Escobar C, Liu H, Burtin D, Lepri O, Christou P (1998) Overexpression of the oat arginine decarboxylase cDNA in transgenic rice (Oryza sativa L.) affects normal development patterns in vitro and results in putrescine accumulation in transgenic plants. Theor Appl Genet 97:246–254 Capell T, Bassie L, Christou P (2004) Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress. Proc Natl Acad Sci U S A 101(26):9909–9914 Champa WAH, Gill MIS, Mahajan BVC, Bedi S (2015) Exogenous treatment of spermine to maintain quality and extend postharvest life of table grapes (Vitis vinifera L.) cv. Flame Seedless under low temperature storage. LWT – Food Sci Technol 60:412–419. https://doi. org/10.1016/j.lwt.2014.08.044 Channarayappa C, Biradar DP (2018) Soil basics, management, and rhizosphere engineering for sustainable agriculture. In: Channarayappa C, Biradar DP (eds) Abiotic stress: plants response to moisture and salt stresses. CRC Press, Boca Raton Chattopadhyay MK, Gupta S, Sengupta DN, Ghosh B (1997) Expression of arginine decarboxylase in seedlings of indica rice (Oryza sativa L.) cultivars as affected by salinity stress. Plant Mol Biol 34:477–483 Chattopadhyay MK, Tiwari BS, Chattopadhyay G, Bose A, Sengupta DN, Ghosh B (2002) Protective role of exogenous polyamines on salinity stressed rice (Oryza sativa) plants. Physiol Plant 116:192–199 Chaves MM, Oliveira MM (2004) Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. J Exp Bot 55:2365–2384 Chaves MM, Flexas J, Pinheiro C (2009) Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann Bot 103:551–560 Chen T, Li W, Hu X, Guo J, Liu A, Zhang B (2015) A cotton MYB transcription factor, GbMYB5, is positively involved in plant adaptive response to drought stress. Plant Cell Physiol 56:917– 929. https://doi.org/10.1093/pcp/pcv019 Chen D, Shao Q, Yin L, Younis A, Zheng B (2019) Polyamine function in plants: metabolism, regulation on development, and roles in abiotic stress responses. Front Plant Sci 9:1945. https:// doi.org/10.3389/fpls.2018.01945 Cheng L, Zou Y, Ding S, Zhang J, Yu X, Cao J, Lu G (2009) Polyamine accumulation in transgenic tomato enhances the tolerance to high temperature stress. J Integr Plant Biol 51:489–499. https://doi.org/10.1111/j.1744-7909.2009.00816.x Cheng X-Q, Zhu X-F, Tian W-G, Cheng W-H, Hakim SJ, Jin S-X, Zhu H-G (2017) Genome- wide identification and expression analysis of polyamine oxidase genes in upland cotton (Gossypium hirsutum L.). Plant Cell Tissue Organ Cult 129:237–249. https://doi.org/10.1007/ s11240-017-1172-0 Damour G, Simonneau T, Cochard H, Urban L (2010) An overview of models of stomatal conductance at the leaf level. Plant Cell Environ 33:1419–1438. https://doi. org/10.1111/j.1365-3040.2010.02181.x de Oliveira LF, Elbl P, Navarro BV, Macedo AF, dos Santos ALW, Floh EIS (2017) Elucidation of the polyamine biosynthesis pathway during Brazilian pine (Araucaria angustifolia) seed development. Tree Physiol 37(1):116–130 de Oliveira LF, Navarro BV, Cerruti GV, Elbl P, Minocha R, Minocha SC, dos Santos ALWS, Floh EIS (2018) Polyamine-and amino acid-related metabolism: the roles of arginine and ornithine are associated with the embryogenic potential. Plant Cell Physiol 59(5):1084–1098 DeScenso RA, Minocha SC (1993) Modulation of cellular polyamines in tobacco by transfer and expression of a mouse ornithine decarboxylase cDNA. Plant Mol Biol 22:113–127 Diao Q-N, Song Y-J, Shi D-M, Qi H-Y (2016) Nitric oxide induced by polyamines involves antioxidant systems against chilling stress in tomato (Lycopersicon esculentum Mill.) seedling. J Zhejiang Univ Sci B 17:916–930. https://doi.org/10.1631/jzus.B1600102 Do PT, Drechsel O, Heyer AG, Hincha DK, Zuther E (2014) Changes in free polyamine levels, expression of polyamine biosynthesis genes, and performance of rice cultivars under salt 312 S. de Sousa Araújo et al.

stress: a comparison with responses to drought. Front Plant Sci 5:182. https://doi.org/10.3389/ fpls.2014.00182 Duan J, Li J, Guo S, Kang Y (2008) Exogenous spermidine affects polyamine metabolism in salinity-stressed Cucumis sativus roots and enhances short-term salinity tolerance. J Plant Physiol 165:1620–1635. https://doi.org/10.1016/J.JPLPH.2007.11.006 Duque AS, de Almeida AM, da Silva AB, da Silva JM, Farinha AP, Santos D, Fevereiro P, Araújo SS (2013) Abiotic stress responses in plants: unraveling the complexity of genes and networks to survive. In: Vahdati K, Leslie C (eds) Abiotic stress – plant responses and applications in agriculture. InTech, Rijeka, pp 49–101 Duque AS, López-Gómez M, Kráčmarová J, Gomes CN, Araújo SS, Lluch C, Fevereiro P (2016) Genetic engineering of polyamine metabolism changes Medicago truncatula responses to water deficit. Plant Cell Tissue Organ Cult 127:681–690.https://doi.org/10.1007/s11240-016-1107-1 Espasandin FD, Maiale SJ, Calzadilla P, Ruiz OA, Sansberro PA (2014) Transcriptional regulation of 9-cis-epoxycarotenoid dioxygenase (NCED) gene by putrescine accumulation positively modulates ABA synthesis and drought tolerance in Lotus tenuis plants. Plant Physiol Biochem 76:29–35. https://doi.org/10.1016/J.PLAPHY.2013.12.018 Espasandin FD, Calzadilla PI, Maiale SJ, Ruiz OA, Sansberro PA (2018) Overexpression of the arginine decarboxylase gene improves tolerance to salt stress in Lotus tenuis plants. J Plant Growth Regul 37:156–165. https://doi.org/10.1007/s00344-017-9713-7 Falahi H, Sharifi M, Chashmi NA, Maivan HZ (2018) Water stress alleviation by polyamines

and phenolic compounds in Scrophularia striata is mediated by NO and H2O2. Plant Physiol Biochem 130:139–147. https://doi.org/10.1016/j.plaphy.2018.07.004 Farooq M, Wahid A, Lee DJ (2009) Exogenously applied polyamines increase drought tolerance of rice by improving leaf water status, photosynthesis and membrane properties. Acta Physiol Plant 31:937–945. https://doi.org/10.1007/s11738-009-0307-2 Filippou P, Antoniou C, Fotopoulos V (2013) The nitric oxide donor sodium nitroprusside regulates polyamine and proline metabolism in leaves of Medicago truncatula plants. Free Radic Biol Med 56:172–183 Flemetakis E, Efrose R-C, Desbrosses G, Dimou M, Delis C, Aivalakis G, Udvardi M-K, Katinakis P (2004) Induction and spatial organization of polyamine biosynthesis during nodule development in Lotus japonicus. Mol Plant Microbe Interact 17:1283–1293 Flores HE (1991) Changes in polyamine metabolism in response to abiotic stress. In: Slocum R, Flores HE (eds) The biochemistry and physiology of polyamines in plants. CRC Press, Boca Raton, pp 214–225 Flores HE, Galston A (1982) Analysis of polyamines in higher plants by high performance liquid chromatography. Plant Physiol 69:701–706 Franceschetti M, Fornale S, Tassoni A, Zuccherelli K, Mayer MJ, Bagni N (2004) Effects of spermidine synthase over-expression on polyamine biosynthetic pathway in tobacco plants. J Plant Physiol 161:989–1001 Gill SS, Tuteja N (2010) Polyamines and abiotic stress tolerance in plants. Plant Signal Behav 5(1):26–33 Gong B, Li X, Vanden Langenberg KM, Wen D, Sun S, Wei M, Li Y, Yang F, Shi Q, Wang X (2014) Overexpression of S-adenosyl- l -methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism. Plant Biotechnol J 12:694–708. https://doi.org/10.1111/ pbi.12173 Groppa MD, Benavides MP (2008) Polyamines and abiotic stress: recent advances. Amino Acids 34:35–45. https://doi.org/10.1007/s00726-007-0501-8 Grover A, Aggarwal PK, Kapoor A, Katiyar-Agarwal S, Agarwal M, Chandramouli A (2003) Addressing abiotic stresses in agriculture through transgenic technology. Curr Sci 84:355–367 Gupta K, Sengupta A, Chakraborty M, Gupta B (2016) Hydrogen peroxide and polyamines act as double edged swords in plant abiotic stress responses. Front Plant Sci 7:1343. https://doi. org/10.3389/fpls.2016.01343 Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 313

Hamill JD, Robins RJ, Parr AJ, Evan DM, Furze JM, Rhodes MJC (1990) Over-expression of a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation. Plant Mol Biol 15:27–38 Hanfrey C, Sommer S, Mayer MJ, Burtin D, Michael AJ (2001) Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity. Plant J 27:551–560 Hanna WW (1995) Centipedegrass- diversity and vulnerability. Crop Sci 35:332–334. https://doi. org/10.2135/cropsci1995.0011183X003500020007x Hanzawa Y, Takahashi T, Michael AJ, Burtin D, Long D, Pineiro M, Coupland G, Komeda Y (2000) ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase. EMBO J 19:4248–4256. https://doi.org/10.1093/emboj/19.16.4248 Harshavardhan VT, Govind G, Kalladan R, Sreenivasulu N, Hong C-Y (2018) Cross-protection by oxidative stress: improving tolerance to abiotic stresses including salinity. In: Kumar V, Wani S, Suprasanna P, Tran LS (eds) Salinity responses and tolerance in plants, vol 1. Springer International Publishing, Cham, pp 283–305 Hassan FAS, Ali EF, Alamer KH (2018) Exogenous application of polyamines alleviates water stress-induced oxidative stress of Rosa damascene Miller var. trigintipetala Dieck. S Afr J Bot 116:96–102 He L, Ban Y, Inoue H, Matsuda N, Liu J, Moriguchi T (2008) Enhancement of spermidine content and antioxidant capacity in transgenic pear shoots overexpressing apple spermidine synthase in response to salinity and hyperosmosis. Phytochemistry 69:2133–2141. https://doi. org/10.1016/J.PHYTOCHEM.2008.05.015 He M, He C-Q, Ding N-Z (2018) Abiotic stresses: general defenses of land plants and chances for engineering multistress tolerance. Front Plant Sci 9:1771. https://doi.org/10.3389/ fpls.2018.01771 Ikbal FE, Hernández JA, Barba-Espín G, Koussa T, Aziz A, Faize M, Diaz-Vivancos P (2014) Enhanced salt-induced antioxidative responses involve a contribution of polyamine biosynthesis in grapevine plants. J Plant Physiol 171:779–788. https://doi.org/10.1016/j.jplph.2014.02.006 Islam MA, Hirata M (2005) Centipedegrass (Eremochloa ophiuroides (Munro) Hack.): growth behavior and multipurpose usages. Grassl Sci 51:183–190. https://doi. org/10.1111/j.1744-697X.2005.00014.x Kakkar RK, Sawhney VK (2002) Polyamine research in plants – a changing perspective. Physiol Plant 116:281–292. https://doi.org/10.1034/j.1399-3054.2002.1160302.x Kasinathan V, Wingler A (2002) Effect of reduced arginine decarboxylase activity on salt tolerance and on polyamine formation during salt stress in Arabidopsis thaliana. Physiol Plant 121:101–107 Kasukabe Y, He L, Nada K, Misawa S, Ihara I, Tachibana S (2004) Overexpression of spermidine synthase enhances tolerance to multiple environmental stresses and up-regulates the expression of various stress-regulated genes in transgenic Arabidopsis thaliana. Plant Cell Physiol 45:712–722 Kasukabe Y, He L, Watakabe Y, Otani M, Shimada T, Tachibana S (2006) Improvement of environmental stress tolerance of sweet potato by introduction of genes for spermidine synthase. Plant Biotechnol 23:75–83. https://doi.org/10.5511/plantbiotechnology.23.75 Khare T, Srivastav A, Shaikh S, Kumar V (2018) Polyamines and their metabolic engineering for plant salinity stress tolerance. In: Kumar V, Wani S, Suprasanna P, Tran LS (eds) Salinity responses and tolerance in plants, vol 1. Springer International Publishing, Cham, pp 339–358 Kolotilin I, Koltai H, Bar-Or C, Chen L, Nahon S, Shlomo H, Levin I, Reuveni M (2011) Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit. Physiol Plant 142:211–223. https://doi.org/10.1111/j.1399-3054.2011.01458.x Kumar A, Taylor MA, Arif SAM, Davies HV (1996) Potato plants expressing antisense and sense S-adenosylmethionine decarboxylase (SAMDC) transgenes show altered levels of polyamines and ethylene: antisense plants display abnormal phenotypes. Plant J 9:147–158. https://doi. org/10.1046/j.1365-313X.1996.09020147.x 314 S. de Sousa Araújo et al.

Kumar A, Altabella T, Taylor MA, Tiburcio AF (1997) Recent advances in polyamine research. Trends Plant Sci 2:124–130 Kumar RR, Sharma SK, Rai GK, Singh K, Choudhury M, Gaurav D, Singh GP, Goswami S, Pathak H, Rai RD (2014) Exogenous application of putrescine at pre-anthesis enhances the thermotolerance of wheat (Triticum aestivum L.). Indian J Biochem Biophys 51(5):396–406 Kumar V, Wani SH, Suprasanna P, Tran L-SP (eds) (2018) Salinity responses and tolerance in plants. Volume 1, Targeting sensory, transport and signaling mechanisms. Springer International Publishing, Cham. https://doi.org/10.1007/978-3-319-75671-4 Kumria R, Rajam MV (2002) Ornithine decarboxylase transgene in tobacco affects polyamine metabolism, in vitro morphogenesis and response to salt stress. J Plant Physiol 159:983–990 Lamaoui M, Jemo M, Datla R, Bekkaoui F (2018) Heat and drought stresses in crops and approaches for their mitigation. Front Chem 6:1–14. https://doi.org/10.3389/fchem.2018.00026 Lesins K, Lesins I (1979) Genus Medicago (Leguminosae): a taxogenetic study. Junk Publishers, The Hague. https://doi.org/10.1007/978-94-009-9634-2 Li K, Xing C, Yao Z, Huang X (2017) PbrMYB21, a novel MYB protein of Pyrus betulaefolia, functions in drought tolerance and modulates polyamine levels by regulating arginine decarboxylase gene. Plant Biotechnol J 15(9):1186–1203. https://doi.org/10.1111/pbi.12708 Liu J-H, Kitashiba H, Wang J, Ban Y, Moriguchi T (2007) Polyamines and their ability to provide environmental stress tolerance to plants. Plant Biotechnol 24:117–126 Liu YH, Offler CE, Ruan YL (2013) Regulation of fruit and seed response to heat and drought by sugars as nutrients and signals. Front Plant Sci 4:282. https://doi.org/10.3389/fpls.2013.00282 Liu J-H, Wang W, Wu H, Gong X, Moriguchi T (2015a) Polyamines function in stress tolerance: from synthesis to regulation. Front Plant Sci 6:827. https://doi.org/10.3389/fpls.2015.00827 Liu M, Chu M, Ding Y, Wang S, Liu Z, Tang S, Ding C, Li G (2015b) Exogenous spermidine alleviates oxidative damage and reduce yield loss in rice submerged at tillering stage. Front Plant Sci 6:919. https://doi.org/10.3389/fpls.2015.00919 Lu S, Zhuo C, Wang X, Guo Z (2014) Nitrate reductase (NR)-dependent NO production mediates

ABA- and H2O2-induced antioxidant enzymes. Plant Physiol Biochem 74:9–15. https://doi. org/10.1016/J.PLAPHY.2013.10.030 Luo J, Liu M, Zhang C, Peipei Z, Jingjing C, Zhenfei G, Shaoyun L (2017) Transgenic centipedegrass (Eremochloa ophiuroides [Munro] Hack.) overexpressing S-adenosylmethionine decar-

boxylase (SAMDC) gene for improved cold tolerance through involvement of H2O2 and NO signaling. Front Plant Sci 8:1655. https://doi.org/10.3389/fpls.2017.01655 Lutts S, Hausman JF, Quinet M, Lefèvre I (2013) Polyamines and their roles in the alleviation of ion toxicities in plants. In: Ahmad P, Azooz MM, Prasad MNV (eds) Ecophysiology and responses of plants under salt stress. Springer, New York, pp 315–353 Lyons JM (1973) Chilling injury in plants. Annu Rev Plant Physiol 24:445–466. https://doi. org/10.1146/annurev.pp.24.060173.002305 Majumdar R, Barchi B, Turlapati AS, Gagne M, Minocha R, Long S, Minocha SC (2016) Glutamate, ornithine, arginine, proline, and polyamine metabolic interactions: the pathway is regulated at the post-transcriptional level. Front Plant Sci 7:78 Martin-Tanguy J (2001) Metabolism and function of polyamines in plants: recent development (new approaches). Plant Growth Regul 34:135–148 Masgrau C, Altabella T, Farras R, Flores D, Thompson AJ, Besford RT, Tiburcio AF (1997) Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants. Plant J 11:465–473. https://doi.org/10.1046/j.1365-313X.1997.11030465.x Mattoo AK, Sobolev AP, Neelam A, Goyal RK, Handa AK, Segre AL (2006) Nuclear magnetic resonance spectroscopy based metabolite profiles of transgenic tomato fruit engineered to accumulate polyamines spermidine and spermine reveal enhanced anabolic nitrogen-carbon interactions. Plant Physiol 142(4):1759–1770 Mattoo AK, Fatima T, Upadhyay RK, Handa AK (2014) Polyamines in plants: biosynthesis from arginine, and metabolic, physiological, and stress-response roles. In: D’Mello JPF (ed) Polyamine biosynthesis in plants. CAB International, Wallingford, pp 177–194 Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 315

Mehta RA, Cassol T, Li N, Ali N, Handa AK, Mattoo AK (2002) Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality and vine life. Nat Biotechnol 20(6):613–618 Michaeli S, Fromm H (2015) Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined? Front Plant Sci 6:419 Minocha R, Majumdar R, Minocha SC (2014) Polyamines and abiotic stress in plants: a complex relationship. Front Plant Sci 5:175 Mohammadi H, Ghorbanpour M, Brestic M (2018) Exogenous putrescine changes redox regulations and essential oil constituents in field-grownThymus vulgaris L. under well-watered and drought stress conditions. Ind Crop Prod 122:119–132 Mohapatra S, Minocha R, Long S, Subhash C, Minocha SC (2009) Putrescine overproduction negatively impacts the oxidative state of poplar cells in culture. Plant Physiol Biochem 47:262–271 Moschou PN, Paschalidis AKA, Roubelakis-Angelakis KA (2008) Plant polyamine catabolism: the state of the art. Plant Signal Behav 3(12):1061–1066 Nayyar H, Kaur S, Singh K, Kumar S, Singh KJ, Dhir KK (2005) Involvement of polyamines in the contrasting sensitivity of chickpea (Cicer arietinum L.) and soybean (Glycine max (L.) Merrill.) to water deficit stress. Bot Bull Acad Sci 46:333–338 Ndayiragije A, Lutts S (2006) Exogenous putrescine reduces sodium and chloride accumulation in NaCl-treated calli of the salt-sensitive rice cultivar I Kong Pão. Plant Growth Regul 48:51–63 Negrão S, Schmöckel SM, Tester M (2017) Evaluating physiological responses of plants to salinity stress. Ann Bot 119:1–11. https://doi.org/10.1093/aob/mcw191 Nunes C, Araújo SS, da Silva JM, Fevereiro MPS, da Silva AB (2008) Physiological responses of the legume model Medicago truncatula cv. Jemalong to water deficit. Environ Exp Bot 63:289–296. https://doi.org/10.1016/j.envexpbot.2007.11.004 Page AF, Minocha R, Minocha SC (2012) Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells. Amino Acids 42(1):295–308 Pál M, Szalai G, Janda T (2015) Speculation: polyamines are important in abiotic stress signaling. Plant Sci 237:16–23. https://doi.org/10.1016/J.PLANTSCI.2015.05.003 Pandey R, Gupta A, Chowdhary A, Pal RK, Rajam MV (2015) Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato. Plant Mol Biol 87:249–260. https://doi.org/10.1007/s11103-014-0273-y Pang C, Wang C, Chen H, Guo Z, Li C (2009) Transcript profiling of cold responsive genes in Medicago falcata. In: Yamada T, Spangenberg G (eds) Molecular breeding of forage and turf. Springer New York, New York, pp 141–150 Patel J, Ariyaratne M, Ahmed S, Ge L, Phuntumart V, Kalinoski A, Morris PF (2017) Dual functioning of plant arginases provides a third route for putrescine synthesis. Plant Sci 262:62–73 Pathak MR, Teixeira da Silva JA, Wani SH (2014) Polyamines in response to abiotic stress tolerance through transgenic approaches. GM Crops Food 5:87–96. https://doi.org/10.4161/ gmcr.28774 Peremarti A, Bassie L, Christou P, Capell T (2009) Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing Datura stramonium S-adenosylmethionine decarboxylase. Plant Mol Biol 70:253–264. https://doi.org/10.1007/ s11103-009-9470-5 Podlešáková K, Ugena L, Spíchal L, Doležal K, De Diego N (2018) Phytohormones and polyamines regulate plant stress responses by altering GABA pathway. New Biotechnol 25:53–65 Prabhavathi VR, Rajam MV (2007) Polyamine accumulation in transgenic eggplant enhances tolerance to multiple abiotic stresses and fungal resistance. Plant Biotechnol 24:273–282. https:// doi.org/10.5511/plantbiotechnology.24.273 Prabhavathi V, Yadav JS, Kumar PA, Rajam MV (2002) Abiotic stress tolerance in transgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitol phosphodehydrogenase gene. Mol Breed 9:137–147. https://doi.org/10.1023/A:1026765026493 316 S. de Sousa Araújo et al.

Purushothaman R, Krishnamurthy L, Upadhyaya HD, Vadez V, Varshney RK (2017) Genotypic variation in soil water use and root distribution and their implications for drought tolerance in chickpea. Funct Plant Biol 44:235–252. https://doi.org/10.1071/FP16154 Radhakrishnan R, Lee I (2013) Spermine promotes acclimation to osmotic stress by modifying antioxidant, abscisic acid, and jasmonic acid signals in soybean. J Plant Growth Regul 32:22–30 Rady MM, El-Yazal MAS, Taie HAA, Ahmed SMA (2016) Response of wheat growth and productivity to exogenous polyamines under lead stress. J Crop Sci Biotechnol 19:363–371. https://doi.org/10.1007/s12892-016-0041-4 Romero FM, Maiale SJ, Rossi FR, Marina M, Ruíz OA, Gárriz A (2018) Polyamine metabolism responses to biotic and abiotic stress. In: Alcázar R, Tiburcio A (eds) Polyamines. Methods in molecular biology, vol 1694. Humana Press, New York, pp 37–49 Rosenzweig C, Iglesias A, Yang XB, Epstein PR, Chivian E (2001) Climate change and extreme weather events; implications for food production, plant diseases, and pests. Glob Change Hum Health 2:90–104. https://doi.org/10.1023/A:1015086831467 Roy M, Wu R (2001) Arginine decarboxylase transgene expression and analysis of environmental stress tolerance in transgenic rice. Plant Sci 160:869–875. https://doi.org/10.1016/ S0168-9452(01)00337-5 Roy M, Wu R (2002) Overexpression of S-adenosylmethionine decarboxylase gene in rice increases polyamine level and enhances sodium chloride-stress tolerance. Plant Sci 163:987– 992. https://doi.org/10.1016/S0168-9452(02)00272-8 Ruelland E, Vaultier M-N, Zachowski A, Hurry V (2009) Chapter 2: Cold signalling and cold acclimation in plants. Adv Bot Res 49:35–150. https://doi.org/10.1016/S0065-2296(08)00602-2 Saha J, Brauer EK, Sengupta A, Popescu SC, Gupta K, Gupta B (2015) Polyamines as redox homeostasis regulators during salt stress in plants. Front Environ Sci 3:21. https://doi. org/10.3389/fenvs.2015.00021 Santa-Catarina C, Silveira V, Scherer GF, Floh EIS (2007) Polyamine and nitric oxide levels relate with morphogenetic evolution in somatic embryogenesis of Ocotea catharinensis. Plant Cell Tissue Organ Cult 90(1):93–101 Sato S, Peet MM, Thomas JF (2000) Physiological factors limit fruit set of tomato (Lycopersicon esculentum Mill.) under chronic, mild heat stress. Plant Cell Environ 23:719–726. https://doi. org/10.1046/j.1365-3040.2000.00589.x Sato S, Peet MM, Gardner RG (2001) Formation of parthenocarpic fruit, undeveloped flowers and aborted flowers in tomato under moderately elevated temperatures. Sci Hortic (Amsterdam) 90:243–254. https://doi.org/10.1016/S0304-4238(00)00262-4 Seifi HS, Shelp BJ (2019) Spermine differentially refines plant defense responses against biotic and abiotic stresses. Front Plant Sci 10:117. https://doi.org/10.3389/fpls.2019.00117 Serafini-Fracassini D, Del Duca S (2008) Transglutaminases: widespread crosslinking enzymes in plants. Ann Bot 102:145–152 Silveira V, Santa-Catarina C, Tun NN, Scherer GFE, Handro W, Guerra MP, Floh EIS (2006) Polyamine effects on the endogenous polyamine contents, nitric oxide release, growth and differentiation of embryogenic suspension cultures of Araucaria angustifolia (Bert.) O. Ktze. Plant Sci 171(1):91–98 Sun P, Zhu X, Huang X, Liu J-H (2014) Overexpression of a stress-responsive MYB transcription factor of Poncirus trifoliata confers enhanced dehydration tolerance and increases polyamine biosynthesis. Plant Physiol Biochem 78:71–79. https://doi.org/10.1016/j.plaphy.2014.02.022 Tajti J, Janda T, Majláth I, Szalai G, Pál M (2018) Comparative study on the effects of putrescine and spermidine pre-treatment on cadmium stress in wheat. Ecotoxicol Environ Saf 148:546–554 Talaat NB, Shawky BT, Ibrahim AS (2015) Alleviation of drought-induced oxidative stress in maize (Zea mays L.) plants by dual application of 24-epibrassinolide and spermine. Environ Exp Bot 113:47–58 Tanou G, Ziogas V, Belghazi M, Christou A, Filippou P, Job D, Fotopoulos V, Molassiotis A (2014) Polyamines reprogram oxidative and nitrosative status and the proteome of citrus plants exposed to salinity stress. Plant Cell Environ 37(4):864–885 Engineering Polyamine Metabolic Pathways for Abiotic Stress Tolerance in Plants 317

Tardieu F, Simonneau T, Muller B (2018) The physiological basis of drought tolerance in crop plants: a scenario-dependent probabilistic approach. Annu Rev Plant Biol 69:733–759. https:// doi.org/10.1146/annurev-arplant-042817-040218 Tiburcio AF, Alcázar R (2018) Potential applications of polyamines in agriculture and plant biotechnology. In: Alcázar R, Tiburcio A (eds) Polyamines: methods in molecular biology, volume 1694. Humana Press, New York Tiburcio AF, Altabella T, Borrell A, Masgrau C (1997) Polyamine metabolism and its regulation. Physiol Plant 100(3):664–674 Tun NN, Santa-Catarina C, Begum T, Silveira V, Handro W, Floh EI, Scherer GF (2006) Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings. Plant Cell Physiol 47(3):346–354 Urano K, Yoshiba Y, Nanjo T, Igarashi Y, Seki M, Sekiguchi K, Yamaguchi-Shinozaki K, Shinozaki K (2003) Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages. Plant Cell Environ 26:1917–1926 Urano K, Yoshiba Y, Nanjo T, Ito T, Yamaguchi-Shinozaki K, Shinozaki K (2004) Arabidopsis stress-inducible gene for arginine decarboxylase AtADC2 is required for accumulation of putrescine in salt tolerance. Biochem Biophys Res Commun 313:369–375 Waie B, Rajam MV (2003) Effect of increased polyamine biosynthesis on stress responses in transgenic tobacco by introduction of human S-adenosylmethionine gene. Plant Sci 164:727–734 Wang W, Liu J-H (2016) CsPAO4 of Citrus sinensis functions in polyamine terminal catabolism and inhibits plant growth under salt stress. Sci Rep 6:31384. https://doi.org/10.1038/srep31384 Wen X-P, Pang X-M, Matsuda N, Kita M, Inoue H, Hao Y-J, Honda C, Moriguchi T (2008) Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers. Transgenic Res 17:251–263. https://doi.org/10.1007/ s11248-007-9098-7 Wen X-P, Ban Y, Inoue H, Matsuda N, Moriguchi T (2009) Aluminum tolerance in a spermidine synthase-overexpressing transgenic European pear is correlated with the enhanced level of spermidine via alleviating oxidative status. Environ Exp Bot 66:471–478. https://doi. org/10.1016/J.ENVEXPBOT.2009.03.014 Wen X-P, Ban Y, Inoue H, Matsuda N, Moriguchi T (2010) Spermidine levels are implicated in heavy metal tolerance in a spermidine synthase overexpressing transgenic European pear by exerting antioxidant activities. Transgenic Res 19:91–103. https://doi.org/10.1007/ s11248-009-9296-6 Wen X-P, Ban Y, Inoue H, Matsuda N, Kita M, Moriguchi T (2011) Antisense inhibition of a spermidine synthase gene highlights the role of polyamines for stress alleviation in pear shoots subjected to salinity and cadmium. Environ Exp Bot 72:157–166. https://doi.org/10.1016/J. ENVEXPBOT.2011.03.001 Wimalasekera R, Tebartz F, Scherer GF (2011) Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses. Plant Sci 181(5):593–603 Wu Y, Zhou H, Que Y-X, Chen R-K, Zhang M-Q (2008) Cloning and identification of promoter Prd29A and its application in sugarcane drought resistance. Sugar Tech 10:36–41. https://doi. org/10.1007/s12355-008-0006-0 Wuddineh W, Minocha R, Minocha SC (2018) Polyamines in the context of metabolic networks. In: Alcázar R, Tiburcio AF (eds) Polyamines: methods and protocols. Humana Press, New York, pp 1–23 Xu W, Cui K, Xu A, Nie L, Huang J, Peng S (2015) Drought stress condition increases root to shoot ratio via alteration of carbohydrate partitioning and enzymatic activity in rice seedlings. Acta Physiol Plant 37:9. https://doi.org/10.1007/s11738-014-1760-0 Xu J, Wolters-Arts M, Mariani C, Huber H, Rieu I (2017) Heat stress affects vegetative and reproductive performance and trait correlations in tomato (Solanum lycopersicum). Euphytica 213:156. https://doi.org/10.1007/s10681-017-1949-6 Yadav SK (2010) Cold stress tolerance mechanisms in plants. A review. Agron Sustain Dev 30:515–527. https://doi.org/10.1051/agro/2009050 318 S. de Sousa Araújo et al.

Yadav JS, Rajam MV (1997) Spatial distribution of free and conjugated polyamines in leaves of Solanum melongena L. associated with differential morphogenetic capacity: efficient somatic embryogenesis with putrescine. J Exp Bot 48(8):1537–1545 Yang W, Li Y, Yin Y, Qin Z, Zheng M, Chen J, Luo Y, Pang D, Jiang W, Li Y, Wang Z (2017) Involvement of ethylene and polyamines biosynthesis and abdominal phloem tissues characters of wheat caryopsis during grain filling under stress conditions. Sci Rep 7:46020.https://doi. org/10.1038/srep46020 Ye B, Muller HH, Zhang J, Gressel J (1998) Constitutively elevated levels of putrescine and putrescine generating enzymes correlated with oxidant stress resistance in Conyza bonariensis and wheat. Plant Physiol 115:1443–1451 Yin L, Wang S, Tanaka K, Fujihara S, Itai A, Den X, Zhang S (2016) Silicon-mediated changes in polyamines participate in silicon-induced salt tolerance in Sorghum bicolor L. Plant Cell Environ 39:245–258 Zapata PJ, Serrano M, García-Legaz MF, Pretel MT, Botella MA (2017) Short term effect of salt shock on ethylene and polyamines depends on plant salt sensitivity. Front Plant Sci 8:855. https://doi.org/10.3389/fpls.2017.00855 Zeid IM, Shedeed ZA (2006) Response of alfalfa to putrescine treatment under drought stress. Biol Plant 50(4):635–640 Zhang Y, Zhang H, Zou ZR, Liu Y, Hu XH (2015) Deciphering the protective role of spermidine against saline-alkaline stress at physiological and proteomic levels in tomato. Phytochemistry 110:13–21 Zhuo C, Liang L, Zhao Y, Guo Z, Lu S (2018) A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover, antioxidant protection, and proline accumulation. Plant Cell Environ 41:2021–2032. https://doi. org/10.1111/pce.13114 Fructan Metabolism in Plant Growth and Development and Stress Tolerance

Alejandro del Pozo, Ana María Méndez-Espinoza, and Alejandra Yáñez

1 Introduction

The basic components for biomass accumulation derive from the assimilation of carbon into carbohydrates via photosynthesis, and these are used for the synthesis of other compounds like organic acids, amino acids and lipids (Medrano and Flexas 2000; Geigenberger et al. 2005). Most plants store sucrose or starch as reserve carbohydrates, whereas about 10–15% of flowering plant species store fructans (Hendry 1993; Vijn and Smeekens 1999; Van den Ende et al. 2004). Among the families that accumulate fructans are dicots of the Asteraceae, Campanulaceae and Boraginaceae and monocots of the Liliaceae and Poaceae (Versluys et al. 2017a). Fructans are abundant in plants from temperate and arid zones (with frost and drought), but are almost absent in tropical and aquatic environments (Hendry 1993). The accumulation of fructans in plant organs has been related to adaptation to abiotic stresses such as freezing and drought (Livingston et al. 2009). In temperate cereals, such as wheat (Triticum aestivum) and barley (Hordeum vulgare), carbohydrates are stored in the stem and leaf sheaths as water-soluble carbohydrates (WSCs). These are composed predominantly by fructans, followed by sucrose and, to a lesser extent, glucose and fructose (Virgona and Barlow 1991; Chalmers et al. 2005). These stem WSCs contribute to grain growth and filling, particularly under water-deficit conditions when leaf

A. del Pozo (*) · A. M. Méndez-Espinoza Centro de Mejoramiento Genético y Fenómica Vegetal, Facultad de Ciencias Agrarias, Universidad de Talca, Talca, Chile e-mail: [email protected] A. Yáñez Facultad de Ciencias Agrarias y Forestales, Universidad Católica del Maule, Curicó, Chile

photosynthesis and the production of assimilates destined for grain filling are inhibited (Blum et al. 1994; Blum 1998; Dreccer et al. 2009). In this chapter we describe briefly the WSCs and examine fructan metabolism, function and dynamics in plants, particularly in temperate cereals. Also, we have analysed the role of fructans in stress tolerance and the expression of genes involved in fructan accumulation and remobilization in temperate cereals.

2 Water-Soluble Carbohydrates

WSCs play a central role in the metabolism of plants as carbon and energy sources in cells. Their levels are continuously adjusted as a result of the balance between supply and demand of carbon at the whole plant level. Plants accumulate WSCs in roots (de Roover et al. 2000; Jiang and Huang 2001), stems (Bogeat-Triboulot et al. 2007; Méndez et al. 2011; del Pozo et al. 2012, 2016), leaves (Kim et al. 2000; Teulat et al. 2001; Cramer et al. 2007) and flowers and fruits (Liu et al.2004 ; Mercier et al. 2009). The metabolism of sugars is enormously dynamic and varies with the stage of development of plants and in response to the environment (Rolland et al. 2006; Ramel et al. 2009; Martínez-Vilalta et al. 2016). In most plants, the primary products of the photosynthetic assimilation of carbon in the leaves are sucrose and starch (Zeeman et al. 2007), and the rate of synthesis of sucrose in the cytosol is coordinated with the rate of CO2 fixation and the synthesis of starch in the chloroplast (Lunn 2007). On the one hand, sucrose is a form of carbon storage that often accumulates in plants exposed to cold or drought (Pérez et al. 2001; Xue et al. 2008a; Méndez et al. 2011), acting as a compatible solute for protecting cell integrity (Xue et al. 2008a). It also acts as a signalling molecule, modulating the expression of genes involved in metabolism and development (Koch 2004; Osuna et al. 2007). Starch, on the other hand, is the main storage carbohydrate in higher plants, and it is deposited in granules in the chloroplasts of photosynthetic tissues, which represent a transitory storage of carbon that is mobilized during the night to maintain respiration, export of sucrose and growth in the dark (Scofield et al. 2009). Alongside sucrose and starch, there is a third reserve of carbohydrate in plants: fructans, which are the main storage carbohydrates in cereals like wheat and barley (Pollock and Cairns 1991). Fructans are linear or branched polymers of fructose derived from sucrose (Lasseur et al. 2006). They consist of a sucrose molecule with additional fructose linked in the β-(2-1) and/or β-(2-6) positions (Gadegaard et al. 2008). They can be found in grains and in vegetative organs – stems, leaves and roots – depending on the plant’s state of development and its surrounding environmental conditions including light intensity, temperature and water availability, as well as the nutritional state of the plant (Morcuende et al. 2004; Yang et al. 2004; Morcuende et al. 2005; Verspreet et al. 2013). Fructan Metabolism in Plant Growth and Development and Stress Tolerance 321

3 Fructan Metabolism and Functions

Fructans are linear or branched polymers of fructose derived from sucrose (Lasseur et al. 2006). Five structural classes of fructans have been distinguished: inulin, levan, mixed levan, inulin neoseries and levan neoseries (Halford et al. 2011). Fructans are synthesized from sucrose in the vacuole by a group of fructosyltransferases belonging to a family of 32 plant glycoside hydrolase enzymes (Van den Ende et al. 2011); they differ in length (degree of polymerization), branching, the types of junctions between adjacent fructose molecules and the position of glucose residues (Halford et al. 2011). The incorporation of a fructose molecule into one of the three primary alcohol groups of sucrose gives rise to one of the three basic trisaccharides, 1-kestose, 6-kestose or 6G-kestose (neokestose), which are the precursors of longer fructans with a higher degree of polymerization. The linkage between the C2 of one fructose and the primary alcoholic group of C1 or C6 of another gives rise to the β-(2-1) (inulin) or β-(2-6) (levan) fructan types, respectively (Chalmers et al. 2005). Inulins are predominantly linear chains of two to 60 fructan units and one glucose unit, and this class is present mainly in dicots species (van Laere and van Den Ende 2002). The levan-type consists mainly of linear fructans containing β-(2-6) linkages found mainly in bacteria and grasses (van Laere and van Den Ende 2002). Graminan-type fructans are branched polymers containing a mixture of β-(2-1) and β-(2-6) linkages, which are present in cereals like wheat and barley (Veenstra et al. 2017). Fructan biosynthesis is mediated by four fructosyltransferase (FT) enzymes (Xue et al. 2008b). First, the sucrose:sucrose 1-fructosyltransferase (1-SST) allows the transfer of fructose from one donor sucrose to another molecule of sucrose to generate 1-kestose and glucose (Vijn and Smeekens 1999; Kawakami and Yoshida 2005). Second, sucrose:fructan 6-fructosyltransferase (6-SFT) transfers one molecule of fructose from a donor sucrose to 1-kestose to form 1,6-kestotetraose; this also facilitates the elongation of fructan molecules by fructosyl transfer from sucrose to fructans with β-(2-6) linkages (Duchateau et al. 1995; Rao et al. 2011; Yue et al. 2015). Fructan:fructan 1-fructosyltransferase (1-FFT) catalyses the elongation of the fructan chain (Kawakami and Yoshida 2005; Van den Ende et al. 2005). Finally, fructan:fructan 6G-fructosyltransferase (6G-FFT) transfers the fructose from a fructan molecule to the glucosyl residue of another fructan or sucrose molecule and facilitates the subsequent elongation of the chain with β-(2-1) or β-(2-6) linked, allowing the respective formation of the inulin neoseries or levan neoseries fructan types (Chalmers et al. 2005; Hou et al. 2018). The degradation of fructans is catalysed by fructan exohydrolase (FEH) enzymes, which release terminal fructose units (Van den Ende et al. 2004). Different isoforms of FEH have been isolated, which include fructan 1-exohydrolases (1-FEH) and fructan 6-exohydrolases (6-FEH), and these hydrolyse fructans with β-(2-1) and β-(2-6) linkages, respectively (Van den Ende et al. 2004). Fructan exohydrolases are 322 A. del Pozo et al. regulated at the transcriptional level, so their transcript levels are related to variations in the fructan content (Xue et al. 2008b). The accumulation and remobilization of fructans in plants involve the concerted action of FT and FEH, which are closely and coordinately regulated alongside invertases (Joudi et al. 2012). There is evidence that the regulation of fructan synthesis in barley leaves is independent of hexokinase, and it seems to depend on sucrose sensitization (Müller et al. 2000). It has been proposed that the existence of a threshold concentration of sucrose is necessary for the induction of fructan synthesis, as evidenced by certain studies with cut leaves of Lolium temulentum (Cairns and Pollock 1988) and barley leaves incubated in low illumination (Simmen et al. 1993) and/or incubated with sucrose in the dark (Wagner 1986) to induce the biosynthesis of fructans. Abscisic acid (ABA) is important for the induction of FEHs in wheat (Ruuska et al. 2008). However, sucrose could be an inhibitor of FEH enzyme activity in chicory, so this sugar could be an inhibitor of fructan hydrolysis during mobilization (Verhaest et al. 2007). Additionally, phosphate decreases sucrose phosphate synthase activity and reduces sucrose levels, which is the substrate for fructan biosynthesis. The principal function of fructans is to reduce the gap between the availability of photosynthates and the demand for them. Along with their role as a carbohydrate reserve, fructans contribute to regrowth (Morvan-Bertrand et al. 2001) and osmotic regulation during floral opening (Le Roy et al.2007 ), they confer tolerance to cold and drought (Pilon-Smits et al. 1995; de Roover et al. 2000; Kawakami et al. 2008; Livingston et al. 2009; Salinas et al. 2016), and they contribute to the maintenance of osmotic potential through the stabilization of cell membranes (Hincha et al. 2007). Indeed, transgenic plants of tobacco and sugar beet that accumulate fructans show a high tolerance to drought (Pilon-Smits et al. 1995; Pilon-Smits et al. 1999). It has also been suggested that they could counteract oxidative stress (Peshev et al. 2013). Fructans and FEH enzymes have been observed in the apoplast (Van den Ende et al. 2005) in response to stress (Livingston and Henson 1998; Yoshida and Kawakami 2013), where they can be degraded by the FEHs, generating sucrose, fructose or other oligosaccharides that could affect membrane stabilization (Livingston and Henson 1998). The production of apoplastic fructans is one of the possible mechanisms that the plant uses to respond to cell rupture, with at least part of the polysaccharide inserted into the lipid head group region of the membrane (Livingston et al. 2009). In other words, fructans play a role in the plant immune system (Versluys et al. 2017b). In addition, inulin-type fructans increase the nutritional value of the grain in cereals because they are a component of the dietary fibre that is easily fermented by colon microbiota (Verspreet et al. 2015). Fructans accumulate in the vegetative tissues of many species including temperate forage grasses and cereals. The study conducted by Pollock and Cairns (1991) in excised leaves of the grass Lolium temulentum showed that sucrose accumulation preceded the synthesis of trisaccharides and more complex fructan molecules, suggesting that the synthesis of fructans begins when the carbohydrate supply exceeds the demand of the plant. The analysis of WSCs in stems of wheat plants showed Fructan Metabolism in Plant Growth and Development and Stress Tolerance 323

25 a 40 b WW WL ) ) 20 -1 -1 30 15 20 10 10 Glucose (mg g

5 Fructose (mg g

0 0 01020304050 -10 01020304050 140 c 250 d 120 ) ) 200 -1 100 -1 80 150

60 100 40 Sucrose (mg g Fructan (mg g 50 20 0 0 01020304050 01020304050 Days after anthesis Days after anthesis Fig. 1 Changes in concentration of glucose (a), fructose (b), sucrose (c) and fructans (d) in the stem, from anthesis to maturity in a spring wheat genotype. Plants were grown in a glasshouse under well-watered (WW) and water-limited (WL) conditions. Values are the mean ± SE of four replicates (Modified from Yañez et al. (2017)) that fructans were the predominant WSCs, followed by sucrose, fructose and glucose (Fig. 1), and they can represent up to 85% of the WSCs in the internodes (Turner et al. 2008). In temperate cereals, stem WSCs are accumulated from stem elongation to the early phase of grain filling (Ehdaie et al. 2006; Dreccer et al. 2009). Usually the lower internodes of the stem contain larger quantities of fructans (Virgona and Barlow 1991; Ruuska et al. 2006; Xue et al. 2008a; Joudi et al. 2012; Khoshro et al. 2014), which suggests that the synthesis of fructans was induced in the stems when the availability of carbon was higher than the demand for other assimilate (Ruuska et al. 2008). McIntyre et al. (2011) suggested that the assimilation of nitrogen into amino acids is an important factor that modulates WSC levels in stems of wheat.

4 Role of Fructans in Stress Tolerance in Temperate Cereals

The accumulation of WSCs in the stem is influenced by environmental factors (Blum et al. 1994; Ehdaie et al. 2006; Ruuska et al. 2006) such as drought (Xue et al. 2008a; Dreccer et al. 2009; del Pozo et al. 2016), low temperatures (Pérez et al. 2001; Del Viso et al. 2009), reduction in the size of the sink (Martínez-Carrasco et al. 1993), deficiency in nitrogen (Wang et al.2000 ; Shiomi et al. 2006), high CO2 (Pérez et al. 2005; Aranjuelo et al. 2011) and biotic stresses such as plant disease (Dreccer et al. 2009; Yue et al. 2015). In the absence of stress, fructans accumulate in stems until approximately 2 weeks post-anthesis (Dreccer et al. 2009); later they are degraded 324 A. del Pozo et al. and partially remobilized to the grain for the synthesis of starch in late stages of grain filling (Fig. 1d). However, under unfavourable environmental conditions, stem fructans can be degraded in the early stages of grain filling to effectively compensate for the decrease in photosynthates and thus help maintain grain filling (Li et al.2013 ). In grasses and cereals, grain filling depends on carbohydrates that originate from two fundamental sources: (1) newly synthesized carbohydrates that are transported directly to the grains from photosynthesis in the leaves and the spike and (2) assimilates stored and distributed to reserve organs from the vegetative tissues (Gebbing et al. 1999; Yang and Zhang 2006; Ehdaie et al. 2008). In spite of the decline in photosynthesis during water-deficit conditions, the concentration of carbohydrates in the different organs of the plant increases, which suggests a decoupling between the supply (photosynthesis) and the carbon demand (growth) that leads to an improvement in the carbon status of the plant (Chaves et al. 2009; Hummel et al. 2010; Müller et al. 2011). An increase in carbohydrate content in plants subjected to water deficit has been shown in several plant species, in different parts of the plant and for different forms of carbon (soluble and structural) (Müller et al. 2011). In wheat, water deficit increased the accumulation of stem glucose, fructose, sucrose and fructans after anthesis, compared to plants grown under well-watered conditions (Fig. 1d). In Mediterranean climate regions, crops are exposed to a progressive water deficit during flowering and grain filling stages, leading to what has been called ‘terminal drought stress’ (Monneveux et al. 2006; Dolferus et al. 2013; del Pozo et al. 2016). The water deficit during these phenological stages reduces leaf photosynthesis and the production of photosynthetic assimilates that are directly transferred to the grain (Monneveux et al. 2006). As a consequence, large changes in stem weight as well as stem WSC concentration and content occur between anthesis and maturity, particularly under water-limited conditions (Fig. 2). It has been estimated that the stem reserves contribute up to 74% and 57% of the kernel weight of barley and wheat, respectively, when the crops are under drought stress after anthesis (Gallagher et al. 1976). Under water-deficit conditions around anthesis, the stem WSC concentration and content has been shown to increase compared to well-watered plants (Ehdaie et al. 2006; del Pozo et al. 2016 for wheat; Méndez et al. 2011; McIntyre et al. 2012; del Pozo et al. 2012 for barley). A positive and significant relationship has been found between the stem WSCs remobilized from anthesis to maturity and the grain yield of 225 wheat genotypes, when plants were grown in rainfed (water stress) conditions, but not under well-watered conditions (Fig. 3). Other studies have reported positive correlations between WSCs and grain yield in both water- deficit and well-watered conditions (Foulkes et al. 2007; McIntyre et al. 2012). Also, the stem WSCs can contribute significantly to the final kernel weight (Schnyder 1993; Gebbing et al. 1999; del Pozo et al. 2016), and positive correlations between WSC content around anthesis and kernel weight at maturity in wheat genotypes support this view (Ruuska et al. 2006; Dreccer et al. 2009; Xue et al. 2008a; del Pozo et al. 2016). Therefore, high WSC concentrations are considered a potentially useful feature for grain weight improvement and productivity in environments where production may be limited by water availability (Blum 1998; Ruuska et al. 2006; Foulkes et al. 2007). Fructan Metabolism in Plant Growth and Development and Stress Tolerance 325

3.5 a 3.0 2.5 2.0 - 56.0% - 40.9% 1.5 1.0 Stem weight (m g) 0.5 0.0

300 b 250 ) -1 200

150 - 79.1% - 71.9% 100 Stem WSC (mg g 50

200 c ) -2 m 150

100 - 75.8% - 87.4%

50 Stem WSC content (mg 0 Anthesis Maturity Anthesis Maturity

SR SR CAU CAU

Fig. 2 Changes in stem weight (a), the stem water-soluble carbohydrate (WSC) concentration (b) and stem WSC content per unit land area (c), between anthesis and maturity. Data are from 225 cultivars and advanced lines of spring bread wheat with similar phenology grown in rainfed Mediterranean conditions at Cauquenes (CAU) and full irrigation at Santa Rosa (SR) in 2012. (Modified from del Pozo et al. 2016( )) 326 A. del Pozo et al.

6 a R2= 0.39 5

4 ) -2 3

GY (g m 2

1 CAU 2011 CAU 2012 0 050100 150200

14 b 12

10 )

-2 8

6 GY (g m 4 SR 2011 2 SR 2012 0 050100 150200 Apparent removilizaon of WSC (g m-2)

Fig. 3 Relationship between the apparent remobilization of the water-soluble carbohydrate (WSC) content per unit of land area (WSCC), between anthesis (a) and maturity (m). The remobilization was calculated as WSCCa – WSCCm. Data are from 225 cultivars and advanced lines of spring bread wheat with similar phenology grown in rainfed Mediterranean conditions at Cauquenes (CAU) and full irrigation at Santa Rosa (SR) in 2012. (Modified from del Pozo et al. (2016))

5 Genetic Viability and Expression of Genes Involved in Fructan Accumulation and Remobilisation in Cereals

The variation in the stem WSCs is one of the phenotypic traits that subsequently affects kernel weight and grain yield under water-deficit conditions (Li et al.2015 ). In wheat, there is wide genetic variability in the accumulation of WSCs in the stems, a characteristic that presents a high heritability (Ruuska et al. 2006; Ehdaie et al. 2006; del Pozo et al. 2016). The genotypic differences in the concentration of WSCs during flowering are attributed mainly to fructans (Ruuska et al. 2006; Xue et al. 2008b; Yue et al. 2015). Differences in the composition of fructans according to their degree of polymerization could possibly depend on the availability of sucrose Fructan Metabolism in Plant Growth and Development and Stress Tolerance 327 for the accumulation of reserves and the capacity of the particular fructans to be transformed (Ruuska et al. 2006). It has been shown that the main difference between the types of fructans that accumulate in the short and long term is the abundance of branches between the fructosyl residues, with β-(2-1) linkages predomi- nating initially and β-(2-6) linkages occurring secondarily (Carpita et al. 1991), and in the second case fructans with high degree of polarization also accumulate (Bancal et al. 1992). The stem WSCs are complex traits associated with multiple quantitative trait loci (QTLs), each one having a small individual effect (McIntyre et al. 2012), but with a relatively high heritability (Ruuska et al. 2006). The identification of QTLs related to stem WSCs in bread wheat has been carried out in biparental populations (e.g. recombinant inbred lines (RILs) or double haploid lines (DHLs)), as well as diverse panels of advanced lines and cultivars. For example, studies conducted in DHLs under two water regimes identified seven additive QTLs for the interaction between WSCs and the environment, in chromosomes 1A, 1D, 2D, 4A, 6B and 7B, with different additive effects and contribution percentages (Yang et al. 2007). Another study in DHLs of wheat grown over different environments and seasons reported five QTLs for WSCs (Snape et al.2007 ). In a RIL population, Salem et al. (2007) identified three QTLs associated with the mobilization of WSCs and the maintenance of grain size. In three mapping populations (Cranbrook/Halberd, Sunco/ Tasman and CD87/Katepwa), Rebetzke et al. (2008) identified four to eight significant QTLs in each population. Further, in a population derived from the cross between durum (Triticum turgidum ssp. durum) and emmer wheat (Triticum turgidum ssp. dicoccoides), 15 and 22 QTLs were reported under well-watered and water-deficit conditions, respectively (Peleg et al.2009 ). Finally, in a collection of 166 wheat cultivars planted in four environments, and using 18,207 SNP markers, 52 significant marker-trait associations were identified on all wheat chromosomes except for 2A, 2D, 4D, 5B, 6A and 6D (Dong et al. 2016). The fructosyltransferase enzymes necessary for the biosynthesis of fructans in higher plants are 1-SST, 1-FFT and 6-SFT (Xue et al. 2008a; Xiang et al. 2010; Hou et al. 2018). Three of these genes (1-SST, 1-FFT and 6-SFT) have been cloned and characterized in wheat (Kawakami et al. 2005; Xue et al. 2008a). The synthesis of fructans occurs in the vacuole of cell stems from sucrose synthesized in the cytosol and moved into vacuole through sucrose transporters. The process involves the synthesis of β-(2-6)-linked fructans by the consecutive action of the enzymes 1-SST and 6-SFT, and β-(2-1)-linked fructans by the activity of the enzymes 1-SST and 1-FFT (Xue et al. 2008a). The predominance of β-(2-6) linkage in the fructose polymers of the stem seems to be related to degradation of the branches by the simultaneous action of fructan exohydrolases during the biosynthesis of fructans (Van den Ende et al. 2003). An analysis of transcript levels of carbohydrate metabolic enzymes of 16 recombinant inbred lines of wheat, differing in stem WSCs, which were grown under water deficit in field conditions, revealed that the expression of the 1-SST and 6-SFT genes was positively correlated with stem WSC and fructan concentrations and negatively correlated with the expression of sucrose synthase and soluble acid invertase genes (Xue et al. 2008a). Under terminal drought stress, the transcript 328 A. del Pozo et al. levels of 1-SST and 6-SFT in stems and roots of wheat increased significantly at 7, 14 and 21 days after anthesis, but this was not the case for the 1-FFT gene (Bagherikia et al. 2019). The strong positive correlation between the expression of the 1-SST and 6-SFT genes and stem WSC concentrations suggests that both enzymes are related to genotypic variation in fructan accumulation (Xue et al. 2008a; Khoshro et al. 2014) and explained the predominance of fructans with β-(2-6) linkages in the stems (Carpita et al. 1991; Bancal et al. 1992; Bancal and Triboï 1993). Comparisons between drought tolerant and susceptible genotypes of wheat have revealed differences in the expression of genes involved in fructan metabolism in response to water deficit. For instance, the drought-tolerant genotype had higher up-regulation of the fructan 1-fructosyltransferase B (1-FFTB) and fructan 1-exohydrolase w2 (1-FEHw2) genes, whereas the susceptible genotype presented an up-regulation of the 6-SFT and fructan 1-exohydrolase w3 (1-FEHw3) genes (Yañez et al. 2017). In another study conducted in two wheat varieties, the drought- tolerant genotype presented higher expression levels of the genes involved in fructan synthesis (1-SST-A1, 1-SST-A2, 1-SST-D, 6-SFT, FFT-A and FFT-B) at 10–20 days after anthesis, whereas the expression levels of the 1-FEH-W3, 6-FEH and INV3 genes were higher at 30 days after anthesis (Hou et al. 2018). The mobilization of fructans from the stem to the grain was accompanied by an increase in the activity of 1-FEH (Van den Ende et al. 2003; Kawakami et al. 2005; Khoshro et al. 2014) and the transcripts levels of the enzyme (Zhang et al. 2009). Therefore, the 1-FEH-w3 and 6-FEH genes seem to play an important role in the metabolism of fructans and could be used to improve the mobilization of fructans and increase kernel weight. Abscisic acid is also important for the induction of FEH in wheat (Ruuska et al. 2008). In addition, other genes have been reported to be involved in the metabolism of fructans: the SPS gene (encoding the enzyme that transforms sucrose 6-P into D-fructose 6-P and vice versa), SPP (encoding the enzyme that transforms sucrose 6-P into sucrose), INV (which transforms sucrose into fructose), SUT1 and SUT2 (transporter genes that move sucrose from the cytosol to the apoplast and vice versa), all of which are related to a high fructan content and remobilization under terminal drought stress (Bagherikia et al. 2019).

6 Conclusion and Future Perspectives

Fructans are an important carbohydrate reserves in plants, particularly in temperate cereals. Remobilization of fructans from the stem to the grain during grain filling contributes to the kernel weight and grain yield of barley and wheat. Under terminal drought conditions, the contribution of stem reserves to grain growth is of paramount importance. A large genetic variability in traits related to stem reserves has been reported in wheat, and QTLs have been associated with these traits. Genotypes with greater remobilization of fructans under drought stress are more productive. Genes related to fructan metabolism and other candidate genes could be used in breeding programmes for selecting drought stress-tolerant cultivars. Fructan Metabolism in Plant Growth and Development and Stress Tolerance 329

References

Aranjuelo I, Cabrera-Bosquet L, Morcuende R, Avice JC, Nogués S, Araus JL, Martínez-Carrasco R, Pérez P (2011) Does ear C sink strength contribute to overcoming photosynthetic acclima-

tion of wheat plants exposed to elevated CO2? J Exp Bot 62:3957–3969 Bagherikia S, Pahlevani M, Yamchi A, Zaynalinezhad K, Mostafaie A (2019) Transcript profiling of genes encoding fructan and sucrose metabolism in wheat under terminal drought stress. J Plant Growth Regul 38(1):148–163. https://doi.org/10.1007/s00344-018-9822-y Bancal P, Carpita NC, Gaudillere JP (1992) Differences in fructan accumulated in induced and field-grown wheat plants: an elongation-trimming pathway for their synthesis. New Phytol 120:313–321 Bancal P, Triboï E (1993) Temperature effect on fructan oligomer contents and fructan-related enzyme activities in stems of wheat (Triticum aestivum L.) during grain filling. New Phytol 123:247–253 Blum A (1998) Improving wheat grain filling under stress by stem reserve mobilisation. Euphytica 100:77–83 Blum A, Sinmena B, Mayer J, Golan G, Shpiler L (1994) Stem reserve mobilisation supports wheat-grain filling under heat stress. Funct Plant Biol 21:771–781 Bogeat-Triboulot M-B, Brosché M, Renaut J, Jouve L, Le Thiec D, Fayyaz P, Vinocur B, Witters E, Laukens K, Teichmann T (2007) Gradual soil water depletion results in reversible changes of gene expression, protein profiles, ecophysiology, and growth performance in Populus euphratica, a poplar growing in arid regions. Plant Physiol 143:876–892 Cairns AJ, Pollock CJ (1988) Fructan biosynthesis in excised leaves of Lolium temulentum L. II. Changes in fructosyl transferase activity following excision and application of inhibitors of gene expression. New Phytol 109:407–413 Carpita NC, Housley TL, Hendrix JE (1991) New features of plant-fructan structure revealed by methylation analysis and carbon-13 nmr spectroscopy. Carbohydr Res 217:127–136 Cramer G, Ergül A, Grimplet J, Tillett R, Tattersall ER, Bohlman M, Vincent D, Sonderegger J, Evans J, Osborne C, Quilici D, Schlauch K, Schooley D, Cushman J (2007) Water and salinity stress in grapevines: early and late changes in transcript and metabolite profiles. Funct Integr Genomics 7:111–134 Chalmers J, Lidgett A, Cummings N, Cao Y, Forster J, Spangenberg G (2005) Molecular genetics of fructan metabolism in perennial ryegrass. Plant Biotechnol J 3:459–474 Chaves MM, Flexas J, Pinheiro C (2009) Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann Bot 103:551–560 de Roover J, Vandenbranden K, van Laere A, van den Ende W (2000) Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.). Planta 210:808–814 del Pozo A, Castillo D, Inostroza L, Matus I, Méndez AM, Morcuende R (2012) Physiological and yield responses of recombinant chromosome substitution lines of barley to terminal drought in a Mediterranean-type environment. Ann Appl Biol 160:157–167 del Pozo A, Yáñez A, Matus I, Tapia G, Castillo D, Araus JL, Sanchez-Jardón L (2016) Physiological traits associated with wheat yield potential and performance under water-stress in a Mediterranean environment. Front Plant Sci 7:987. https://doi.org/10.3389/fpls.2016.00987 Del Viso F, Puebla A, Hopp H, Heinz R (2009) Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in the cold tolerant Patagonian species Bromus pictus. Planta 231:13–25 Dreccer MF, van Herwaarden AF, Chapman SC (2009) Grain number and grain weight in wheat lines contrasting for stem water soluble carbohydrate concentration. Field Crops Res 112:43–54 Dolferus R, Powell N, Ji X, Ravash R, Edlington J, Oliver S, van Dongen J, Shiran B (2013) Chapter 8: The physiology of reproductive-stage abiotic stress tolerance in cereals. In: Rout G, Das A (Eds) Molecular stress physiology of plants. Springer India, pp 193–218 330 A. del Pozo et al.

Dong Y, Liu J, Zhang Y, Geng H, Rasheed A, Xiao Y, Cao S, Fu L, Yan J, Wen W, Zhang Y, Jing R, Xia X, He Z (2016) Genome-wide association of stem water soluble carbohydrates in bread wheat. PLoS One 11:e0164293. https://doi.org/10.1371/journal.pone.0164293 Duchateau N, Bortlik K, Simmen U, Wiemken A, Bancal P (1995) Sucrose:Fructan 6-Fructosyltransferase, a key enzyme for diverting carbon from sucrose to fructan in barley leaves. Plant Physiol 107:1249–1255 Ehdaie B, Alloush GA, Madore MA, Waines JG (2006) Genotypic variation for stem reserves and mobilization in wheat: II. Postanthesis changes in internode water-soluble carbohydrates. Crop Sci 46:2093–2103 Ehdaie B, Alloush GA, Waines JG (2008) Genotypic variation in linear rate of grain growth and contribution of stem reserves to grain yield in wheat. Field Crops Res 106:34–43 Foulkes MJ, Sylvester-Bradley R, Weightman R, Snape JW (2007) Identifying physiological traits associated with improved drought resistance in winter wheat. Field Crops Res 103:11–24 Gadegaard G, Didion T, Folling M, Storgaard M, Andersen CH, Nielsen KK (2008) Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT. J Plant Physiol 165:1214–1225 Gallagher JN, Biscoe PV, Hunter B (1976) Effects of drought on grain growth. Nature 264:541–542 Gebbing T, Schnyder H, Kühbauch W (1999) The utilization of pre-anthesis reserves in grain filling

of wheat. Assessment by steady-state 13CO2/12CO2 labelling. Plant Cell Environ 22:851–858 Geigenberger P, Kolbe A, Tiessen A (2005) Redox regulation of carbon storage and partitioning in response to light and sugars. J Exp Bot 56:1469–1479 Halford NG, Curtis TY, Muttucumaru N, Postles J, Mottram DS (2011) Sugars in crop plants. Ann Appl Biol 158:1–25 Hendry GAF (1993) Evolutionary origins and natural functions of fructans -a climatological, bio- geographic and mechanistic appraisal. New Phytol 123:3–14 Hincha DK, Livingston DP III, Premakumar R, Zuther E, Obel N, Cacela C, Heyer AG (2007) Fructans from oat and rye: Composition and effects on membrane stability during drying. Biochim Biophys Acta Biomembr 1768:1611–1619 Hou J, Huang X, Sun W, Du C, Wang C, Xie Y, Ma Y, Ma D (2018) Accumulation of water-soluble carbohydrates and gene expression in wheat stems correlates with drought resistance. J Plant Physiol 231:182–191. https://doi.org/10.1016/j.jplph.2018.09.017 Hummel I, Pantin F, Sulpice R, Piques M, Rolland G, Dauzat M, Christophe A, Pervent M, Bouteillé M, Stitt M, Gibon Y, Muller B (2010) Arabidopsis plants acclimate to water deficit at low cost through changes of carbon usage: an integrated perspective using growth, metabolite, enzyme, and gene expression analysis. Plant Physiol 154:357–372 Jiang Y, Huang B (2001) Osmotic adjustment and root growth associated with drought preconditioning-enhanced heat tolerance in kentucky bluegrass. Crop Sci 41:1168–1173 Joudi M, Ahmadi A, Mohamadi V, Abbasi A, Vergauwen R, Mohammadi H, Van den Ende W (2012) Comparison of fructan dynamics in two wheat cultivars with different capacities of accumulation and remobilization under drought stress. Physiol Plant 144:1–12 Kawakami A, Sato Y, Yoshida M (2008) Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance. J Exp Bot 59:793–802 Kawakami A, Yoshida M (2005) Fructan:fructan 1-fructosyltransferase, a key enzyme for biosynthesis of graminan oligomers in hardened wheat. Planta 223:90–104 Kawakami A, Yoshida M, Van den Ende W (2005) Molecular cloning and functional analysis of a novel 6&1-FEH from wheat (Triticum aestivum L.) preferentially degrading small graminans like bifurcose. Gene 358:93–101 Khoshro H, Taleei A, Bihamta M, Shahbazi M, Abbasi A, Ramezanpour S (2014) Expression analysis of the genes involved in accumulation and remobilization of assimilates in wheat stem under terminal drought stress. Plant Growth Regul 74:165–176 Kim J-Y, Mahé A, Brangeon J, Prioul JL (2000) A maize vacuolar invertase, IVR2, is induced by water stress. Organ/tissue specificity and diurnal modulation of expression. Plant Physiol 124:71–84 Fructan Metabolism in Plant Growth and Development and Stress Tolerance 331

Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr Opin Plant Biol 7:235–246 Lasseur B, Lothier J, Djoumad A, De Coninck B, Smeekens S, Van Laere A, Morvan-Bertrand A, Van den Ende W, Prud’homme M-P (2006) Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.). J Exp Bot 57:2719–2734 Le Roy K, Lammens W, Verhaest M, De Coninck B, Rabijns A, Van Laere A, Van den Ende W (2007) Unraveling the difference between invertases and fructan exohydrolases: A single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase. Plant Physiol 145:616–625 Li H, Cai J, Jiang D, Liu F, Dai T, Cao W (2013) Carbohydrates accumulation and remobilization in wheat plants as influenced by combined waterlogging and shading stress during grain filling. J Agric Crop Sci 199:38–48 Li W, Zhang B, Li R, Chang X, Jing R (2015) Favorable Alleles for Stem Water-Soluble Carbohydrates Identified by Association Analysis Contribute to Grain Weight under Drought Stress Conditions in Wheat. PLoS One 10:e0119438 Liu F, Jensen CR, Andersen MN (2004) Drought stress effect on carbohydrate concentration in soybean leaves and pods during early reproductive development: its implication in altering pod set. Field Crops Res 86:1–13 Livingston DP, Henson CA (1998) Apoplastic sugars, fructans, fructan exohydrolase, and invertase in winter oat: Responses to second-phase cold hardening. Plant Physiol 116:403–408 Livingston DP, Hincha D, Heyer A (2009) Fructan and its relationship to abiotic stress tolerance in plants. Cell Mol Life Sci 66:2007–2023 Lunn JE (2007) Compartmentation in plant metabolism. J Exp Bot 58:35–47 Martínez-Carrasco R, Cervantes E, Pérez P, Morcuende R, Del Molino IMM (1993) Effect of sink size on photosynthesis and carbohydrate content of leaves of three spring wheat varieties. Physiol Plant 89:453–459 Martínez-Vilalta SA, Asensio D, Galiano L, Hoch G, Palacio S, Piper FI, Lloret F (2016) Dynamics of non-structural carbohydrates in terrestrial plants: a global synthesis. Ecol Monogr 86:495–516 McIntyre C, Casu R, Rattey A, Dreccer M, Kam J, van Herwaarden A, Shorter R, Xue G (2011) Linked gene networks involved in nitrogen and carbon metabolism and levels of water-soluble carbohydrate accumulation in wheat stems. Funct Integr Genomics 11:585–597 McIntyre CL, Seung D, Casu RE, Rebetzke GJ, Shorter R, Xue GP (2012) Genotypic variation in the accumulation of water soluble carbohydrates in wheat. Funct Plant Biol 39:560. https://doi. org/10.1071/fp12077 Medrano H, Flexas J (2000) Fijación del dióxido de carbono y biosíntesis de fotoasimila- dos. In: Azcón-Bieto J, Talón M (eds) Fundamentos de Fisiología Vegetal. McGraw-Hill- Interamericana, Barcelona, España, pp 173–187 Méndez AM, Castillo D, Del Pozo A, Matus I, Morcuende R (2011) Differences in stem soluble carbohydrate contents among recombinant chromosome substitution lines (RCSLs) of barley under drought in a Mediterranean-type environment. Agron Res 9:433–438 Mercier V, Bussi C, Lescourret F, Génard M (2009) Effects of different irrigation regimes applied during the final stage of rapid growth on an early maturing peach cultivar. Irrig Sci 27:297–306 Monneveux P, Rekika D, Acevedo E, Merah O (2006) Effect of drought on leaf gas exchange, carbon isotope discrimination, transpiration efficiency and productivity in field grown durum wheat genotypes. Plant Sci 170:867–872 Morcuende R, Kostadinova S, Pérez P, IMM DM, Martínez-Carrasco R (2004) Nitrate is a negative signal for fructan synthesis, and the fructosyltransferase-inducing trehalose inhibits nitrogen and carbon assimilation in excised barley leaves. New Phytol 161:749–759 Morcuende R, Kostadinova S, Pérez P, Martínez-Carrasco R (2005) Fructan synthesis is inhibited by phosphate in warm-grown, but not in cold-treated, excised barley leaves. New Phytol 168:567–574 332 A. del Pozo et al.

Morvan-Bertrand A, Boucaud J, Le Saos J, Prud’homme MP (2001) Roles of the fructans from leaf sheaths and from the elongating leaf bases in the regrowth following defoliation of Lolium perenne L. Planta 213:109–120 Müller B, Pantin F, Génard M, Turc O, Freixes S, Piques M, Gibon Y (2011) Water deficits uncou- ple growth from photosynthesis, increase C content, and modify the relationships between C and growth in sink organs. J Exp Bot 62:1715–1729 Müller J, Aeschbacher RA, Sprenger N, Boller T, Wiemken A (2000) Disaccharide-mediated regulation of sucrose:fructan-6-fructosyltransferase, a key enzyme of fructan synthesis in barley leaves. Plant Physiol 123:265–274 Osuna D, Usadel B, Morcuende R, Gibon Y, Bläsing OE, Höhne M, Günter M, Kamlage B, Trethewey R, Scheible W-R, Stitt M (2007) Temporal responses of transcripts, enzyme activities and metabolites after adding sucrose to carbon-deprived Arabidopsis seedlings. Plant J 49:463–491 Peleg Z, Fahima T, Krugman T, Abbo S, Yakir D, Korol AB, Saranga Y (2009) Genomic dissection of drought resistance in durum wheat x wild emmer wheat recombinant inbreed line population. Plant Cell Environ 32:758–779 Pérez P, Morcuende R, Martı́n del Molino I, Martı́nez-Carrasco R (2005) Diurnal changes of Rubisco in response to elevated CO2, temperature and nitrogen in wheat grown under temperature gradient tunnels. Environ Exper Bot 53:13–27 Pérez P, Morcuende R, Martín del Molino I, Sánchez de la Puente L, Martínez-Carrasco R (2001) Contrasting responses of photosynthesis and carbon metabolism to low temperatures in tall fescue and clovers. Physiol Plant 112:478–486 Peshev D, Vergauwen R, Moglia A, Hideg É, Van den Ende W (2013) Towards understanding vacuolar antioxidant mechanisms: a role for fructans? J Exp Bot 64:1025–1038 Pilon-Smits E, Ebskamp M, Paul MJ, Jeuken M, Weisbeek PJ, Smeekens S (1995) Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol 107:125–130 Pilon-Smits EAH, Hwang S, Mel Lytle C, Zhu Y, Tai JC, Bravo RC, Chen Y, Leustek T, Terry N (1999) Overexpression of ATP sulfurylase in indian mustard leads to increased selenate uptake, reduction, and tolerance. Plant Physiol 119:123–132 Pollock CJ, Cairns AJ (1991) Fructan metabolism in grasses and cereals. Annu Rev Plant Biol 42:77–101 Ramel F, Sulmon C, Gouesbet G, Couée I (2009) Natural variation reveals relationships between pre-stress carbohydrate nutritional status and subsequent responses to xenobiotic and oxidative stress in Arabidopsis thaliana. Ann Bot 104:1323–1337 Rao RSP, Andersen JR, Dionisio G, Boelt B (2011) Fructan accumulation and transcription of candidate genes during cold acclimation in three varieties of Poa pratensis. J Plant Physiol 168:344–351 Rebetzke GJ, van Herwaarden AF, Jenkins C, Weiss M, Lewis D, Ruuska S, Tabe L, Fettell NA, Richards RA (2008) Quantitative trait loci for water-soluble carbohydrates and associations with agronomic traits in wheat. Aust J Agric Res 59:891–905 Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signaling in plants: conserved and novel mechanisms. Annu Rev Plant Biol 57:675–709 Ruuska SA, Lewis DC, Kennedy G, Furbank RT, Jenkins CLD, Tabe LM (2008) Large scale transcriptome analysis of the effects of nitrogen nutrition on accumulation of stem carbohydrate reserves in reproductive stage wheat. Plant Mol Biol 66:15–32 Ruuska SA, Rebetzke GJ, van Herwaarden AF, Richards RA, Fettell NA, Tabe L, Jenkins CLD (2006) Genotypic variation in water-soluble carbohydrate accumulation in wheat. Funct Plant Biol 33:799–809 Salinas C, Handford M, Pauly M, Dupree P, Cardemil L (2016) Structural modifications of fructans in Aloe barbadensis Miller (aloe vera) grown under water stress. PLoS One 11:e0159819 Salem K, Röder M, Börner A (2007) Identification and mapping quantitative trait loci for stem reserve mobilisation in wheat (Triticum aestivum L.). Cereal Res Commun 35(3):1367–1374 Fructan Metabolism in Plant Growth and Development and Stress Tolerance 333

Scofield GN, Ruuska SA, Aoki N, Lewis DC, Tabe LM, Jenkins CLD (2009) Starch storage in the stems of wheat plants: localization and temporal changes. Ann Bot 103:859–868 Schnyder H (1993) The role of carbohydrate storage and redistribution in the source-sink relations of wheat and barley during grain filling-a review. New Phytol 123:233–245 Shiomi N, Benkeblia N, Onodera S, Yoshihira T, Kosaka S, Osaki M (2006) Fructan accumulation in wheat stems during kernel filling under varying nitrogen fertilization. Can J Plant Sci 86:1027–1035 Simmen U, Obenland D, Boller T, Wiemken A (1993) Fructan synthesis in excised barley leaves (identification of two sucrose-sucrose fructosyltransferases induced by light and their separation from constitutive invertases). Plant Physiol 101:459–468 Snape JW, Foulkes MJ, Simmonds J, Leverington M, Fish LJ, Wang Y, Ciavarrella M (2007) Dissecting gene x environmental effects on wheat yields via QTL and physiological analysis. Euphytica 154:401–408. https://doi.org/10.1007/s10681-006-9208-2 Teulat B, Borries C, This D (2001) New QTLs identified for plant water status, water-soluble carbohydrate and osmotic adjustment in a barley population grown in a growth-chamber under two water regimes. Theor Appl Genet 103:161–170 Turner LB, Cairns AJ, Armstead IP, Thomas H, Humphreys MW, Humphreys MO (2008) Does fructan have a functional role in physiological traits?. Investigation by quantitative trait locus mapping. New Phytol 179:765–775 Van den Ende W, Clerens S, Vergauwen R, Van Riet L, Van Laere A, Yoshida M, Kawakami A (2003) Fructan 1-exohydrolases. β-(2,1)-Trimmers during graminan biosynthesis in stems of wheat? purification, characterization, mass mapping, and cloning of two fructan 1-exohydrolase isoforms. Plant Physiol 131:621–631 Van den Ende W, Coopman M, Clerens S, Vergauwen R, Le Roy K, Lammens W, Van Laere A (2011) Unexpected presence of graminan- and levan-type fructans in the evergreen frost-hardy eudicot Pachysandra terminalis (Buxaceae): Purification, cloning, and functional analysis of a 6-SST/6-SFT enzyme. Plant Physiol 155:603–614 Van den Ende W, De Coninck B, Van Laere A (2004) Plant fructan exohydrolases: a role in signaling and defense? Trends Plant Sci 9:523–528 Van den Ende W, Yoshida M, Clerens S, Vergauwen R, Kawakami A (2005) Cloning, characterization and functional analysis of novel 6-kestose exohydrolases (6-KEHs) from wheat (Triticum aestivum). New Phytol 166:917–932 Van Laere A, Van Den Ende W (2002) Inulin metabolism in dicots: chicory as a model system. Plant Cell Environ 26:803–813 Lynn D. Veenstra, Jean-Luc Jannink, Mark E. Sorrells, (2017) Wheat Fructans: A Potential Breeding Target for Nutritionally Improved, Climate-Resilient Varieties. Crop Science 57(3):1624 Veenstra LD, Jannink J, Sorrells ME (2017) Wheat fructans: A potential breeding target for nutritionally improved, climate-resilient varieties. Crop Sci 57:1624–1640 Verhaest M, Lammens W, Le Roy K, De Ranter CJ, Van Laere A, Rabijns A, Van den Ende W (2007) Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor. New Phytol 174:90–100 Versluys M, Kirtel O, Toksoy Öner E, Van den Ende W (2017a) The fructan syndrome: Evolutionary aspects and common themes among plants and microbes. Plant Cell Environ 41:16–38. https:// doi.org/10.1111/pce.13070 Versluys M, Tarkowski ŁP, Van den Ende W (2017b) Fructans As DAMPs or MAMPs: Evolutionary prospects, cross-tolerance, and multistress resistance potential. Front Plant Sci 7:2061 Verspreet J, Cimini S, Vergauwen R, Dornez E, Locato V, Le Roy K, De Gara L, Van den Ende W, Delcour JA, Courtin CM (2013) Fructan metabolism in developing wheat (Triticum aestivum L.) kernels. Plant Cell Physiol 54:2047–2057 Verspreet J, Dornez E, Van den Ende W, Delcour JA, Courtin CM (2015) Cereal grain fructans: Structure, variability and potential health effects. Trends Food Sci Technol 43:32–42 Vijn I, Smeekens S (1999) Fructan: More than a reserve carbohydrate? Plant Physiol 120:351–360 Virgona J, Barlow E (1991) Drought stress induces changes in the non-structural carbohydrate composition of wheat stems. Funct Plant Biol 18:239–247 334 A. del Pozo et al.

Wagner W (1986) Regulation of fructan metabolism in leaves of barley (Hordeum vulgare L. cv Gerbel). Plant Physiol 81:444 Wang C, Van den Ende W, Tillberg J-E (2000) Fructan accumulation induced by nitrogen deficiency in barley leaves correlates with the level of sucrose:fructan 6-fructosyltransferase mRNA. Planta 211:701–707 Xiang GAO, She MY, Yin GX, Yang YU, Qiao WH, Du LP, Ye XG (2010) Cloning and characterization of genes coding for fructan biosynthesis enzymes (FBEs) in Triticeae plants. Agric Sci China 9:313–324 Xue G, McIntyre CL, Jenkins CLD, Glassop D, van Herwaarden AF, Shorter R (2008a) Molecular dissection of variation in carbohydrate metabolism related to water-soluble carbohydrate accumulation in stems of wheat. Plant Physiol 146:441–454 Xue W, Xing Y, Weng X, Zhao Y, Tang W, Wang L, Zhou H, Yu S, Xu C, Li X, Zhang Q (2008b) Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nat Genet 40:761 Yang DL, Jing RL, Chang XP, Li W (2007) Identification of quantitative trait loci and environmental interactions for accumulation and remobilization of water-soluble carbohydrates in wheat (Triticum aestivum L.) stems. Genetics 176:571–584. https://doi.org/10.1534/ genetics.106.068361 Yang J, Zhang J (2006) Grain filling of cereals under soil drying. New Phytol 169:223–236 Yang J, Zhang J, Wang Z, Zhu Q, Liu L (2004) Activities of fructan- and sucrose-metabolizing enzymes in wheat stems subjected to water stress during grain filling. Planta 220:331–343 Yañez A, Tapia G, Guerra F, del Pozo A (2017) Stem carbohydrate dynamics and expression of genes involved in fructan accumulation and remobilization during grain growth in wheat (Triticum aestivum L.) genotypes with contrasting tolerance to water stress. PLoS One 12(5):e0177667. https://doi.org/10.1371/journal.pone.0177667 Yoshida M, Kawakami A (2013) Molecular analysis of fructan metabolism associated with freezing tolerance and snow mold resistance of winter wheat. In: Imai R, Yoshida M, Matsumoto N (eds) Plant and microbe adaptations to cold in a changing world. Springer, New York, NY Yue A, Li A, Mao X, Chang X, Li R, Jing R (2015) Identification and development of a functional marker from 6-SFT-A2 associated with grain weight in wheat. Mol Breed 35:1–10 Zeeman SC, Delatte T, Messerli G, Umhang M, Stettler M, Mettler T, Streb S, Reinhold H, Kötting O (2007) Starch breakdown: recent discoveries suggest distinct pathways and novel mechanisms. Funct Plant Biol 34:465–473 Zhang J, Dell B, Conocono E, Waters I, Setter T, Appels R (2009) Water deficits in wheat: fructan exohydrolase (1-FEH) mRNA expression and relationship to soluble carbohydrate concentrations in two varieties. New Phytol 181:843–850 Index

A Agrobacterium rhizogenes, 57 ABA responding element (ABRE), 292, 300 ALDH10 isoenzymes, 127 Abiotic stresses, 1 Amino acid/auxin permease (AAAP), 48 and biotic, 288, 291, 308 Amino acid permease (AAP), 48, 202 degradation genes, 235 Amino acid–polyamine–choline (APC) drought stress, 106 family, 202 exogenous GB-mediated modulation, 148 Amino acid proline, 42 photosynthetic machinery, 145–146 4-Aminobutanal (ABAL), 291 plant hormones, 146–147 1-Aminocylopropane-1-carboxylic acid types, 141, 142 (ACC), 293 exogenous Put application, 293 Anhydrobiotic organisms, 181 extreme temperatures, 111 Anthocyanin, 259 metabolic changes, 233–235 Antioxidant defense system, 114, 115 on nutrient status, 112, 113 Antioxidant enzymes, 88, 89 osmolyte priming, 261, 262 Antioxidants PAs, ROS and NRS, 292 activity, 258, 259 without phenotypic alterations, 227, 229, 236 overproduction, 263 with phenotypic alterations, 225–227 and ROS scavenging, 257 physiological and biochemical processes, stress imprint, 262 106–109 Antioxidative defence, 143, 144 in plants, 258 Arginine decarboxylase (ADC), 289, 290, 292, plant-water relations, 111 293, 295, 303 polyamines, 293 priming compounds, 259–261 salinity stress, 111 B seed priming, 263 Bacterial P2CRs, 43 transcriptional changes, 232, 233 Bacteroides, 271, 272, 277, 280 trehalose biosynthesis, 235 Betaine aldehyde dehydrogenase (BADH), 14, Abiotic stress-tolerant traits, 206 20, 23, 24, 157, 201, 209, 210, Abscisic acid (ABA), 51, 146, 158, 193, 232, 246–248 261, 273, 276, 303, 322 GB synthesis, 124, 127 AdoHcy hydrolase, 210 Betaine homocysteine methyl transferase AdoMet synthetase, 210 (BHMT), 128 Agmatine iminohydrolase (AIH), 274 Betaines, 3 AGP-glucose pyrophosphorylase (AGPase), 183 Bioinformatic, 5, 30

Biomass accumulation, 319 Endogenous GB Biotic stresses, 1 abiotic stresses, 153 Brassinosteroids (BRs), 276, 277 ambient temperature, 153 cellular adaptive responses, 154 chloroplast targeted transgenesis, 165, 168 C CMO and BADH isoenzymes, 165 Cadaverine (Cad), 273, 274, 277–279 crop yield, 154 Cadmium (Cd), 144 global agricultural land area, 153 Calvin cycle, 270 heat and cold stress, 153 Carbamoil putrescine aminohydrolase metabolic routes, 168 (CPA), 274 osmoprotectants (see Osmoprotectants) Carbohydrates, 15–16 osmotic stress, 159, 160 Carbonyl cyanide m-chlorophenylhydrazone plant abiotic stress responses, 157, 158 (CCCP), 211 primary stress phase, 154 Carotenoid, 259 ROS and plant hormones, 154 Cereals, 319 saline soils, 154 fructans, role, 320, 322 and temperature stress, 160–162 grain filling, 324 transgene approaches, 162 Chaperones, 2, 4 transgenesis, 162–165 Choline monooxygenase (CMO), 157, 209, transgenic plants, 168 210, 246–248 Energy, 41 cis-acting regulatory elements, 128 Environmental stresses, 1, 2 CMO1 and CMO2, 127 Enzymes Cys-181, 126 FEH, 321, 322, 328 GB synthesis, 124 glycoside hydrolase, 321 gene sequences, 126 Ethylene biosynthesis, 290 SAMS transcript, 130 European Nucleotide Archive (ENA), 5 Chromatin remodelers, 2 Exogenous application, GB Compatible solutes, 141, 246, 248, 249, 252 abiotic oxidative stress tolerance, 142 CPA (N-carbamoylputrescine amidohydrolase) in cultured tobacco, 143 gene, 15 in fine rice, 142 Cyanobacteria, 41 lentil seedlings, 143 maize, 142 in marigold, 143 D in mung bean seedlings, 143 Decarboxylated S-adenosylmethionine nitrogen deficiency and Cd stress, 144 (dcSAM), 290, 301 in perennial ryegrass, 143 DEOP database, 2 on rice seedlings, 144 DHS (deoxyhypusine synthase) gene, 15 on tea buds, 143 Diamine oxidase (DAO), 273, 274, 276, 277, 291 on tomato, 143 1,4-Diaminobutane, 287 water-stressed wheat seedlings, 142 1,5-Diaminopentane, 287 gene expression, 147–148 1-3 Diaminopropane (DAP), 273 photosynthetic machinery, 145–146 DNA Data Bank of Japan (DDBJ), 5 plant hormones and metabolites, 146–147 Drought, 1, 295, 298, 305, 309 plant resistance, 148 stress, 106, 292–294, 299–302 ROS scavenging and detoxification, 148 tolerance, 231, 232, 294, 298 Exogenous proline Drought-responsive element (DRE), 292, 300 under abiotic stress (see Abiotic stress) Durum wheat, 159 antioxidant defense system, 114, 115 concentrations, 116 growth and yield quantity and quality, 115 E photosynthetic performance, 113, 114 Ectoine, 3 Extracellular trehalose, 190 Embryogenesis, 61, 62 Extreme temperatures, 305–307, 309 Index 337

F exogenous treatment, 247 FAD-dependent AOs (FAD-AOs), 291 gene expression, 210 Fructan exohydrolase (FEH), 321 genetic engineering, 215, 216 Fructans, 3, 27 inter-organ transport, 216 ABA, 322 metabolism accumulation, 319, 322 BHMT, 128 biosynthesis, 321 cellular components, 128 degradation, 321 cellular volume maintenance, 123 expression of genes, 327, 328 CMO and BADH, 124 and FEH enzymes, 322 direct and/or indirect participation, 124 genetic variability, 326 Hcy/Met cycle, 123 inulins, 321 plant growth and development, 123, levan-type, 321 129, 130, 132, 133 metabolism, 328 plant tissue, 129 mobilization, 328 quaternary amine, 123 principal function, 322 synthesis pathway, 124, 126–128 remobilization, 322 stress-related metabolites, 248 in stress tolerance, 323–326 stress-responsive genes, 249 structural classes, 321 translocation, 210, 212 in temperate cereals, 323 Grain filling, 324 vegetative tissues, 322 Graminan-type fructans, 321 WSCs, 320 Fructosyltransferase (FT), 321, 327 H Heat shock proteins, 2, 27 G Hexamines, 273 Gamma aminobutyric acid (GABA), 48, 202, High temperatures, 1 259, 274, 276, 291, 309 Histone modifiers, 2 GenBank, 5 Homospermidine (HomSpd), 273 Gene expression Homospermidine synthase fructans, roles, 324, 327, 328 (HSS), 278 GB, exogenous application, 147–148 Homospermine (HomSpm), 273

proline metabolism, 49, 50, 53, 54 Hydrogen peroxide (H2O2), 278 Genetically modified (GM) plants, 22–23, 25–28 Hydropriming, 257, 262 Genomic-scale approach, 5 Hypoxia, 188 Gibberellic acid (GA), 261 Glucose-6-phosphate (G6P), 225, 249 Glutamate (Glu), 274 I Glutamate-semialdehyde (GSA), 11, 44, 46, 201 Indole acetic acid (IAA), 147 Glutathione reductase (GR), 304 In silico genome mapping, 19 Glycine betaine (GB), 6, 9, 14, 20, 22–24, analysis, 17 154, 156–168, 201–204 cysteine, 17 abiotic and biotic stresses, 248 GB, 17 accumulation in plants, 208, 209 grasses, 18 bacteria-specific glycine methylation legumes and soybean, 17, 18 pathway, 246 myo-inositol, 17 beneficial effects, 142 plant species, mapping, 17 biosynthesis, 209, 210 proline, 17 codA-and BADH-transgenic tomato, 248 as transgenes as compatible solutes, 141 amino acid proline, 20 crop plants, 247 carbohydrates, 26, 27 description, 141 GB, 20–24 exogenous application, 141, 212, 214, 215 sugar alcohols, 28 (see also Exogenous application, GB) trehalose, 17 338 Index

Inulins, 321 Ornithine-delta-aminotransferase (OAT), 43, Ionic stress, 270 44, 46, 48, 53, 57, 201 Osmolytes chemical/molecular chaperones, 4 K potential pathways, 6 KEGG (Kyoto Encyclopedia of Genes and protective, 3 Genomes), 6 Osmoprotectant degradation, 7 Osmoprotectant-related genes analysis, in plant species, 8 L and associated pathways, 6–8 Late embryogenesis abundant (LEA), 262 drought stress, 11 Legume-rhizobia symbiosis experimental assays, 8 agrarian, 271 expression PAs, 277–279 amino acid proline, 11–14 salinity, 272, 273 carbohydrates, 15, 16 salt stress, 272, 279 GB, 14 Lipoxygenase (LOX), 249 polyamines (PA), 14–15 Low temperature-responsive (LTR), 292 sugar alcohols, 16–17 Lysine decarboxylase (LDC), 274, 278 salinity stress, 8 Lysine histidine transporter (LHT), 48, 202 in silico genome mapping, 17–20 stress application methods, 11 Osmoprotectants, 201, 202, 272, M 275, 280 Malondialdehyde (MDA), 294 abiotic stresses, 155 Maltooligosyl trehalose synthase (MTS), antioxidant defense system, 156 229–231 classes, 2 Maltooligosyl trehalose trehalohydrolase as compatible solutes, 2 (MTH), 229, 230 definition, 2 Marker-assisted selection (MAS), 30 external stress, 155 MEDLINE, 20 groups, 156 Metabolism, 247–249 metalloenzyme superoxide Metabolites, 235, 236 dismutase, 156 MetaCyc databases (Metabolic Pathway optimal K+/Na+ ratio, 157 Database), 6 organic solutes, 156 Methionine synthase, 210 osmotic stress, 4 Methylglyoxal (MG) detoxification system, 143 plant abiotic stress tolerance, 155 Mitogen-activated protein kinase (MAPK), 158 plant hormones, 155 Mung bean, 160 plant stress tolerance, 155 Myo-inositol, 16, 17, 29 plants under abiotic stress, 3, 4 salinity and water deficit, 157 transcriptomic study, 4–6 N water efflux, 155 Nitrogen fixation, 271, 272, 279 Osmoprotection Non-photosynthetic organisms, 41 cellular water potential (ΨW), 77 Norspermidine (NorSpd), 273 compatible osmolyte, 77, 79 enzymes and membranes, 79, 81 free proline concentration, 77 (see also O ROS scavenging) Oligosaccharides, 3 Osmoprotective osmolytes, 2, 5 Ornithine aminotransferase (OAT), 274 Osmotic adjustment, 242, 244, 252 Ornithine cyclodeaminase (OCD), 42, 43, Osmotic stress, 3, 4, 270 57, 58 Oxidative pentose phosphate pathway (OPPP), Ornithine decarboxylase (ODC), 278, 289, 57, 58 290, 295, 299 Oxidative stress, 308 Index 339

P Ca+2-dependent calmodulin, 244 P5C dehydrogenase (P5CDH), 43, 46, 48, 50, catabolic enzymes, 53–55 53, 55 catabolism, 100 P5C-proline cycle, 86, 87 Cu/ZnSOD and MnSOD encoding P5C reductase (P5CR), 244 genes, 246 P5C synthetase (P5CS), 44, 45, 47, 49, 52, 244 to drought stress, 11 Pentamines, 273 endogenous, 206 Phosphoenolpyruvate carboxylase (PEPC), exogenous treatment, 246 249–251 GABA synthesis, 101 Phosphoethanolamine N-methyltransferase glutamate pathway, 244 (PEAMT), 215 inter-organ transport, 204, 216 3-Phosphoglycerate dehydrogenase intracellular transport, 205 (PGDH), 215 metabolism and regulation, 104, 106 Phospholipase-D (PLD), 249 nonessential proteinogenic amino Photosynthesis, 41, 145 acid, 100 Photosynthetic machinery osmotic adjustment, 244 under abiotic stress, exogenous GB, OsP5CS2 and OsP5CR promoters, 246 145–146 P5CS and P5CR genes, 244, 245 Photosystem II (PSII), 145, 146 pathways, 100 Plant growth and development, T6P/trehalose, PDH expression, 11 181–185 physiological role, 205 Plant growth regulators (PGRs), 259 in plant at organization levels, 103, 104 Plant hormones ROS signaling pathways, 103 exogenous GB, 146, 148 to salt stress, 11 Plant osmoprotectants, 3 stress marker genes, 245 See also Osmoprotectants stress response and tolerance Plants processes, 244 under abiotic stress, 3 stress-responsive transcription factors, 244 under stress, 1 uptake in plants, 202, 204 Plasmodesmata, 49 Proline accumulation Polyamine oxidase (PAO), 274, 275 ABA production, 76 Polyamines (PAs), 3, 7, 287–295, 298–309 ABRE, 77 abiotic stress regulators, 275–277 antioxidant enzymes, 88, 89 ADC, ODC and OAT genes, 14 C and N source, 89, 90 biosynthesis, 278 CRE, 77 CPA and DHS genes, 15 de novo biosynthesis, 74 functions, 14 HY5, 76

legume-rhizobia symbiosis, 277–279 hydrogen peroxide (H2O2), 76 metabolism, 273, 274 mitochondrial oxidation, 74 in nature, 273 osmoprotection (see Osmoprotection) in plants, 273 P5CR, 75 putrescine (Put), 14 photosynthetic tissues, 74 and salinity stress, 302–305 plant response, abiotic stresses, 74 salt stress, 274, 275 preliminary evidence, 73 spermidine (Spd), 14 ProDH, 76 and transgenic plants, 24, 26 putative cis-regulatory elements, 74 (see Polyamines metabolism, 290 also Redox state) Polyols, 16 role, 73 Proline, 3, 22 ROS, 76 abiotic stress responses, 246 stress-induced, plant species, 74, 75 abscisic acid, 102 Proline biosynthetic enzymes accumulation, 244 OCD, 42, 43 biosynthesis, 100, 102 P2CRs, 43 biosynthesis enzymes, 49–53 P5C, 44 340 Index

Proline degradation enzymes ROS scavenging P5C, 46 abiotic and biotic stresses, 81 ProDH, 45 cultured tobacco BY2 cells, 82 Proline dehydrogenase (ProDH), 44–46, 53 GABA precursor, 83 Proline metabolism, 47–49 hydroxyproline, 83 duplications and functional diversification, 49 MDA formation, 81 embryo development, 61–62 mitochondrial electron leakage, 82 flowering, 58–60 non-enzymatic antioxidants, 82 plant development, 56–57 physiological conditions, 83 pollen fertility, 60–61 putative antioxidant properties, 82 regulatory mechanisms, 49 TEMP oxidation, 82 root growth, 58 RT-qPCR (real-time reverse transcription- seed germination, 57–58 polymerase chain reaction) transcription factors (TFs), 49 technique, 7 under stress, 62–63 Proline oxidase, 45 Proline-responsive element (PRE), 245 S Proline transport S-adenosylmethionine decarboxylase intercellular transport, 48–49 (SAMDC), 278, 290, 292, 301, intracellular transport, 47 306, 307 and metabolism, 47–48 S-adenosylmethionine (SAM), 276, 278, 309 Proline transporter (ProT), 48, 61, 211 Salicylic acid (SA), 146, 147, 276 Putrescine (Put), 14, 25–26 Salinity, 258, 259, 262, 272, 273, 293, 295, Pyrroline-2-carboxylate reductase (P2CR), 298, 302–304, 309 42, 43 Salinity stress, 111 Pyrroline-5-carboxylate (P5C), 43–46, 48, 49, Salt stress, 3, 9–10, 14–16 55, 201 brassinosteroids, 277 Pyrroline-5-carboxylate dehydrogenase GABA, 280 (P5CDH), 245 harmful effect, 270 Pyrroline-5-carboxylate reductase (P5CR), legume-rhizobia symbiosis, 272, 279 44–47, 52, 61, 62 in legumes, 276 Pyrroline-5-carboxylate synthetase (P5CS), 201 polyamines, 274, 275 root nodules of Medicago truncatula, 272 Salt-stressed bread wheat, 159 Q Serine hydroxymethyltransferase (SHMT), 215 Quantitative trait loci (QTLs), 327 Signaling process, 1 Quaternary ammonium compounds (QACs), 2 Soil water content (SWC), 300 Spd by spermidine synthase (SPDS), 273, 290, 292, 295, 301, 304 R Spermidine (Spd), 14, 24–26 Reactive oxygen species (ROS), 4, 154, 257, Spermidine/spermine N-1 acetyl transferase 258, 262, 270, 272, 275 (SSATs), 291 under cold stress, 24 Spermidine synthase (SPDS), 278 regulation, 4 Spermine (Spm), 24–26 T6P/trehalose role, 188 Spinacia BADH protein, 167 Red beet, 159 Spm synthase (SPMS), 273, 274, 278, 290, 307 Redox state Stress stimulus, 1 catabolism and generation under stress, 87, 88 Stress-tolerant transgenic plants, 228, 230–231 P5C-proline cycle, 86, 87 Sucrose non-fermenting-related kinase-1 proline-glutamate interconversions, 85, 86 (SnRK1), 184 Regulatory proteins, 2 Sugar alcohols, 3, 16–17 Relative water content (RWC), 294, 299, 300 Sugar beet, 210, 212 Reserve carbohydrates, 319 Sugars, 11 RNA interference (RNAi), 2, 295 Superoxide dismutase (SOD), 259, 304 Index 341

T compatible solutes, 249 Temperature stress, 258 description, 175 Terminal drought stress, 324, 327, 328 as elicitor of plant defense responses, 189–190 Thiobarbituric acid reactive substances endogenous, 208 (TBARS), 304 environmental stresses, 249 Tolerance exogenous treatments, 252 Al stress, 304 genome sequencing, 177 biotic and abiotic stress, 291 glucose monomers, 250 dehydration, 302 inter-organ transport, 216 drought, 301, 304 metabolism, 249 osmotic and ionic, 302 in mycorrhizal fungi-plant relationships, 189 and polyamines, 305–307 origin, 175 Put and Spd levels, 295 osmoprotectants, 225 salinity, 303 pathway, 225 salt and cadmium stress, 305 in plant growth and development, 181–185 Spd-induced NO, 292 R2R3-type MYB family, 251 Spm and Spd levels, 300 SnRK1-upregulated genes, 250 stress, 293, 309 stomata, 231, 232 thermotolerance, 294 as stress protectant, 181

WT and GhPAO3 transgenic line, 305 stress-related genes, 249 TPS-TPP pathway, 176 TPP proteins, 180 Transcription, 232, 233 TPS proteins, 177–180 ABA-regulated stress-related genes, 235 TRE proteins, 180–181 Transcription factors (TFs), 2 trehalase (TRE), 177 bZIP, 245, 250 trehalose-6-phosphate phosphatase, 249 CREs site, 244 uptake in plants, 206, 208 MYB102 and WRKY, 251 Trehalose synthase (TreS), 176, 227, 228, 234 MYB2, 244 pathway, 176 proline-, glycinebetaine- and trehalose- Trehalose-6-phosphate (T6P) induced, 243 in abiotic stress tolerance and stress-responsive genes, 242 drought stress, 186–187 Transcriptomic study hypoxia, 188 description, 4 oxidative stress, 188 genomic-scale approach, 5 salt stress, 185–186 global expression of genes, 5 temperature stress, 187–188 molecular targets, 5 applications, 191–193 osmoprotectants and potential related in biotic stress tolerance pathways, 5, 6 as elicitor of plant defense response, statistical analysis (p-values), 5 189–190 transcriptomic libraries, 4 pathogen attack, 190–191 Transfer DNA (T-DNA), 57 and plant-microorganism symbiosis, 189 Transgenic plants, 20, 24, 26, 27, 293, 295, description, 182 300–304, 306–308 in embryo development, 182 abiotic stress-tolerant, 234 flowering time and inflorescence drought-tolerant, 227 architecture, 184 Gaff’s hypothesis, 231 on glucose, 178 leaves of, 225 on meristem development, 184 and non-transgenic plants, 231 normal growth, 182 Trehalase (TRE), 3, 15, 16, 27, 180–181 on plant development, 182 abiotic stress (see Abiotic stress) plant growth, 183 anomers, 175 SnRK1 by T6P, 184 applications, 191–193 sucrose level, 183 Attre1-1 and Attre1-2 mutants, 251 sugar levels, 184 biosynthesis, 201, 234, 235, 252 Trehalose-6-phosphate phosphatase (TPP), 176, biosynthesis pathways, 176 180, 202, 225–227, 230, 231, 234 342 Index

Trehalose-6-phosphate synthase (TPS), W 176–180, 201, 225, 226, 229, 230, Water-deficit stress, 12–13 233, 249–251 Water-soluble carbohydrates (WSCs), TPS-TPP pathway, 176 325, 326 TreZ-TreY pathway, 176 grain growth and filling, 319 identification, QTLs, 327 role, plants metabolism, 320 U sucrose and starch, 320 UDP-glucose (UDPG), 225 variation, 326 Uridine diphosphate glucose (UDP-Glc), 249 in vegetative stems, 322 Usually multiple amino acids move in and out water-deficit conditions, 324 transporters (UMAMITs), 49 wheat and barley, 319