University of Udine Dept. of Agricultural, Food, Animal and Environmental Sciences Doctoral course in Agricultural Science and Biotechnology (ASB) Cycle XXIX, Coordinator: prof. Giuseppe Firrao FLAVONOID ROLE IN PLANT STRESS RESPONSES Supervisor PhD student prof. Enrico Braidot Antonio Filippi Co-supervisor dott. Elisa Petrussa I This thesis was presented by Antonio Filippi with the permission of the Dept. of Agricultural, Food, Animal and Environmental Sciences, University of Udine, for public examination and approved by the supervisor: prof. Enrico Braidot II Alla mia mamma III IV ABSTRACT FLAVONOID ROLE IN PLANT STRESS RESPONSES Flavonoids are the most powerful bioactive plants metabolites, able to interact with both plant and animal metabolism. They have occurred in terrestrial plants since their land colonization and are part of mammalian diet since millions of years. Flavonoids exert many different biological activities both in plants (UV-protection, ROS scavenging, enzymatic activity modulation, flower and fruit coloration, signalling and cellular communication) and in mammals (antioxidant activity, cancer cell proliferation inhibition, enzymatic activity modulation). Flavonoid biological activities are strongly connected to plant cellular ability to transport, store, excrete and sequester them into specific cellular compartments. The scientific community has debated upon flavonoid metabolism many times in the last 30 years, trying to obtain a complete overview of the synthesis, the transport systems and the role in plants, but up to date a full understanding of such a complicated mechanism is far from being elucidated. This PhD thesis aims to provide a contribution to the comprehension of flavonoid function in plants, particularly considering the role of quercetin (QC), the most abundant flavonoid in plant kingdom, in different physiological contests. The role of flavonoids has been explored starting from pathogen attack, up to the senescence, and from cellular transport, up to the role in heavy metal detoxification. V VI TABLE OF CONTENTS Frontispiece ............................................................................................I Abstract ................................................................................................. V Table of contents ................................................................................. VII Summary.............................................................................................. IX Introduction...................................................1 Flavonoids..................................................................2 Flavonoid transport................................................13 Flavonoid role in plants.........................................29 Flavonoid beyond the plant system....................43 Chapter 1.......................................................55 In vivo assay to monitor flavonoid uptake across plant cell membranes Chapter 2.......................................................63 Flavonoid facilitated/ passive transport: characterization of quercetin microsomial uptake Chapter 3.......................................................79 Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity Chapter 4.......................................................97 Green synthesis of silver nanoparticles: analysis of the role of flavonoid quercetin Discussion..................................................109 Bibliography..............................................117 Poster presentations..........................................127 Appendix 1..........................................................133 Appendix 2..........................................................143 VII VIII SUMMARY Plant flavonoids are one of the most studied members of natural polyphenolic family due to their biochemical, physiological and pharmacological effects both in plants and humans. The observation that the flavonoid metabolic pathway has remained unaffected for millions of years is consistent with the view that natural selection favoured the conservation of these phenolic metabolites thanks to their numerous functional roles in plants. Flavonoids encompass more than 10,000 molecules present in plant tissues at different molar concentrations and differentially produced during all the developmental stages. The enormous variability of biological effects ascribed to flavonoids lies mainly on their specific and, at the same time, variable chemical structure. For all these reasons, nowadays a complete and clear vision of the role of flavonoids in plants is really far to be elucidated and new discoveries are constantly made upon these important metabolites. For these reasons, in my PhD thesis I focused the attention on flavonoids and their role in plant stress response. Initially the work focused mainly on the identification of proteins involved in flavonoid binding that are expressed in mature tissues, such as seed and berry skin of Vitis vinifera. Their detection was initially performed on microsomes through Western Blot (WB) assays using a monoclonal antibody targeting a flavonoid-binding site of a mammalian organic anion carrier. The immuno-purification and sequencing were performed on the proteins that cross-reacted with that antibody. Chitinase was one of the proteins identified by amino acid sequencing. I then investigated the modulation of its activity by two different classes of flavonoids (quercetin as a flavonol and catechin as a flavan-3-ol). The second line of research focused on the development of a new methodology for the analysis of in vivo transport of quercetin. To this purpose, I used first the cellular model of V. Vinifera liquid suspension cultures and then on microsomal vesicles obtained from Pisum sativum stems. This work has allowed us to propose a new easy-to-use and fast flavonoid transport assay. In the third line of research, biochemical investigations were carried out on barley plants to evaluate the contents of ATP and reactive oxygen species (ROS), and assessing the impact of nanoparticles of cerium and titanium on cellular energy processes. Finally, I was involved in the study of quercetin role as a reducing agent, in order to obtain silver nanoparticles (NPs) from salt solutions. The purpose was to finely control the process of "green biosynthesis" and produce nature-friendly NPs, nanomaterial that has huge interest in medical and agricultural fields. IX X INTRODUCTION Introduction - Flavonoids Flavonoids Plant adaptation to land and evolution of flavonoid pathway The multicellular algae that moved from their marine environment to invade the harsh terrestrial land had to face lots of negative factors, including higher oxygen concentration, desiccation, increasing gravity, damaging heat and UV light, greater daily and seasonal fluctuations of temperatures, chances to be infected and eaten by new pathogens and grazers, and need to establish communications with organisms of the same and even other kingdoms. Also the co-evolution with other living organisms (the insects, in particular) forced plants to develop new strategies for the recruitment of pollinators, leading the appearance of the first colorful flowers and the synthesis of a large amount of volatile chemicals. Today, more than 500 million years later, we observe modern plants representing the most evolutionarily successful and longevous eukaryotic species, colonizing all parts of our planet, spanning from North to South poles. This evolutionary success has been possible thanks to the biosynthesis of numerous new bioactive metabolites. Secondary metabolites synthesis, including flavonoids, represents an important step in the colonization process of Earth’s terrestrial environment by vascular plants. Physiological functions of flavonoids and the reasons for their ubiquitous existence have been widely discussed. A short list of these functions includes: (i) protection against insect predation and defence against microbes; (ii) action as sunscreens to absorb UV radiation and strong light, thus replacing mycosporine-like amino acids usually present in algae; (iii) attraction of insect pollinators through production of colourful anthocyanins, absorbing different spectra of visible light; (iv) action as antioxidants, inhibiting the generation of reactive oxygen species (ROS), by maintaining their concentration within a sub-lethal range; (v) involvement in pollen germination; (vi) involvement in biological communication in the rhizosphere; (vii) action as developmental regulators, involved in auxin transport and catabolism; (viii) modulation of enzymatic activities. Under an evolutionary point of view, small quantities of flavonoids may have existed also in the primordial algae. Several studies have actually demonstrated that algae are capable of forming p-coumaric acid, the precursor of the flavonoid synthesis, having many essential flavonoid enzymes such as chalcone synthase or chalcone isomerase, although genes for other important enzymes such as flavanone-3- hydroxylase or flavonol synthase have not yet been detected in their genomes (Goiris et al. 2014). 2 Introduction - Flavonoids It is most probable that genes involved in primary metabolism pathways could later evolve into codifying sequences for enzymes of phenylpropanoid pathway via gene duplication. Alternatively, they could be acquired via horizontal gene transfer during plant symbioses with bacteria and fungi that are known to