CIRCADIAN CHARACTERIZATION of ACCUMULATION and UTILIZATION of NON-STRUCTURAL CARBOHYDRATES in Erythrina Velutina DURING LATE ESTABLISHMENT

MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE PRÓ-REITORIA DE PÓS-GRADUAÇÃO UNIDADE ACADÊMICA ESPECIALIZADA EM CIÊNCIAS AGRÁRIAS - UAECIA ESCOLA AGRÍCOLA DE JUNDIAÍ - EAJ PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FLORESTAIS

Nº 081

CIRCADIAN CHARACTERIZATION OF ACCUMULATION AND UTILIZATION OF NON-STRUCTURAL CARBOHYDRATES IN Erythrina velutina DURING LATE ESTABLISHMENT

MARYELLE CAMPOS SILVA

Macaíba/RN July of 2020

MARYELLE CAMPOS SILVA

CIRCADIAN CHARACTERIZATION OF ACUMULATION AND UTILIZATION OF NON- STRUCTURAL CARBOHYDRATES IN Erythrina velutina DURING LATE ESTABLISHMENT

Dissertation presented to Programa de Pós-Graduação em Ciências Florestais of Universidade Federal do Rio Grande do Norte to obtain the degree of Master in Forest Science (Area of Concentration in Forest Sciences - Research Line: Seeds, Propagation and Physiology of Forest Species).

Advisor: Prof. Dr. Eduardo Luiz Voigt

Macaíba/RN July of 2020

Universidade Federal do Rio Grande do Norte - UFRN

Sistema de Bibliotecas - SISBI

Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Rodolfo Helinski Escola Agrícola de Jundiaí - EAJ

Silva, Maryelle Campos. Circadian characterization of acumulation and utilization of non-structural carbohydrates in Erythrina velutina during late establishment / Maryelle Campos Silva. - 2020. 57f.: il.

Dissertation (Master) Universidade Federal do Rio Grande do Norte, Unidade Acadêmica Especializada em Ciências Agrárias, Programa de Pós-Graduação em Ciência Florestais, Macaíba, RN, 2020. Orientador: Dr. Eduardo Luiz Voigt.

1. Carbon partitioning - Dissertation. 2. Heterotrophy- autotrophy transition - Dissertation. 3. Seedling growth - Dissertation. 4. Soluble sugars - Dissertation. 5. Source-sink relationship - Dissertation. 6. Starch - Dissertation. I. Voigt, Eduardo Luiz. II. Título.

RN/UF/BSPRH CDU 549.21

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CIRCADIAN CHARACTERIZATION OF ACUMULATION AND UTILIZATION OF NON- STRUCTURAL CARBOHYDRATES IN Erythrina velutina DURING LATE ESTABLISHMENT

Maryelle Campos Silva

Dissertation presented to Programa de Pós-Graduação em Ciências Florestais of Universidade Federal do Rio Grande do Norte to obtain the degree of Master in Forest Science (Area of Concentration in Forest Sciences -Research Line: Seeds, Propagation and Physiology of Forest Species) and approved by the examining board on July 31, 2020.

Examining Board

______Prof. Dr. Eduardo Luiz Voigt DBG/CB/UFRN President

______Prof. Dr. Mauro Vasconcelos Pacheco UAECIA/UFRN Internal Examiner

______Prof. Dr. Sergio Luiz Ferreira da Silva UFRPE External Examiner

Macaíba/RN July of 2020

To my family for always supporting me in my dreams, giving me all the necessary strength to reach them.

DEDICATION

ACKNOWLEDGEMENTS ______

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. I thank the Programa de Pós-Graduação em Ciências Florestais da Universidade Federal do Rio Grande do Norte (UFRN) and the Departamento de Biologia Celular e Genética do Centro de Biociências da UFRN for the funding and structure that made this work possible. To Professor Eduardo Luiz Voigt for the impeccable guidance, dedication on the elaboration and execution of the research project, for the wisdom of transmitting your knowledge clearly, and for being an example of professionalism and ethics to be followed. To all the team of the Laboratório de Estudos em Biotecnologia Vegetal (LEBV), who helped in data and samples collection for the execution of this project, especially Danilo Flademir for the generosity, dedication, constant guidance and availability. To colleagues at Programa de Pós-Graduação em Ciências Florestais for the support during the course of the classes, for the mutual support and companionship in other activities. To my friends, who encouraged, inspired, and motivated me in many ways to persist in the journey of scientific research, helping me to get through the everyday adversities and reaffirming how they believe in my potential. To my family members, Adoniran, Jason, Juliana, Otto and Rosemary for always being by my side, stimulating, supporting, and giving me emotional support to continue my professional qualification and for legitimising my choices by valuing my efforts to achieve my goals.

GENERAL ABSTRACT ______

CIRCADIAN CHARACTERIZATION OF ACUMULATION AND UTILIZATION OF NON- STRUCTURAL CARBOHYDRATES IN Erythrina velutina DURING LATE ESTABLISHMENT

We carried out a circadian characterization of the non-structural carbohydrate dynamics in the different organs of Erythrina velutina seedlings at late establishment. Seeds were incubated under controlled conditions for 9 days and then seedlings were hydroponically grown in a greenhouse for 8 days. Gas exchanges were measured in the cordiform leaves every 2 h during the daytime (12 h) and seedlings were harvested every 4 h during the day-night cycle (24 h) to assess the contents of non-structural carbohydrates and the activities of amylases and invertases. Photosynthetic net rate was highly synchronized with transpiration rate under high irradiance, when the cordiform leaves assumed the paraheliotropic position and water use efficiency increased. Diel patterns of non-structural carbohydrate content were identified in the photosynthetic organs, in which the turnover of starch operated far from its depletion at dawn. In the heterotrophic organs, however, changes in the starch content over 24 h may have maintained the supply of soluble sugars, buffering transient fluctuations in carbon availability. Although it seems that amylase activity was not influenced by the circadian rhythm in the different seedling organs, diel patterns of invertase activity were recognized in the leaves and roots.

Keywords: Carbon partitioning, heterotrophy-autotrophy transition, seedling growth, soluble sugars, source-sink relationship, starch.

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RESUMO GERAL

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CARACTERIZAÇÃO CIRCADIANA DA ACUMULAÇÃO E DA UTILIZAÇÃO DOS CARBOIDRATOS NÃO ESTRUTURAIS EM Erythrina velutina DURANTE O ESTABELECIMENTO TARDIO

A dinâmica dos carboidratos não estruturais foi caracterizada em escala circadiana nos diferentes órgãos da plântula de Erythrina velutina durante o estabelecimento tardio. As sementes foram incubadas em condições controladas por 9 dias e as plântulas foram então crescidas hidroponicamente em casa de vegetação por 8 dias. As trocas gasosas foram mensuradas nas folhas cordiformes a cada 2 h durante o dia (12 h) e as plântulas foram coletadas a cada 4 h durante o ciclo dia-noite (24 h) para acessar os conteúdos de carboidratos não estruturais e as atividades de amilases e invertases. A taxa líquida de fotossíntese foi altamente sincronizada com a taxa de transpiração sob alta irradiância, quando as folhas cordiformes assumiram a posição paraheliotrópica e a eficiência do uso da água foi aumentada. O conteúdo de carboidratos não estruturais nos órgãos fotossintéticos apresentou um padrão diário de variação, no qual o ciclo síntese-degradação do amido operou distante da depleção ao amanhecer. Nos órgãos heterotróficos, entretanto, alterações no conteúdo de amido ao longo de 24 h podem ter mantido o aporte de açúcares solúveis, tamponando as flutuações transientes na disponibilidade de carbono. Embora pareça que a atividade de amilases não seja influenciada pelo ritmo circadiano nos diferentes órgãos da plântula, a atividade de invertases apresentou um padrão diário de variação nas folhas e nas raízes.

Palavras-chave: Açúcares solúveis, amido, crescimento da plântula, partição de carbono, relação fonte-dreno, transição heterotrofia-autotrofia.

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SUMMARY ______

Page 1. GENERAL INTRODUCTION...... 1 2. GENERAL OBJECTIVES ...... 4 3. LITERATURE REVIEW ...... 6 3.1. Caatinga ...... 6 3.2. Erythrina velutina Willd...... 9 3.3. Reserve mobilization ...... 10 3.4. Circadian clock ...... 14 4. MATERIALS AND METHODS ...... 18 4.1. Plant material ...... 18 4.2. Gas exchange measurements ...... 19 4.3. Quantification of non-structural carbohydrates...... 20 4.4. Enzyme assays ...... 20 4.5. Experimental design and statistical analysis ...... 21 5. RESULTS AND DISCUSSION ...... 23 5.1. Results ...... 23 5.2. Discussion ...... 29 6. CONCLUSIONS ...... 36 7. CITED LITERATURE ...... 38

FIGURE LIST ______

Figure 1. Chemical composition of amylose and amylopectin and graphic representation of the double and single helicoidal structures of amylose and amylopectin ...... 12

Figure 2. Influence of source-sink relation on the mobilization of storage reserves in cucumber cotyledons...... 14

Figure 3. Schematic representation of the relationships between the circadian clock and the environmental and metabolic processes...... 15

Figure 4. Hydroponic system arrangement in distilled water of E. velutina seedlings kept in plastic pots under greenhouse conditions...... 19

Figure 5. Distribution of harvest times to circadian characterization of the physiological and biochemical markers related to the dynamics of non-structural carbohydrates in different organs of E. velutina seedlings during late establishment...... 19

Figure 6. Diaheliotropic and paraheliotropic movements during daytime in E. velutina cordiform leaves during late establishment...... 24

Figure 7. Content of starch (a, c), total soluble sugars, and non-reducing sugars (b, d) in the cordiform leaves (a, b) and in the cotyledons (c,d) of E. velutina seedlings hydroponically cultivated in a greenhouse during the daily cycle...... 25

Figure 8. Content of starch (a, c, e), total soluble sugars, and non-reducing sugars (b, d, f) in the roots (a, b), in the hypocotyl (c,d) and in the epicotyl + first trifoliate leaf (c,d) of E. velutina seedlings hydroponically cultivated in a greenhouse during the daily cycle...... 26

Figure 9. Activity of amylases and invertases in the cotyledons (a, b) and in the cordiform leaves (c, d) of E. velutina seedlings hydroponically cultivated in a greenhouse during the daily cycle...... 27

Figure 10. Activity of amylases and invertases in the roots (a, b), in the hypocotyl (c,d), and in the epicotyl + first trifoliate leaf (e, f) of E. velutina seedlings hydroponically cultivated in a greenhouse during the daily cycle...... 28

TABLE LIST ______

Table 1. Daily course of photosynthetically active radiation (PAR), net CO2 assimilation (A), leaf transpiration (E), and water use efficiency (WUE) in the cordiform leaves of E. velutina seedlings during late establishment...... 23

ABBREVIATION LIST ______

AINV – Acid invertases BFG – The Brazil Flora Group CCA1 – Circadian clock associated 1 CINV – Cytosolic invertases CWINV – Cell-wall invertases DBH – Diameter at breast height IBGE – Brazilian Institute of Geography and Statistics, from Portuguese Instituto Brasileiro de Geografia e Estatística INPE – National Institute of Space Research, from portuguese Instituto Nacional de Pesquisas Espaciais IRGA – Infrared gas analyzer LHY – Late elongated hypocotyl MMA – Ministry for the Environment, from Portuguese Ministério do Meio Ambiente NINV – Neutral invertases NRS – Non-reducing sugars NSC – Non-structural carbohydrates PAR – Photosynthetically active radiation PSVs – Protein storage vacuoles RFOs – Raffinose-family oligosaccharides RS – Reducing sugars TAGs – Triacylglycerols TDF – Tropical dry forest TSS – total soluble sugars VINV – Vacuolar invertases WUE – Water-use efficiency

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General Introduction

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1. GENERAL INTRODUCTION ______

The Caatinga is a dry tropical forest biome located mainly in the Northeast of Brazil and it occupies about 1.330.000 km2, where more than 31.000.000 of people live (SOUZA et al., 2015). This biome is described by an average temperature higher than 25°C and annual rainfall less than 800 mm poorly distributed throughout the year, the presence of a dry season, and the prevalence of deciduous trees (FERREIRA et al., 2016). Despite the fact the Caatinga has a high biodiversity, including endemic species, it is one of the less studied and most threatened biomes due to unsuitable agricultural practices, such as deforestation and burning (SANTOS et al., 2014). Moreover, several areas from Caatinga are affected by or are susceptible to desertification (RIBEIRO et al., 2016), and it is estimated that 25% of irrigated areas are salinized (BESSA et al., 2017). Thus, it is essential to create recovery and/or restoration programs in these degraded areas. One of the most constraints in forest restoration is the lack of knowledge about native seedling establishment, given that this is a vital step in the plant lifecycle (SORIANO et al., 2013). Seedling establishment consists of a transition phase during seedling development, in which the reserve-dependent heterotrophic metabolism leads the progression to photoautotrophic activity (WINGLER, 2018). In this process, the insoluble reserves stocked in storage tissues are mobilized, generating soluble metabolites that are transported to the embryonic axis supporting seedling growth (GOMMERS and MONTE, 2018). Recent studies have demonstrated the central role of non-structural carbohydrates (NSC) in the responses of seedlings exposed to environmental stresses, indicating that these compounds are strictly related to their performance under natural conditions (VILLAR- SALVADOR et al., 2015; MAGUIRE and KOBE, 2015). In dicots, NSC corresponds to starch and soluble sugars. Starch is mostly regarded as a storage carbohydrate, whereas soluble sugars perform numerous functions, including energy supply and acting as biosynthetic precursors, intermediate metabolites, transport of carbon, signalling molecules, and as osmolytes (MARTINEZ-VILALTA et al., 2016). Considering that NSC are the main products of photosynthetic activity, their dynamics is strongly influenced by the circadian rhythm. In leaves, it is well established that carbon allocation to starch accumulation during the day, as well as the degradation of starch for the growth maintenance during the night, is regulated by the endogenous circadian oscillator (GREENHAM and MCCLUNG, 2015). Nevertheless, the involvement of the circadian rhythm in reserve mobilization in storage organs and in the cycle of deposition and utilization of starch in other parts of the seedling, such as hypocotyl and roots, still remains unclear. In this sense, efforts in the circadian characterization of the dynamics of NSC during seedling establishment

1 may expand the understanding about the importance of this phenomenon to environmental colonization. Several works have characterised the NSC distribution in seedlings and in young plants, especially in species of temperate climates (VILLAR-SALVADOR et al., 2015). Thus, the literature still lacks studies that aim to comprehend the role of NSC during seedling establishment in tropical species. In this work, Erythina velutina Willd. was used as a model, since this legume tree is a pioneer species in the Caatinga, with potential for recovery of degraded areas, reforestation, and as raw material for the production of herbal medicines (PEREIRA et al, 2014).

General Objective

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2. OBJECTIVES ______

2.1. General Objective The general objective of this study was to investigate the involvement of the circadian rhythm in the dynamics of NSC in different organs of E. velutina seedling during late establishment.

2.2. Specific Objectives

• Verification of changes in the net CO2 assimilation, transpiration, and water use efficiency of expanded cordiform leaves within 24 hours. • Determination of the content of soluble sugars and starch, as well as the activity of amylases and invertases in different seedling organs, during 24 hours.

Literature Review ______

3. LITERATURE REVIEW

3.1. Caatinga

There are many types of forests in the world, and they are classified according to their floristic and edaphoclimatic conditions, in which the Tropical Dry Forests1 (TDFs) are included. TDFs constitute 42% of the tropical forests in the world, and they are located in areas that experience extensive drought periods, where the rainfall is poorly distributed and less than 800 mm/year (SOUZA et al., 2015; FERREIRA et al., 2016); moreover, they possess elevated temperatures, whose values are higher than 25°C. These forests are present in areas of Australia, Africa, Central and South America, India, and Southeast Asia, as confirmed by Ferreira et al. (2016). The Brazilian Institute of Geography and Statistics (IBGE) released, in the year of 2012, the Technical Manual of Brazilian Vegetation (Manual Técnico da Vegetação Brasileira), which defines the Caatinga as a Brazilian Esteppe Savanna, but the term internationally spread still remains as TDF. The Caatinga is an exclusively Brazilian biome, which implies the fact that it is a biome of unique occurrence, formed by a diverse biological area and an exceptional fauna and flora, which occupies about 10% of the Brazilian territory and approximately 70% of the Northeast region (SANTOS et al., 2014; INPE, 2015; FERREIRA et al., 2016). Alagoas, Bahia, Ceará, Maranhão, Pernambuco, Paraíba, Rio Grande do Norte, Piauí, and Sergipe are the North- eastern states where the Caatinga occurs. The high richness of genetic resources and vegetation of the Caatinga is composed by woody, herbaceous, cactaceous, and bromeliad species. The Caatinga area is 1.330.000 km2, with an estimated 31 million inhabitants, and this categorises it as the most inhabited semiarid region in the world. This particularity causes an intense process of degradation and, consequently, desertification processes (SANTOS et al., 2014; SOUZA et al., 2015). According to DANTAS et al. (2014), the anthropic pressure due to subsistence activities by the population is one of the reasons that intensifies the desertification, causing the increase of degraded areas. Studies performed by SOUZA et al., (2015) demonstrated that economic activities essential to the population of these areas exploit the land, turning these populations even more dependent on natural resources. Agriculture, outdated management practices, livestock, goat production, and plant extractive activity are crucial for the spatial distribution of

1 Forests that occurs in tropical areas characterized by pronounced seasonality in the rainfall distribution, resulting in several dry months. The forests that develop under such climate conditions share a very similar structure and physiognomy (MOONEY et al., 1995 apud MILES et al., 2006).

6 plants and the reduction of plant diversity. Overgrazing, as well as burning and deforestation practices for the production of firewood and charcoal are referred to as the main factors of vegetation cover degradation (SANTOS et al., 2014; INPE, 2015). In agreement with the geoprocessing group of the Regional Centre of Northeast (CRN) from the National Institute of Space Research (INPE), using images captured by satellite monitoring carried out in the years of 2013 and 2014, it was possible to attest that, in an area that corresponds nearly 10% of all Caatinga biome, most of them are considered as degraded areas (45%), followed by preserved areas (40%). The other identified areas consist of exposed soil (7,2%), crops (6,5%), and less than 1% of water bodies (INPE 2015). Desertification is a serious problem, especially in areas from Caatinga, since the impact on the its floristic composition is not yet elucidated. This type of degradation leads to soil loss by erosion, impacting areas that were previously productive. Degraded areas require special attention by researchers in order to their conservation and restoration, considering multiple aspects. The accelerated loss of the vegetation cover allied to the economic and social relevance of endemic species set up reasons that justify the elucidation of means that promote their restoration to avoid the progress of desertification (MMA, 2006; FAJARDO et al., 2013). It is necessary to consider that researches involving different types of forests have given less attention to TDFs; a review stated that for every 100 published articles on the topic, 64 are related to humid forests and only 7 discuss TDFs (MELI, 2003 apud FAJARDO et al., 2013). Several aspects contribute to the intensification of the process that are materialising the desertification of huge areas of Caatinga biome, highlighting the land structure, periodic droughts and the extractive and predatory character of exploitation practices of the natural resources (ARAÚJO FILHO, 2013). The Ministry for the Environment (MMA), by the Atlas of Susceptible to Desertification in Brazil, mapped desertified areas or lands in desertification process, the so-called Desertification Nuclei, including four nuclei located in Gilbués (PI), Irauçuba (CE), Seridó (RN and PB) and Cabrobó (PE), Brazil. According to Perez-Martin et al (2013), there are still two more Desertification Nuclei located in Cariris Velhos (PB) and in Sertão do São Francisco (BA), which did not receive appropriated attention by the MMA. The Seridó Desertification Nuclei has 2.987 km² of compromised area, where live nearly 260.000 inhabitants (PEREZ-MARINS et al., 2013). The Seridó Nuclei includes the cities of Currais Novos, Cruzeta, Equador, Carnaúba dos Dantas, Acari, Parelhas, Caicó, Jardim do Seridó, Ouro Branco, Santana do Seridó, São João do Sabugi, Santa Luzia, and Várzea (MMA, 2007). In these cities, temperature, and irregular and low rainfall affect the soil quality and depth, acting directly in the spontaneous desertification process and they are intensified by anthropic action due to tree felling for firewood production, wood extraction because of a

7 strong presence of the ceramics industry in these cities and an extensive livestock as reported by PEREZ-MARINS et al.(2013). Thus, more efforts are required to elucidate the desertification process and its evolution mechanism, the ways to slow down and recover degraded and desertified areas, so that promote the maintenance of the environment, the economic activities and specially the survival and the development of the local population (SANTOS et al., 2017). The concept of endemic species is trivialised by a taxon that is exclusive to a particular geographic location, as stated by Zappi et al. (2015). In agreement with these authors, the Caatinga has a total of 4.657 species and approximately 20% of them are considered as endemic, which means that more than 900 species are unique to the Caatinga biome. A study performed in 2015 compared the evolution of the Brazilian biomes in relation to the identification of new species and it attested that the Caatinga had an 22.7% increase in the number of endemic species (169), between 2010 and 2015 (FORZZA et al., 2010), being higher than that from other biomes such as Mata Atlântica (6%) and Cerrado (2.5%), which clearly shows the elevated level of the endemism in the Caatinga and the need to improve studies to characterize this biome (ZAAPI et al., 2015). Data from INPE (2015) establish a total of 932 species in the Caatinga, and around 41% of this total are considered endemic species, in disagreement with the counting of endemic species carried out by The Brazil Flora Group in 2015, corroborating data from MAIA et al. (2017), which declared that “it is notable the existence of divergences in the literature regarding the amount of the species belonging to this biome”. This group also affirms that the increase of the endemic species percentage in the Caatinga occurred due to academic studies at the local botany, among other factors. The angiosperms are the most numerous species in the Caatinga and one of the main families having endemic species is the Fabaceae family, with 605 species, followed by Poaceae with 205 species (ZAPPI et al., 2015). The study of endemic species of the Caatinga is indispensable, since it works as adaptation models to mechanisms of survival under abiotic stress conditions and in degraded and strongly anthropized areas. The native population of the Caatinga extracts products from its biodiversity to assure their survival, food, forage for animals, medicines, wood for building and energy production, all of them based on extractivism. With a low technological level, the agriculture is performed mostly in rainfed conditions with few areas that utilize irrigation systems and, in these areas, soil salinization can become a problem if no proper management is done, as described by MAIA et al. (2017). According to ARAÚJO FILHO (2013), there are eight plant types that offer products that are beyond the traditional extractive paths of the Caatinga, besides the techniques previously mentioned, namely: plants that produce wax, oils and tannins, known as non-wood forest products, fruit plants, beekeeping activities, as well as plants that are rich in plant fibres, and ornamental and medicinal plants.

SOUZA et al., (2015) carried out studies in the Paraíba Caatinga using different forest species, relating them to deserted and non-deserted areas. The results showed that the pioneer species (76.35%) were those which contributed mostly to the composition of deserted areas, however in non-deserted areas there was high richness of species, more than double for deserted areas. Thus, it is clear the importance of the study of pioneer species as an instrument for the elaboration of strategies toward the forest recovery of degraded areas.

3.2. Erythrina velutina Willd.

The Erythrina velutina species is angiosperm belonging to Fabaceae family and to Faboideae subfamily. It is a native, pioneer, and abundant tree from the Caatinga biome that is characterized for being aculeate, presenting trifoliate composed leaves and deciduous behaviour; of dichotomous branching, having a wide, open, and round crown; in the adult phase, this species reaches around 10 m of height and 80 cm of diameter at breast height (DBH). Its fruit is a dehiscent, non-septate legume with one to three oblong and bi-coloured seeds, called mimetics, exhibiting a dark-red and orange-red colouring. The fruit and seed dispersal are authocoric. Its flowers are wide, presenting a coral red colour arranged in racemes panicles with powdery rachis that are formed after the tree loses all its leaves (CARVALHO, 2008; PALUMBO et al., 2016). The species is popularly known as bucaré (CE), mulungu (CE, PB, PE, RN, SP and SE), mulungu-da-flor-vermelha (CE), mulungu-da-flor- amarela (CE), muchoco (MG) and mulungá (MG), and it is geographically distributed in the Brazilian states of Bahia, Ceará, Maranhão, Minas Gerais, Paraíba, Pernambuco, Piauí, Rio Grande do Norte and Sergipe. It is found mainly in colluvial soils of humid, alluvial, sandy or clay nature (CARVALHO, 2008). E. velutina species is indicated to heterogeneous reforestation, recovery of degraded and desert areas due to its rapid growth and great adaption to environmental semi-arid conditions; it is also utilized as wood source in small properties and in carpentry, its flowers are edible and used as a dye and its seeds are used as raw material for handicrafts. The tree is ornamental and it is employed in the afforestation of streets and avenues, and it can be used in beekeeping; its bark has tannin properties and it is also used as a source of bioactive compounds for pharmacological uses (alkaloids, flavonoids, and terpenes) (CARVALHO, 2008; PALUMBO et al., 2016; RODRIGUES et al., 2018). According to CARVALHO et al. (2008), the flowering of E. velutina occurs between the months of July to February, changing relative to the geographic region, and the fruiting occurs during the months of September to March. The seeds from this species present a coat- imposed dormancy, being necessary a pre-germinative scarification treatment to allow the germination process. Regarding to storage conditions, E. velutina seeds has a satisfactory

9 response to germination for a period of more than 10 months, presenting high reproducibility in laboratory and in a greenhouse conditions (CARVALHO et al., 2008; PEREIRA et al., 2014). Some works have previously demonstrated the importance of elucidating the E. velutina behaviour in relation to the plant establishment for large-scale cultivation focusing its using in the recovery of degraded areas, reforestation, and as raw material for producing herbal medicines (PEREIRA et al., 2014). The same authors verified that seed germination has great success after the seeds were mechanically scarified, potentializing the imbibition and germination processes between the fourth and 15th day. Moreover, E. velutina showed great shoot height and stem diameter during the first four months of cropping, demonstrating an establishment potential higher than other plants from the same genus. Thus, the low complexity of large-scale production of E. velutina in a greenhouse or in the field allows to meet demands of herb medicine market and the recovery of degraded areas (PEREIRA et al., 2014).

3.3. Reserve mobilization – Carbohydrates, lipids, and proteins

Seed germination and seedling establishment are fundamental steps for the reproductive success of native and cultivated species in the environment colonization and in the establishment of crops, respectively, presenting ecologic and economic relevance (BEWLEY et al., 2013). Germination starts with imbibition, involving the resumption of metabolism and it ends when the embryonic axis emerges, commonly the primary root or radicle. From this moment on, seedling establishment begins (ROSENTAL et al., 2014). Seedling establishment consists of a transitory phase of development, in which the reserve-dependent heterotrophic metabolism enables the progression to photoautotrophic metabolism. In these process, insoluble reserves within the cells of the storage tissues are mobilized, generating soluble metabolites that can be transported to the embryonic axis, supporting seedling growth (WINGLER, 2018). The storage tissues and organs accumulate mostly some carbon source, such as carbohydrates and/or lipids, besides some nitrogen (N) and sulphur (S) reserve in the form of proteins. During the mobilization process, these reserves are used to provide energy and material for seedling growth. In addition to major reserves, phytin is also considered as a food stock, which provides mineral nutrients, being the main source of phosphate, magnesium (Mg2+), calcium (Ca2+) and potassium (K+) (BEWLEY et al., 2013). The reserve carbohydrates include sucrose, raffinose family oligosaccharides (RFOs), starch and cell wall storage polysaccharides (BEWLEY et al., 2013). Sucrose and RFOs are non-reducing sugars (NRS) widely distributed among the angiosperms, yielding 2 to 6% of seed dry mass. Sucrose is constituted by one unit of D-glucose and one of D-fructose

10 connected by (12) glycosidic linkage. RFOs, in turn, are galactosyl sucroses, made up of D-galactose units connected to sucrose by (1→6) glycosidic linkages. Therefore, raffinose corresponds to monogalactosyl sucrose, stachyose consist of digalactosyl sucrose, and verbascose is composed of trigalactosyl sucrose (HELDT and PIECHULLA, 2011). It is assumed that sucrose and RFOs are primarily accumulated in the cytosol of the embryonic axis cells, where they can contribute to stabilize the cell membranes during the dessication process at the maturation phase and during imbibition at the beginning of germination in orthodox seeds. Complementarily, sucrose and RFOs can also act as the main cellular fuels in the early steps of germination, allowing the repair of damaged cell structures, especially membranes and chromatin (BUCKERIDGE et al., 2004b). RFOs are hydrolyed by -galactosidases, releasing D-galctose and sucrose, whereas the latter is cleaved by invertases, producing D-glucose and D-fructose. It was well accepted that free hexoses are primarily utilized as cellular fuels in the tissues where they are generated (BUCKERIDGE et al., 2004b). Among the reserve carbohydrates, starch is the polysaccharide most commonly found in Angiosperms. In cereals and legumes, starch can compose about 80 and 56%, respectively, of seed mass after harvest. In addition, starch can correspond to 28% of seed fresh weight in oil seeds (BLACK et al., 2006). Starch consists in two polymers of glucose: amylose and amylopectin. Amylose is a predominantly straight-chain polysaccharide, constituted by D- glucose residues connected by (1→4) glycosidic linkages. In comparison, amylopectin is a highly branched polysaccharide, presenting residues linked by (1→4) glycosidic bonds in the main chain and the branch chains are (1→6) glycosidic linkages (HELDT; PIECHULLA, 2011) (Figure 1). Starch stored in seeds is found in the amyloplast matrix forming inclusions known as granules. Starch granules are composed of about 25% amylose and 75% amylopectin. The amylose and amylopectin molecules can be arranged in concentric semicrystalline structures (MACNEILL et al., 2017).

Figure 1 – Chemical composition of amylose and amylopectin and graphic representation of the double and single helicoidal structures of amylose and amylopectin.

Source: Adapted from BEWLEY et al. (2013).

In cereals, it is well established that the enzymes that initially attack starch granules are α-amylases, endoglucanases that randomly hydrolyse the α(1→4) glycosidic links between the amylose and amylopectin polymers, releasing maltose and dextrins. Exoglucanases attack the non-reducing units producing maltose and glucose-1-phosphate by -amylases and starch phosphorylases, respectively. Dextrins presenting α(1→6) linkages are substrates for debranching enzymes (pullulanases, isoamylases, and dextrinases), and the resulting maltose is degraded by maltases (HELDT and PIECHULLA, 2011). In oil seeds, storage lipids are the most abundant carbon source, which accounts for 17 to 64% of seed mass after harvest. Storage lipids, also known as oils, are complex mixtures of triacylglycerols (TAGs) that contain high quantities of unsaturated fatty acids chains in their

12 composition (BLACK et al., 2006). These TAG reserves are accumulated in organelles called oleosomes or oil bodies, which are bounded by a phospholipid monolayer membrane containing structural proteins and presenting a core filled by TAGs (HUANG, 2018). TAGs are mobilised by inter-organelle interactions involving oil bodies, glyoxysomes, and mitochondria. By the action of lipases, the three fatty acyl ester bonds are cleaved, releasing glycerol and fatty acids. The latter are activated to acyl-CoA and transported to the glyoxysome; the acyl-CoA are degraded to acetyl-CoA by -oxidation and the acetyl radical is metabolized by the glyoxylate cycle. In the glyoxylate cycle, succinate is produced and transported to the mitochondria. By the Krebs cycle, succinate is converted to malate, which is transferred to the cytosol and used as gluconeogenesis precursor (EASTMOND et al., 2015). Sucrose resulting from gluconeogenesis is then distributed to seedling growing parts (BEWLEY et al., 2013). In higher plants, seed storage proteins correspond to the main sources of nitrogen, providing 9 to 50% of seed fresh weight (BLACK et al., 2006). Among these reserves, the albumins and globulins have a greater representation in dicots, mainly in legumes (BEWLEY et al., 2013). According to the sedimentation coefficient, seed storage proteins from dicots can also be termed as 2S albumins, 11S globulins (legumins), and 7S globulins (vicilins). Regarding the structure, 2S albumins are commonly constituted by two different polypeptide chains connected by a disulphide bond. The 11S globulins present six subunits that are joined by non-covalent bonds and each subunit contains an acidic polypeptide and a basic polypeptide that are linked by a single disulphide bond. Finally, the 7s globulins are composed of three polypeptide chains that are linked by non-covalent interactions (HELDT and PIECHULLA, 2011). Proteases are the enzymes responsible for the hydrolysis of storage proteins and, in dicots, are deposited in the protein storage vacuoles (PSVs). These enzymes are synthetized by the biosynthetic-secretory pathway in the form of proenzymes, being activated by proteolysis. There are three types of proteases according to the location in which their substrates are hydrolysed: endopeptidases (internal peptide bonds), aminopeptidases (N- terminal of the peptide), and carboxypeptidases (C-terminal of the peptide). The mobilization of storage proteins is performed by the coordinated action of the different proteases. The initial action of some proteases produces peptides that are degraded by carboxypeptidases within the PSVs. The produced peptides are then transported to cytosol, where they are degraded by aminopeptidases, generating free amino acids (TAN-WILSON and WILSON, 2012). The end products of hydrolysis can be utilized in the biosynthesis of new proteins, as substrate for respiratory pathways or they can be converted into transport amides (glutamine and asparagine) (BEWLEY et al., 2013).

Reserve mobilization can be regulated by mechanisms involving hormonal signalling and the source-sink relation. The hormonal mechanisms are well described in cereal grains, and it involves the action of gibberellins produced by the embryo in the expression and release of hydrolases from the aleurone layer to the starchy endosperm, where they degrade the reserves, specially starch and storage proteins (YU et al., 2015). According to the source-sink relation, which is well known in dicots, the embryonic axis acts as a sink, consuming the products of mobilization transported from storage tissues, which act as a source. Thus, the embryonic axis regulates the activity of hydrolytic enzymes through negative feedback (BEWLEY et al., 2013) (Figure 2). Despite the source-sink mechanism is closely related to the success of the heterotrophic-autotrophic transition, allowing seedling establishment, the literature still lacks studies to elucidate the involvement of circadian clock in this mechanism.

Figure 2 – Influence of source-sink relation on the mobilization of storage reserves in cucumber cotyledons.

Source: Adapted from BEWLEY et al. (2013).

In the legumes Pisium and Vicia faba, starch is the major reserve, yielding 50% or more of dry weight, as attested by HALMER (1985). In the seeds of another legume, Caesalpinia peltophoroides, it was determined that, regarding the dry weight, lipids provide up to 50% of reserve content, followed by soluble carbohydrates (32%), starch (7.7%), and soluble proteins (6.8%) (CORTE et al., 2006).

3.4. Circadian clock

The circadian clock is an endogenous mechanism that generates rhythms with periods of approximately 24 hours, providing the synchronization of the physiology and the temporal metabolism of plants determining, for example, control of the flowering time, growth, stomatal aperture, transpiration, and metabolic processes (FARRÉ; WEISE, 2012). Plants synchronize their physiology and metabolism according to environmental conditions mainly due to the

14 perception of changes in temperature and the alternance between light and darkness; hence, the circadian clock plays a significant role on plant growth regulation via the control of the photosynthesis during day (GRAF et al., 2010).

In the presence of light, plants fix atmospheric CO2 into organic compounds through photosynthesis and, in the dark period, they depend mainly on stored non-structure carbohydrates to maintain the basal metabolism and the developmental processes. In many species, starch accumulates in the presence of light and it is degraded in order to provide sugars that are metabolised during night (HAYDON et al., 2013). In the model species Arabidopsis thaliana, in which the circadian clock influence is widely described, it is known that the starch consumption rate reaches about 95% in the darkness (GRAF et al., 2010). In addition, in Phaseolus leaves kept in continuous light, CO2 assimilation is under circadian control and it do not change along with alterations in the stomatal conductance (FARRÉ; WEISE, 2012). According to FARRÉ and WEISE (2012), in A. thaliana, the transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are expressed at dawn, repressing the transcription of evening transcriptional regulators (TIMING OF CAB 1, TOC1; EARLY FLOWERING 3, ELF3; EARLY FLOWERING 4, ELF4; and LUX ARRYTHMO, LUX). These evening regulators activate indirectly the transcription of CCA1 and LHY and they are also capable of performing a feedback mechanism with the pseudo- response regulators PRR5, PRR7 e PRR9 (figure 3), whose peak occurs in different moments of the day.

Figure 3 – Schematic representation of the relationships between the circadian clock and the environmental and metabolic process.

Source: Adapted from FARRÉ and WEISE (2012).

The circadian clock is essential to assure the establishment and survival of plants, ensuring an adaptive benefit, since this endogenous oscillator acts in the primary metabolism and optimises the growth and the development of these organisms (HAYDON et al., 2013). However, few efforts have been made to explain the influence of the circadian clock in reserve mobilization in non-crop species or in native species of the Caatinga. Therefore, it is a challenge to elucidate the mechanisms that can clarify the circadian oscillations on the accumulation and utilization of NSC in these species.

Material and Methods

______

4. MATERIAL AND METHODS ______

4.1. Plant Material

Fruits of E. velutina were collected from ten mother trees located at Acari, Rio Grande do Norte, Northeast of Brazil; these fruits were processed manually, and intact seeds (without visual damages) were separated and kept under refrigeration (10°C) for approximately 40 days. After mechanical scarification, seeds were immersed in commercial detergent diluted 1:500 for 30 s and then rinsed under running water. Disinfection was performed with 70% (v/v) etanol for 30 s followed by 0.25% (w/v) NaClO for 3 min under continuous mixing and rinsed three times in sterile distilled water. After these steps, seeds were imbibed in distilled water for 2 h and sown in towel paper humidified with sterile distilled water in the proportion of 10% of paper weight. The papers were rolled up and kept under controlled conditions (28 ± 2 °C, 12 h light/12 h dark photoperiod, and photosynthetically active radiation (PAR) of 80 μmol m-2 s-1) for 9 days in a growth chamber. Paper rolls were inspected daily in order to remove contaminated and/or defective seedlings and were humidified with distilled water to maintain the optimum microclimate for seed germination. Nine-day-old seedlings were transferred to distilled water in hydroponics in plastic pots with 1 L capacity (Figure 4), submitted to manual aeration using a glass stick and kept in a greenhouse for 8 days. Harvests were carried out at 4 h intervals starting at the dawn, during a period of 24 h (Figure 5), which corresponds to the physiological stage when seedlings presented a pair of expanded cordiform leaves. Measurements of daily gas exchange curve were performed on the 8th day. After this, seedlings were harvested and divided into cordiform leaves, cotyledons, hypocotyl, epicotyl + first trifoliate leaf, and root. Then, samples were frozen at -20°C for biochemical determinations.

Figure 4 – Hydroponic system arrangement in distilled water of E. velutina seeds kept in plastic pots under greenhouse conditions.

Source: Picture taken by the author (2020).

Figure 5 – Distribution of harvest times to circadian characterization of the physiological and biochemical markers related to the dynamics of NSC in different organs of E. velutina seedlings during late establishment.

Dawn Dawn

Dusk

5h30 9h30 13h30 21h30 1h30 5h30 17h30

Source: Figure elaborated by the author (2020).

4.2. Gas exchange measurements

The determination of PAR, net CO2 assimilation (A), and leaf transpiration (E) in one of the cordiform leaves of each seedling every 2 h during the daytime (12h) was obtained by using an Infrared Gas Analyzer (IRGA), LCpro-SD model, from ADC BioScientific Ltd. In addition, the water used efficiency (WUE) was calculated by the A/E ratio. During the gas exchange measurements, the CO2 concentration was 400 ppm.

4.3. Quantification of NSC

NSC were extracted from 200 mg of frozen tissues with 80% (v/v) ethanol at 60°C for 60 min. Total soluble sugars (TSS) were determined with the anthrone reagent (MORRIS, 1948; YEMM E WILLIS, 1954), using D-glucose as standard. The quantification of non- reducing sugars was performed using the anthrone method with modifications (VAN HANDEL, 1968), employing sucrose as standard. Both TSS and NRS were expressed mg g-1 DW. Starch was extracted from the residues obtained after the removal of ethanolic fractions. These residues were macerated with chilled 30% (v/v) perchloric acid. Samples were centrifuged at 10,000 xg for 15 min at 4°C, the supernatants were collected and the pellets were re-extract twice. Starch was measured using the anthrone reagent (MORRIS, 1948; YEMM e WILLIS, 1954) utilizing D-glucose as standard. The values were multiplied by 0.9 to convert glucose to starch (MCCREADY et al., 1950) and expressed as mg g-1 DW.

4.4. Enzyme assays

The activity of amylases was determined by the releasing of reducing sugars (RS) from soluble starch (ELARBI et al., 2009) and RS was assessed using the dinitrosalicylic (DNS) method (MILLER, 1959). Enzymes were extracted from 100 mg of frozen tissues by maceration with chilled 100 mM potassium acetate buffer (pH 6.0) containing 5 mM CaCl2. After centrifugation at 10,000 xg for 20 min at 4°C, supernatants were collected and utilized as enzyme extracts. In the enzyme assay, 100 μL of extract were added to 400 μL of 100 mM potassium acetate buffer (pH 6.0) supplemented with 0.5% (w/v) soluble starch and 5 mM

CaCl2. The reaction was performed at 55 °C for 10 min. The reaction was interrupted by adding 1 mL of the DNS reagent. Amylase activity was expressed as μmol RS g-1 MS min-1. Invertase activity was estimated by RS liberation from sucrose OLIVEIRA et al., 2006) and DNS method (MILLER, 1959) was employed in order to access the concentration of RS in the reaction medium. For enzyme extraction, 100 mg of frozen tissues were extracted with ice cold 50 mM potassium phosphate buffer (pH 7.5) containing 5 mM MnSO4 and 1 mM β- mercaptoethanol. Homogenates were centrifuged at 10,000 xg for 20 min at 4°C and the supernatants were used as enzyme extracts. In order to measure the neutral invertase (NINV) activity, 100 μL of extract were added to 800 μL of 50 mM potassium phosphate buffer (pH 7.0) and 100 μL of 500 mM sucrose. The reaction was performed at 37 °C for 30 min and stopped by adding 1 mL of the DNS reagent. The reaction buffer was altered to 50 mM potassium acetate (pH 5.0) to determine the activity of acid invertases (AINV). The NINV and AINV activities were expressed as μmol RS g-1 MS min-1.

4.1. Experimental design and statistical analysis

The experiments were conducted according to a completely randomised design. For metabolite analysis and enzymatic activity, treatments consisted of seven harvest times carried out every 4 h during 24 h, whereas the gas exchange measurements comprised seven samplings performed every 2 h during 12 h. Each treatment consisted of five replicates and the experimental unit consisted of a pot containing one seedling. Results were submitted to polynomial regression analysis using the R version 3.6.1 software.

Results and Discussion ______

5. RESULTS AND DISCUSSION ______

5.1 Results

During 12 h of measurements, it was possible to notice synchrony between the different variables estimated, regarding the gas exchanges (Table 1). The PAR was relatively low from dawn to 07:30 a.m. PAR values increased about 13-fold starting at 09:30 a.m. and it remained elevated in the periods of higher illumination until 03:30 p.m., when it progressively decreased, becoming null at the last measurement.

Table 1 – Daily course of photosynthetically active radiation (PAR), net CO2 assimilation (A), leaf transpiration (E), and water use efficiency (WUE) in the cordiform leaves of E. velutina seedlings during late establishment. Values represent mean ± standard deviation.

PAR A E WUE

-2 -1 -2 -1 –2 –1 -1 TIME μmol m s µmol CO2 m s mol H2O m s µmol CO2 mol H2O

05:30 a.m. 13.2 ± 1.6 -0.60 ± 0.16 1.28 ± 0.15 -0.47 ± 0.13 07:30 a.m. 44.2 ± 7.2 1.22 ± 1.29 2.11 ± 0.23 0.54 ± 0.56 09:30 a.m. 607.2 ± 28.1 7.27 ± 0.47 7.04 ± 0.35 1.03 ± 0.06 11:30 a.m. 656.0 ± 19.8 7.75 ± 0.52 7.66 ± 0.49 1.02 ± 0.12 01:30 p.m. 485.6 ± 66.3 6.42 ± 1.03 6.25 ± 0.25 1.03 ± 0.16 03:30 p.m. 136.8 ± 34.8 0.99 ± 0.93 4.52 ± 0.42 0.23 ± 0.22 05:30 p.m. 0.0 ± 0.0 -1.63 ± 0.03 0.89 ± 0.24 -2.00 ± 0.65

At dawn, A was negative, increasing to a value more than 7-fold higher until 09:30 a.m. and maintained this pattern until 01:30 p.m. (Table 1). From 3:30 p.m., it was noticed a reduction of almost 16% in A, which at the last harvest returned to present negative value. Relating PAR to A, it was observed that the times of higher irradiance coincides with the period of higher A. In addition, when the values of PAR were very low or close to zero, negative values were found in A.

The E was in synchronization with A throughout all the daily course (Table 1). During

2 -1 the first harvest, E was equivalent to 1.28 mol H2O m s and it more than doubled at the second harvest. Between 09:30 a.m. and 01:30 p.m., the E increased approximately 6-fold

23 compared to the first measurement. After this period, E values dropped and, at the last harvest, presented values as low as those observed at dawn.

Considering the negative values of A to both dawn and dusk, the WUE also presented negative values at these moments of the day (Table 1). However, the value measured at 07:30

-1 a.m. augmented to 0.54 µmol CO2 mol H2O and it reached values close to 1.00 µmol CO2

-1 mol H2O between 09:30 a.m. and 01:30 p.m., indicating that seedlings showed high WUE in the period of higher illumination. At 03:30 p.m., following the decrease of A, the WUE

-1 decreases again, reaching 0.23 µmol CO2 mol H2O.

Figure 6 – Diaheliotropic and paraheliotropic movements during daytime in E. velutina cordiform leaves during late establishment.

At the times with greater light supply, that is, of higher irradiance, it was noticed that the cordiform leaves performed a heliotropic movement. During the 05:30 a.m. harvest the pair of leaves was diaheliotropically positioned (Figure 6), when the leaf blade remains perpendicular to sun rays. As the day progressed, the leaf blade gradually changed its position and, in fact, at 01:30 p.m., it presented paraheliotropic movement, regulating the interception of direct solar radiation on the leaf blade, orienting itself parallel of the sun rays. This combination of movements, diaheliotropic and paraheliotropic, in the course of the day allows an escape form direct solar radiation, balancing the absorption of PAR and reducing the loss of water due to transpiration in the periods of higher abiotic stress.

Cordiform leaves and cotyledons, organs considered as sources, demonstrated changes related to the accumulation pattern of the different NSC in the course of 24 h (Figure 7). Although there was a progressive accumulation of starch in both organs during the illumination period and a decrease of its content during the night period, it is notable that the cordiform leaves accumulated starch until 09:30 p.m., reaching values 76% higher than those measured at dawn (Figure 7a) and presented similar value to the dawn of the day before at 01:30 a.m. Additionally, it is important to highlight that the cotyledons presented values about

80% greater than those verified in the cordiform leaves regarding the starch content (Figure 7a, c).

The content of TSS in the cordiform leaves reached a peak at 01:30 p.m. (Figure 7b), when A presented one of the highest values measured during the daily cycle. In the cotyledons, by contrast, this peak occurred at 05:30 p.m. (Figure 7d), when A was very low in the cordiform leaves. Apparently, in these organs the maintenance of the content of NRS occurs both during the day and the night (Figure 7b, d).

Figure 7 – Content of starch (a, c), total soluble sugars, and non-reducing sugars (b, d) in the cordiform leaves (a, b) and in the cotyledons (c,d) of E. velutina seedlings hydroponically cultivated in a greenhouse during the daily cycle. Dots represent means and vertical bars represent standard deviations and ns denotes not significant.

The organs considered as sinks, such as roots, hypocotyl, and epicotyl + first trifoliate leaf, also showed differences regarding the accumulation of NSC throughout the experiment. In the roots, starch content gradually declined during the illumination period (Figure 8 a), starting from 55.8 mg/g DW at 05:30 a.m. and reaching the lowest value at 09:30 p.m., equivalent to 33.6 mg/g DW. From then on, starch content remained almost unchanged until 05:30 a.m. the day after. The opposite occurred in the hypocotyl, because the starch content progressively increased during the day and reached a peak at 09:30 p.m. (Figure 8c), in which the value was more than the double that was measured at dawn. After 09:30 p.m., starch

25 content reduced, reaching 63.00 mg/g DW in the last measurement. In the epicotyl + first trifoliate leaf, from the beginning of the day to 09:30 p.m. there was an accumulation of starch of the order of 53% (Figure 8e). At this time until dawn of the day after, there was a decrease of the starch content, which value was similar to those measured at the beginning of the day before. It is noteworthy that the hypocotyl presented higher starch content related to the others sink organs and the epicotyl + first trifoliate leaf demonstrated an accumulation profile similar to that of the source organs.

Figure 8 – Content of starch (a, c, e), total soluble sugars, and non-reducing sugars (b, d, f) in the roots (a, b), in the hypocotyl (c,d) and in the epicotyl + first trifoliate leaf (c,d) of E. velutina seedlings hydroponically cultivated in a greenhouse during the daily cycle. Dots represent means and vertical bars represent standard deviations and ns denotes not significant.

In the roots, TSS content maintained around 297 mg/g DW during the measured periods, except the 09:30 a.m. harvest, in which the content of these metabolites decreased, reaching the lowest value (Figure 8b). Similarly, in the hypocotyl, TSS content presented values about 285 mg/g DW from dawn to 01:30 p.m., but in the last measurement, at 05:30

26 a.m. the following day, the content dropped 35% in relation to those quantified at dawn of the day before (Figure 8d). TSS content in the epicotyl + first trifoliate leaf exhibited a similar pattern to that of cordiform leaves, showing a peak at 01:30 p.m. and, at the other times, the values measured were decreasing until dawn of the day after, when TSS content was 50% lower than that estimated at 01:30 p.m. (Figure 8f).

The NRS content in the roots augmented gradually from dawn to the last harvest at 05:30 a.m. of the following day, although at the 01:30 p.m. measurement it was observed a decrease of approximately 32% in this content in relation to that measured at the beginning of the experiment (Figure 8b). In the hypocotyl, NRS content increased during the experiment until 01:30 p.m. and it maintained almost unaffected until 09:30 p.m., with values 5-fold higher than those verified at the beginning of the day (Figure 8d). Although there was a reduction of the NRS content until the end of the experiment, the values observed were about 3-fold greater than that measured at the beginning of the previous day. In the epicotyl + first trifoliate leaf, it was observed a gradual increase in the content of NRS until 05:30 p.m., whose value was 4.6- fold greater than that shown at dawn. After this time, NRS content was decreasing until reaching value close to the measured at 05:30 a.m. the day before (Figure 8f).

Figure 9 – Activity of amylases and invertases in the cotyledons (a, b) and in the cordiform leaves (c, d) of E. velutina seedlings hydroponically cultivated in a greenhouse during the daily cycle. Dots represent means and vertical bars represent standard deviation and ns denotes not significant.

Figure 10 – Activity of amylases and invertases in the roots (a, b), in the hypocotyl (c,d), and in the epicotyl + first trifoliate leaf (e, f) of E. velutina seedlings hydroponically cultivated in a greenhouse during the daily cycle. Dots represent means and vertical bars represent standard deviations and and ns denotes not significant.

The activity of amylases did not show significant alterations in the cotyledons (Figure 9a), cordiform leaves (Figure 9c), and roots (Figure 10a) in the course of the experiment, even though the starch content varied in these organs (Figure 7a and c, Figure 8a). Curiously, the activity of these enzymes reached a peak from 01:30 p.m. to 05:30 p.m. in the epicotyl + first trifoliate leaf (Figure 10e) and at 09:30 p.m. in the hypocotyl (Figure 10c), coinciding with starch accumulation (Figure 8c and e). For example, the activity of amylases increased 90% from 05:30 a.m. to 01:30 p.m. in the epicotyl + first trifoliate leaf (Figure 10e) and it augmented 60% starting at 05:30 p.m. to 09:30 p.m. in the hypocotyl (Figure 10c).

In comparison with the activity of amylases, the activity of AINV and NINV showed higher variations during day-night cycle in the different seedling organs (Figures 9 and 10). Indeed, the activity of both enzymes exhibited two peaks in the cordiform leaves (Figure 9d), epicotyl + first trifoliate leaf (Figure 10f), and roots (Figure 10b) in the course of the experiment; the first peak occurred under high PAR and the second happened in the dark. It is worthy to mention that the activity of both invertases decreased when A was still high in the cordiform leaves (Table 1) and TSS was accumulated in the cordiform leaves (Figure 7b) and epicotyl + first trifoliate leaf (Figure 8f). In the cotyledons (Figure 9b) and hypocotyl (Figure 10d), the activity of NINV did not show significative alterations during harvests. On the other hand, the activity of AINV was high at dawn in the cotyledons (Figure 9b), whereas it decreased under high PAR and reached the peak at 09:30 p.m. in the hypocotyl (Figure 10d), coinciding with starch accumulation (Figure 8c).

5.2. Discussion

Despite the NSC are a topic widely discussed in the literature due to its fundamental role in plant metabolism and in plant performance under natural conditions (VILLAR- SALVADOR et al., 2015; MAGUIRE and KOBE, 2015), this topic has been revisited because many gaps about the knowledge regarding the accumulation and utilization of NSC among the different plant organs during the daily course still persist. Even though some efforts have been made to elucidate these gaps in plants during vegetative growth (GRAF et al., 2010; FARRÉ and WEISE, 2012; HAYDON et al., 2013), less is known about the circadian accumulation and utilization of NSC during the transition from heterotrophy to autotrophy that enable seedling establishment. In this sense, the work shown here presents an approach that seeks to integrate gas exchanges in the first leaves and the NSC contents in different organs, in order to comprehend how the carbon dynamics is involved in this phase transition in E. velutina.

The pattern of A (Table 1) in the cordiform leaves of E. velutina seedlings during the day is similar to those observed in others tropical woody species. For example, in the leaves of Annona x atemoya (BARON et al., 2018) and Hevea brasiliensis (NÓIA-JÚNIOR et al., 2018), the photosynthetic activity presented higher values in the morning, decreasing around noon, when the illumination conditions are more severe. In comparison, in woody species of temperate climate, such as Fagus sylvatica, Quercus petraea (ARANDA; GIL; PARDOS, 2000) and Quercus suber (TENHUNEN et al., 1984), the decreasing of the photosynthetic activity occurs later, accompanying the light dimming. Therefore, more studies may characterize this pattern of photosynthetic activity verified in tropical woody species as a response related to the reduction of possible damages during the most stressful periods of the day (SANTOS et al., 2014a).

Although the decreasing of A has been registered at 03:30 p.m., there was no concomitant decreased in E (Table 1), so it is not possible to attribute the reduction of A to stomata closure, but it might be linked to the decreasing of PAR. Opposite results were found in other species from warm climates, such as Cynophalla flexuosa, Poincianella pyramidalis (SANTOS et al., 2014c), Oenothera biennis (KOYAMA; TAKEMOTO, 2015), and Ziziphus joazeiro (SANTOS et al. 2014b), in which the limitation of the photosynthetic activity correlates with the reduction of E and the stomatal conductance.

Plants can also use photoprotection strategies to balance the light absorption with its use. Light capture can be modulated by adjusting leaf interception, light penetration in chloroplasts and by leaf positions in relation to the sun rays (DEMMIG-ADAMS; ADAMS, 2018). In the legume Styrax camporum (HABERMANN et al., 2008), it was demonstrated that A and E were higher in paraheliotropic leaves (parallel to sun rays) than in diaheliotropic leaves (perpendicular to sun rays), indicating that the former can prevent damages to photosynthetic activity in times of high illumination. During the measurements, it was noticed that, at times of high PAR, the cordiform leaves presented paraheliotropism (Figure 8). In a previous work, it was described the presence of pulvinus in the base of its cordiform leaves, which are involved in tropic movements (SILVA et al., 2008). Thus, the paraheliotropism may have been a response to high irradiance to reduce possible damages to the photosynthetic apparatus, explaining, at least in parts, why it is possible to maintain high A under this condition.

The WUE indicates the amount of biomass produced per unit of water used. Stomatal conductance (gs) is primarily responsible for regulating WUE and, in moderate water stress conditions, generally increases due to low gs and E, as described in wheat (OWEIS; ZHANG; PALA, 2000) and in bean (ARAÚJO, 2015). However, under severe water deficit, WUE can decrease (TAMBUSSI; BORT; ARAUS, 2007). Surprisingly, in E. velutina seedlings during late establishment, in the moments of the day under high PAR it occurs an increase in WUE (Table 1), which it maintained almost unaffected from 09:30 a.m. to 01:30 p.m. Conversely, in other species, such as the legume Styrax camporum, a common shrub species from Cerrado (HABERMANN et al., 2008) and in Annona x atemoya (BARON et al., 2018), a tropical woody species, WUE decreases in the moments of high PAR. In this way, it seems that the gs adjustement combined with paraheliotropism in the cordiform leaves might be involved in a mechanism that allows to increase WUE in the moments of higher exposition to water stress in E. velutina seedlings during late establishment.

Considering that plant growth is based on the products from photosynthetic activity, the NSC play a central role in the responses given by plants to environmental stresses and they are closely related to their performance under natural conditions (VILLAR-SALVADOR;

USCOLA; JACOBS, 2015; MAGUIRE; KOBE, 2015). In dicots, starch and soluble sugars are the main NSC. Starch mostly plays the function of reserve to support metabolism and to the night-time growth (GRAF et al., 2010), whereas soluble sugars develop diverse functions, mainly the continuous supply of carbon and energy required to metabolism maintenance (MARTINEZ-VILALTA et al., 2016). In leaves, it is well reported that carbon allocation for starch synthesis during daytime, as well as its degradation in order to maintain the growth during night-time, is regulated by circadian rhythm (GREENHAM; MCCLUNG, 2015).

In E. velutina seedlings there is an accumulation of starch and TSS in the organs considered as sources, the cordiform leaves and cotyledons, during the period of high illumination (Figure 7), extending beyond this period. In these organs, starch contents at the dawn of the first and second days are similar, demonstrating a constancy of this content, being a temporary reserve that seems to remain within a threshold, avoiding its exhaustion (MARTINEZ-VILALTA et al., 2016). A pattern similar to that of E. velutina was verified in young plants of Ceiba pentandra, a tree of fast growth, native to South and Central America and Western Africa (OREN et al., 2019). In Arabidopsis, it is clear that the starch degradation rate in leaves at night is fundamentally linear and basically all starch is consumed until dawn (GRAF et al., 2010). Similarly, in Prunus dulcis leaves (TIXIER et al., 2018), there is an accumulation of starch during the day and starch is degraded until reaches the exhaustion at night-time.

In the cordiform leaves, NRS content (Figure 7b) remained almost unchanged in the course of the day, but a different pattern was noticed in the cotyledons (Figure 7d), in which there was a peak of these metabolites in the period of higher PAR, indicating an accumulation of the products from photosynthetic activity. TSS content in the cotyledons remained almost unaffected during all the day, being the metabolites that presented more stable values from dawn of the first day until dawn the following day. However, in the cordiform leaves, there was a peak of TSS during the period of higher illumination, indicating a response to the photosynthetic activity. Similar pattern was verified in leaves of young plants of Ceiba pentrandra (OREN et al., 2019), and an opposite pattern was observed in leaves of Prunus dulcis (TIXIER et al., 2018), whose sugar content remained unaltered in the course of the day. In the cotyledons, the increase of NRS is probably related to a limited photosynthetic activity and/or mobilization of storage lipids (data not shown). These results suggest that it might exist a minimum threshold to the content of starch and soluble sugars in source organs, indicating that seedlings utilize these metabolites in a way that the exhaustion does not occurs in these organs, especially at night.

Although the epicoyl + first trifoliate leaf can be comprehended as sink organs, they showed a profile similar to source organs, namely cordiform leaves and cotyledons, related to

NRS accumulation (Figure 8e, f). Even though, in E. velutina seedlings, these organs did not reach the source condition, since the complete expansion of the first trifoliate leaf did not occur yet, it is possible to verify that the NSC dynamic is more similar to those in sources than those observed in sinks. Considering that the accumulation of TSS and NRS in epicotyl + first trifoliate leaf (Figure 8f) is synchronized with the photosynthetic activity in the cordiform leaves, epicotyl + first trifoliate leaf might play a role as a minor source during the transition from sink to source in the late establishment of E. velutina.

It is relevant to emphasize that on the day before the harvests the climate conditions were cloudy, while the harvest was carried out in a sunny day. This condition may have influenced the NSC profile in roots and hypocotyl. Since the root is the sink most distant from cotyledons and cordiform leaves, it was possible that the diurnal degradation of starch (Figure 8a) might be a strategy utilised to the maintenance of TSS and NRS levels in this organ (Figure 8b) until the photosynthates from the assimilation during the measurements can be translocated. Indeed, during the following night there was a stabilisation of starch stocks in the roots (Figure 8a). The opposite is observed in roots of Ceiba pentandra (OREN et al., 2020), cultivated in a greenhouse, and Macroptiliut lathyroides (ASANO et al., 2000), cultivated in pots in an experimental field, in which occur an increase in the content of starch in the course of the day and degradation during the night.

Regarding the accumulation of starch and NRS in sink organs during the harvests, the roots and hypocotyl presented antagonistic responses. In opposition to what was mentioned about the root, there was an accumulation of starch and NRS in the hypocotyl during the day (Figure 8c, d). Taking into account that the hypocotyl is a sink closer to cotyledons and cordiform leaves, it is reasonable to suggest that the starch synthesis throughout the day and, most of all, during the night is a response to the availability of NRS from photosynthetic activity and/or reserve mobilisation. Thus, the increase of starch content at the end of the day and at the beginning of night is probably related to a strategy that avoids the accumulation of TSS as a mechanism to escape an osmotic effect that could potentially alter the water relations of the hypocotyl (MARTINEZ-VILALTA et al., 2016).

The amylases are essential in the process of starch depolymerisation in free sugars, which are utilised in the metabolism as energy source for the plant maintenance and development (BEWLEY et al., 2013). The α-amylases (endoamylases) randomly hydrolise the (α1→4) glycosidic linkages in the extension of amylose and amylopectin polymers producing branched and linear oligosaccharides such as malto-oligossacharides and dextrins. The β- amylases and phosphorylases (exoamylases) catalyse, successively, the hydrolysis of

32 nonreducing ends in (α1→4) glycosidic linkages, releasing maltose and glucose-1-phosphate, respectively (HELDT; PIECHULLA, 2011).

Despite the activity of amylases in E. velutina seedlings, the enzymes are not activated in all moments of the day in some organs. It is notable that the activity of amylases remained almost unchanged in the cotyledons (Figue 9a), cordiform leaves (Figure 9c), and roots (Figure 10a) of seedlings throughout the experiment, even though the content of starch varied during this period in these organs (Figures 7a and c, 8a). In cordiform leaves the activity of the enzyme was continuous in the course of the day and showed an expression more active only at dawn. Considering that amylases play a fundamental role in transitory starch degradation in photosynthetic organs and starch plays a role as food reserve in heterotrophic organs (SMIRNOVA et al., 2015), it was expected that its activity increases when starch is degraded. As this trend is not observed in the results obtained, it is possible that the activity of amylases in the cotyledons, cordiform leaves, and roots is regulated by post-transcriptional mechanisms. Regardless of these mechanisms are still poorly understood, there is evidence that amylases can undergo redox regulation mediated by thioredoxins (SKRYHAN et al., 2018) and allosteric modulation promoted by sugars (LI et al., 2017). On the contrary, the activity of amylases increases in the epicotyl + first trifoliate leaf (Figure 10e) and in the hypocotyl (Figure 10c) when an accumulation of starch in these organs occurs (Figure 8c and e). This counterintuitive results may be explained by the probable action of isoamylases during the production of starch granules. These enzymes are responsible for trimming excessive ramifications produced during amylopectin biosynthesis, allowing it to be compacted in concentric layers (BEWLEY et al., 2013). In epicotyl + first trifoliate leaf, there is an increase of all measured parameters (TSS, NRS, and starch), indicating strongly that there is a circadian influence. The increase of the activity of amylases in these organs probably is because of an augment of isoamylase activity, due to the contribution of hydrolytic enzymes in the formation of starch granules BEWLEY et al., 2013).

Although the activity of amylases has not shown typical circadian variations in the different parts of E. velutina seedlings, the activities of acid and neutral invertases demonstrated synchronized changes in the cordiform leaves (Figure 9d), epicotyl + first trifoliate leaf (Figure 10f), and roots (Figure 10b) in the course of 24 h. Invertases are the main enzymes that degrade sucrose, and acid invertases are found in the cell wall (CWINV) and in the vacuole (VINV), whereas neutral invertases occurred mainly in the cytosol (CINV). CWINV are involved in the phloem apoplastic unloading and are being considered as sink strength markers, VINV plays some role in cellular growth and CINV are implicated in sugar homeostasis (WAN et al., 2018). Taking into account that the activities of acid and neutral invertases reach a first peak during the daytime and a second at night in the abovementioned

33 organs of E. velutina seedlings, these activities may be regulated at transcriptional level. There are evidences that invertases are targets of transcriptional regulation mediated by light, sugars and hormones (LI et al., 2017b), and also a probable connection with the circadian clock (PROELS and HÜCKELHOVEN, 2014). In the cordiform leaves (Figure 9b) and in the epicotyl + first trifoliate leaf (Figure 10f), the activities of acid and neutral invertases decreased under high irradiance (Table 1), when there is an accumulation of TSS in these organs (Figures 7b and 8f); in the roots, the activities of these enzymes decrease in parallel with starch degradation (Figure 8a). Therefore, it is possible that sugar availability affects the activity of acid and neutral invertases in these organs. In the cotyledons (Figure 9b) and hypocotyl (Figure 10d), the activity of NINV does not show a daily pattern of variation. Moreover, the activity of AINV is lower in the cotyledons than other organs (Figures 9 and 10), indicating that the cotyledons may still act as sources during late establishment. In the hypocotyl, the increase of AINV activity (Figure 10d) coincides with starch accumulation (Figure 8c) and, thus, is probable that these enzymes have contributed to sucrose capture and carbon storage.

Conclusions

______

5. CONCLUSIONS ______

It is possible to conclude that the heliotropic movements of the cordiform leaves can contribute to the maintenance of photosynthetic activity and the increase of WUE under high irradiance. There is evidence that NSC dynamics is influenced by the circadian rhythm in the photosynthetic organs and that the starch synthesis-degradation daily cycle operates far from exhaustion in these organs. In the heterotrophic organs, NSC dynamics appears to be clearly related to buffering variations in carbon availability. Although the starch synthesis-degradation daily cycle in the main source organs (cotyledons and cordiform leaves) is not linked to circadian alterations in the activity of amylases, the activity of invertases shows a daily pattern of variation that coincides in the cordiform leaves, epicotyl + first trifoliate leaf and roots of E. velutina seedlings.

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