SÃO PAULO STATE UNIVERSITY – UNESP JABOTICABAL CAMPUS THE ROLE OF PHYTOCHROME B1 OF TOMATO (Solanum lycopersicum L.) IN THE REPRODUCTIVE STAGE DURING DROUGHT STRESS Carlos Alberto Silva Junior Agronomist engineer 2021 SÃO PAULO STATE UNIVERSITY – UNESP JABOTICABAL CAMPUS THE ROLE OF PHYTOCHROME B1 OF TOMATO (Solanum lycopersicum L.) IN THE REPRODUCTIVE STAGE DURING DROUGHT STRESS MSc Student: Carlos Alberto Silva Junior Advisor: Prof. PhD. Rogério Falleiros Carvalho Co-advisor: Prof. PhD. Luiz Fabiano Palaretti Dissertation submitted to the School of Agricultural and Veterinary Sciences – Unesp, Campus of Jaboticabal, in partial fulfillment of the requirement for the degree of Master of Science in Agronomy (Crop production) 2021 S586r Silva Junior, Carlos Alberto The role of phytochrome B1 of tomato (Solanum lycopersicum L.) in the reproductive stage during drought stress / Carlos Alberto Silva Junior. -- Jaboticabal, 2021 88 p. : il., tabs., fotos Dissertação (mestrado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal Orientador: Rogério Falleiros Carvalho Coorientador: Luiz Fabiano Palaretti 1. Fisiologia vegetal. 2. Fotomorfogênese. 3. Deficit hídrico. 4. Estresse vegetal. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Jaboticabal THE ROLE OF PHYTOCHROME B1 OF TOMATO (Solanum lycopersicum L.) IN THE REPRODUCTIVE STAGE DURING DROUGHT STRESS TÍTULO DA DISSERTAÇÃO: CERTIFICADO DE APROVAÇÃO AUTOR: CARLOS ALBERTO SILVA JUNIOR ORIENTADOR: ROGÉRIO FALLEIROS CARVALHO COORIENTADOR: LUIZ FABIANO PALARETTI Aprovado como parte das exigências para obtenção do Título de Mestre em AGRONOMIA (PRODUÇÃO VEGETAL), pela Comissão Examinadora: Prof. Dr. ROGÉRIO FALLEIROS CARVALHO (Participaçao Virtual) Departamento de Biologia Aplicada a Agropecuaria / FCAV UNESP Jaboticabal Prof. Dr. LUCAS APARECIDO GAION (Participaçao Virtual) UNIMAR / Marília/SP Profa. Dra. MARINA ALVES GAVASSI (Participaçao Virtual) Instituto de Biociências de Rio Claro-IB-UNESP / Rio Claro/SP Jaboticabal, 26 de outubro de 2021 Faculdade de Ciências Agrárias e Veterinárias - Câmpus de Jaboticabal - Via de Acesso Professor Paulo Donato Castellane, s/n, 14884900, Jaboticabal - São Paulo https://www.fcav.unesp.br/#!/pos-graduacao/programas-pg/agronomia-producao-vegetalCNPJ: 48.031.918/0012-87. P/ P/ P/ AUTHOR’S CURRICULUM INFORMATION Carlos Alberto Silva Junior – was born to Carlos Alberto Silva and Odilza Ana da Costa Silva, on January 4, in 1995, in Cuiabá, Mato Grosso, Brazil. He joined the Agronomy course at “Federal University of Mato Grosso – Cuiabá campus” in November 2012. During his graduate course, he was monitor in the discipline of forage production from the Department of Animal Science and Rural Extension in 2016. He was member of study groups that aimed to discuss the most advanced knowledge published in papers, such as NERP (Ruminants and Pasture Studies Nucleus) and NESA (Agronomic Solutions Studies Nucleus). He was a Scholarship holder in the scientific initiation modality by the National Council for Scientific and Technological Development (CNPq) during three consecutive years, from 2014 to 2017. In the last semester, he did an agricultural internship in the United States of America, where lived and worked in a livestock farm named Reynolds Livestock between May 2017 to May 2018 and participated in lectures at Iowa State University. From November 2018 to February 2019, he carried out a curricular internship in a soybean farm located in Tabaporã – Mato Grosso, named Lunil Farms I. He obtained the title of Agronomist Engineer in May 2019. In August 2019, he started his master’s course in the Graduate Program in Agronomy (Crop Production), at the “São Paulo State University – Jaboticabal campus”. The master’s project was funded by CNPq and São Paulo Research Foundation (FAPESP), under the advising of Prof. PhD. Rogério Falleiros Carvalho and co-advising of Prof. PhD. Luiz Fabiano Palaretti. “Explaining nature is too difficult a task for any man or for any time. It's much better to do a little and definitely leave the rest to the others who come after you” Isaac Newton DEDICATION I dedicate this work to my parents, Carlos Alberto Silva and Odilza Ana da Costa Silva, my endless sources of inspiration, for all the support, care, and advice during my journey. ACKNOWLEDGMENT To God, that no matter what, always provided me the strength required to keep the focus in the way. To Prof. PhD. Rogério Falleiros Carvalho, for all knowledge, friendship, support, guidance, and for rose in myself the reading habit, a legacy that a have passed to all people around me. To Prof. PhD. Luiz Fabiano Palaretti, for all knowledge, friendship and support in the experiment. To “São Paulo State University (UNESP) – School of Agricultural and Veterinary Science of Jaboticabal (FCAV)” and all employees, for providing the infrastructure and material needed to conduct the work. To the National Council for Scientific and Technological Development (CNPq, 148342/2019-1) and São Paulo Research Foundation (FAPESP, 2019/25737-2) for their grant of master level scholarship. To the professors of the Graduate Program in Agronomy (Crop Production) at UNESP/FCAV that humbly transferred their huge knowledge to contribute to my scientific formation. To the Dissertation Defense Examining Board members, Lucas Gaion and Marina Gavassi for accepting our invitation and for their contribution to the work. To my graduate classmates and friends Reginaldo de Oliveira, Daniel Dalvan, Jéssica Caixeta, Mariana Bomfim, Maria José, Marcilene Machado, Jonathan Viana, Regiara Croelhas, Eduarda Reis and João Carlos who were crucial to overcoming the difficulties, helping me in the academic activities, for the distracting moments, and friendship, filling the room left for family distance. To my family, my safe harbor, that even far away, was always present in every single moment, giving their support with everything I needed, encouraging and motivating me to go after my dreams. i SUMMARY Page ABSTRACT .................................................................................................................. iii CHAPTER 1 – Phytochrome type B family: the abiotic stress response signaller in plants ... 1 1.INTRODUCTION ........................................................................................................ 1 2.LITERATURE REVIEW ............................................................................................... 3 2.1. Drought stress .................................................................................................. 3 2.2. Salt stress ........................................................................................................ 9 2.3. Low and high-temperature stress ..................................................................... 13 2.4. High light stress .............................................................................................. 15 2.5. Heavy metal stress ......................................................................................... 22 2.6. Epilogue ........................................................................................................ 23 3.REFERENCES ......................................................................................................... 24 CHAPTER 2 - Phytochrome B1 type of tomato (Solanum lycopersicum L.) modulates responses to drought stress in vegetative and reproductive stage growth .......................... 42 1.INTRODUCTION ...................................................................................................... 43 2.MATERIAL AND METHODS ...................................................................................... 45 2.1. Plant material and growth conditions ................................................................ 45 2.2. Experimental design and irrigation treatment ..................................................... 46 2.3. Determination of vegetative parameters ............................................................ 47 2.4. Determination of reproductive parameters ......................................................... 47 2.5. Fruit production, quality and morphometric analysis ........................................... 48 2.6. Data analyses ................................................................................................ 48 3.RESULTS ................................................................................................................ 49 3.1. Water deficit stress in vegetative growth stage .................................................. 49 3.1.1. Vegetative growth analysis ............................................................................. 49 3.1.2. Reproductive parameters................................................................................ 52 3.1.3. Fruit production, quality and morphometric analysis ....................................... 52 3.2. Water deficit stress in reproductive growth stage ............................................... 55 3.2.1. Vegetative growth analysis ............................................................................. 55 3.2.2. Reproductive parameters................................................................................ 58 3.2.3. Fruit production, quality and morphometric analysis ....................................... 58 4.DISCUSSION ........................................................................................................... 61 ii 4.1. Responses to drought in early developmental stages are mediated by phyB1 ...... 61 4.2. Drought stress in reproductive stage changes source-sink partitioning by phyB1 dependent manner ................................................................................................... 66 5.CONCLUSION .......................................................................................................... 68 6.REFERENCES ......................................................................................................... 69 APPENDICES ............................................................................................................. 75 Appendix A. Supplementary material for chapter 2 ...................................................... 76 iii THE ROLE OF PHYTOCHROME B1 OF TOMATO (Solanum lycopersicum L.) IN THE REPRODUCTIVE STAGE DURING DROUGHT STRESS ABSTRACT – Photoreceptors are primarily known as key photomorphogenic modulators of various physiological events during plant development. Although there are different groups of photoreceptors, the phytochrome B (phyB) family mediates developmental responses in a wide range of plant species, from seed germination to flowering. In addition, these molecules also regulate abiotic stress acclimation responses, such as salinity, drought, low/high temperature, high light, and heavy metals. The signalling pathways mediated by phyB could enhance plant resistance to environmental stresses, as phyB mutants reduced leaf transpiration through lowering of stomatal conductance, increased the antioxidant system, enhanced protective pigments, and increased the expression of genes related to plant stress acclimation. Therefore, the scope of chapter one is to compile and discuss the evidence on abiotic stress response in plants that are modulated by the phytochrome type B family. In addition, chapter two aimed to elucidate the responses mediated by phyB1 in tomato fruit production comparing the effects of drought stress in vegetative and reproductive stages. The water deficit treatment was performed in two different stages growth: in vegetative (start at 26 days after sowing [DAS]); end at 36 DAS) and reproductive (start at 33 DAS; end at 41 DAS) stage growth. Keywords: fruit yield, fruit quality, evapotranspiration, red-light influence, source-sink relationship, water deficit 1 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 CHAPTER 1 – Phytochrome type B family: the abiotic stress response signaller in plants 1. INTRODUCTION Light controls plant development via complex photoreceptor systems that perceive different wavelengths of light. The light causes changes in photoreceptor molecule conformation, allowing it to signal to the nucleus and regulate transcriptional activity of the genome (Voitsekhovskaja, 2019). The activation of photosensory receptors by light triggers responses in several processes during the plant life cycle from seed germination to flowering (Casal, 2013). For example, cryptochromes and phototropins are photoreceptors that primarily detect wavelengths of blue light (300- 500 nm) (Christie et al, 2015; D’Amico-Damião and Carvalho, 2018) and were reported as a typical regulator of flowering (Guo et al., 1998) and stomata opening (Inoue et al., 2010), respectively. The photoreceptor UVR8 (UV resistance locus 8) perceives and signals ultraviolet (UV)-B light, modulating plant growth under UV-light. UVR8 interacts with the E3 ubiquitin ligase complex that is also regulated by other photoreceptors (e.g., constitutive photomorphogenic 1 [COP1]), mediating responses, such as hypocotyl length reduction, under UV-B light (Favory et al., 2009). However, the phytochrome (phys) family contains well-characterised photoreceptors, showing absorption peaks in red (R; ~660 nm) and far-red (FR; ~ 730 nm) light wavelengths. Phytochromes are dimeric proteins (~124 kDa) (Jones and Quail, 1986) covalently linked to a phytochromobilin, which is a linear tetrapyrrole with an open chain that acts as a chromophore (Rockwell et al., 2006). Studies concerning phys began in the 1950s, showing that R irradiation induced germination of lettuce (Lactuca sativa) seeds, while FR reversed this induction (Borthwick et al., 1952). The interconvertible property of phys triggers plant responses with the active (Pfr – Phytochrome far-red) and inactive (Pr – Phytochrome red) form present after exposure to R and FR, respectively (Rockwell et al., 2006). Some decades latter phy gene sequences were described in Arabidopsis (Arabidopsis thaliana) plants and five phys genes were identified, including PHYA, PHYB, PHYC, PHYD and PHYE that encode their own apoproteins (PHYA to PHYE) (Clack et al., 1989). Biosynthesis of phyA to phyE occurs when each apoprotein binds to its respective chromophore. Phylogenetically, phy 2 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 arose from gene duplication of an ancestral phytochrome gene, but among plant species, different phy types can be found and have not undergone the same phylogenetic evolution. Since their split 140-200 million years ago (Mya), monocots and dicots began evolving independently, and presented separation of PHYA from PHYC and PHYB/D from PHYB/E (Alba et al., 1997; Mathews and Sharrock, 1997). For example, phyA to phyC is present in rice plant (Oryza sativa) (Dehesh et al., 1991) and phyA, phyB1, phyB2, phyE and phyF in tomato (Solanum lycopersicum) (Alba et al., 2000). Briefly, some phytochrome subfamilies control similar traits, such as the primary function in seedling de-etiolation of Arabidopsis and tomato mediated by both phyA and phyB (Casal, 2013). However, the individual role of phys, even in the same species, seems to be truly intricate because phyB1 and the homologous phyB2 originated by gene duplication and have non redundant roles. For example, in maize (Zea mays), phyB1 plays a substantial role in seedling traits (e.g, hypocotyl length) and phyB2 is fundamental to repress flowering under long-day (LD) photoperiods (Sheehan et al., 2007). In tomato, phyB1 showed a major role in light and auxin responses (gravitropism and phototropism), while phyB2 acted antagonistically to phyB1, promoting photosynthesis (Carlson et al., 2020). Thus, regarding these multifaceted aspects of the phyB family, it is not a surprise that the responses modulated by these photoreceptors include complex signalling pathways. The activation of phy causes changes in molecule conformation that triggers the phy-regulated photomorphogenic responses. Upon light excitation, the phy molecule suffers an isomerization, which induces a conformation change that exposes nuclear localization sequences allowing the active phys (Pfr) to migrate to the nucleus (Hiltbrunner et al., 2005). When accumulated in the nucleus, Pfr inhibits the transcription of protein groups that act as repressor of photomorphogenesis (e.g., COP/DET/FUS, constitutive photomorphogenic/deetiolated/FUSCA; suppressor of PHYA1 [SPA1]; and phytochrome interacting factors [PIFs]) (Favero, 2020; Lau and Deng, 2012; Leivar and Quail, 2011; Xu et al., 2015), allowing the accumulation of many transcriptional factor (TIFs). Thus, these TIFs promote photomorphogenesis (e.g., HY5 – elongated hypocotyl 5) and trigger the expression of various genes, which leads to the modulation of phytochrome-mediated traits as cited above (Huang et al., 3 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 2014; Balcerowicz, 2020). Recently, it has been observed that responses to abiotic stress are related to photomorphogenesis repressors, such as COP1 and PIF (Kim et al., 2016; Qiu et al., 2020). Thus, it is not surprising that phys were previously described to regulate responses to diverse abiotic and biotic stresses (Carvalho et al., 2011a) such as salt stress (Indor et al., 2007), drought stress (Kraepiel et al., 1994), low and high-temperature stress (Williams et al., 1972; Foreman et al., 2011) and high light (Wellmann et al., 1984). Highlighting the phy type B family show some similar responses among species and many research groups have targeted their efforts to understand phyB-modulated stress responses, because such responses could be altered in the same species, e.g., phyB1 and phyB2 gene duplication controlling different stress acclimation responses (Arico et al., 2019; Gavassi et al., 2017; Kreslavski et al., 2015; kwon et al., 2018; Yoo et al., 2017). Therefore, in this review we summarized the phyB-modulated stress responses to elucidate future research for plant breeding. 2. LITERATURE REVIEW 2.1. Drought stress Drought stress is a major limiter of crop production and has become even more worrying because of rapid global climate changes in recent decades (Lesk et a., 2016; Garcia e al., 2020). Plants naturally display resistance mechanisms that help them to cope with drought conditions, such as alteration in morphological, physiological, and molecular characteristics (Hussain et al., 2018). The mechanisms could be classified as escape strategies (e.g., earlier flowering) and drought tolerance (e.g., transpiration rate, osmoprotectant content and root-to-shoot ratio) (Meyre et al., 2001). Studies of physiological adaptive changes to drought suggest that phy modulates water stress responses since a former study using Nicotiana tabacum mutants deficient in phy chromophores (Kraepiel et al., 1994) and later studies using Arabidopsis phyB mutants (Boccalandro et al., 2009; Boggs et al., 2010) showed changes in the control of leaf transpiration. In addition, leaf water loss is highly correlated with stomatal conductance and its negative regulator abscisic acid (ABA) (Kriedemann et al., 1972). In some species, studies have corroborated the hypothesis that stomatal conductance is 4 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 negatively regulated by phyB in drought stress conditions. For example, in Arabidopsis (Boggs et al., 2010; González et al., 2012) and tomato (D’Amico-Damião et al., 2015), phyB mutants (phyB1 and phyB2 in tomato) presented lower stomatal conductance than the wild-type (WT) in drought stress conditions and this may be related to the induction of ABA synthesis in Arabidopsis (Boggs et al., 2010; González et al., 2012). However, González et al. (2012) attributed the lower drought tolerance of Arabidopsis phyB mutants to lower ABA sensitivity, instead of ABA content, as the WT and phyB mutants showed similar ABA levels. Moreover, the reduction in expression of ABA- signalling genes, such as ABCG22 (ATP-binding cassette) (Kuromori et al., 2011) and PYL5 (family of pyrabactin resistance) (Santiago et al., 2009; Park et al., 2009), in phyB mutants (González et al., 2012) likely influence the lower sensitivity of phyB mutants to ABA. Taken together, these reports show that phyB plays an important role in droght stress tolerance and in the avoidance of water-loss via stomatal closure. However, stomatal closure followed by decreased conductance may also reduce the photosynthetic rate, as shown by Boccalandro et al. (2009). These authors observed that Arabidopsis phyB mutants exhibited lower stomata density, resulting in a lower transpiration rate that decreased the CO2 uptake. Although drought-tolerance improvement via the reduction of water-loss seems positive, the lower CO2 uptake may be dangerous for plants under high photosynthetic active radiation exposure. For example, the excess of excitation energy not used in the photochemistry step of the photosynthesis reaction (because of low CO2 concentrations) may be transferred to molecular oxygen, synthesizing harmful reactive oxygen species (ROS) if not sufficiently dissipated by the fluorescence or heat (Niyogi, 1999). These studies suggest that phyB mutants would be susceptible to drought stress. However, other studies showed that phyB was part of other signalling pathway in the drought stress response, such as the antioxidant system. Indeed, ascorbate peroxidases (APXs) and catalases (CATs) were upregulated in rice phyB mutant genotypes in drought stress conditions compared to the WT, and this upregulation had a positive effect on the phyB mutant plants, which acquired a drought-tolerance phenotype (Yoo et al., 2017). 5 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 These elucidations may contribute to the maintenance of grain yield in rice phyB mutants, even with a decreased photosynthesis rate. Liu et al. (2012) found a lower stomatal density and net CO2 uptake with no effect on rice production in phyB mutant drought-stressed plants. phyB may also modulate drought tolerance in tomato plants, specifically the phyB1 mutant. Gavassi et al. (2017) exposed 7-day-old seedlings of tomato to a low water potential solution and detected higher shoot and root lengths in phyB1 mutants compared to WT, as well as higher content of proline and glycine- betaine. Proline and glycine-betaine are helpful osmoprotectants that actively support the osmotic regulation of cells, which helps plants cope with drought injury (Abro et al., 2019). The aforementioned escape strategy also contributes to give importance to phyB as drought stress mediator. Rice phyB mutants exhibited earlier flowering than the WT after water-deficit treatment and exhibited the two mechanisms of perception, both associated with flowering-related gene expression, but some were ABA- dependent and others were ABA-independent (Du et al., 2018). Taken together, the majority of data suggests that phyB is a negative regulator of drought tolerance acting on different mechanisms (Table 1), such as the antioxidant system, accumulation of osmoprotectants, stomatal movement, regulation of phytohormones and expression of stress-related genes. 6 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 Table 1 – Summarized data of characteristic phyB-regulated in salt stress, drought stress and low temperature stress. SALT STRESS Plant specie Treatment Type of phyB Effect of phyB on plant (phyB1 in tomato). References Nicotiana tabacum 800 Mm NaCl phyB Increase: MDA, eletrolyte leakage. Yang et al. (2018) Reduce: Chlorophyll, proline, antioxidant activity, Net photosynthesis rate, ABA content, JA content. Tomato 100 nM NaCl phyB1 and phyB2 Increase: MDA. Gavassi et al. (2017) Reduce: root lenght, chlorophyll, carotenoids and proline 100 nM NaCl phyB1 Increase: MDA, H2O2. Cao et al. (2018) Reduce: Chlorophyll, proline, antioxidant activity, Net photosynthesis rate. Rice 200 Mm NaCl phyB Increase: Na+ content, Na+/K+ ratio. Kwon et al. (2018) Reduce: Chlorophyll, OsHTKs expression, Fresh weight, survival rate. 7 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 DROUGHT STRESS Arabidopsis 4-6% volumetric water content phyB Increase: stomatal conductance Boggs et al. (2010) Reduce: ABA concent. Arabidopsis 7 days without watering phyB Increase: stomatal conductance, ABCG22 and PYL5 expression, drought tolerance phenotype. González et al. (2012) Tomato 5 days without watering phyB Increase: stomatal conductance. D’Amico- Damião et al. (2015) Tomato PEG at Ψw of −0.3 MPa phyB1 and phyB2 Increase: MDA. Gavassi et al. (2017) Reduce: shoot lenght, Root lenght, chlorophyll, carotenoids, proline, glycine-betaine. Rice 16 days without watering phyB Increase: leaf area, stomatal density, transpiration rate, Net CO2 uptake. Liu et al. (2012) Reduce: proline content, recovery plants. Rice 4 days without watering phyB Increase: H2O2. Yoo et al. (2017) Reduce: antioxidant activity. Rice 10-20% soil water content phyB Increase: days to flowering. Du et al. (2018) 8 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 LOW TEMPERATURE STRESS Arabidopsis Cold (-9°C) for 1h or cold (- 5°C) for 0,5h phyB Increase: survival rate, CBF expression, COR expression. Jiang et al. (2020) Reduce: ion leakage Tomato Cold (4°C) for 7 d phyB Increase: eletrolyte leakage. Wang et al. (2016) Reduce: survival rate, CBF1 expression, COR expression, ABA, JA. Rice Cold (4°C) for 4 d phyB Increase: MDA, eletrolyte leakage. He et al. (2016) Reduce: survival rate, CBF/DREB1s expression. Rice Cold (4°C) for 24 h phyB Increase: photoinibition. Yang et al. (2013) Reduce: USFA, chlorophyll. 9 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 2.2. Salt stress Soil salinity is an increasing problem in agriculture, which drastically affects plants growth (Machanda and Garg, 2008). Many efforts have been made to alleviate salinity conditions through the improvement of soil quality (Hasini et al., 2020). However, physiological alteration in plants is also a potential tool to improve tolerance in these environmental conditions because salt stress activates complex downstream signalling pathways (Holm et a., 2001; Trifunović-Momčilov et al., 2020; Wani et al., 2020). These pathways are primarily related to the increase in ROS, which is associated with oxidative damage in salt-stressed plants (Akyol et al., 2020). Various studies have shown that ROS (e.g., hydrogen peroxide and superoxide) are increased in plant tissue during salt stress, as well as upregulation of antioxidant ezymes under longer stressors conditions (e.g., CAT, APX, superoxide dismutase [SOD], glutathione reductase, guaiacol peroxidase [GPOX] and peroxidase [POD]) (Asrar et al., 2020; Cheng et al., 2020; Fadzilla et al., 1997; Hossain and Dietz, 2016; Li et al., 2017). Therefore, ROS and antioxidant enzymes are indicators of plant stress acclimation. Higher concentration of ROS may lead to cell damage via lipid peroxidation in cellular membranes (Kwiecien et al., 2014), which is a critical reason for plants to avoid high increments of these compounds. Plants exhibit protective mechanisms for survival under salt stress conditions via the synthesis of ROS scavengers. Many ROS scavengers have already been described in plants (Mittler, 2002). Other compounds, synthesized via the conversion of heme catalyzed by heme oxygenase (HO) (Mahawar and Shekhawat, 2017; Tenhunen et al., 1968), have similar antioxidant functions, such as biliverdin (BV), which also acts as a radical trap (Stocker et al., 1987). For example, the activity of GPOX and SOD were increased in soybeans leaves after BV pretreatment in salt-stressed plants (Balestrasse et al., 2008). Evidence indicates that HO in controlled by phy, since a phytochrome chromophore-deficient mutant (pcd1) of Pisum sativum plants lacked HO activity (Weller et al., 1996). Certainly, these results suggests that phy play a role in the regulation of ROS scavengers through HO activity (e.g., GPOX and SOD), but which phy is responsible for these effects is unknown. Additionally, reciprocal regulation between phy and HO was observed since and Arabidopsis mutant, deficient in HO (hy1 10 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 mutant), was completely insensitive to R/FR and exhibit long hypocotyl in white light (Terry et al., 2002). Such circumstances entail the cooperative influence of phy and HO to induce salt stress tolerance through antioxidant system enhancement. In addition to the tight and complex relationship between ROS scavengers (HO) and phy synthesis in salt-stressed plants, phy-mediated light signalling could also alter the transcription of proteins that were previously described to confer salt tolerance in plants. For example, the protein salt tolerance-related (STO) improved root growth in Arabidopsis transgenic plants overexpressing STO under salt stress (Nagaoka and Takano, 2003). Thus, phyB likely interacts with STO since STO was described to be repressed by COP1 (Indorf et al., 2007) and the SPA1-COP1-PIF1 (PIF1) kinase regulatory complex, which is negatively regulated by phyB (Paik et al., 2019). In other words, phyB repressed the SPA-COP1-PIF1 complex, allowing transcription of STO, which in turn induced root growth under salt stress conditions (Figure 1). Recent data corroborated the role of phyB in salt stress regulation but as negative regulator. For example, seedlings of tomato phyB1 mutants showed lower oxidative damage after stress treatment, as demonstrated by the reduction of malondialdehyde (MDA) content (Gavassi et al., 2017). Similarly, Cao et al. (2018) manipulated light quality to increase inactive phyB1-form content, which mimics the behavior of phyB1 mutants; these authors observed that the plants were salt-tolerant after exposure to low R:FR ratios with improvement of many desirable traits, such as increased ROS scavenger activity (CAT, POD and SOD) and decreased ROS content (superoxide and hydrogen peroxide). 11 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 Figure 1. Schematic of the role of phyB in the acclimation responses to cold, heat, salt and heavy metal stress. From the red- light signalling, phyB coordinates the negative regulation of COP1-SPA-dependent degradation of PIF1 transcription factors, which triggers changes in stress response target genes expression. phyB coordinates other TIFs with no reported evidence of a role for the COP1-SPA complex. Note that these are key pathways in the induction of genes related to signalling and/or the biosynthesis of ROS scavengers and stress components. Although the complex signalling of phyB in stress acclimation involves some unknown components, this photoreceptor plays a crucial role in red-dependent stress responses. Arrows indicate upregulation, T-bars indicate down-regulation, and dotted lines indicate unknown (?) signalling routes. COP1, constitutive photomorphogenic 1; COR, cold-responsive gene; DREB1, dehydration-responsive element binding protein 1; HKTs, high-affinity K+ transporters genes; PIF1, phytochrome interacting factor 1; PIFs, phytochrome interacting factors; SPA1, suppressor of PHYA-105; STO, salt tolerance-related gene. 12 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 phyB and its complexity in salt tolerance improvement is not limited to oxidative stress and related compounds. Other proteins with different modes of action were described as salt tolerance-inductors, which were also mediated by phyB. For example, the transporter high-affinity K+ transporters (HKTs) played a pivotal role in avoiding the accumulation of harmul Na+ concentration and enhanced salinity tolerance (Deinlein et al., 2014). In rice, phyB repressed the expression of putative OsHKTs gene, a convenient regulation that was effective on plant salt-tolerance induction, considering the lower content of Na+ found in rice phyB mutants (Kwon et al., 2018). Therefore, these perspectives open a new avenue for up-stream activity of phyB family in salt stress response. Similar to other abiotic stresses, the relationship between adaptative salt stress mechanisms and phytohormonal dynamics must not be overlooked, as ABA and JA are correlated with salt stress alleviation in plants (Kang et al., 2005; Khan et al., 2019; Zhang et al., 2006; Zhu et al., 2019). The exogenous application of both phytohormones to strawberry plants (Fragaria x ananasa Duch.) activated protective mechanisms against salt stress, such as antioxidant capacity and phenolic compounds (Jamalian et al., 2020) Nevertheless, the phytohormonal influence on salt stress tolerance adjustments suggests that phyB plays a role in salt tolerance via phytohormonal modulation (ABA and JA). For instance, salt stress treatment increased the ABA and JA content in phyB mutants of Nicotiana tabacum, which ameliorated many salt-tolerance characteristics, decreasing MDA content and electrolyte leakage, while increasing the antioxidant system (Yang et al., 2018). Although phyB-regulated responses differed in past studies as a positive or negative regulator of salt stress tolerance, most current evidence highlights that phyB is a negative regulator of salt stress (Table 1). However, many defence-associated genes were up-regulated in responses to salt stress in Nicotiana tabaum phyB mutants, such as NtLEA5 (late embryogenesis abundant protein), NtER10C (early responsive dehydration) and NtABF2 (ABA-responsive element binding) (Grover et al., 2001; Yang et al., 2018). Thus, further studies are required to identify the phyB-regulated mechanisms that affect acclimation responses to salinity. 13 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 2.3. Low and high-temperature stress Temperature is an environmental factor that affects crucial responses during plant growth, from seed germination to flowering. Plants naturally display strategies to perceive environmental temperature fluctuations and then trigger seed germination, which could guarantee ideal conditions for seedlings to rise. For example, low/high temperatures could break seed dormancy (Kozlowski and Pallardy, 2002) and interestingly, phyB contributes to breaking cold-induced dormancy (Donohue et al., 2008). In addition to seed responses to temperature stress, heat and cold tolerance were described as a great evolutionary characteristic that allowed plants to thrive along the Earth ages. Notably, plants show different responses to heat and cold stress based on the incident light quality, influencing fundamental characteristics for cell survival. The stability and fluidity of cell membranes is a crucial aspect in temperature acclimation responses, the main target of which is the thylakoid membrane, which upon cold stress can suffer damage (Yordanov, 1992), leading to electron leakage from the photochemical reaction and ROS formation (Niyogi, 1999). Composition of the plasma membrane in terms of the higher unsaturated fatty acid (USFA) to saturated fatty acid (SFA) ratio (USFA:SFA ratio) was closely correlated with the stability and fluidity of cell membranes and, thus, to chilling tolerance (Ishizaki et al., 1996; Szalontai et al., 2003). Indicators, such as higher electrolyte leakage (Murray et al., 1989) and increased MDA content (Jouve et al., 1993), are largely used to identify injured cell membranes. In a pioneering work, Williams et al. (1972) demonstrated that a night break with R suppressed cold acclimation in Cornus stolonifera, as shown by higher leaching of the electrolyte content. However, the suppression was relieved when the R was followed by FR. Although this work did not identify the main photoreceptors involved, the results suggest that phytochromes were part of the cold stress response. In fact, phytochromes have also been shown to be thermosensor pigments, specially phyB, as demonstrated by Legris et al. (2016), who proposed that the main mechanism underlying phyB responses was the quick reversion to Pr triggered by temperature in a light independent-manner. For example, rice plants lacking phyB (phyB mutants) showed increased cold tolerance verified by lower electrolyte leakage and higher 14 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 seedling survival rate compared to WT plants (He et al., 2016). This may be assigned to higher stability of USFA synthesis that also alleviated the photoinhibition caused by chilling (Yang et al., 2013). However, contradictory data have also been registered, in which after freezing treatment, Arabidopsis phyB mutants and phyB-overexpression presented higher and lower electrolyte leakage, respectively (Jiang et al., 2020). Cold tolerance positively regulated by phyB seemed to involve a complex molecular mechanism, especially those related with the expression of cold-related (COR) genes, such as cor14b (Crosatti et al., 1999) and cor15a (Kim et al., 2002). In Arabidopsis, expression of the latter cold-related genes was activated by transcription factors C repeat binding factors and drought response element binding factor 1 (CBFs/DREB1s) that were phyB- induced during cold exposure (Kim et al., 2002). Therefore, downstream action of the CBFs/DREB1s transcriptional factor was suggested in cold acclimation responses since He et al. (2016) reported that cold tolerance in rice was negatively regulated by phyB, demonstrating that CBFs/DREB1s was negatively regulated by phyB, conferring a cold resistant phenotype in rice phyB mutants. However, the underlying mechanisms of chilling tolerance mediated by phyB are complex and involve other key molecules that comprise a huge number of pathway cascades that may include phytohormone regulation. Similar to rice plants, lack of phyB in tomato (mutants phyB1 and phyB2) increased the expression of COR genes, but it was suggested to be dependent on ABA and JA, since these hormones act to up-regulate the transcript levels of COR genes. Additionally, the levels of ABA and JA were increased in tomato phyB1 and phyB2 mutants after cold treatment (Wang et al., 2016). Temperature stress regulated by phyB is not limited to lower temperatures and also extends to heat stress. Gavassi et al. (2017) reported longer shoot length in phyB1 mutants compared to the WT after heat stress exposure. This mutant also presented lower MDA content (a product of lipid peroxidation), suggesting a presumptive phyB role in alleviating the deleterious effect of ROS. Notably, phyB signalling of heat stress may occur via the negative control of putative basic helix–loop–helix transcription 15 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 factors, such as PIFs (Leivar et al., 2012; Lorrain et al., 2008; Martínez et al., 2018). Among the known PIFs, phytochrome interacting factors 4 and 5 (PIF4 and PIF5) (Koini et al., 2009; Stavang et al., 2009), as well as phytochrome interacting factor 7 (PIF7) (Fiorucci et al., 2020), have been shown to have a crucial role in the hypocotyl elongation of Arabidopsis during high temperature stress. Qiu et al. (2019), demonstrated a substantial reduction of hypocotyl growth in Arabidopsis pif4-2 mutants compared to the WT; the authors clearly showed that the thermo-sensing responses to heat were more pronounced under R light and LD conditions, which suggests the role of phyB in thermosensory machinery to warm conditions. Such evidence was also supported by Song et al. (2017) in Arabidopsis phyB mutant seedlings that exhibited a higher survival rate and lateral root length after heat stress treatment compared to WT seedlings. These responses, shown by phyB mutants, were directly related to an increase in PIF accumulation, since Arico et al. (2019) demonstrated that the thermotolerance acquired by Arabidopsis phyB mutants (lower proportion of damaged plants after heat shock) was not pronounced in pifq mutants (pif1, pif3, pif4 and pif5), which exhibited no difference from WT plants. In contrast to cold stressed plants, heat stressed plants decreased the levels of USFA, which can reduce the targets of oxidative damage (double bonds between C-atoms) and increase the thermotolerance to heat stress (Das and Roychoudhury, 2014). In addition, higher thermotolerance could be attributed to lower USFAs in phyB mutants (Arico et al., 2019). As discussed above, the TFs involved in heat stress acclimation are apparently distinct from the TFs involved in cold stress acclimation (Figure 1), which may explain how the same photoreceptors help plants to cope with two antagonistic stresses. 2.4. High light stress Light is one of the most important factors for plants, controlling growth through photosynthesis and photomorphogenesis processes (Carvalho et al., 2011b). However, light could also be a stressor agent when its quantity is higher than photosynthetic capacity. High light provides an excess of excitation energy that leads to electron leakage from photochemical reactions and the electron transport system, which induces ROS generation and cell damage (Kimura et al., 2003; Niyogi, 1999). To cope with the deleterious effects of an excess of light, plants have established 16 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 several mechanisms, including increased activity of ROS scavenger, accumulation of accessory pigments, and anthocyanins to decrease light damage in shoot tissues (Asada, 1996; Gould et al., 2010). Interestingly, phytochrome involvement in alleviating high light stress has been suggested because of the negative regulation of chlorophyll (component of antenna complex) biosynthesis by PIF1, since chlorophyll accumulation was increased in Arabidopsis pif1 mutants and decreased in phyB mutants (Huq et al., 2004). Additionally, PIF4 and PIF5 were reported to negatively regulate anthocyanin biosynthesis under R (Liu et al., 2015), as these results attributed a positive role to phyB in high light stress tolerance (in terms of intensity), preventing the formation of ROS that would be increased if these pigments (chlorophyll and anthocyanin) were suppressed. Accordingly, phyB1 and phyB2 photoreceptors showed an important function during tomato plant exposition to high light stress; the phyB1phyB2 double mutant had a strong decrease in quantum yield (Fv/fm) and photosynthesis rate compared to WT plants, in addition to pigment (chlorophyll, carotenoids and anthocyanins) reduction in the phyB2 mutant after 2 hr of exposure to high-intensity light (900 μmol m−2 s−1 of white light) (Kreslavski et al., 2020). High light stress is also related to the light quality that comprise the short- wavelengths bands of the electromagnetic spectrum, such as UV-C (220–280 nm), UV-B (280–320 nm) and UV-A (320–400 nm) (Frederick et al., 1989). These short- wavelengths or UV have claimed scientific attention over the years, given the current scenarios of climate change and ozone layer damage that allow higher proportions of hazardous radiation to reach the earth's surface (Bernhard et al., 2020). Plants suffer serious injuries when exposed to a high proportion of UV, which limits their production potential as a result of the negative effect on important physiological mechanisms, such as the photosynthetic apparatus (PA), which includes damage to photosystem II (PSII), lower ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) regeneration, low Fv/fm and reduced photosynthetic pigment accumulation (Teramura and Sullivan, 1994). Therefore, two mechanisms are used to cope with the detrimental effects of high UV proportions: (a) enhanced repair mechanisms and (b) UV screening pigment synthesis. The enhancement of repair mechanisms included UV-damaged DNA restoration upon light-dependent photoreactivation by photolyases and 17 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 acclimation responses (Britt, 1996), including an increase in ROS scavengers (Rao et al., 1996). An increase in the synthesis of UV screening pigments includes flavonoids and carotenoids (Cen and Bornman, 1990). These physiological alterations are the most studied and reported responses, with huge potential to improve the UV stress- relieving mechanisms. Research showed an interaction between UV-B and R light wavelengths in the induction of cotyledon opening; exposure of UV enhanced the phyB perception of R light radiation and phyB mutants had impaired cotyledon opening under UV-B and R (Boccalandro et al., 2001). Therefore, a possible interaction between phyB and a specific UV photoreceptor could be suggested; for example, UVR8 interacted with the central regulator of light signalling COP1, since both proteins accumulate in the nucleus under UV exposure in Arabidopsis (Rizzini et al., 2011), which resulted in a reduction in hypocotyl length (Favory et al., 2009). Moreover, UVR8 activity in the promotion of UV tolerance was largely reported in different species, mainly through induction of flavonoids and anthocyanin synthesis in Arabidopsis (Kliebenstein et al., 2002; Favory et al., 2009) and tomato (Liu et al., 2020). In addition, both UVR8 and phyB regulate COP1 activity. Gao et al. (2018) showed that phyB mutants of Arabidopsis grown under UV-B light increased UVR8 photoreceptor gene expression. In other words, phyB likely negatively regulates UV-tolerance through suppression of UVR8 genes, which are known promotors of UV-tolerance (Figure 2). In fact, the evidence that UV-tolerance is negatively regulated by phyB has also been reported in tomato, but not on a molecular level. For example, phyB1 and phyB2 mutants were more tolerant to UV-B exposure and showed higher shoot and root dry weight and shoot and root length, as well as a decreased MDA content (Gavassi et al., 2017). However, these insights are preliminary and more studies are required to elucidate the crosstalk mechanisms between phyB and URV8, considering that phyB1 and phyB2 mutants also exhibited lower anthocyanin content and anthocyanin accumulation that is an UV-tolerance characteristic, contradicting the positive role of phyB. 18 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 Figure 2. Schematic of the role of phyB in acclimation responses to high radiation (intensity), UV and drought stress. From the red-light signalling, phyB coordinates the regulation of unknown TIFs and triggers changes in some stress response gene expression. Note that these are key pathways in the induction of genes related to signalling and/or the biosynthesis of components related to acclimation response. Although the complex signalling of phyB in stress acclimation involves some unknown components, this photoreceptor plays a crucial role in red-dependent stress responses. Arrows indicate upregulation, T-bars indicate down-regulation, and dotted lines indicate unknown (?) signalling routes. PIFs, phytochrome interacting factors; UVR8, UV-resistance locus 8. 19 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 Whether phys act as negative or positive regulators of UV tolerance is not well established (Table 2). Other findings also showed that phytochromes are positive regulators of UV-tolerance since Arabidopsis phyAphyB double mutants had lower PA resistance and a decreased photosynthesis rate after UV-B exposure (Kreslavski et al., 2017). However, positive or negative regulation of UV stress tolerance by phyB seems to be species specific, since PHYB-ovx plants of potato (Solanum tuberosum – tolerant) and Arabidopsis (susceptible) showed opposite responses for Fv/fm and pigment content (both increased in potato and decreased in Arabidopsis) during exposure to UV-B light in potato and UV-A light in Arabidopsis (Kreslavski et al., 2015, 2016). phyB also acts on other UV light tolerance strategies. For example, programmed cell death (PCD) is a mechanism that maintains tissue homeostasis during plant growth under stress condition and enables nutrient remobilization from dying cells (Nawkar et al., 2013). Exposure to UV can trigger ROS generation, which can damage cells, resulting in PCD (De Pinto et al., 2011). Under UV, phyB was described to regulate PCD through unknown mechanisms as Arabidopsis phyB mutants showed a reduction in ROS and ROS scavenger (SOD, CAT and APX) compared to the WT plants under UV-C, but did not restrain PDC progression (Rusaczonek et al., 2015). Thus, phyB- dependent ROS production under UV is not a key factor involved in PCD, and a multitude of phyB-dependent TIFs may be associated with the response to UV stress (Chen et al., 2013), suggesting that further studies are required on UV stress to unravel the involvement of other phyB-regulated mechanisms. 20 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 Table 2. Summarized data of characteristics phyB-regulated in high temperature stress, high light stress, and heavy metal stress. HIGH TEMPERATURE STRESS Plant species Treatment Type of phyB Response on plant Reference Arabidopsis Heat (37°C) for 3 d phyB Reduce: survival rate, lateral roots length. Song et al. (2017) Arabidopsis Heat (45°C) for 45 min phyB Increase: electrolyte leakage and USFA. Arico et al. (2019) Reduce: plants without damage (%), Plant survival (%), SFA Tomato Heat (42°C) for 6h d-1, during 3 d phyB1 and phyB2 Increase: anthocyanin and MDA. Gavassi et al. (2017) HIGH-LIGHT STRESS Arabidopsis Rc (10 molm−2s−1) phyB Increase: anthocyanin content. Liu et al. (2015) Arabidopsis UV-B (10.08 kJ m- 2) for 8 h d-1, during 15 d phyB Reduce: UVR8 expression, hypocotyls length, petiole length. Gao et al. (2018) Arabidopsis UV-A (12 W m-2) phyB Increase: PA resistance, photosynthesis rate. Kreslavski et al. (2017) Arabidopsis UV-A (10 W m-2) phyB Increase: Fv/Fm, chlorophyll, carotenoids. Kreslavski et al. (2016) Arabidopsis UV-C (200 mJ cm- 2) phyB Increase: ROS, antioxidant activity Rusaczonek et al. (2015) 21 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 Tomato WL (900 μmol m-2 s-1) phyB1 and phyB2 Increase: Fv/Fm, Photosynthesis rate. Kreslavski et al. (2020) phyB2 Increase: chlorophyll, carotenoids, anthocyanin. Tomato UV-B for 8h d-1, during 3 d phyB1 and phyB2 Increase: anthocyanin and MDA. Gavassi et al. (2017) Reduce: shoot dry weight, root dry weight, shoot length, root length, chlorophyll, carotenoids. Potato UV-B (12 W m-2) for 45 min phyB Increase: Fv/Fm, chlorophyll, carotenoids, UV-absorbing substances. Kreslavski et al. (2015) HEAVY METAL STRESS Tomato 65 mM CdCl2 phyB1 and PhyB2 Increase: MDA. Gavassi et al. (2017) Reduce: shoot length, Shoot dry weight, carotenoids (only phyB1). 22 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 2.5. Heavy metal stress Heavy metal (HM) contamination in soil is one of the main topics in the debate about food security and food safety worldwide (Kong, 2014; Tóth et al., 2016). Although agriculture has produced food for the growing population demand in a more sustainable way, the high use of inorganic fertilisers has contributed to the accumulation of HMs in the soil (Atafar et al., 2010; Wang et al., 2020). In fact, fertilisers are contaminated by HMs (Gimeno-García et al., 1995; Mortvedt, 1996; Wang et al., 2020) and their use could affect crop yield by high HM accumulation in the soil. Plant exposure to HMs results in toxicity, represented by rapid growth inhibition and decreased PA activity (Skórzyńska-Polit and Baszyński, 1997; Alaoui-Sossé et al., 2004), related to oxidative stress induced by HM (Mithöfer et al., 2004). Therefore, a strategy that could mitigate the deleterious effects of unavoidable HM toxicity in cash crops reaches physiological alterations that comprises the phytochrome role. On a molecular level, evidence has shown that As (arsenic) treatment (120 μM of sodium arsenate - Na2HAsO4) in Arabidopsis down-regulated the expression of PIF3, a known component in the phytochrome signalling pathway (Shukla et al., 2018). Despite being elusive, such influences indicate that phytochromes may also have a part in the signalling pathways of HM stress acclimation. Gururani et al. (2016) studied the influence of a high level of Zn (20 nM ZnCl2) and reported that Agrostis stolonifera L. transgenic lines (S599A-14 and S5994-18 – hyperactive of phyA) exhibited lower hydrogen peroxide (H2O2) content and increased proline content, antioxidant activity, and plant dry weight compared to the WT. Moreover, this was likely associated with phytochrome positive regulation of ROS-scavenger via HO activity (discussed in salt stress topic), which was also reported to alleviate HM stress in Medicago sativa (Cui et al., 2012). Conversely, phyB-mediated HM tolerance seems to vary among species or between developmental growth stages. In 7-day-old phyB1 and phyB2 mutant tomato seedlings, Gavassi et al. (2017) reported higher dry weight and lower MDA than the WT after cadmium (Cd) treatment (65 mM of CdCl2). However, both tomato mutants did not show any changes compared to the WT in 21-day-old plants under Cd treatment (150 mM of CdCl2) (Gaion et al., 2018). However, further studies are needed 23 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 to elucidate whether phyB influences HM stress responses, considering the lack of reports about this topic. 2.6. Epilogue This paper is an overview of how phyB regulates various environmental stresses that negatively affect plant development under these adverse growth conditions. The well-known role of phyB as a negative regulator of photomorphogenesis reveals characteristics of interest for improving plant tolerance to both abiotic and biotic stresses, mainly through phyB-mediated physiological and/or morphological changes in stressed plants. However, the stress responses regulated by phyB seem to be complex, since phyB could modulate the characteristics of tolerance to different stresses, positively or negatively, and depending on the plant species studied. The majority of data suggests that for increasing tolerance to drought, salinity, high temperature, and low temperature the suppression of phyB-signalling seems to be beneficial, whereas to increase tolerance to heavy metals, induction of phyB-signalling is required. However, no pattern between species was observed for phyB-mediated high light and UV stress tolerance characteristics. Thus, a meticulous assessment of a wide range of plant species is necessary to better understand the underlying phyB- dependent mechanisms that affect high light and UV stress responses in plants. Taken together with world climate changes, these results suggest the importance of the development of plants that are tolerant to the most diverse environmental stresses and that phyB photoreceptor may be yet another target molecule for plant breeding. Thus, manipulation of the phyB family (overexpression or knockout) resulted in traits of agronomic interest for the improvement of plant tolerance to environmental stress, but more comprehensive studies on different plant species, stages of plant development, and combinations of various stressors are important to show the consistency of phyB- mediated responses during biotic and abiotic stress. 24 This chapter was published in the Annals of Applied Biology 178: 135 – 148, 2021 Acknowledgements The first author acknowledges São Paulo Research Foundation (FAPESP) grant no 2019/25737-2 and the National Council for Scientific and Technological Development (CNPq) grant no 148342/2019-1 for his MSc scholarship. A Ph.D. fellowship was granted to Victor D’Amico-Damião, financed by the Coordination for the Improvement of Higher Education Personnel – Brazil (CAPES) (Finance Code 001) and FAPESP (grant number 2017/26130-9). 3. 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Thus, the mutants phytochrome B1 deficient (phyB1) and its isogenic line wild-type (WT) were cultivated in a greenhouse under two reposition of daily water evapotranspiration (ETP): 100% and 50% ETP, performed in two moments: (I) in vegetative stage growth – starting at 26 days after sowing (DAS) and ending at 36 DAS; (II) in reproductive stage growth – starting at 33 DAS and ending at 41 DAS. At the end of ETP treatments were performed analysis of dry weight of leaf, stem, root as well as leaf area, stem diameter/length and root length. After the last harvest (135 DAS) were evaluated fresh and dry weight of fruits, diameters and soluble solid (°Brix). When water deficit occurred in the vegetative stage growth, phyB1 presented higher vegetative biomass accumulation than WT, but WT presented higher root development and fruit yield. In the other hand, phyB1 showed lower fruit size and higher °Brix in fruit when water deficit was performed in reproductive stage growth. Therefore, to raise tomato plants that keep their vegetative growth in water shortage without concern about fruit yield, overexpression of phyB1 would be advantageous. In the opposite manner, if the priority is fruit quality, phyB1 knockout would be beneficial. Keywords: Biomass accumulation, drought tolerance, tomato yield, Source-sink partitioning, Evapotranspiration, °Brix. 43 1. INTRODUCTION The light controls the development of plants through a complex system of photoreceptors, which participate in almost all responses from germination to flowering. For example, cryptochromes and phototropins, mainly detect the wavelengths of blue light (320–400 nm), and phytochromes, present absorption peaks in the red (V; ~ 660 nm) and extreme red (VE; ~ 730 nm) range of the light spectrum (Sancar, 2003; Carvalho et al., 2011). However, phytochromes are the most characterized and exponentially studied. These pigments are dimeric proteins (~130 KDa) covalently linked to a phytocromobilin, a linear open-chain tetrapyrrole that acts as a chromophore. Evidence that angiosperms have several species of phytochromes, encoded by a small family of genes, was initially verified in studies with Arabidopsis thaliana. Five phytochrome genes have been isolated in this species: PHYA, PHYB, PHYC, PHYD and PHYE, which code for the apoproteins PHYA, PHYB, PHYC, PHYD and PHYE (Sharrock and Quail, 1989). Such apoproteins, after binding to the chromophore, form the phytochromes phyA, phyB, phyC, phyD and phyE. Currently, the molecular characterization of phytochromes has been carried out for several species, including tomato (Solanum lycopersicum L.), one of the most important vegetables in the world. In this species, five genes for apoproteins were also found: PHYA, PHYB1, PHYB2, PHYE and PHYF (Pratt et al., 1997). Since the discovery of phytochromes, studies in several species have revealed important functions of these photoreceptors during plant development, such as seed germination (Dechaine et al., 2009; Oh et al., 2009), flowering (Andres et al., 2009; Brock et al., 2010), hypocotyl elongation (Yang et al., 2009; Kunihiro et al., 2010) and flavonoid and carotenoid synthesis (Carvalho et al., 2010; Toledo-Ortiz et al., 2010). Furthermore, recent discoveries have revealed an important role of phytochromes in modulating responses to biotic and abiotic stresses. For example, it has been shown that phytochromes are part of the signalling responses to herbivory (Howe & Jander, 2008; Ballaré, 2009), low or high temperatures (Donohue et al., 2008; Foreman et al., 2011), excess harmful radiation (eg. Ultra-Violet-B or UV-B) (Boccalandro et al., 2001), salinity (Balestrasse et al., 2008; Datta et al., 2008) and more elusively, heavy metals (Cui et al., 2011; Gaion et al., 2018; Gavassi et al., 2017). 44 However, it was brought to attention the fact that phytochromes are part of the responses to stress induced by water deficit, the most worrying stressor for agronomic cultivation. For example, during water deficit, the pew1 mutant of Nicotiana plumbaginifolia, which is deficient in phytochrome chromophore biosynthesis, showed increased levels of abscisic acid (ABA) (Kraepiel et al., 1994), one of the most important hormones involved in this kind of stress. However, because the chromophore is common to all types of phytochromes, this response appears to be quite variable and species dependent. For example, Ferreira Júnior et al. (2018) observed that in the tomato mutant, also deficient in chromophore biosynthesis (aurea or au mutant), it was more sensitive to water deficit. The specificity of phytochromes has become increasingly evident, especially to the type B family of these photoreceptors. For example, compared to the control, Arabidopsis phyB mutants, which are deficient in phytochrome B biosynthesis, showed a reduction in stomatal conductance under water deficit conditions (Boggs et al., 2010). Furthermore, in the same species, Allen et al., (2019) observed an increase in dehydration tolerance in mutants with deficiency in phytochrome B due to the lower relative water loss in detached leaves. Interestingly, this evidence also reaches plants with agronomic interest, since, in rice (Oriza sativa L. cv Nipponbare), plants deficient in phyB showed greater tolerance to water deficit compared to the control genotype, and despite having shown a reduction in net carbon dioxide assimilation due to reduced leaf area, stomatal density and transpiration, the decrease in photosynthetic rate did not affect grain production (Liu et al., 2012). In tomato, Gavassi et al. (2017) observed that 10-day-old seedlings of the phyB1 mutant, submitted to low osmotic potential, presented an increase in the concentration of chlorophylls and carotenoids, as well as the greater length of hypocotyl and root in relation to control genotype. Interestingly, part of these responses was followed by a greater accumulation of proline and glycine-betaine in the phyB1 mutant. According to the above evidence, although the molecular mechanisms depending on the phytochrome B family are poorly understood, so far this type of phytochrome seems play an important role in control responses to water deficit. Thus, it is reasonable to conjecture that the phyB type of phytochromes can modulate the 45 responses to water deficit during different stage growth, a fact that is highly important for agronomic species such as tomato, but which is not yet known. In this species, to date, the knowledge about the effects of the phyB1 mutant shows that, under well hydrated conditions, there was an increase in production of 74% and 39% of fruits both in the greenhouse and in the field, respectively, with no decrease in physicochemical quality compared to the control genotype (Alba et al., 1999). In addition, previous studies demonstrated that phytochromes are part of responses to water deficit in tomato (D’Amico-Damião et al., 2015), with phyB1 being one of the main modulators (Gavassi et al., 2017). However, so far it is not known whether these favorable responses identified in tomato seedlings deficient in phyB1 will have any effect on fruit production or even on their physicochemical characteristics. Thus, considering the economic importance of tomatoes, this unprecedented proposal will make it possible to elucidate the participation of phyB1 in tomato production under water deficit conditions, the stressor that has been a matter of concern due to evident climate changes. Moreover, considering the plant as a complex system that perceives environmental changes, especially water deficit, we hope not only to confirm or reject the presented hypothesis, but to raise new questions that can be explored by the scientific community, and create a new approach for the plant improvement. The objective of this work is to verify whether phytochrome B1 of tomato is part of the responses to water deficit in fruit production. 2. MATERIAL AND METHODS 2.1. Plant material and growth conditions The use of mutants in phytochromes is a great tool to understand the functions of these photoreceptors throughout vegetative and reproductive development, given the unique effect of genetic components (alteration in phytochrome biosynthesis). Therefore, for this proposal, was used the tomato (Solanum lycopersicum L.) mutant deficient in phyB1 biosynthesis (temporary red light insensitive – tri) which presents as phenotypical characteristic etiolated seedings under red light (van Tuinen et al., 1995) and its isogenic line wild-type (WT) of cv Moneymaker. Seeds were kindly provided by the “Tomato Genetics Resource Center” (TGCR; Davis – California). To avoid any 46 contamination, seeds were pre-treated with a 5% sodium hypochlorite solution for 10 min and subsequently well washed before sown. The sowing was realized in trays containing a 1:1 (v/v) mixture of a commercial substrate (BioPlant, Brazil) and expanded vermiculite, and then, left to grow in a chamber with artificial illumination and 12h photoperiod. Fifteen days after sowing (15 DAS), seedlings were transferred to 12 L pots filled with 12 kg of typical dystrophic oxisol supplemented with 4.8 g P2O5, 0.018 g B, 0.36 g N, and 1.8 g K2O. The maintenance fertilizations were made once a week, for 16 weeks applying 1/16 part of the total requirement of N (300 kg ha-1) and K2O (240 kg ha-1), totalizing 0.1125 g pot-1 N and 0.09 g pot-1 K2O in each fertilization, following the