RESSALVA Atendendo solicitação do(a) autor(a), o texto completo desta tese será disponibilizado somente a partir de 12/09/2025. i UNIVERSIDADE ESTADUAL PAULISTA (UNESP) FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL INTEGRATIVE EFFECTS OF SELENIUM AND SULFUR ON NUTRITIONAL AND PHYSIOLOGICAL RESPONSES OF COWPEA (Vigna unguiculata (L.) Walp) GENOTYPES M.Sc. VINICIUS MARTINS SILVA Agronomist, Master in Agronomy 2023 UNIVERSIDADE ESTADUAL PAULISTA (UNESP) ii FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL INTEGRATIVE EFFECTS OF SELENIUM AND SULFUR ON NUTRITIONAL AND PHYSIOLOGICAL RESPONSES OF COWPEA (Vigna unguiculata (L.) Walp) GENOTYPES M.Sc. Vinicius Martins Silva Supervisor: Prof. Ph.D. André Rodrigues dos Reis Co-supervisor: Prof. Ph.D. Arthur Bernardes Cecílio Filho Co-supervisor: Prof. Ph.D. Priscila Lupino Gratão Thesis presented to the School of Agricultural and Veterinary Studies (FCAV) – UNESP as part of the requirements to obtain the title of Doctor in Agronomy (Crop Production). 2023 S586i Silva, Vinicius Martins Integrative effects of selenium and sulfur on nutritional and physiological responses of Cowpea (vigna unguiculata (l.) Walp) genotypes / Vinicius Martins Silva. -- Jaboticabal, 2023 185 p. Tese (doutorado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal Orientador: André Rodrigues dos Reis 1. Selenium. 2. Sulfur. 3. Plant metabolism. 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. iii UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Jaboticabal INTEGRATIVE EFFECTS OF SELENIUM AND SULFUR ON NUTRITIONAL AND PHYSIOLOGICAL RESPONSES OF COWPEA (Vigna unguiculata (L.) Walp) GENOTYPES TÍTULO DA TESE: CERTIFICADO DE APROVAÇÃO AUTOR: VINICIUS MARTINS SILVA ORIENTADOR: ANDRÉ RODRIGUES DOS REIS COORIENTADOR: ARTHUR BERNARDES CECILIO FILHO COORIENTADORA: PRISCILA LUPINO GRATÃO Aprovado como parte das exigências para obtenção do Título de Doutor em Agronomia (Produção Vegetal), pela Comissão Examinadora: Prof. Dr. ANDRÉ RODRIGUES DOS REIS (Participaçao Virtual) Departamento de Engenharia de Biossistemas / Faculdade de Ciencias e Engenharia UNESP Prof. Dr. JOSÉ LAVRES JUNIOR (Participaçao Virtual) Laboratório de Isótopos Estáveis / CENA USP Piracicaba/SP Prof. Dr. FELIPE KLEIN RICACHENEVSKY (Participaçao Virtual) Universidade Federal do Rio Grande do Sul (UFRGS) / Porto Alegre/RS Prof. Dr. PEDRO LUÍS DA COSTA AGUIAR ALVES (Participaçao Virtual) Departamento de Biologia / FCAV UNESP Jaboticabal Prof. Dr. LEANDRO BORGES LEMOS (Participaçao Virtual) Departamento de Ciencias da Producao Agricola / FCAV UNESP Jaboticabal Jaboticabal, 12 de setembro de 2023 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. iv v DADOS CURRICULARES DO AUTOR VINICIUS MARTINS SILVA – Nascido em 31 de maio de 1991, Natural de Ilha Solteira, São Paulo. Em 2009, ingressou no curso de agronomia na Faculdade de Engenharia de Ilha Solteira (UNESP), foi bolsista de iniciação cientifica da Fundação de Amparo à Pesquisa do Estado de São Paulo (11/00412-1) sob orientação do professor Dr. Salatier Buzetti. Graduou-se em 2014 e após breve período trabalhando no setor sucroalcooleiro, em 2016 iniciou mestrado em Agronomia (Sistemas de Produção) na Faculdade de Engenharia de Ilha Solteira (UNESP), sob orientação do Prof. Dr. André Rodrigues dos Reis e Coorientação do Prof; Dr. Marco Eustáquio de Sá. Foi bolsista de mestrado pela Fundação de Amparo à Pesquisa do Estado de São Paulo (16/19773-8) e bolsista BEPE (17/21950-8) como pesquisador visitante na University of Nottingham (Inglaterra) sob supervisão do Prof. Dr. Martin Broadley. Em 2019 defendeu o Mestrado e iniciou o doutorado em Agronomia (Produção Vegetal) na Faculdade de Ciências Agrárias e Veterinárias (UNESP), sob orientação do Prof. Dr. André Rodrigues dos Reis e Coorientação do Prof. Dr. Arthur Bernardes Cecílio Filho e da Profa. Dra. Priscíla Lupíno Gratão. Foi bolsista de doutorado pela Fundação de Amparo à Pesquisa do Estado de São Paulo (18/18936-6) e bolsista BEPE (20/12218-4) como pesquisador visitante na Cornell University (Estados Unidos) sob supervisão da Profa. Dra. Li Li. Foi laureado como melhor aluno da 38ª turma do curso de agronomia da Faculdade de Engenharia de Ilha Solteira. Junto ao seu orientador André Rodrigues dos Reis, foi laureado (3º lugar) com o Prêmio Péter Murányi 2020 – Alimentação, pelo trabalho realizada com Biofortificação de Feijão-Caupi. Atuando nas áreas de nutrição vegetal, biofortificação e fisiologia de plantas, publicou mais de 30 artigos em periódicos revisados por pares, é revisor dos periódicos Scientific Reports, Gesunde Pflanzen e The Brazilian Journal of Biosystems Engineering. De acordo com o banco de dados da Web of Science, sua produção científica soma 384 citações e resulta em índice H 11 (19/08/2023). vi “Somewhere, something incredible is waiting to be known.” - Sharon Begley, Jornalist August 15, 1977, Newsweek magazine DEDICO A minha parceira, companheira, a mulher que escolhi para estar ao meu lado durante toda a minha vida, Líria Nakamiti Novaes. Que me apoia em cada decisão, me motiva a ser cada vez melhor e mais forte, para poder apoiá-la também. Aos meus pais Amarildo Aparecido da Silva e Eloisa Maria Bíglia Martins Silva, que sempre foram exemplos de honestidade, raça e simplicidade, sem deixar de lado a alegria e inteligência emocional necessárias para lidar com os percalços da vida. E ao meu irmão caçula, Vitor Martins Silva, companheiro de conversas e conselhos. A todos os demais membros da minha família, em especial meu Avô Eloi Cruz Martins e avó Ana Rita Bíglia Martins (em memória), avô Antônio Aparecido da Silva (em memória) e avó Maria Madalena da Silva. ACKNOWLEDGMENTS I would like to thank Prof. Ph.D. André Rodrigues dos Reis, my supervisor, for the opportunities and teaching that helped me to grow in my career. Who gave me so many valuable advice and examples and was not only a leader but also a friend. Another great mentor to my scientific career that deserves all my gratitude, is Prof. Ph.D. Li Li, who welcomed me at Cornell University and USDA, received me in her lab for an entire year. advised me and taught me so much, helping me to turn into a better and more prepared professional and scientist. I would like to thank Professor Olena Vatamaniuk, who opened the possibility for my staying at Cornell under Dr. Li’s tutelage. vii Thank you to my co-advisors, Prof. Ph.D. Arthur Bernardes Cecílio Filho and Priscila Gratão, who welcomed me in Jaboticabal and helped me with advises and opportunities to learn. As my Ph.D is a continuation of my master, I also would like to thank two great mentos I had in my preparation for my Ph.D, during my master: Prof. Ph.D Marco Eustáquio de Sá, my co-advisor during my master, and Prof. Ph.D. Martin R. Broadley who accepted me in his lab at Nottingham University. Prof. Ph.D. Rouverson Pereira da Silva, who advised me and helped me in hard times, was an example of dedication and benevolence that I would like to bring with me to my whole life. I’m glad for the opportunity to work with two great institutions: UNESP and FAPESP (process number 2018/18936-6 and 2020/12218-4) that provided me all that I needed to properly carry out my research. I appreciate the efforts and work of all the staff of UNESP, that gave me all the support necessary to perform my work. A special thanks to the staff member Claudio Roselli Batista de Andrade, who helped me a lot with all the official process required to finish my thesis. I appreciate the support of Ph.D. Maurisrael de Moura Rocha, researcher of Embrapa Meio-Norte, for providing me the cowpea genotypes used in my research. I also appreciate the company of my co-workes from GEFA and friends from Tupã: Nandhara, Maria Gabriela, Matheus Cunha, Lara, Andressa, Maycon, Julio and Thais, Cleverson, Tauan, Renato, Pedro, Kamille, Hilário, Carol, Marco, Marcio, Izaias, Júlia, Eduardo Marcandali, Rafael Lawandovski, and Priscilla. Great people who were all moved to provide scientific results in Andre’s lab. I have learned a lot with these guys and hope to keep in touch with them for long time. This also applies to my friends and Co-Workers in Priscilla’s and Arthur’s labs: Mirela, Gilmar, Soninha, Carolina, Camila, Beliza, and Maria José, that were part of my everyday life while I was working in Jaboticabal, helping me with my research and teaching me something new. I would like to thank my roommates and friends in Jaboticabal: Hugo, Emanuel, Gustavo end Antônio, who were also part of my everyday life, and people with whom I have shared many funny moments. viii I’m glad for the opportunity to spend time with my cohort during Ph.D classes, thank you for the friendship Mariana Bonfim, José Lucas, Luís, João, Antônio, Luís Felipe and Gabita, friends that were around during my first year as a Ph.D student. I’m grateful for the opportunity to work with Li’s Lab group: Sombir Rao, Tianhu, Shuesong, Jiping, and the entire staff of Robert W. Holley Center, who provided me the knowledge and structure to perform my research abroad. I’m glad for the opportunity to cross paths with all the amazing people I’ve befriended in Ithaca during my time at Cornell University. Monna Lysa, Filipe, Keeley, Antonio, Renato, Gabi, Letícia, Ananda, Adriana, Gustavo, Mari, Roberto and Biel, my co-workers and friends Andy, Emalee and Kay; Bebel, Naná, Marcella, Talitha, Fernanda and Jan, Shabeeb, Swatah, Debbie, Olya, Vale, Paloma and Nathalia. These people were my family during the entire year of 2022, and I’ll carry a piece of each and one of you with me. To my friends in science, Prof. Dr. Fernando Shintate Galindo and Dr. João William Bossolani, for all the conversations and pieces of advice. Thanks to my long-term friends, Douglas, Guilherme, João, Kauã, Marcelo, Marco Túlio, Matheus and Yuji, the “tero lifestyle” group, friends I have for so long that I’m pretty sure will be along for life. Thank you guys for all the small talk that helped me to relieve my mind during stressful moments. And last, but definitely not least. I would like to express my immense gratitude to my partner in life, Líria, and my parents, Amarildo and Eloisa, who gave me the strength necessary to carry on during the hardest times. INTEGRATIVE EFFECTS OF SELENIUM AND SULFUR ON NUTRITIONAL AND PHYSIOLOGICAL RESPONSES OF COWPEA (Vigna unguiculata (L.) Walp) GENOTYPES RESUMO – Selênio (Se) e enxofre (S) são elementos similares que afetam a absorção e assimilação um do outro pelas plantas, principalmente na forma de selenato e sulfato, pois compartilham transportadores nas plantas. Essa tese objetivou investigar mais a fundo a interação entre Se e S no metabolismo de plantas, estudando o feijão-caupi (Vigna unguiculata), uma importante fonte de proteína e nutrientes em países em desenvolvimento. Na revisão de literatura (capítulo 1), foi observado que a interação entre Se e S pode afetar produtividade das culturas, concentração de metabólitos e que o S pode atenuar a toxicidade de plantas a exposição ao Se. Muitos trabalhos mencionam a importância dos transportadores de sulfato (SULTRs) na absorção e assimilação de Se e S, mas apenas alguns estudos de fato estudam o efeito desses elementos combinado na expressão desses genes. No capítulo dois, o efeito da aplicação de selenito e selenato no sistema antioxidante e na concentração de pigmentos e açúcares foi avaliado em experimento de feijão- caupi cultivado em condições de campo. A aplicação de doses baixas de selenato (2,5 e 5 g ha-1) resultou em notável aumentos na concentração de pigmentos e açúcares, bem como estimulou o sistema antioxidante. Nos capítulos três e quatro, 29 genótipos de feijão-caupi sob aplicação de Se foram avaliados para observação da partição do Se pelos tecidos (cap 3) e o seu efeito na biossíntese de açucares e ureídeos, bem como a produtividade (cap 4). E os resultados observados demonstram que BRS- Guariba é um bom candidato de acordo com sua resposta para aplicação de Se. No capítulo 5, um experimento foi realizado visando observar a interação entre selenato e sulfato em condições de campo. Os resultados reportados demonstram que a interação entre S e Se no campo pode gerar aumentos de proteínas, amino ácidos e açúcares, sem comprometer a produtividade. Enfim, no capítulo 6, foi observado o efeito da interação entre Se e S na expressão gênica, nodulação e síntese de flavonoides no genótipo guariba. A interação entre Se e S resultou no aumento da expressão de genes transportadores de sulfato (SULTRs), enquanto a nodulação e síntese de flavonoides foram afetadas negativamente pela aplicação combinadas desses elementos. Palavras-chave: Vigna unguiculata, variação genotípica, selenato, sulfato, nodulação, flavonoides, SULTRs INTEGRATIVE EFFECTS OF SELENIUM AND SULFUR ON NUTRITIONAL AND PHYSIOLOGICAL RESPONSES OF COWPEA (Vigna unguiculata (L.) Walp) GENOTYPES ABSTRACT – Selenium (Se) and Sulfur (S) are similar elements that affect each other uptake and assimilation in plants, mainly Selenate and Sulfate since both share transporters in plants. In this thesis, it was aimed to further investigate S and Se interaction in plant metabolism, studying cowpea (Vigna unguiculata), an important source of protein and nutrients in developing countries. In the literature review (Chapter 1), it was observed that S and Se affect crop yield, and the concentration of metabolites, in addition was observed that S application can increase plant’s tolerance to Se toxicity. Many studies mention sulfate transporters' importance in Se and S uptake and assimilation, but just a few studies focus on transporters under the application of both elements. In chapter two, the effect of selenite and selenate in pigment concentration, antioxidant system, and sugar content were evaluated in cowpea plants under field conditions. The application of low rates of selenate (2.5 and 5 g Se ha-1) enhances pigments, sugar concentration, and antioxidant activity in plants. In chapters three and four, 29 cowpea genotypes under Se application were studied to observe this element partitioning (Ch3) and its effect on yield, and on sugars and ureides biosynthesis (Ch4). Considering the observed results, BRS-Guariba is a good genotype regarding response to Se. In chapter 5, a field experiment was carried out to observe sulfate and selenate interaction. The reported results demonstrate that Se and S interaction in the field might lead to increased sugars, proteins, and amino acids concentration in seeds without impairing crop yield. Finally, in chapter 6 the effect of Se and S interaction on gene expression, root nodulation, and flavonoid synthesis was reported in cowpea genotype BRS Guariba. The interaction between Se and S resulted in the up-regulation of SULTRs genes, while nodulation and flavonoid biosynthesis were negatively affected by Se and S combined application. Keywords: Vigna unguiculata, genotypic variation, selenate, sulfate, nodulation, flavonoid, SULTRs 1 CHAPTER 1 – INTRODUCTION: INTEGRATIVE EFFECTS OF SELENIUM AND SULFUR ON NUTRITIONAL AND PHYSIOLOGICAL RESPONSES OF COWPEA (Vigna unguiculata (L.) Walp) GENOTYPES ABSTRACT Selenium (Se) is a nutrient for animals and a beneficial element for plants, and sulfur (S) is a macronutrient for all organisms. Both elements share a plethora of similarities and, mainly as sulfate and selenate, are absorbed and assimilated in plants through the same pathways. Those similarities lead to interactions between Se and S that turn into plant responses in the uptake and assimilation of these elements and these compounds as well as physiological and growth alterations. This review aimed to compile studies about Se and S interaction and how the elements affect plant metabolism, yield, and elemental assimilation and uptake. Since sulfate and selenate are absorbed by sulfate transporters (SULTRs) in plant roots, most works focus on the observations regarding sulfate and selenate in plants, however, some papers reported the effect of S on selenite. In general S application decreases Se uptake by roots which is detrimental when the focus is Se biofortification but might be beneficial to avoid Se toxicity. Selenium on the other hand usually increases S uptake, mostly likely by enhancing SULTRs expression in plant roots. A minority of reports observed S enhancing Se uptake, and Se hindering S uptake, indicating that complex interaction between Se and S could be affected by plant species, Se accumulation pattern and soil type. Due to the effect of Se and S interaction in plant metabolism, some studies reported the interaction of those elements affecting crop quality, such as increase in sugar content and decrease in erucic acid in plants seeds. Although many studies mention sulfate transporters’ influence on Se and S uptake and assimilation, a minority of reports focus on investigating those genes expression under Se and S interaction. Further investigation of Se and S interaction should consider environmental and plants- specific aspects to better understand the complex behavior of Se and S within crops. The effect of Se and S interaction in sulfate transporters should be further investigated. Keywords: selenate, sulfate SULTR, elemental interaction, nutrient uptake, nutrient assimilation 2 1. INTRODUCTION: INTEGRATIVE EFFECTS OF SELENIUM AND SULFUR ON NUTRITIONAL AND PHYSIOLOGICAL RESPONSES OF COWPEA (Vigna unguiculata (L.) Walp) GENOTYPES Sulfur (S) is a nutrient for all organisms (Kopriva et al., 2019), is part of essential amino acids, thus plays a role in protein formation in every living being (Nakai et al., 2020). In plants, sulfur is a macronutrient, and is considered the fourth most required for plant nutrition after N, P and K (Li et al., 2020), S is also related to several metabolic processes in plants such as sugar and lipids synthesis, as well as respiration (Liang et al., 2022). Selenium (Se) is a nutrient for animals and humans (Ekuma et al., 2022). Due to the Se-containing aminoacid Selenocysteine (SeCys) being incorporated in proteins Se plays many roles in human health (Rayman, 2000). The element is important in central nervous system maintenance (Steinbrenner & Sies, 2013),Se also plays a role in the thyroid gland function (Derumeaux et al., 2003). And considering Se effect as an antioxidant, the element protects sperm cells from oxidative damage, thus being important to male fertility (Chen et al., 2013), as well as protecting DNA from oxidative damage (Giray & Hincal 2002). While in plants Se is a beneficial element that can enhance antioxidant enzymes activity (Silva et al., 2020; Lanza & Reis 2021), pathogens resistance (Schiavon & Pilon-Smits, 2017) and nodulation in Fabaceae (Cunha et al., 2022). Selenium can also increase the concentration of pigments in plant leaves (Silva et al., 2020) as well as enhance the sugar accumulation in grains and the N metabolism (Silva et al., 2023). Se and S share chemical similarities, especially sulfate and selenate (Cabannes et al., 2011; Natasha et al., 2018), since both Se and S are in the same group in the periodic table (group 16), thus, these elements share similarities in chemical and physical properties (Dauphas, 2013; Wang and Becker, 2013). These similarities lead to S and Se interaction in soil (Song et al., 2022), plants (Silva et al., 2022), and other organisms (Gilbert et al., 2022). And those interactions generate responses on plants that affects the elements uptake and accumulation (Silva et al., 2022; Guimarães et al., 2022) as well as metabolism such as senescence and antioxidative system (Cheng et al., 2016). The effect of Se and S interaction also affects the availability of those 3 elements in soil solution and how they are reduced by microorganisms (Zher & Oremland; 1987; Dowdle & Orelamd, 1998). Cowpea (Vigna unguiculata [L.] Walp) is a Fabaceae plant with high protein concentration in its grains and high tolerance to drought, high temperature and low fertility. Thus it is a crucial crop to provide protein for low income regions and countries. Cowpea also has a very wide genetic variability and previous studies demonstrate it has a high response to Se application considering biofortification potential as well as the physiological responses of Se to mitigate oxidative stress. The aim of this thesis was to evaluate the effects of Se and its interactions with S in cowpea metabolism, S and Se uptake, nodulation as well as genotypic response of cowpea to Se application as sodium selenate.. 177 5. CONCLUSION Selenate and sulfate application in the present study did not affect cowpea growth, indicating that the Se application is not unsafe for the plant’s development under the present condition. Although Se application was not detrimental to S accumulation in plants, S application impaired Se accumulation. 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