UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL O PAPEL DOS FITOCROMOS B NAS RESPOSTAS AO ESTRESSE INDUZIDO PELA DEFICIÊNCIA DE NPK EM TOMATEIRO Mariana Bomfim Soares Engenheira Agrônoma 2022 UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL THE ROLE OF PHYTOCHROMES B IN RESPONSES TO STRESS INDUCED BY NPK DEFICIENCY IN TOMATO Mariana Bomfim Soares Orientador: Prof. Dr. Renato de Mello Prado Coorientador: Prof. Dr. Rogério Falleiros Carvalho Tese apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Doutor em Agronomia (Produção Vegetal). 2022 DADOS CURRICULARES DA AUTORA Mariana Bomfim Soares, nascida em Teixeira de Freitas, Bahia, no dia 01 de maio de 1993. Filha de Maria Rita Teixeira Bomfim e José Rocha Soares. Iniciou o curso de Agronomia em outubro de 2011, na Universidade Estadual de Santa Cruz onde concluiu em fevereiro de 2017. Durante a graduação foi bolsista do Conselho Nacional de Desenvolvimento Científico e tecnológico (CNPq) por quatro anos, onde desenvolveu pesquisa na área de nutrição de plantas e propagação vegetativa do cacaueiro, no Centro de Pesquisas do Cacau (Cepec/Ceplac), e recebeu o prêmio de “Melhor apresentação oral” por dois anos consecutivos. Ainda na graduação realizou estágio não obrigatório na empresa Agrícola Conduru LTDA, onde acompanhou atividades desde o plantio ao beneficiamento de amêndoas de cacau e executou projeto de paisagismo. Em 2017 ingressou no mestrado no Programa de Pós- Graduação em Produção Vegetal na Universidade Estadual de Santa Cruz, sendo bolsista da Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), desenvolvendo o projeto “Miniestacas ortotrópicas: enraizamento, crescimento e qualidade de mudas de cacaueiros”, sob orientação do professor George Andrade Sodré, com conclusão em fevereiro de 2019. Em março de 2019, iniciou o curso de Doutorado na Universidade Estadual Paulista “Júlio de Mesquita Filho” (Unesp), no programa de Pós-Graduação em Agronomia (Produção Vegetal) sendo bolsista da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), onde desenvolveu pesquisas nas áreas de nutrição de plantas e fisiologia vegetal, sob orientação do professor Renato de Mello Prado. “Por vezes sentimos que aquilo que fazemos não é senão uma gota de água no mar. Mas o mar seria menor se lhe faltasse uma gota.” Madre Teresa de Calcutá À minha mãe, Maria Rita Teixeira Bomfim (in memoriam) pelo amor incondicional, dedicação, esforço e por sempre ter acreditado em mim. Dedico AGRADECIMENTOS A Deus e a espiritualidade amiga, por todo amparo durante a minha vida, por nunca ter me deixado abater pelas dificuldades. A minha família, por todo apoio e amparo. Aos meus pais Maria Rita Teixeira Bomfim (in memoriam) e José Rocha Soares por nunca terem medido esforços para me criar e me educar, aos meus irmãos Ariza e Filipe por todo amor, apoio e companheirismo. A Ana Virgínia por todo amor, companheirismo, incentivo e apoio. À Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias (Unesp/FCAV) por todo conhecimento e oportunidades que me proporcionou, à Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela concessão da bolsa de estudos. Ao meu orientador, o professor Renato de Mello Prado e ao meu coorientador, o professor Rogério Falleiros Carvalho, pela paciência, dedicação, por terem me orientado e me dado todo o suporte necessário, por terem me ajudado a ser uma profissional melhor e por serem exemplos de profissionais a ser seguido. Aos membros da banca examinadora por terem aceitado o convite e pela contribuição com o trabalho. Aos professores da Pós-Graduação em Agronomia (Produção Vegetal) e Ciência do Solo UNESP/FCAV, em especial o professor Arthur Bernardes Cecílio Filho, o professor Pedro Luís da Costa Aguiar Alves, e a professora Mara Cristina Pessôa da Cruz, por todo conhecimento passado, paciência em me auxiliar e contribuição com os meus experimentos. Ao Departamento de Ciências da Produção Agrícola, Setor de Ciência do Solo, ao Departamento de Biologia e todos os funcionários que me auxiliaram, em especial as técnicas do laboratório de Fisiologia Vegetal e de Nutrição de Plantas, Sonia Maria R. Carregari e Claudinha . Aos amigos e colegas da Pós-Graduação, Lucas, Luis Felipe, Gelza, Rafael, Kamila, Dilier, Alexander, Lívia, Gabriela, Antônio, Marcilene, Eduarda, Clebson, Kleve, Carlos, Regis, Regiara, Victor, Kolima, Vanessa, João, Leonardo, Luís Fernando e Vinicius por terem feito dos meus dias mais leves, por terem me auxiliado com a pesquisa, pelos momentos de descontração, por todo carinho e por terem feito o meu caminhar mais leve. A todos aqueles que cruzaram o caminho com o meu e que de alguma forma contribuíram com o meu crescimento pessoal e profissional. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. SUMÁRIO RESUMO.................................................................................................................... iii ABSTRACT ................................................................................................................ iv CAPÍTULO 1 – Considerações Gerais ........................................................................ 1 1. Introdução ............................................................................................................... 1 2. Revisão de literatura ............................................................................................... 3 2.1. Fotomorfogênese .............................................................................................. 3 2.2. Fitocromos: aspectos gerais ............................................................................. 3 2.2.1. Caracterização morfofisiológica dos mutantes em phyB de tomateiro ........... 5 2.3. Tomateiro como planta modelo alternativo a Arabidopsis Thaliana .................. 5 2.4. Fitocromos no controle da nutrição de plantas.................................................. 6 2.5. Deficiência nutricional ....................................................................................... 7 2.6. Enxertia como ferramenta de estudo da comunicação entre parte aérea-raiz .. 8 3. Referências ............................................................................................................. 9 CAPÍTULO 2 - Tomato phytochromes B1 and B2 are part of the responses to the nutritional stress induced by NPK deficiency ............................................................. 13 1. Introduction ............................................................................................................ 14 2. Materials and methods .......................................................................................... 15 2.1. Growth conditions and plant material .............................................................. 15 2.2. Treatments and experimental design .............................................................. 16 2.3. Performed analyses ........................................................................................ 17 2.4. Statistical analysis ........................................................................................... 18 3. Results .................................................................................................................. 19 3.1. Complete nutrient solution .............................................................................. 19 3.2. Nitrogen deficiency (-N) .................................................................................. 19 3.3. Phosphorus deficiency (-P) ............................................................................. 22 3.4. Potassium deficiency (-K) ............................................................................... 24 4. Discussion ............................................................................................................. 27 4.1. Complete nutrient solution .............................................................................. 28 4.2. Nitrogen deficiency (-N) .................................................................................. 29 4.3. Phosphorus deficiency (-P) ............................................................................. 30 4.4. Potassium deficiency (-K) ............................................................................... 32 References ............................................................................................................. 34 CAPÍTULO 3 - O Fitocromo B1 de tomateiro modula as respostas a deficiência de N, P e K através da comunicação parte aérea-raiz ........................................................ 39 Resumo ..................................................................................................................... 39 1. Introdução ............................................................................................................. 39 2. Material e Métodos ................................................................................................ 41 2.1. Condições de crescimento, material vegetal e enxertia .................................. 41 2.2. Tratamentos e delineamento experimental ..................................................... 42 2.3. Análises realizadas ......................................................................................... 43 2.4. Análise estatística ........................................................................................... 45 3. Resultados ............................................................................................................ 45 3.1. Solução completa ............................................................................................ 45 3.2. Deficiência de nitrogênio (-N) .......................................................................... 50 3.3. Deficiência de fósforo (-P) ............................................................................... 51 3.4. Deficiência de potássio (-K) ............................................................................ 53 4. Discussão .............................................................................................................. 54 4.1. Solução completa ............................................................................................ 54 4.2. Deficiência de nitrogênio (-N) .......................................................................... 56 4.3. Deficiência de fósforo (-P) ............................................................................... 58 4.4. Deficiência de potássio (-K) ............................................................................ 59 5. Referências ........................................................................................................... 62 APÊNDICE ................................................................................................................ 65 Apêndice A – Material suplementar para o capítulo 3 ............................................ 66 iii O PAPEL DOS FITOCROMOS B NAS RESPOSTAS AO ESTRESSE INDUZIDO PELA DEFICIÊNCIA DE NPK EM TOMATEIRO RESUMO – A luz é o sinal ambiental que influencia o crescimento e desenvolvimento das plantas, desde a germinação ao florescimento. As plantas percebem, interpretam e traduzem os sinais luminosos por meio dos fotorreceptores, entre eles os fitocromos, que absorvem comprimentos de onda do vermelho ao vermelho extremo e controlam diversas respostas no ciclo de vida das plantas, incluindo a nutrição. Tem se tornado cada vez mais evidente a participação desse fotorreceptor nas respostas das plantas ao estresse causado pela deficiência nutricional. Entretanto, embora haja algumas evidências na literatura, ainda é pouco conhecida a interação dos fitocromos B com os mecanismos de respostas ao estresse por deficiência de nitrogênio (N), fósforo (P) e potássio (K), que são os nutrientes considerados mais responsivos pelas plantas. Essa pesquisa foi realizada com o objetivo de estudar o papel dos fitocromos B nas respostas nutricionais, fisiológicas e de crescimento do tomateiro ao estresse por deficiência de N, P e K. Inicialmente, plantas dos mutantes com perda de função de phyB1 (phyB1) e phyB2 (phyB2) e do seu genótipo controle (Solanum lycopersicum L. cv Moneymaker) foram cultivadas sob deficiência de N, P e K. A deficiência de phyB1 e phyB2 diminuiu o acúmulo de N, P e K, a concentração de clorofilas e carotenoides e a produção de massa seca das plantas sob suficiência nutricional. Em plantas deficientes em N, phyB1 regula positivamente a absorção de N, a síntese de pigmentos e produção de massa seca, enquanto apenas a absorção de N está sob controle de phyB2. A deficiência de phyB1 mitigou os danos causados pela deficiência de P, pois aumentou a produção de massa seca das plantas em relação ao genótipo controle. Sob deficiência de K, phyB1 aumenta a absorção de P e síntese de clorofilas e carotenoides e phyB2 diminui a peroxidação lipídica das membranas. Posteriormente, a fim de avaliar se o controle de phyB1 nas respostas do tomateiro a deficiência de N, P e K ocorre reciprocamente a partir da comunicação entre parte aérea e raiz, foram utilizados enxertos recíprocos e combinações de enxertia entre phyB1 e o genótipo controle sob condições de deficiência de N, P e K. O fitocromo B1 da parte aérea está envolvido na absorção de N e P e regula positivamente a condutância estomática e transpiração do tomateiro. A utilização do genótipo controle como enxerto e phyB1 como porta-enxerto aliviou os danos causados pela deficiência de N, evidenciado pelo aumento na massa seca. Esses resultados sugerem que o controle das respostas nutricionais, fisiológicas e de crescimento do tomateiro é realizado pelos fitocromos através da comunicação entre parte aérea e raiz. Palavras-chave: Solanum lycopersicum L., deficiência nutricional, mutante, phyB iv THE ROLE OF PHYTOCHROMES B IN RESPONSES TO STRESS INDUCED BY NPK DEFICIENCY IN TOMATO ABSTRACT - It is already well known that light is an environmental signal that influences the growth and development of plants from germination to flowering. Plants perceive, interpret, and translate light signals through a complex system of photoreceptors, including phytochromes, which absorb wavelengths from red to far red and control several responses in the life cycle of plants, including nutrition. The participation of this photoreceptor in plant responses to stress caused by nutritional deficiency has become increasingly evident. However, although there is some evidence in the literature, little is known about the interaction of phytochromes B (phyB) with the mechanisms of stress responses due to nitrogen (N), phosphorus (P) and potassium (K) deficiency, which are the nutrients considered most responsive by plants. This research was carried out with the objective of studying the role of phytochromes B in the nutritional, physiological and growth responses of tomato to N, P, and K deficiency. Initially, phyB1 and phyB2 tomato mutants (deficient in phyB1 and phyB2) and their control genotype (Solanum lycopersicum L. cv Moneymaker) were cultivated under sufficiency and N, P and K deficiency. In N-deficient plants, phyB1 upregulates N uptake, pigment synthesis and dry weight production, while only N uptake is under the control of phyB2. phyB1 deficiency mitigated the damage caused by P deficiency, as it increased the plant dry weight production in relation to the control genotype. Under K deficiency, phyB1 increased P uptake, chlorophyll, and carotenoid synthesis and phyB2 decreases membrane lipid peroxidation. Afterwards, to verify whether the interaction between phyB1 and N, P, and K occurs reciprocally to the communication between shoots and roots; reciprocal grafts and graft combinations between the control genotype and phyB1 were used under nutritional sufficiency and individual deficiencies of N, P, and K. It was verified that shoot phytochrome B1 is involved in the absorption of N and P and regulates stomatal conductance and transpiration of tomato. The grafting combination between the control genotype as scion and phyB1 as rootstock alleviated the damages of nitrogen deficiency by increasing dry weight production. These results suggest that the control of nutritional, physiological and growth responses of tomato is performed by phytochrome through shoot-root communication. Keywords: Solanum lycopersicum L., nutritional deficiency, mutant, phyB 1 CAPÍTULO 1 – Considerações Gerais 1. Introdução O nitrogênio (N), fósforo (P) e potássio (K) são apontados como os nutrientes que mais restringem o crescimento das culturas (De Souza Osório et al., 2020; Teixeira et al., 2021). Cada espécie quando submetida a deficiência nutricional apresenta diferentes mecanismos fisiológicos e taxas de absorção de nutriente que influenciam o seu crescimento (De Souza Osório et al., 2020). Devido a esses elementos serem considerados como essenciais, as plantas desenvolveram mecanismos para otimizar a sua absorção e utilização. A luz é um sinal ambiental chave que regula o crescimento e desenvolvimento das plantas incluindo a germinação de sementes, desestiolamento, expansão foliar, alongamento do caule, fototropismo, evitação à sombra, ritmo circadiano, florescimento e a nutrição (Li et al., 2011; Xu et al., 2021). As plantas monitoram a direção, duração, quantidade, qualidade, e através desse monitoramento, conseguem dinamicamente absorver e utilizar os nutrientes em resposta às flutuações ambientais (Xu et al., 2021). As informações contidas na luz são obtidas por um complexo sistema de fotorreceptores, que são caracterizados pelo comprimento de onda que percebem (Carvalho et al., 2011). Até o momento, foram identificados cinco classes de fotorreceptores nas plantas superiores, entre eles, os fitocromos (phys), que absorvem comprimentos de onda na faixa do vermelho (600-700 nm) e vermelho extremo (700- 750 nm) (Mawphlang e Kharshiing, 2017). Embora haja diferentes fotorreceptores, os fitocromos são os mais estudados, e, a cada ano, um número considerável de pesquisas utilizando tratamento com luz vermelha/vermelho extremo, bem como mutantes fotomorfogenéticos, são publicadas descrevendo novas funções bioquímicas e moleculares (Carvalho et al., 2011). A caracterização molecular dos fitocromos tem sido feita para várias espécies, incluindo o tomateiro (Solanum Lycopersicum L.), uma das hortaliças mais importantes do mundo. Nessa espécie, foram identificados cinco genes de membros da família dos 2 fitocromos, entre eles PHYA, PHYB1, PHYB2, PHYE e PHYF que codificam para as apoproteínas PHYA, PHYB1, PHYB2, PHYE e PHYF (Hauser et al., 1995). Embora a participação dos fitocromos tenha sido amplamente explorada, desde a germinação de sementes (Dechaine et al., 2009; Oh et al., 2009) até o florescimento, (Andres et al., 2009; Brock et al., 2010), tem sido revelada a participação desses fotorreceptores em respostas inerentes aos estresses abióticos, a exemplo da deficiência nutricional (Carvalho et al., 2016; Sakuraba e Yanagisawa, 2018). No que diz respeito a participação dos fitocromos na nutrição de plantas, foi revelado em estudo com Arabidopsis thaliana que os fitocromos influenciam a capacidade do boro em estimular o crescimento do hipocótilo (Kocábek et al., 2009). Além disso, Sakuraba et al. (2018), na mesma espécie, observaram que phyB regula favorecendo a absorção de fósforo pelas raízes. Já Carvalho et al. (2016) observaram que phyA pode participar da nutrição de tomateiro. Nesse sentido, é importante verificar se mecanismos similares também controlam a absorção e utilização de nutrientes em outras espécies e membros da família dos fitocromos, especialmente em condições de déficit nutricional, que limita a produtividade das culturas. Existem indicações a partir de combinações de enxertia e de mutantes fotomorfogenéticos, que os fitocromos são parte da comunicação entre parte aérea e raiz. Por exemplo, em Arabidopsis as combinações entre o wild type (WT) e o mutante deficiente no fitocromo B (phyB-9) (e.g WT/phyB-9 e phyB-9/WT) absorveram menos fósforo em relação ao genótipo controle WT/WT, indicando que ambos fitocromos, da parte aérea e da raiz estão envolvidos na absorção desse elemento nas raízes (Sakuraba et al., 2018). Dessa forma, o controle de phyB sobre a nutrição dependente da comunicação entre raiz e parte aérea levantam questões importantes sobre o controle fotomorfogenético da nutrição, principalmente no que diz respeito a espécies agronomicamente importantes, como tomateiro. Por exemplo: i) as respostas do tomateiro à deficiência de NPK são moduladas pelos fitocromos B1 e B2? ii) a interação entre phyB1 de tomateiro e N, P, e K ocorre, reciprocamente, a partir da comunicação entre a parte aérea e raiz? Para responder essas questões, no presente trabalho nós realizamos a técnica de enxertia utilizando mutantes em phyB de tomateiro. 3 2. Revisão de literatura 2.1. Fotomorfogênese A luz é essencial para o crescimento vegetal, não estando restrita ao processo de fotossíntese, pois também atua na transferência de informações para controlar uma série de respostas fisiológicas ao longo do ciclo de vida das plantas e garantir a sua sobrevivência às flutuações ambientais (Kami et al., 2010). A fotomorfogênese é um processo chave que controla o desenvolvimento das plantas desde a germinação de sementes até o florescimento e senescência; nos vegetais é governada pelo espectro de luz que chega na superfície da terra, se estendendo aproximadamente do violeta (~380 nm) ao vermelho (~700 nm). A qualidade, intensidade, direção e duração da luz são aspectos percebidos por um complexo sistema de fotorreceptores, que juntos traduzem a informação da luz em sinais bioquímicos. Cinco classes de fotorreceptores estão presentes nas plantas superiores, e cada um possui um comprimento de onda específico de absorção da luz, dentre estes podemos citar: os fitocromos (phys), que são os mais importantes reguladores da fotomorfogênese; os receptores da luz azul, que são representados pelos criptocromos (crys), fototropinas (phots) e zeitlupes os “UV Resistance Locus 8” (UVR8) (Carvalho et al., 2016; Mawphlang e Kharshiing, 2017). 2.2. Fitocromos: aspectos gerais Os fitocromos são os principais fotoreceptores do espectro vermelho e vermelho extremo (600 a 750 nm) da luz solar e estão presentes em algas, cianobactérias, musgos, samambaias e plantas superiores. Além disso, desempenha papel fundamental na regulação do crescimento e desenvolvimento das plantas, a exemplo da germinação de sementes, fotomorfogênese, ritmo circadiano, alongamento do hipocótilo, florescimento e mais recente, a nutrição (Wang, 2015; Sakuraba et al., 2018; Sakuraba e Yanagisawa, 2018; Soares et al., 2021; Wang et al., 2022). 4 Esse fotorreceptor é composto por uma apoproteína, que é sintetizada no citosol, e um cromóforo, no qual é um tetrapirrol linear sintetizado no plastídeo, e que juntos formam uma holoproteína (Wang et al., 2022). A união do cromóforo com a apoproteína ocorre no citoplasma, entretanto, a ativação do fitocromo ocorre no núcleo, onde há a interação da forma ativa com os componentes de sinalização e regulação da transcrição de genes dependentes da luz, e que estão relacionados com o início das respostas fisiológicas controladas pelos fitocromos (Klose et al., 2020). A atividade fotosensorial do fitocromo resulta da sua capacidade de mudar a conformação da molécula. Em plantas cultivadas no escuro, os fitocromos são sintetizados na forma inativa (Fv, absorve luz vermelha), entretanto, a exposição do tecido vegetal a luz faz com que a forma inativa seja interconvertida para a forma ativa (Fve, absorve vermelho extremo) (Figura 1) (Li et al., 2011). Essa propriedade permite que os fitocromos estabeleça o equilíbrio dinâmico dependendo das condições de luz. Figura 1. Esquema ilustrando fotoconversão dos fitocromos da forma inativa (Fv) para forma ativa (Fve). Em tomateiro, foram identificados cinco genes que codificam para as apoproteínas, denominados PHYA, PHYB1, PHYB2, PHYE e PHYF (phyA, phyB1, phyB2, phyE e phyF) (Pratt et al., 1997). Os diferentes membros da família dos fitocromos monitoram o mesmo sinal luminoso, contudo, desencadeiam respostas fisiológicas distintas (Bae e Choi, 2008). Foi observado em plântulas e plantas adultas de tomateiro maior abundância do gene de PHYA, seguido por PHYB1, PHYE, PHYB2 e PHYF (Hauser et al., 1997). A caracterização dos fitocromos e o entendimento dos seus papeis fisiológicos tem sido possível através de análises transgênicas e utilização de mutantes em diversas espécies, incluindo o tomateiro. 5 2.2.1. Caracterização morfofisiológica dos mutantes em phyB de tomateiro De modo geral, os mutantes com perda de função de phyB (phyB) são caracterizados pela sua incapacidade de desestiolar sob luz vermelha contínua, resultando no alongamento do hipocótilo e menores cotilédones, além disso, as plantas apresentam leve diminuição na concentração de clorofilas quando cultivadas sob luz branca (Van Tuinen et al., 1995). Em estudo recente, o cultivo de phyB1 e phyB2 sob luz solar resultou em plantas com menor produção de biomassa total, concentração de clorofilas a e b, condutância estomática e transpiração (Mereb et al., 2020). 2.3. Tomateiro como planta modelo alternativo a Arabidopsis Thaliana O tomateiro (Solanum lycopersicum L.) é uma das espécies mais consumidas mundialmente. Isso está relacionado ao seu fruto ser uma importante fonte de vitamina C, ácido fólico e carotenoides, a exemplo do licopeno (Perveen et al., 2015). Aliado a isso, essa planta tem sido considerada como modelo adicional à Arabidopsis para estudo da fotomorfogênese. Assim como em Arabidopsis, o tomateiro possui genoma relativamente pequeno e a maior parte são genes de cópia única e rapidamente transformáveis e regeneráveis (Pratt et al., 1997). Além disso, existem outros fatores que reforçam a utilização do tomateiro como planta modelo a exemplo: essa espécie já é bem caracterizada geneticamente; existe uma grande comunidade de cientistas trabalhando com ele; já existem diversos mutantes em fitocromos disponíveis; o tomateiro ainda oferece como vantagem a produção elevada de sementes e plântulas que facilitam a realização de análises; formação de frutos climatéricos, folhas compostas e raízes micorrízicas (Pratt et al., 1997; Carvalho et al., 2011). 6 2.4. Fitocromos no controle da nutrição de plantas Na literatura é bem conhecida e explorada a modulação dos fitocromos desde germinação de sementes (Dechaine et al., 2009; Oh et al., 2009) até o florescimento (Andres et al., 2009; Brock et al., 2010). Além disso, tem sido revelada a participação desses fotorreceptores em alguns aspectos da nutrição de plantas. Em um estudo com arroz, a iluminação das plântulas com a luz vermelha promoveu o aumento na atividade da enzima nitrato redutase. Além disso, esse efeito estimulador da luz vermelha foi contrabalanceado pela iluminação com luz vermelha extrema, indicando que a indução da atividade da nitrato redutase é modulada pelos fitocromos (Sasakawa e Yamamoto, 1979). Já em Arabidopsis, a análise de mutantes fotomorfogenéticos revelou a interação entre a sinalização da luz e o boro durante o crescimento e desenvolvimento das plantas (Kocábek et al., 2009). Os autores observaram que a habilidade do boro em estimular o alongamento do hipocótilo foi alterada pela luz azul e vermelha. Isso é, juntas, ambas as luzes amplificaram o estímulo provocado pelo boro no crescimento do hipocótilo, indicando que ambos fitocromos e criptocromos modulam a ação do boro no estímulo ao crescimento do hipocótilo em Arabidopsis (Kocábek et al., 2009). Com a finalidade de verificar se o status nutricional do tomateiro é afetado pelo fitocromo A (phyA), Carvalho et al. (2016) realizaram um estudo utilizando o mutante deficiente em phyA, que foi submetido a deficiência de macronutrientes. Os autores verificaram que em condições de suficiência nutricional, phyA acumulou mais N, Ca, Mg e S na planta inteira, resultando em maior produção de massa seca. Quando o mutante foi submetido a deficiência de N, apresentou maior altura e já sob deficiência de P aumentou o índice de coloração verde. Além disso, sob deficiência de K as plantas acumularam menos N, P e S na planta inteira e que resultou em plantas com menor altura, área foliar, ICV e massa seca. Os resultados desse estudo revelaram que o mutante em phyA apresentou comportamento variado quanto a nutrição e crescimento em condições de deficiência nutricional, indicando que esse fotorreceptor é parte da sinalização de respostas a nutrição do tomateiro (Carvalho et al., 2016). 7 Ainda em Arabidopsis, foi observado que a luz vermelha estimulou a expressão de genes que codificam os transportadores de amônio e de nitrato, indicando que a sinalização da luz promove a absorção de nitrogênio por meio da regulação dos genes relacionados a transportadores que podem afetar a absorção desse elemento (Sakuraba e Yanagisawa, 2018). A avaliação de mutantes de Arabidopsis deficientes nos fitocromos B-9 E B-10 (phyB-9 e phyB-10) revelou que esse fotorreceptor é um regulador positivo da absorção de P e que essa regulação ocorre através do controle da expressão de genes que codificam para os transportadores de P. Além disso, através de combinações de enxertia entre os mutantes em phyB e o WT, os autores ainda observaram que ambos fitocromos, da parte aérea e da raiz, estão envolvidos na regulação da absorção de P nas raízes e que esse controle ocorre de forma mais expressiva sob condições de deficiência desse elemento (Sakuraba et al., 2018). 2.5. Deficiência nutricional A deficiência nutricional é um dos fatores que mais comprometem o crescimento e a produtividade das culturas (Patel et al., 2020). Aliado a isso, sabe-se que de modo geral, o N, P e K são os nutrientes mais responsivos pelas plantas cultivadas, e isso se deve à função metabólica desempenhada por esses elementos (Marschner, 2012; Prado, 2021). Por exemplo, o N é componente de aminoácidos, proteínas, coenzimas, bases nitrogenadas, pigmentos e participa de processos fundamentais como a absorção de nutrientes, fotossíntese, respiração e multiplicação e diferenciação celular (Prado, 2021). Já o P faz parte dos fosfolipídios das membranas e do armazenamento e transferência de energia na forma de ATP. Além disso, esse elemento também atua no processo de absorção de nutrientes, fotossíntese, síntese de proteínas, multiplicação e divisão celular (Marschner, 2012). O K desempenha papeis em diversos processos, incluindo atividade enzimática, pressão de turgor, síntese de proteínas e crescimento celular (Shin, 2017). Em plantas de tomate os estudos indicam que o N, P e K são os nutrientes mais limitantes para o crescimento das plantas (Maia et al., 2019; De Andrade et al., 2021) causando desordens nutricionais que evoluem até atingir nível de tecidos e, portanto, sendo visíveis. A deficiência de N em plantas de tomate inicia nas folhas mais velhas 8 causando clorose uniforme (Maia et al., 2019). As plantas deficientes em P apresentam folhas velhas com coloração púrpura, que mais tarde desenvolve necrose nas margens (Maia et al., 2019; De Andrade et al., 2021). Enquanto a deficiência de K se inicia nas folhas mais velhas na forma de clorose marginal, evoluindo para necrose (De Andrade et al., 2019). As respostas das plantas a deficiência nutricional envolvem alterações moleculares, bioquímicas e fisiológicas específicas para cada nutriente (Marschner, 2012). 2.6. Enxertia como ferramenta de estudo da comunicação entre parte aérea- raiz A enxertia consiste na união de duas partes de plantas distintas que se fundem, crescem e se desenvolvem em uma única planta. A parte que irá originar a parte aérea é chamada de enxerto e o sistema radicular é o porta-enxerto. Essa técnica vem sendo utilizada na agricultura para promover ganhos em qualidade, resistência a doenças, produtividade das culturas e para aumentar a tolerância aos estresses bióticos e abiótico (Thomas e Frank, 2019). Desde a descoberta de que existe uma interação e influência do porta-enxerto no enxerto, vários experimentos foram realizados para tentar estabelecer a base do transporte de substâncias entre ambas as partes (Albacete et al., 2015; Thomas e Frank, 2019). Além disso, a enxertia vem ganhando atenção como ferramenta de pesquisa, principalmente no estudo da comunicação entre parte aérea e raiz, pois permite que os pesquisadores separem e remontem o sistema de transporte a longa distância, permitindo a descoberta da comunicação intraorganismos (Thomas e Frank, 2019). Independente da sua aplicação na agricultura, a enxertia vem sendo utilizada para estudo da fotomorfogênese através da utilização de mutantes. Por exemplo, chama a atenção o fato que, por meio de combinações de enxertia entre plantas do mutante de tomateiro high pigment1 (hp1), que amplifica o sinal da luz, e o mutante aurea, deficiente na biossíntese do fitocromo, foi mostrado que durante sinalização de resposta ao déficit hídrico houve participação desse fotorreceptor na biossíntese de prolina (Ferreira Júnior et al., 2018). Em um trabalho com Arabidopsis, os autores utilizaram da técnica de enxertia para verificar qual fitocromo, da parte aérea e raiz, estava associado com a regulação da absorção de P, utilizando plantas do genótipo 9 controle (WT) e do mutante em phyB-9 (phyB-9) (Sakuraba et al., 2018). Foi observado que ambos fitocromos estão envolvidos na regulação da absorção de P pelas raízes, isso por que tanto a combinação WT/phyB-9 quanto phyB-9/WT apresentaram menor absorção desse elemento em relação a WT/WT (Sakuraba et al., 2018). 3. 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Wang H, Wang H (2015) Phytochrome signaling: Time to tighten up the loose ends. Molecular Plant 8: 540–551. Wang P et al. (2022) Photomorphogenesis in plants: The central role of phytochrome interacting factors (PIFs). Environmental and Experimental Botany, 194:104 13 Este capítulo corresponde ao artigo científico publicado na revista Physiologia Plantarum 173: 2238-2247, 2021. CAPÍTULO 2 - Tomato phytochromes B1 and B2 are part of the responses to the nutritional stress induced by NPK deficiency ABSTRACT - Phytochromes are red-light photoreceptors that play an important role in regulating many responses of plants, including its nutritional control. Nutrient deficiency in plants has become a constraint for agricultural production; thus, we investigated the role of phytochromes B1 and B2 in the nutritional, physiological and growth changes of the control genotype (WT) and both phyB1 and phyB2 tomato mutants (deficient in phyB1 and phyB2) under nutritional sufficiency and individual deficiency of N, P and K. Under complete solution, the plants of phyB1 and phyB2 had a decreased N, P and K accumulation compared to WT and consequently a reduced content of chlorophyll and carotenoids, and dry weight production. In the condition of N deficiency, phyB1 had decreased N absorption, pigments concentration and plant dry weight, while increased oxidative stress of membranes (MDA content). Similarly, phyB2 also had reduced N absorption. The deficiency of phyB1 mitigated the effects of P deficiency as phyB1 mutant had improved nutritional and physiological responses, increasing plant dry weight production. In contrast, phyB2 reduced N accumulation, quantum efficiency of photosystem II (Fv/Fm) and the concentration of pigments, while it increased MDA. Under K deficiency, phyB1 displayed a reduced P accumulation, as well as the total concentration of chlorophylls and carotenoids and K use efficiency. An increased concentration of MDA was found in phyB2 plants, as well as a reduction in chlorophylls concentration and in the use efficiency of K. Together, these results indicate a new perspective on the control of phytochromes in the nutrition of tomato plants under nutritional stress. 14 1. Introduction The understanding of the mode of action of photoreceptors on plant development and responses to abiotic stress has been growing in recent years due to analysis of photomorphogenic mutants (Franklin and Quail 2010). Phytochromes are red-light photoreceptors that absorb wavelengths within the range of red (600-700 nm) and extreme red (700-750 nm) (Carvalho et al. 2016; Mawphlang and Kharshiing 2017). The molecular characterization of phytochromes was made for several species, including one of the most important vegetables in the world: tomato (Solanum lycopersicum L.). This species is considered as an alternative model plant to Arabidopsis thaliana for the study of photomorphogenesis (Pratt et al. 1997). In tomato plants, phytochromes are encoded by five genes: PHYA, PHYB1, PHYB2, PHYE, and PHYF (Hauser et al. 1995); however, the individual functions of phytochromes in plants cultivated under abiotic stresses were poorly investigated. The deficiency of phyB1 and phyB2 in tomato plants results in reduced chlorophyll content and biomass production during the vegetative phase, indicating that this photoreceptor acts in the control of chlorophyll synthesis in addition to dry weight production (Mereb et al. 2020). For instance, the participation of phytochromes in plant nutrition has been shown in Arabidopsis, where phytochrome B (phyB) induces the activation of genes encoding for phosphorus transporters. PhyB participation was found to be more physiologically relevant in P-deficient conditions (Sakuraba et al. 2018). Nutrients participate actively in multiple physiological processes in plants to increase dry matter production and, consequently, the growth and development of crops (Prado 2020). Nitrogen (N), phosphorus (P), and potassium (K) are the most limiting nutrients for tomato plants (Maia et al. 2019). In regions with poor-income settings, there are restrictions on the use of chemical fertilizers, which limits plant growth. Hence, adapted genotypes are required to this kind of environmental condition. NPK deficiencies trigger a series of physiological, biochemical, and metabolic disorders related to NPK function in the plant metabolism. For example, plants with inadequate supply of NPK usually exhibit stunted growth, lower pigment content and 15 quantum efficiency of photosystem II, and high levels of lipid peroxidation in chloroplasts (Kumar Tewari et al. 2007; Chen et al. 2018). To the author’s knowledge, the only study that reported the mode of action of phytochromes in tomatoes, specifically phytochrome A (phyA) under conditions of nutritional sufficiency and macronutrients deficiency, was performed by Carvalho et al. (2016). In that study, phyA deficiency favored N absorption and the production of dry weight under a nutrient sufficiency condition. It was also observed that phyA deficiency had no effect on N- and P-deficient plants. However, reduced N and P absorption lead to a lower dry matter production in K-deficient plants (Carvalho et al. 2016). The responses of plants mediated by phytochromes have already been extensively investigated in Arabidopsis, and the findings indicate that this photoreceptor can modulate positive or negative responses in conditions of macronutrient sufficiency and deficiency. In this sense, we hypothesized that phyB1 and phyB2 participate in the signaling responses of tomato plants triggered by NPK deficiency. We assumed that the deficiency of these photoreceptors would affect the absorption of NPK, the nutritional efficiency, physiology, and growth of tomatoes. To our knowledge, this is the first study that presents such a broad characterization of phyB1 and phyB2-deficient mutants, focusing on plant nutrition. In addition, based on the rudimentary knowledge of plant responses to phytochrome deficiency, it will provide bases for agricultural practice to improve the use of more adapted genotypes to agricultural environment and increase crop production. 2. Materials and methods 2.1. Growth conditions and plant material The experiment was conducted inside a greenhouse at the São Paulo State University (Unesp), in Jaboticabal, Brazil, and lasted for 37 days after sowing (DAS). We used the Solanum lycopersicum L. cv. Moneymaker as wild-type (WT) and photomorphogenic mutants defective for the genes PHYB1 and PHYB2 that encode 16 PHYB1 (phyB1) and PHYB2 (phyB2) apoproteins in the cv Moneymaker background (van Tuinen et al. 1999). The air temperature and humidity data were monitored inside the growth room, with the observed mean temperature day/night of 24 ± 2°C and relative humidity 40 ± 5%. The photon flux density was approximately 70 µmol of photons m-2 s-1, and the photoperiod was maintained at 12h/12h. Seeds of phyB1, phyB2 and WT were placed in polystyrene trays to germinate, which were filled with a mixture of the commercial substrate BioPlant® (composed of sphagnum peat, vermiculite, coconut fiber, rice husk, husk pine, and additives containing calcium), and expanded vermiculite in a volumetric proportion 1: 1 (v: v). At 20 DAS, the plants were transferred to propylene pots (2 L) containing the nutrient solution of Hoagland and Arnon (1950). The solution was initially diluted to 25% ionic strength and after ten days, the concentration was increased to 50%, remaining at this concentration until the end of the experiment. The nutrient solution was prepared with deionized water, continuously aerated with the aid of the water pump and changed at 30 DAS. The pH value of the solution was measured on a daily basis and maintained at 5.5 ± 0.5, with the aid of sodium hydroxide and hydrochloric acid solutions, both at 1%. 2.2. Treatments and experimental design Three experiments were carried out in a 3x2 factorial scheme, with the first factor being the mutant cultivars of tomato plants (WT, phyB1 and phyB2) and the second factor defined as the nutrient solution, which was either complete (containing 15 mmol L-1 of N, 1 mmol L-1 of P and 6 mmol L-1 of K) , deficient in N (1 mmol L-1 of N using NH4NO3 as source, 1 mmol L-1 of P and 6 mmol L-1 of K), P (15 mmol L-1 of N, 0 mmol L-1 of P and 6 mmol L-1 of K) and K (15 mmol L-1 of N, 1 mmol L-1 of P and 0 mmol L-1 of K) (Table 1). A completely randomized design with six replications was adopted, and one replicate was formed by four plants. 17 2.3. Performed analyses 2.3.1. Quantum efficiency of PSII (Fv/Fm) and pigments content The quantum efficiency of PSII was measured between 7:00 and 8:00 h. The third fully expanded leaves were dark-adapted for 30 min using clips, and then the minimal (F0) and maximal fluorescence (Fm), as well as the Fv/Fm were obtained using a portable fluorometer (Opti-Sciences, Os30P). Total chlorophyll and carotenoids contents were determined according to the methodology described by Lichtenthaler (1987). Fresh samples (0.025 - 0.030 g) of the leaves were collected from tomato plants of each treatment, and the readings were performed in a Beckman DU 640 spectrophotometer at 663, 647 and 470 nm to acquire the respective contents of chlorophyll a, b and carotenoids, based on fresh weight. The total chlorophyll content was a result of the sum between the concentrations of chlorophyll a and b. Tabela 1. Composition of nutrient solutions used to induce N, P, and K deficiencies in Solanum lycopersicum L. The values are correspondent to a 100% nutrient solution which was diluted to 25% and 50%. Treatment Sources Complete -N -P -K mol L-1 mL L-1 KH2PO4 1 1 - - KNO3 5 - - - Ca (NO3)2. 5H2O 5 - 5 5 MgSO4 2 2 2 2 KCL - 5 6 - CaCl2. 2H2O - 5 - - NH4H2PO4 - - - 1 NH4NO3 - 0.5 2.5 2 Micronutrients* 1 1 1 1 Fe-EDDHA** 1 1 1 1 *In 1L: 2.86 g H3BO3; 1.81 g MnCl2. 4H2O; 0.10 g ZnCl2; 0.04 g CuCl2; 0.02 g H2MoO4H2O. ** In 1L: 83.33 g Fe-EDDHA 2.3.2. Lipidic peroxidation Lipid peroxidation was determined in shoots samples (stem and leaves), which were collected, washed, immersed in liquid N2, and stored at -80ºC, following the methodology of thiobarbituric acid described by Heath and Packer (1968). The reactive 18 metabolites of cell membranes, especially malondialdehyde (MDA), react with thiobarbituric acid and can be quantified with spectrophotometry. Briefly, shoot tissue (0.3g) was homogenized with 1 mL trichloroacetic acid and polyvinylpyrrolidone (20%). Subsequently, the samples were transferred into tubes and centrifuged at 10,000 g for 15 min. The supernatant was blended with 1 mL of trichloroacetic acid 20% (m/v) and thiobarbituric acid 0.5% (m/v), and then incubated in a water bath at 95°C for 30 min. After this period, samples were centrifuged at 11,000 g for 5 min. The readings were performed in a spectrophotometer at 535 and 600 nm, the concentration of MDA was determined using the coefficient 1.55 × 10−5 mol−1cm−1 (Gratão et al. 2012), and the results were expressed as nmol g−1 of fresh weight. 2.3.3. Growth parameters The plants were harvested at 37 DAS and separated into shoots and roots. The plant material was washed in running water with a detergent solution (0.1% v/v), followed by an HCl solution (0.3% v/v), and deionized water. Subsequently, the material was dried in an oven with forced air circulation (65 ± 5°C) until constant weight to determine the shoot, root and plant dry weight. 2.3.4. Nutrient concentration and use efficiency The concentrations of N, P, and K in the shoots were determined as described by Bataglia et al. (1983). Based on the N, P, and K concentrations and the shoot dry weight, nutrient accumulation was calculated. The nutrient use efficiency was calculated according to the equation (shoot dry weight)2/ (accumulation of the respective nutrient) (Siddiqi & Glass 1981). 2.4. Statistical analysis All data were submitted to a bidirectional analysis of variance (ANOVA) by the F test (p≤0.05) after checking for homogeneity of variances (teste W de Shapiro-Wilks). When significant, the Tukey multiple comparison test of was applied to compare the 19 means of the genotypes under each nutrient solution at a 5% probability level. The same test was used to compare the deficiency of each nutrient with the complete nutrient solution, within each genotype, using the statistical software AgroEstat®. 3. Results 3.1. Complete nutrient solution The phytochromes deficiency altered the accumulation of nutrients in tomato plants (Fig. 1). Both phyB1 and phyB2 mutant plants accumulated less N, P and K in comparison to WT (Fig. 1). However, none of the mutants differed from WT in terms of MDA concentration (Fig. 2A). In contrast, phyB1 and phyB2 displayed a lower content of Chl a+b and carotenoids (Fig. 2B, C) than WT grown under the complete nutrient solution. These alterations in the contents of pigments did not alter the Fv/Fm ratio as none of the mutants Fv/Fm ratio differed from WT (Fig. 2D). Reductions in the use efficiency of N, P and K were also observed in both phyB1 and phyB2 plants (Fig. 3), which resulted in lower shoot and plant dry weights than WT (Fig. 4A, C). There was no difference between the root dry weight of phyB2 and WT. However, these genotypes showed higher root dry weight than phyB1 plants (Fig. 4B). 3.2. Nitrogen deficiency (-N) A comparison of means revealed that the interaction between genotypes (phyB1, phyB2 and WT) and N deficiency was significant on P and K accumulations (Fig. 1B,C), MDA, carotenoids, Fv/Fm (Fig. 2A,C,D), N use efficiency (Fig. 3A), shoot, root and plant dry weight (Fig. 4). Regardless of the genotype, the N accumulation by plants cultivated under N deficiency was lower than plants grown under complete nutrient solution. Conversely, phyB1 and phyB2 plants had lower N accumulation than WT (Fig. 1A). Both mutants and WT accumulated less P and K (Fig. 1B, C) in comparison to plants subjected to 20 the complete solution; however, no differences were observed between phyB1, phyB2 and WT. The plants of phyB1 and phyB2 cultivated under N deficiency had an increased MDA concentration (Fig. 2A), which was not verified in WT, compared to the complete nutrition treatment. However, phyB1 mutant showed higher levels of MDA than WT and phyB2 when cultivated under N deficiency (Fig. 2A). The concentrations of Chl a+b and carotenoids were reduced in N-deficient plants (Fig. 2B, C). However, a more severe reduction was observed in phyB1 compared to WT. Interestingly, N deficiency did not affect the Fv/Fm ratio in WT and phyB1, but the phyB2 mutant displayed a lower Fv/Fm in comparison to the phyB2 plants cultivated under complete solution and compared to phyB1 and WT in all treatments (Fig. 2D). 21 Figure 1. Nutrient accumulation in the shoots of tomato mutants deficient in phyB1, phyB2, and the WT grown under complete or deficient (-) nutrient solution. (A) N, (B) P, and (C) K. Values are the means ± SE of each treatment (n = 4 replicates, containing four plants each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Tukey's test (p < 0.05). G represents genotype, S represents nutrient solution, and G x S represents the interaction between factors. A significant and interactive effect caused by N deficiency was observed in the utilization of N by the mutant cultivars and WT. Both phyB1 and phyB2 plants and WT had a reduced use efficiency of N compared to the plants cultivated under complete solution (Fig 3A); nevertheless, no differences were observed between genotypes. The use efficiencies of P and K were lower in N-deficient plants, regardless of the genotype. 22 However, phyB1 presented lower P and K use efficiency (Fig. 3B, C) compared to WT. Conversely, no differences were observed in the use efficiency of P between all plants in N-deficient conditions, while phyB2 had a lower use efficiency of K than WT (Fig 3C). The shoot and plant dry weights of the mutants phyB1, phyB2, and WT decreased when these were cultivated under N deficiency (Fig. 4A, C). In addition, shoot and plant dry weights were similar in mutants and WT, and the root dry weight decreased only in phyB2 and WT plants grown under N deficiency; however, phyB1 and phyB2 mutants presented lower root dry weight than WT (Fig. 4B). 3.3. Phosphorus deficiency (-P) The ANOVA revealed a significant interaction (p ≤ 0.05) between genotype and nutrient solution for all variables evaluated (Figs 1-4). When exposed to P deficiency, all genotypes accumulated less N, P, and K than plants cultivated under the complete nutrient solution (Fig. 1). None of the phytochrome mutants differed from the WT in the levels of N and P under P deficiency (Fig. 1A,B). However, phyB1 accumulated more N than phyB2. Phytochrome B1 appeared to play an important role in K accumulation, as the phyB1 mutant showed a higher accumulation of this macronutrient than WT and phyB2 (Fig. 1C). The plants of phyB1, phyB2 and WT had an increased MDA concentration when cultivated under P deficiency compared to the treatment with complete solution; however, phyB1 presented higher levels of MDA than WT and phyB2 under P deficiency (Fig. 2A). Nevertheless, all genotypes displayed reduced concentrations of Chl a+b and carotenoids (Fig. 2B, C), leading to a decreased Fv/Fm ratio (Fig. 2D). A more severe reduction in pigment accumulation and Fv/Fm was observed in phyB2 mutant compared to WT and phyB1. On the other hand, phyB1 plants showed a similar accumulation of pigments than WT, as well as a higher Fv/Fm ratio than WT and phyB2 under P deficient conditions. 23 Figure 2. (A) Malondialdehyde (MDA), (B) chlorophyll a + b, (C) carotenoid content and (D) quantum efficiency of PSII (Fv/Fm) of tomato mutants deficient in phyB1, phyB2, and the WT grown under complete or deficient (-) nutrient solution. Values are the means ± SE of each treatment (n = 4 replicates, containing four plants each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Tukey's test (p < 0.05). G represents genotype, S represents nutrient solution, and G x S represents the interaction between factors. The use efficiency of N was lower in phyB1, phyB2 and WT plants grown under P deficiency compared to plants cultivated under complete solution (Fig. 3A). P- stressed phyB1 mutant had a higher N use efficiency than WT and phyB2 (WT and phyB2 having similar results). Under P deficiency, the use efficiency of N increased as well bith in the mutants and WT; however, phyB1 presented a higher efficiency than WT and phyB2 (Fig. 3B). It was possible to observe that shoot and plant dry weights were reduced because of P deficiency in all genotypes compared to plants cultivated under complete solution (Fig. 4A, C). In addition, P-stressed phyB1 plants had a higher root dry weight than the plants cultivated under complete solution, while phyB2 plants displayed a reduced root dry weight under the same condition (Fig. 4B). Both mutants showed similar results in relation to WT, but phyB1 presented a higher root dry weight than phyB2. However, phyB1 displayed a higher shoot dry weight than WT and phyB2. 24 3.4. Potassium deficiency (-K) The ANOVA (p ≤ 0.05) showed that the genotypes behaved differently in solutions with/without K for the variables N, P and K accumulation (Fig. 1), Chl a+b (Fig. 2B), N, P and K use efficiency, shoot and plant dry weights (Fig. 4A,C). Plants cultivated under K deficiency accumulated less N, P and K in relation to the ones grown under complete solution (Fig. 1). In addition, phyB1 and phyB2 mutants grown in this deficient condition showed similar K accumulation than WT (Fig. 1C). While phyB2 had a lower N accumulation than WT, phyB1 showed a lower accumulation of P (Fig. 1B). 25 Figure 3. Nutrient use efficiency of tomato mutants deficient in phyB1, phyB2, and the WT grown under complete or deficient (-) nutrient solution. (A) N, (B), and (C) K. Values are the means ± SE of each treatment (n = 4 replicates, containing four plants each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Tukey's test (p < 0.05). SDW represents shoot dry weight; G represents genotype, S represents nutrient solution, and G x S represents the interaction between factors. 26 Figure 4. (A) Shoot, (B) root, and (C) plant dry weight of tomato mutants deficient in phyB1, phyB2, and the WT grown under complete or deficient (-) nutrient solution. Values are the means ± SE of each treatment (n = 4 replicates, containing four plants each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Tukey's test (p < 0.05). G represents genotype, S represents nutrient solution, and G x S represents the interaction between factors. Regardless of the genotype, K deficiency increased lipid peroxidation, as revealed by the analysis of MDA concentration in plants. The mutant phyB1 showed higher levels of MDA than WT and phyB2, and phyB2 was similar to WT (Fig. 2A). When submitted to a K-deficiency condition, phyB1, phyB2 and WT displayed reduced chlorophyll a+b levels compared to plants cultivated under complete solution. Interestingly, the phytochrome B mutants phyB1 and phyB2 had lower concentrations 27 of Chl a+b (Fig. 2B) than WT plants grown under K deficiency. Regardless of the genotype, the plants grown under K deficiency had lower carotenoid content and Fv/Fm ratio (Figs. 2C, D) than the ones grown under complete solution. Moreover, the carotenoid content and Fv/Fm ratio were lower in the phytochrome mutants than WT. K deficiency negatively affected the use efficiency of N and P in phyB1, phyB2 and WT compared to the complete treatment (Fig. 3A, B). On the other hand, in all genotypes, K deficiency increased the use efficiency of this nutrient (Fig. 3C). Although the phytochrome mutants used in this study presented similar statistical results concerning the use efficiency of N and P (Fig. 3A, B), phyB1 and phyB2 showed a lower K use efficiency under K deficiency than WT (Fig. 3C). Regardless of genotype, K-stressed plants presented lower root dry weight than plants grown under complete solution (Fig. 4B). Furthermore, phyB1 plants displayed a lower production of root dry weight compared to WT and phyB2, and phyB2 was statistically similar to WT (Fig. 4B). On the other hand, shoot and plant dry weights were reduced due to K deficiency in all genotypes (Fig. 4A, C); however, phyB1, phyB2, and WT displayed statistically similar results concerning these variables. 4. Discussion In order to improve the nutritional management of crops of high economic importance, such as tomatoes, research is needed to elucidate the relationships between absorbed nutrients, their accumulation, and their effects on plant metabolism, both under nutritional sufficiency and deficiency. Moreover, studies demonstrated that phytochromes can be an important part of the plant’s responses to nutritional deficiency. For instance, under nutritional sufficiency, a negative control of phyA in tomatoes was observed as N absorption and accumulation and the production of dry weight were reduced in phyA (Carvalho et al. 2016). Carvalho et al (2016) also observed that the role of phyA on nutritional relationships in tomatoes is complex because other types of phytochromes may be part of these responses. For example, the phyB family has been shown to participate to many abiotic stress responses (Junior et al. 2021). Thus, we hypothesized that phyB1 and phyB2 are part of the tomato plants’ response to nutrient deficiency. 28 4.1. Complete nutrient solution One of the roles of the red light that is perceived by phytochromes is related to N, P, and K absorption by plants (Sakuraba & Yanagisawa 2018). Our results suggest that tomatoes phyB1 and phyB2 are positive regulators of N, P, and K absorption under nutritional sufficiency, as their accumulations were lower in the mutants in comparison to WT (Fig. 1). No differences were observed in MDA concentration and Fv/Fm ratio between mutants and WT (Fig. 2A, D) supplied with a complete nutrient solution. However, phyB1 and phyB2 had a reduced concentration of Chl a+b and carotenoids (Fig. 2B, C) compared to WT. The interaction between phytochromes and pigments was reported in previous studies, in which the authors suggested that the lower concentration of chlorophyll in phyB1 mutants is due to the participation of this photoreceptor in chlorophyll biosynthesis, and the development of chloroplasts (Zhao et al. 2013). Previous studies also reported that phytochromes suppress chlorophyll degradation and the levels of lipid peroxidation in thylakoids (Joshi et al. 1991). Those results pointed out the positive role of phyB in chlorophylls, in addition to the synthesis of carotenoids and their accumulation in plants grown under nutritional sufficiency. The deficiency of PHYB1 and PHYB2 caused a reduction in the use efficiency of N, P, and K, which might indicate that these phytochromes participate in the regulation of the activity of nitrate reductase, the enzyme responsible for the metabolism of N (Sakuraba & Yanagisawa 2018). Moreover, the lower N accumulation may have reduced the absorption of other nutrients, such as P and K due to the interaction between these nutrients (Cavalcante et al. 2019), and led to a reduction in N use efficiency and consequently of the other nutrients (Fig. 3). It was also observed that phyB1 and phyB2 decreased the shoot and whole plant biomass production (Fig. 4A, C), highlighting the importance of tomato phyB as a positive regulator of pigment production, nutritional efficiency and growth. This was an expected result, seen that active phytochromes control the allocation of biomass, and its deficiency may reduce vegetative biomass accumulation in tomato plants (Mereb et al. 2020). 29 4.2. Nitrogen deficiency (-N) The biological functions of N are directly related to the process of ionic absorption, given its part in the structural composition of amino acids, proteins and chlorophyll, in addition to its role in the synthesis of antioxidant compounds such as enzymes (Marschner 2012; Prado 2020; Zhang et al. 2017). Therefore, N deficiency led to a reduced acumulation of this element by shoots, causing a ionic imbalance due to a reduction in the absorption of P and K in both mutants and the control compared to the complete nutrient solution (Fig. 1). In addition, the nutritional imbalance promoted an increase in the lipid peroxidation of membranes (MDA) of phyB1 and phyB2 and a decrease in the production of photosynthetic pigments, as shown in the results of the mutants and WT (Fig. 2). The strong relationship found between N and the metabolism of P and K led to a reduction in the use efficiency of N, P and K in all genotypes studied (Fig. 3); thus, the plants converted a lower amount of nutrients into biomass, as pointed out in the results of the shoots, roots, and the whole plants’ dry weight (Fig. 4). The deficiency of phyB1 and phyB2 reduced N absorption, suggesting that regardless of the status of N in the plant, phytochromes act on N absorption. In addition, the similar results of P accumulation observed in phyB1 and phyB2 in comparison to WT indicate that these photoreceptors are not part of the signaling for P absorption in conditions of N deficiency (Fig. 1). On the other hand, in phyB mutants, K accumulation was also similar to the control genotype, which suggests that the capacity of K absorption in N-deficient plants is also not under the control of this photoreceptor. Plants phyB1 in N-deficient conditions displayed an increased level of MDA and reduced concentration of photosynthetic pigments (Fig. 2A, C), whereas phyB2 mutants had an increased concentration of MDA and decreased chlorophylls (Fig. 2B, D) regardless of N deficiency. These results suggest different mechanisms of action of phyB1 and phyB2 in the antioxidant system, as well as a production of photosynthetic pigments, where phyB1 acts in association with N, while phyB2 does not. These nutritional and physiological changes triggered by N deficiency were not sufficient to cause damages in the functioning of the photosynthetic apparatus, as 30 shown by the Fv/Fm ratio in all genotypes (Fig. 2D). In N-deficient plants, the functions displayed by phyB1 did not seem to compromise the absorption and metabolism of N and K as the use efficiency of both elements by mutant plants was equivalent to that of the control genotype (Fig. 3A, C). However, the deficiency of this photoreceptor caused a reduction in the use efficiency of P (Fig. 3B). Thus, phyB1 plants deficient in N did not modify the dry weight of shoots or whole plants compared to WT (Fig. 3A, C). However, the results of root dry weight suggest that phyB1 deficiency diminished the root system’s development (Fig. 3B). In tomato plants cultivated under N deficiency, phyB2 did not compromise the N metabolism as the N use efficiency was similar between phyB2 and control (Fig. 3A). In contrast, the deficiency of this photoreceptor reduces the use efficiency of K and the dry weight of the roots (Fig. 4). 4.3. Phosphorus deficiency (-P) One of the main functions of P in plants is energy transfer and storage in the form of ATP, which requires inorganic phosphate for its formation. This energy is used by plants to pump nutrients into cells by active absorption (Marschner 2012). In this sense, plants grown on lower P content had a decreased accumulation of P that caused an antagonistic effect on the absorption of N and K (Fig. 1). In P-deficient plants, phyB1 accumulated more N than phyB2; however, the mutants showed a similar accumulation of N and P than WT (Fig. 1A, B). This suggests that the phyB family is not part of N and P absorption signaling under P deficiency. In this study, it was found that K absorption is under the control of phyB1 as the deficiency of this photoreceptor resulted in a higher accumulation of K (Fig. 1C), which was significantly higher in phyB1 than WT and phyB2. The low availability of P increased MDA concentration (Fig. 2A), which in this study can be associated with the decrease in chlorophyll and carotenoids contents (Fig. 2B,C), thus resulting in lower efficiency of the energy transfer (Fv/Fm; Fig. 2D) compared to a complete nutrient solution (Patel et al. 2020b; Shi et al. 2020). However, under the same condition, lipid peroxidation in phyB1 plants exceeded the values obtained in WT and phyB2. This result shows the beneficial control of phyB1 in lipidic peroxidation and cell membrane integrity, which was significantly higher than phyB2. Previous studies demonstrated that increased levels of hydrogen peroxide (signaling 31 molecule) might be necessary for enabling plants to activate their defense system and restore the redox homeostasis, thus favoring plants’ growth and enhancing their resistance to abiotic stress (Hung et al., 2005; Niu & Liao, 2016). Therefore, we assumed that this may also occur for MDA based on our phyB1 results (increase in MDA and shoot dry weight), an observation that needs to be clarified in the future. The higher increase in lipidic peroxidation observed in phyB1 mutant plants was not enough to induce modifications in the concentration of photosynthetic pigments in comparison to WT (Fig. 2B, C), which increased the efficiency of photosystem II (Fig. 2D). However, phyB2 decreased the accumulation of pigments and the Fv/Fm ratio, suggesting that, under natural conditions, phyB2 positively participates in the synthesis of chlorophyll and carotenoids and consequently modulates the energy transfer to the photosystem II under P deficiency. In this study, we verified that all genotypes had a lower use efficiency of N when plants were cultivated under P deficiency (Fig 3A). However, there was an increase in P use efficiency (Fig. 3B) that can be an adaptive strategy of the genotypes to maintain their metabolic functions in response to P starvation (Patel et al. 2020b). Furthermore, phyB2 and WT plants showed a reduced use efficiency of K compared to plants grown under complete nutrient solution. However, P deficiency did not affect K-use efficiency in phyB1 plants (Fig. 3C). Based on these results, it is reasonable to say that phyB1 acts as a negative regulator of the nutritional responses and the efficiency of photosynthetic apparatus in P-deficient tomato plants. On the other hand, phyB2 contributed positively in controlling the damages of lipid peroxidation, pigment accumulation, efficiency of photosystem II, and the use of P and K in the metabolism. As expected, there was a reduction in shoot and plant dry weights in phyB1, phyB2 and WT grown under P-deficiency (Figs. 4A and C). In contrast, the root dry weight was lower in phyB2 mutants and higher in phyB1, maybe due to an adaptive strategy developed by phyB1 mutant to improve the absorption of this element in conditions of nutritional deficiency (Cavalcante et al. 2019). It was observed that the deficiency of phyB1 increased shoot biomass production (Fig. 4A), which could support the hypothesis of phyB1 negative control of shoot biomass production in response to P deficiency. This result is of paramount importance as it brings a new perspective on the use of tomato mutants in agriculture, considering that the deficiency of phyB1 32 mitigated the damages of P deficiency. Furthermore, the results showed that phyB1 negatively controls root dry weight in a more intensive manner than phyB2. It is also suggested that phyB2 is negatively involved in the regulation of plant biomass production under low P supply. 4.4. Potassium deficiency (-K) Considering that K stimulates the uptake and transport of nitrate as well as the transcriptional levels of phosphates transporters genes (Ma et al. 2012; Rubio et al. 2014; Santos et al. 2017), a deficiency condition of K might result in reduced absorption rates of K, N and P by phyB mutants, as well as in WT. In this study, the deficiency of phyB1 did not cause variations in N absorption, but the results revealed that phyB1 positively regulates P absorption as the mutant had reduced P accumulation compared to WT (Fig. 1B). On the other hand, phyB2 plants had a reduced N accumulation under K-deficiency and none of the phyB mutants caused variations in K accumulation (Fig. 1C). These results reveal that phyB1 induces the accumulation of P under natural conditions, while phyB2 is involved in N and P accumulation in the K-dependent pathway. Thus, this can be associated with the phytochromes participation in the synthesis and regulation of nitrate reductase activity, which in this study seemed to be controlled by phyB2. In addition, K participates in the synthesis of ATP that is used by plants in active absorption of P (Hafsi et al. 2014). Previous studies reported that an adequate supply of K enables plants to maintain membrane integrity, produce secondary metabolites and defense-related compounds that favor the antioxidant mechanism (Wang et al. 2013). K deficiency increased lipid peroxidation of membranes, as evidenced by the higher production of MDA in all genotypes (Fig. 2A), especially in phyB1 and phyB2, suggesting the involvement of these photoreceptors in the maintenance of membrane integrity. Furthermore, all genotypes decreased chlorophyll and carotenoids content, as well as the efficiency of photosystem II (Figs. 2B-D) under K deficiency; however, this decrease was more severe in phyB1 and phyB2 mutants. In a study conducted with cotton plants, K deficiency reduced the concentration of chlorophylls and altered the ultrastructure of chloroplasts, which showed fewer grana (Zhao et al. 2001). Furthermore, it was reported that phyB plays a major role in the synthesis of 33 chlorophylls, development of chloroplasts and formation of grana (Zhao et al. 2013), thus justifying the results obtained in this study. In a previous study with phyA, phyB1, and phyB2 tomato mutants, Kreslavski et al. (2020) reported that the deficiency of these phytochromes reduced the photosystem II activity, and the reduction was proportional to the number of missing phytochromes, which might also be attributed to a lower concentration of photosynthetic pigments. The plants of all genotypes grown under K deficiency displayed lower N- and P- use efficiency (Fig. 3A, B). However, phyB1, phyB2 and WT had a higher K-use efficiency (Fig. 3C), probably as a defense strategy developed by plants given the low amount of K in plant tissues. Consequently, plants reduced their production of shoot, root and whole plant dry weights because of the lower absorption and capacity to metabolize and convert nutrients in dry matter. In addition, under a K-deficient condition, N- and P-use efficiency of phyB mutants remained similar to WT, as well as shoot and plant dry weights (Fig. 4A, C), even though the deficiency of phyB1 and phyB2 led to a reduction of the K-use efficiency (Fig. 3C). We suggest that red light-mediated phyB1 positively regulates P absorption and accumulation as well as the photosynthetic pigments in K-deficient tomatoes. On the other hand, the responses of phyB2 mutant suggest that this photoreceptor is involved in inducing N absorption. However, the molecular, biochemical, and physiological mechanisms in which phytochromes control the nutrition, physiology, and growth of tomato plants grown under nutritional deficiency remain unclear. So far, the pieces of evidence from our study indicate that phyB1 and phyB2 mediated by red light signaling positively modulate the growth of tomato plants, ensuring proper nutrition by promoting the absorption and metabolism of N, P and K, the concentration of photosynthetic pigments, and dry matter production. However, when cultivated under nutrient deficiency, the nature of the responses induced by these photoreceptors varies in function of the deficient nutrient. We also believe that our findings are novel and can contribute to breeding programs identifying new cultivars that are less sensitive to NPK deficiency. Acknowledgements 34 The support of the São Paulo State University (UNESP) is gratefully acknowledged. Authors would like to extend their sincere appreciation to Pedro Luís da Costa Aguiar Alves for providing the greenhouse of his laboratory, where the experiment was carried out. 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Rice Sci 20: 243–248 39 CAPÍTULO 3 - O Fitocromo B1 de tomateiro modula as respostas a deficiência de N, P e K através da comunicação parte aérea-raiz Resumo Embora os fitocromos estejam envolvidos na expressão de genes de transportadores de nutrientes e participem da sinalização de respostas em plantas sob deficiência nutricional, nós verificamos se a interação entre o fitocromo B1 (phyB1) e N, P e K ocorre reciprocamente a partir da comunicação entre parte aérea e raiz. Para isso, utilizamos combinações de enxertia entre o genótipo controle (WT) e o mutante deficiente em phyB1 de tomateiro (phyB1) sob suficiência nutricional e deficiência individual de N, P e K. Em condição de suficiência nutricional, phyB1 da parte aérea estimula a absorção de N e P nas raízes além de aumentar a condutância estomática, transpiração e produção de massa seca, enquanto phyB1 radicular regula a produção de clorofilas na parte aérea. Sob deficiência de N, as plantas enxertadas com perda de função de phyB1 da parte aérea transpiraram mais e foram menos eficientes no uso da água. Entretanto a combinação entre WT/phyB1 atenuou os danos provocados pela deficiência de N, pois aumentou a massa seca da planta inteira. Sob deficiência de P, a perda de função de phyB1 radicular diminuiu a absorção de N e aumentou a a produção de malondialdeído (MDA), enquanto a deficiência de ambos phyB1 prejudicou a eficiência de uso da água das plantas deficiente em P. Em tomateiros sob deficiência de K, a absorção de N e K está sob controle de ambos fitocromos, da parte aérea e da raiz; enquanto phyB1 da parte aérea regula produção de MDA e aumenta a fotossíntese. Nós concluímos que phyB1 faz parte da comunicação entre parte aérea-raiz no controle das respostas nutricionais, fisiológicas e de crescimento do tomateiro. Palavras-chave: Solanum lycopersicum L., luz vermelha, phyB1, mutante, deficiência nutricional 1. Introdução Um dos principais fatores que limita a produção agrícola no mundo é a deficiência nutricional (Patel et al., 2020a). De modo geral, o nitrogênio (N), fósforo (P) e potássio (K) são os elementos que mais limitam o crescimento das plantas devido esses elementos são considerados os mais responsivos pelas plantas (Marschner, 2012). Compreender as respostas das culturas à deficiência de N, P e K torna-se fundamental para a melhoria da eficiência do uso desses elementos e a redução da adubação. 40 A deficiência de N diminui a sua absorção e concentração em todas as partes das plantas de citrus e amendoim, causando desbalanço nutricional, inibição no crescimento, diminuição da concentração de pigmentos fotossintéticos e assimilação de CO2 (De Souza Osório et al., 2020; Patel et al., 2020b; Huang et al., 2021). Além disso, o P é requerido em diversos processos metabólicos na planta, a exemplo da formação da molécula de ATP, utilizada como fonte de energia celular (Prado, 2021). Foi relatado que em plantas deficientes em P houve diminuição da fotossíntese e da condutância estomática além da formação de espécies reativas de oxigênio (ROS) nos cloroplastos (Hernández e Munné-Bosch, 2015). Já a deficiência de K é conhecida por causar danos na fotossíntese, transpiração e condutância estomática (Pandey e Mahiwal, 2015). Não é surpresa que a luz modula a nutrição em plantas (Carvalho et al., 2016; Sakuraba e Yanagisawa, 2018; Soares et al., 2021; D’Amico-Damião et al., 2022), e, recentemente, tem se tornado cada vez mais evidente a função dos fotorreceptores nessa resposta. A família dos fitocromos B (phyB), está envolvida nas respostas ao estresse por deficiência nutricional (Carvalho et al., 2016; Sakuraba et al., 2018; Soares et al., 2021), e em estudo prévio, a perda de função de phyB-9 e phyB-10 resultou na redução da absorção de P em plantas de Arabidopsis Thaliana. Os mesmos autores também verificaram diminuição na expressão de genes dos transportadores de alta afinidade de P, indicando que a luz vermelha faz parte da ativação da absorção de P, especialmente sob deficiência desse nutriente (Sakuraba et al., 2018). Mostramos recentemente que phyB1 de tomateiro é um regulador positivo da absorção de N, síntese de pigmentos e produção de massa seca sob deficiência de N (Soares et al., 2021). Além disso, a perda de função desse fotorreceptor atenuou os danos provocados pela deficiência de P, uma vez que as plantas do mutante aumentaram a produção de massa seca comparado ao genótipo controle (Soares et al., 2021). Sob deficiência de K, phyB1 mostrou ser regulador positivo da absorção de P bem como da produção de pigmentos (Soares et al., 2021). Isso sugere um forte controle de phyB1 sobre as respostas nutricionais induzidas por N, P e K. Dessa forma, para realização do presente trabalho, hipotetizamos que a interação entre phyB1 e N, P e K ocorre, reciprocamente, a partir da comunicação entre a parte aérea 41 e raiz. Pela primeira vez, nós exploramos o papel do gene PHYB1 de tomateiro nas respostas a deficiência de N, P e K utilizando o mutante fotomorfogenético phyB1 e enxertia para entender a participação desse fotorreceptor na comunicação parte aérea-raiz. 2. Material e Métodos 2.1. Condições de crescimento, material vegetal e enxertia O experimento foi conduzido em sala de crescimento e casa de vegetação da Universidade Estadual Paulista, Jaboticabal, Brasil, por 65 dias após a semeadura (DAS). Foram utilizadas plantas de Solanum lycopersicum L. cv. Moneymaker como tipo selvagem (MM) e do mutante fotomorfogenético phyB1, que é defectivo no gene PHYB1 que codifica a apoproteína PHYB1 (Van Tuinen et al., 1995). Sementes de phyB1 e MM foram colocadas em bandejas de poliestireno para germinar, as quais foram preenchidas previamente com uma mistura do substrato comercial BioPlant® (composto de turfa de esfagno, vermiculita, fibra de coco, casca de arroz, casca de pinus e aditivos contendo cálcio) e vermiculita expandida na proporção volumétrica 1:1 (v:v). Dezessete DAS, no mesmo substrato, as plantas foram transferidas para vasos de 200 mL (uma planta por vaso) e realizada a enxertia. Plantas com dezessete dias foram enxertadas pelo método de garfagem em fenda simples, com auxílio de lâmina de bisturi e clipes de enxertia para obter as seguintes combinações de enxertia: MM/MM, phyB1/phyB1, MM/phyB1, phyB1/MM (enxerto/porta-enxerto). Após a enxertia, a base dos vasos foi submersa em água (em câmara úmida flutuante a 25ºC ± 2ºC e alta umidade relativa: 85% ± 10%) sob fotoperíodo de 12 h a 70 μmol de fótons m-2 s- 1, até a cicatrização completa do ponto de enxerto (20 dias após a enxertia, DAE) (Figura 1A). Após a cicatrização da enxertia, as raízes das plantas enxertadas foram lavadas com água deionizada para retirar o excesso do substrato, e as plantas foram transferidas para vasos de 1,7 dm³ contendo areia de textura média, que foi previamente descontaminada com solução de ácido clorídrico 0,1 mol L-1 e lavada 42 com água deionizada. Os vasos foram transferidos para casa de vegetação e irrigados diariamente com solução nutritiva de Hoagland e Arnon (1950) para nutrir as plantas e manter a umidade próxima à capacidade máxima de retenção de água. A solução nutritiva foi inicialmente diluída em 25% de força iônica e após dez dias, a concentração foi aumentada para 50%, permanecendo nesta concentração até o final do experimento. A solução nutritiva foi preparada com água deionizada, e o valor de pH da solução foi mantido em 5,5 ± 0,5 utilizando soluções de hidróxido de sódio e ácido clorídrico, ambos a 1%. Os dados de temperatura e umidade do ar foram monitorados diariamente dentro da casa de vegetação. Houve variação na umidade relativa máxima (85,1 ± 9 ºC) e mínima (33 ± 12 ºC), e na temperatura máxima (28,7 ± 10 ºC) e mínima (14,7 ± 7 ºC). 2.2. Tratamentos e delineamento experimental Três experimentos foram realizados em esquema fatorial 4 x 2, sendo o primeiro fator composto pelas combinações de enxertia entre as plantas de tomateiro (MM/MM, phyB1/phyB1, MM/phyB1, phyB1/MM), e o segundo fator definido como a solução nutritiva, que era completa (contendo 15 mmol L-1 de N, 1 mmol L-1 de P e 6 mmol L-1 de K) , deficiente em N (1 mmol L-1 de N utilizando como fonte NH4NO3), omissão de P (0 mmol L-1), ou com omissão de K (0 mmol L-1) na solução nutritiva (Tabela 1). Foi adotado o delineamento em blocos casualizados, com quatro repetições e a unidade experimental formada por uma planta. 43 Tabela 2. Composição da solução nutritiva utilizada para induzir as deficiências de N, P e K em Solanum lycopersicum L. Os valores são correspondentes a uma solução a 100% que foi diluída para 25% e 50% de força iônica. Tratamentos Fontes Completa -N -P -K mol L-1 mL L-1 KH2PO4 1 1 - - KNO3 5 - - - Ca (NO3)2. 5H2O 5 - 5 5 MgSO4 2 2 2 2 KCL - 5 6 - CaCl2. 2H2O - 5 - - NH4H2PO4 - - - 1 NH4NO3 - 0.5 2.5 2 Micronutrientes* 1 1 1 1 Fe-EDDHA** 1 1 1 1 *Em 1L: 2,86 g H3BO3; 1,81 g MnCl2. 4H2O; 0,10 g ZnCl2; 0,04 g CuCl2; 0,02 g H2MoO4H2O. ** Em 1L: 83,33 g Fe-EDDHA 2.3. Análises realizadas 2.3.1. Trocas gasosas A atividade fotossintética, condutância estomática e a transpiração foram medidas considerando a terceira folha completamente expandida a partir do ápice, utilizando o analisador de gás infravermelho (Li-6400, Licor, EUA) com radiação fotossintéticamente ativa de 900 µmol m-2 s-1 e temperatura foliar a 25 oC. A eficiência do uso da água foi calculada dividindo-se atividade fotossintética pela transpiração. 2.3.2. Eficiência quântica do FS