UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS – RIO CLARO unesp PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLOLÓGICAS (BIOLOGIA VEGETAL) Da germinação à regeneração: o papel do alumínio (Al) e sua interação com os processos fisiológicos de espécies acumuladoras de Al do Cerrado MATHEUS ARMELIN NOGUEIRA Rio Claro – SP 2023 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS – RIO CLARO unesp PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (BIOLOGIA VEGETAL) Da germinação à regeneração: o papel do alumínio (Al) e sua interação com os processos fisiológicos de espécies acumuladoras de Al do Cerrado MATHEUS ARMELIN NOGUEIRA Tese apresentada ao Instituto de Biociências do Câmpus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas (Biologia Vegetal). Orientador: Dr. Gustavo Habermann Coorientador: Dr. Jean Carlos Cardoso Rio Claro – SP 2023 N778g Nogueira, Matheus Armelin Da germinação à regeneração: o papel do alumínio (Al) e sua interação com os processos fisiológicos de espécies acumuladoras de Al do Cerrado / Matheus Armelin Nogueira. -- Rio Claro, 2023 94 p. : tabs. Tese (doutorado) - Universidade Estadual Paulista (Unesp), Instituto de Biociências, Rio Claro Orientador: Gustavo Habermann Coorientador: Jean Carlos Cardoso 1. Plantas acumuladoras de Al. 2. germinação de sementes. 3. organogênese. 4. espécies do Cerrado. 5. micropropagação. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca do Instituto de Biociências, Rio Claro. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. UNIVERSIDADE ESTADUAL PAULISTA "Da germinação à regeneração: o papel do alumínio e sua interação com os processos fisiológicos de plantas acumuladoras do cerrado" TÍTULO DA TESE: CERTIFICADO DE APROVAÇÃO AUTOR: MATHEUS ARMELIN NOGUEIRA ORIENTADOR: GUSTAVO HABERMANN COORIENTADOR: JEAN CARLOS CARDOSO Aprovado como parte das exigências para obtenção do Título de Doutor em Ciências Biológicas (Biologia Vegetal), área: Biologia Vegetal pela Comissão Examinadora: Prof. Dr. GUSTAVO HABERMANN (Participaçao Virtual) Departamento de Biodiversidade / Instituto de Biociencias de Rio Claro Unesp Profª. Drª. MARINA ALVES GAVASSI (Participaçao Virtual) Departamento de Ciências Biológicas / UNESP / Bauru Prof. Dr. CLEBERSON RIBEIRO (Participaçao Virtual) Departamento de Biologia Geral / Universidade Federal de Viçosa Profª. Drª. ANNA CAROLINA GRESSLER BRESSAN (Participaçao Virtual) Departamento de Ecologia / Universidade de São Paulo - Instituto de Biociências Prof. Dr. LUIZ ALFREDO RODRIGUES PEREIRA (Participaçao por Parecer Circunstanciado) Departamento de Botânica / Universidade de Brasília Rio Claro, 26 de outubro de 2023 Instituto de Biociências - Câmpus de Rio Claro - Av. 24-A no. 1515, 13506900 http://ib.rc.unesp.br/#!/pos-graduacao/secao-tecnica-de-pos/programas/biologia-vegetal/apresentacao/CNPJ: 48.031.918/0018-72. Título alterado para: Da germinação à regeneração: o papel do alumínio (Al) e sua interação com os processos fisiológicos de espécies acumuladoras de Al do Cerrado. Agradecimentos Este trabalho é dedicado à memória dos meus avós, Dona Maria e Seu Lorival. A influência de vocês em minha vida vai além das palavras. Agradeço aos meus pais, Regina e José, que sempre estiveram ao meu lado me encorajando e me orientando com sabedoria e paciência. Vocês são a minha inspiração e o meu exemplo de vida. Eu espero poder retribuir um pouco do que vocês fizeram por mim. À minha companheira, Juliana. Sua coragem e determinação não apenas me inspiram, mas também me dão a força necessária para enfrentar os desafios da vida. Seu amor e apoio foram e são essenciais nesta jornada. Amo você! Aos meus queridos amigos Caio, Carol, Felipe, Thiago e Vitor, que sempre acreditaram em mim e sempre e me apoiaram. Cada palavra de encorajamento e cada gesto de amizade contribuíram para a realização deste trabalho. Às minhas amigas do grupo de pesquisa Anna, Brenda, Giselle, Luá e Marina por todo apoio, amizade e incentivo. Ao meu orientador, Gustavo, agradeço a oportunidade de aprender e crescer academicamente. Ao meu coorientador, Jean. Sua orientação e apoio foram fundamentais para a realização deste trabalho. Agradeço também a todo o grupo do Laboratório de Fisiologia Vegetal e Cultura de Tecidos da UFSCar. A colaboração, o companheirismo e a troca de conhecimentos dentro do laboratório enriqueceram não apenas este trabalho, mas também a minha jornada acadêmica. Ao Ntaps (Núcleo de Atenção Psicossocial do campus da UNESP), especialmente à Débora e ao Rafael. Com grande empatia e profissionalismo, me orientaram e ajudaram a me colocar de volta nos trilhos. Sou grato à Reserva Biológica de Mogi-Guaçu e a toda sua equipe. Um agradecimento especial ao João, cuja ajuda constante tornou o trabalho mais leve e agradável. Agradeço ao Laboratório de Pós-colheita de Produtos Hortícolas-USP/ESALQ-LPV por todo apoio e assistência. Cada um dos nomes citados tem um lugar especial neste trabalho e em meu coração e agradeço a vocês por terem acreditado em mim e por terem me apoiado, mesmo agora, em cada passo desta jornada. Este doutorado é tanto meu quanto de vocês. Ao Instituto de Biociência da Unesp de Rio Claro pelo apoio e infraestrutura. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (#14056/2019-3). Resumo O Al é o metal mais abundante na crosta terrestre e, quando presente em solos ácidos (pH < 5,0), torna-se solúvel e tóxico para a maioria das espécies de plantas. Por outro lado, existem plantas que conseguem prosperar em solos com alta disponibilidade de Al e são chamadas de acumuladoras. Essas plantas acumulam grandes quantidades de Al em seus tecidos sem apresentar sinais de toxicidade. No capítulo 1 utilizamos as espécies Qualea grandiflora e Vochysia tucanorum (Vochysiaceae), plantas acumuladoras de Al nativas do Cerrado, para testarmos diferentes hipóteses. Neste estudo, testamos a germinação de sementes em meio de cultura com concentrações crescentes de Al e sob a influência do etileno (Et). As sementes foram cultivadas in vitro em meio MS (Murashige e Skoog 1962) a ¼ de força contendo 0, 90, 180, 270 e 320 μM de Al. As sementes foram tratadas com água e Et para o cultivo. Aqui evidenciamos que o Al impacta significativamente a germinação e o crescimento de Q. grandiflora e V. tucanorum e que o Et induziu a germinação em sementes de V. tucanorum sem Al, sugerindo um efeito substitutivo. O capítulo 2 fornece insights valiosos sobre o papel do Al na organogênese e na produção de compostos fenólicos nessas espécies. O impacto do alumínio (Al) na organogênese e na produção de compostos fenólicos dessas espécies foi observado em Al a 320 μM e ausência de BAP (6-Benzilaminopurina), resultou na regeneração de raízes adventícias em ambas. No entanto, a adição de BAP sem Al não gerou respostas de regeneração. A regeneração e o desenvolvimento de brotos foram observados quando 0,44 e 0,88 μM de BAP foram adicionados ao meio de cultura com 320 μM de Al. Além disso, ambas as espécies exibiram um notável conteúdo fenólico, que foi ainda mais aprimorado pela adição de Al e/ou BAP. A capacidade antioxidante dos extratos também aumentou significativamente com a adição de Al e/ou BAP. Essas abordagens podem fornecer perspectivas valiosas para estratégias de conservação e propagação dessas espécies do Cerrado. Palavras-chave: Plantas acumuladoras, germinação de sementes, organogênese, espécies do Cerrado, micropropagação. Abstract Aluminum (Al) is the most abundant metal in the Earth's crust and, when present in acidic soils (pH < 5.0), becomes soluble and toxic to most plant species. Conversely, some plants thrive in soils with high Al availability and are termed accumulators. These plants accumulate large amounts of Al in their tissues without showing signs of toxicity. In Chapter 1, we employed the species Qualea grandiflora and Vochysia tucanorum (Vochysiaceae), Al-accumulating plants native to the Cerrado, to test different hypotheses. In this study, we examined seed germination in culture medium with increasing Al concentrations and under the influence of ethylene (Et). Seeds were cultured in vitro on 1/4-strength MS medium (Murashige and Skoog 1962) containing 0, 90, 180, 270, and 320 μM of Al. Seeds were treated with water and Et for cultivation. We highlighted that Al significantly impacts the germination and growth of Q. grandiflora and V. tucanorum, and Et induced germination in V. tucanorum seeds without Al, suggesting a substitutive effect. Chapter 2 provides valuable insights into the role of Al in organogenesis and phenolic compound production in these species. The impact of aluminum (Al) on organogenesis and phenolic compound production was observed at 320 μM Al and the absence of 6-Benzylaminopurine (BAP), resulting in the regeneration of adventitious roots in both species. However, the addition of BAP without Al did not generate regeneration responses. Regeneration and shoot development were observed when 0.44 and 0.88 μM BAP were added to the culture medium with 320 μM Al. Additionally, both species exhibited a notable phenolic content, further enhanced by the addition of Al and/or BAP. The antioxidant capacity of the extracts also significantly increased with the addition of Al and/or BAP. These approaches can provide valuable insights for conservation and propagation strategies for these Cerrado species. Key words: Al-accumulating plants, seed germination, organogenesis, Cerrado species, micropropagation SUMÁRIO Introdução geral 8 Referências 16 CAPÍTULO I Is aluminum (Al) essential for embryos germination of Qualea grandiflora and Vochysia tucanorum, Al-accumulating species? 28 Abstract Introduction 30 Material and methods 33 Study area and plant material 33 Aluminum concentration in donor plants 33 Experimental strategy 34 Culture medium 34 In vitro seed germination essays 35 Embryo germination parameters 35 Ethylene measurements 36 Data analysis 36 Results 37 Aluminum concentration in leaves, fruits, and seeds 37 Aluminum and ethylene effect on seed germination 37 Aluminum and ethylene effect on root and shoot growth 38 Ethylene production in vitro 39 Discussion 39 References: 43 Tables 49 Figures 51 Supplementary material 56 CAPÍTULO II 57 In vitro organogenesis, content phenols and antioxidant capacity of two aluminum accumulator plants species from the Cerrado region, Brazil 57 Abstract 58 Introduction 59 Material and Methods 61 Plant Material 61 Aluminum concentration 62 In vitro seed germination and seedling development 62 Organogenesis Induction from Cotyledonary Leaves Segments of Germinated Seedlings 63 Biometric Parameters 63 Total Phenolics and Antioxidant Activity 64 Statistical Analysis 66 Results 67 In Vitro Seed Germination 67 Effects of BAP and Al Treatments on Shoot and Root Proliferation 67 Total Phenolic Contents of Essential Extracts 68 DPPH Radical Scavenging Assay of Extracts 69 Discussion 69 Conclusions 74 References 74 Tables 83 Figures 84 Supplementary material 90 Considerações finais 92 8 Introdução geral A vegetação do Cerrado, amplamente conhecida como ‘savana brasileira’, apresenta entre 1000 e 2000 espécies por ha e se caracteriza por ser um dos hotspots mundiais de biodiversidade (COLLI; VIEIRA; DIANESE, 2020; MYERS et al., 2000). Além disso, é o segundo maior bioma brasileiro (COUTINHO, 2002), sendo superado em área apenas pela Amazônia, ocupando 21% do território nacional (KLINK; MACHADO, 2005; RATTER; BRIDGEWATER; RIBEIRO, 2003). O Cerrado é considerado um mosaico de fisionomias vegetais incluindo florestas, formações savânicas (campo sujo, campo cerrado) e formas campestres bem abertas (campo limpo), que juntas são referidas como Cerrado sensu lato (FERREIRA et al., 2003). Espécies lenhosas são distribuídas em fisionomias mais densas, florestais, como é o caso do ‘Cerradão’. Já no cerrado sensu strictu (s. str.) as espécies constituem um tipo de fisionomia savânica, contendo uma vegetação mais arbustiva e árvores de sub-bosque, com alta irradiância ao nível do solo (KISSMANN et al., 2012). Contudo, muitas espécies endêmicas têm se perdido nas últimas décadas devido principalmente à grande expansão da agricultura e intensa exploração local de produtos nativos (KLINK; MACHADO, 2005; SANO et al., 2010). Essas atividades antrópicas têm provocado a degradação e a perda de habitats desse ecossistema, colocando em risco a sua conservação e o seu potencial biotecnológico (SANO et al., 2019) Cerca de metade dos 2 milhões de km² originais do Cerrado foram transformados em pastagens plantadas, culturas anuais e outros tipos de uso de solo (GOMES et al., 2019). Além disso, estudos que contemplam espécies nativas do Cerrado ainda são incipientes se comparados aos de espécies de outros biomas brasileiros, intensificando a perda de material genético vegetal nativo (STRASSBURG et al., 2017). Dessa forma, a conservação e preservação de tais espécies se torna essencial devido ao cenário atual. 9 Os solos sob o cerrado são geralmente pobres, ácidos (pH < 5,0), bem drenados, profundos e apresentam altos níveis de alumínio trocável (QUEIROZ NETO, 1982; REATTO; CORREIA; SPERA, 1998). O alumínio (Al) pode competir com outros elementos pelos mesmos sítios químicos nas partículas do solo, sendo o terceiro elemento químico mais abundante na crosta terrestre e, em solos ácidos, encontrado principalmente na forma trivalente (Al3+), que é tóxico para a maioria das espécies de plantas (MATSUMOTO, 2000; VARDAR; ÜNAL, 2007). Os solos ácidos representam 30-40% das terras livres de gelo (KOCHIAN; HOEKENGA; PINEROS, 2004; VON UEXKÜLL; MUTERT, 1995) sendo, portanto, juntamente com o Al, ameaças potenciais à agricultura, com impactos negativos para os rendimentos. O primeiro sintoma da toxicidade do Al é a diminuição do crescimento das raízes (HORST; WANG; ETICHA, 2010; SILVA et al., 2019). Em espécies sensíveis, aproximadamente 80-90% do Al absorvido é retido no sistema radicular (SILVA et al., 2023; VITORELLO; CAPALDI; STEFANUTO, 2005). Além dos efeitos diretos nas raízes, o Al pode causar efeitos ‘indiretos’ em órgãos acima do solo, como crescimento reduzido de brotos (JIANG et al., 2009), baixa troca gasosa foliar e respostas fotoquímicas em Coffea arabica (KONRAD et al., 2005), Zea mays (LIDON et al., 1999) e Citrus spp. (BANHOS; DE SOUZA; HABERMANN, 2016; JIANG et al., 2009; JIANG, 2008; SILVA et al., 2018). No entanto, apesar da toxicidade, a flora desta região prospera nestes solos ácidos (HABERMANN; BRESSAN, 2011; HARIDASAN, 2008; NOGUEIRA et al., 2019; SOUZA, et al., 2017). Algumas espécies possuem mecanismos de tolerância ao Al, exsudando ácidos orgânicos como citrato, malato, oxalato e succinato através das membranas celulares de suas raízes. Esses ácidos formam complexos não tóxicos com o Al, prevenindo sua absorção ainda na rizosfera (RYAN et al., 2011). Paralelamente, 10 outras espécies conseguem se desenvolver em solos com alta concentração de Al (m%), acumulando Al em seus tecidos e órgãos sem apresentar danos ou sinais de toxicidade que possam interferir em seu metabolismo ou desenvolvimento (BRESSAN, et al., 2021; DE ANDRADE et al., 2011; DELHAIZE; RYAN, 1995; HARIDASAN, 1982). Exemplos dessas espécies incluem Fagopyrum esculetum Moench (Polygonaceae), Hydrangea macrophylla L. (Hydrangeaceae) e Camellia sinensis (L.) Kuntze (Theaceae), conforme relatado por MA et al, 2001 e WATANABE e OSAKI, 2002. Plantas que armazenam mais de 1000 mg de alumínio por kg de massa foliar seca são chamadas de acumuladoras de Al (CHENERY, 1948). Algumas dessas espécies lenhosas acumuladoras de Al são nativas da vegetação do Cerrado na América do Sul (HARIDASAN, M., 1982) e pertencem às famílias Melastomataceae (gênero Miconia), Rubiaceae, Simplocaceae e Vochysiaceae (gêneros Vochysia e Qualea) (BRESSAN; COAN; HABERMANN, 2016; DE ANDRADE et al., 2011; HARIDASAN, 1982; MALTA et al., 2016; SOUZA, et al., 2015). No entanto, nesta vegetação, essas espécies acumuladoras de Al podem apresentar entre 4.000 e até 20.000 mg Al por kg de massa seca (HARIDASAN, 1982; HARIDASAN, 2008; NOGUEIRA et al., 2019), demonstrando uma grande variação na acumulação de Al. Além das folhas, essas plantas também podem reter Al nas sementes (HARIDASAN, 1982; SCALON; HARIDASAN; FRANCO, 2013). Qualea grandiflora Mart. e Vochysia tucanorum Mart. são espécies de plantas nativas do Cerrado que acumulam Al em suas folhas e raízes (BRESSAN et al., 2021; BRUNNER; SPERISEN, 2013; HARIDASAN, 1982; NOGUEIRA et al., 2019; SILVA, et al., 2023). Pesquisas anteriores mostraram que este metal pode apresentar uma resposta benigna neste grupo de plantas, aumentando a biomassa vegetal e o alongamento das raízes em C. sinensis, Melastoma malabathricum L. (Melastomataceae) e Quercus 11 serrata Murray (Fagaceae) (BOJÓRQUEZ-QUINTAL et al., 2017). As relações específicas e o impacto potencial do Al nos parâmetros fisiológicos que explicam a influência positiva do alumínio no crescimento das plantas em espécies acumuladoras de alumínio são raramente documentadas ou não comumente relatadas. Outro padrão de distribuição entre essas plantas envolve a dependência do Al, como Vochysia tucanorum (Vochysiaceae). Esta espécie não cresce bem em solo calcário e apresenta folhas necróticas e raízes atrofiadas após 45 dias em comparação com um desenvolvimento perfeito em solo ácido rico em Al (SOUZA et al., 2017). O mesmo padrão foi observado quando esta espécie perdeu suas folhas e apresentou raízes necróticas em solução nutritiva sem Al (BRESSAN et al., 2021). Qualea grandiflora segue o mesmo comportamento em condições sem Al (CURY et al., 2020). Apesar desses relatos, nenhum papel fisiológico foi atribuído ao Al nessas plantas ainda. Além de sua habilidade de acumular Al e se adaptar às condições adversas, algumas plantas típicas do Cerrado também têm demonstrado a capacidade de produzir compostos fenólicos (ARRUDA; ARAÚJO; JUNIOR, 2022) como forma de adaptação para resistir ao estresse oxidativo causado por condições ambientais extremas (FARIAS et al., 2013; MOREIRA-ARAÚJO et al., 2019). Os ácidos fenólicos são os metabólitos secundários mais abundantes nas plantas e desempenham papéis importantes em diversos processos fisiológicos (ZHANG et al., 2022). Eles desempenham um papel importante nos mecanismos de defesa das plantas, sendo depositados nas paredes celulares após a infecção por patógenos (KUMAR, Santosh et al., 2020). Quando são excretados pelos sistemas radiculares das plantas, podem inibir o crescimento na rizosfera adjacente e afetar a flora bacteriana do solo (CHOWDHARY et al., 2021). Eles também podem atuar como moduladores do desenvolvimento das plantas, regulando o catabolismo do ácido indol acético (IAA). Eles são eficazes na regulação do crescimento das plantas, 12 diferenciação celular e organogênese (KUMAR et al., 2020; KUMAR et al., 2023). Eles são conhecidos por suas propriedades antioxidantes (FARIAS et al., 2013), e além de sustentarem a sobrevivência dessas plantas em um ambiente hostil, também despertaram o interesse na indústria farmacêutica e alimentícia devido aos benefícios potenciais para a saúde humana (ALBUQUERQUE et al., 2021; SOARES, 2002). A interação complexa entre o ambiente do Cerrado, incluindo a presença de alumínio, pode influenciar a presença de compostos fenólicos e sua atividade antioxidante (NOGUEIRA et al., 2023). Investigar os impactos do Al nessas plantas pode ser um desafio, pois propagá-las e cultivá-las em condições ex situ que isolam os efeitos do Al de outros elementos é uma tarefa complexa (CARDOSO; DA SILVA, 2013; LEITE et al., 2021; LIMA et al., 2022). A cultura de tecidos pode ser uma técnica valiosa para conservar, cultivar e propagar espécies do Cerrado sob condições controladas, permitindo o isolamento e teste de fatores, como o Al, no meio de cultura. Ela é utilizada para a propagação de plantas quando a regeneração através da germinação de sementes é desafiadora. Técnicas de propagação in vitro vem sendo utilizadas como alternativas para propagação de espécies nativas do Cerrado, especialmente para fins econômicos e de conservação (FRANÇA et al., 1995; PEREIRA et al., 2005; REZENDE et al., 2019). Elas oferecem diversas possibilidades para a propagação de plantas podendo se utilizar, por exemplo, de materiais vegetativos para a indução de organogênese e regeneração sob condições assépticas e controladas (CARDOSO; SHENG GERALD; TEIXEIRA DA SILVA, 2018). Entre as técnicas normalmente utilizadas na cultura de tecidos vegetais, a micropropagação tem sido amplamente utilizada para propagar rapidamente várias espécies de interesse econômico, tais como Eucalyptus spp., Citrus spp, (Caryocar brasiliense), o cajuzinho-do-cerrado (Anacardium othonianum Rizz.), o araticum 13 (Annona crassiflora) e o baru (Dipteryx alata Vogel.), (AYALA et al., 2019; DANIELLE SOUSA et al., 2017; RIBEIRO et al., 2009; SANTOS et al., 2006; SILVEIRA; DA SILVA; SIBOV, 2022). A micropropagação consiste em técnicas de propagação vegetativa com o objetivo de obter multiplicação vegetal através: a) embriogênese somática, b) indução direta ou indireta de calos para formação de partes aéreas, e c) indução do desenvolvimento de botões apicais/axilares (AL-MAYAHI, 2019). Em comparação com os métodos tradicionais de propagação vegetativa de plantas, ela apresenta diversas vantagens, como a obtenção de plantas geneticamente uniformes, livres de pragas e doenças, com alto vigor e qualidade, em um curto espaço de tempo e em um pequeno espaço físico (CARDOSO; SHENG GERALD; TEIXEIRA DA SILVA, 2018; MURASHIGE; SKOOG, 1962). Essa técnica pode contribuir para o estudo da fisiologia, da bioquímica e da genética das plantas do cerrado, bem como para o seu uso sustentável e a sua valorização econômica (NIEMEYER, 2017; SANTOS et al., 2006). Além disso, as técnicas de micropropagação não dependem das condições climáticas e são especialmente úteis em espécies que apresentam sementes recalcitrantes, que perdem rapidamente a sua viabilidade; sendo o caso de várias espécies perenes tropicais (LITZ; JAISWAL, 1991). A micropropagação é também importante para os esforços de conservação a longo prazo para preservar a biodiversidade de espécies e ecossistemas ameaçados (CHOKHELI et al., 2020). Apesar dessas vantagens, a micropropagação de plantas do Cerrado também enfrenta alguns desafios como a escassez de informações sobre as exigências nutricionais e fisiológicas das espécies, a baixa taxa de multiplicação e enraizamento in vitro, a ocorrência de anormalidades morfológicas e fisiológicas nas plântulas, a dificuldade de aclimatização e a falta de protocolos padronizados para cada espécie. 14 A germinação de plantas do Cerrado apresenta características distintas, refletindo a resiliência e a adaptabilidade dessas espécies ao seu ambiente único (FRANCO; HARIDASAN; FERREIRA, 2008; ZAIDAN; CARREIRA, 2008). Existem sementes de certas espécies (ou até mesmo dentro da mesma espécie) que não conseguem germinar ou encontram dificuldades para fazê-lo, mesmo quando incubadas em condições que pareçam ser favoráveis. Essas sementes são classificadas como dormentes e não são capazes de germinar nas mesmas condições (BEWLEY, 1997; CORBINEAU et al., 1995). A maioria das sementes de espécies arbóreas do Cerrado exibe um estado de dormência, com a dormência física sendo a mais prevalente (BASKIN; BASKIN, 2001). Estratégias de regeneração dessas espécies envolvem germinação, no entanto, a dormência de sementes é um fenômeno comum na maioria dessas espécies (ZAIDAN; CARREIRA, 2008). Especificamente, o etileno (Et) regula a germinação e a dormência de várias espécies por meio de uma complexa rede de sinalização hormonal (ARC et al., 2013; MATILLA; MATILLA-VÁZQUEZ, 2008). No contexto das sementes de V. tucanorum, foi demonstrado que o Et tem a capacidade de interromper a dormência das sementes e promover a germinação (PEREIRA et al., 2011), embora as justificativas fisiológicas para este fenômeno ainda não estejam completamente esclarecidas. Considerando esse cenário, o presente trabalho utilizou-se de duas espécies nativas do Cerrado acumuladoras de Al: Q. grandiflora e V. tucanorum. No capítulo 1, considerando a dificuldade em obter mudas de espécies lenhosas do Cerrado que acumulam Al, e uma possível interação entre o Et e a dormência das sementes dessas espécies, foram realizados testes de germinação em meio de cultura com concentrações crescentes de Al sob a influência de Et, avaliando-se parâmetros de crescimento e performance germinativa. A hipótese é que seus embriões dependem do Al para retomar o crescimento e desenvolvimento e que o Et poderia melhorar o desempenho da 15 germinação. No capítulo 2, testamos a hipótese da relevância do Al e sua possível interação com a citocinina 6-benzilaminopurina (BAP) na promoção da organogênese de brotos e raízes adventícias dessas espécies. Além disso, investigou-se se a presença de Al e BAP tem uma relação com o conteúdo total de ácidos fenólicos e a atividade antioxidante em extratos dessas duas espécies do Cerrado. Essa abordagem integrada permite uma compreensão mais profunda das interações complexas entre essas espécies do Cerrado, o Al, o BAP e o Et. Dado que não há estudos existentes que empregam o Al em meio de cultura para plantas lenhosas nativas do Cerrado, a importância deste estudo reside em sua capacidade de abrir portas para novas linhas de pesquisa futuras sobre o assunto. Ele oferece uma nova perspectiva e um ponto de partida valioso para explorar ainda mais o potencial e as aplicações do Al na cultura de tecidos de plantas nativas do Cerrado. 16 Referências AL-MAYAHI, Ahmed Madi Waheed. Effect of aluminum on the growth of the in vitro culture tissues of the date palm (Phoenix dactylifera L.) cv. Um-Aldehin. Folia Oecologica, vol. 46, no. 2, p. 164–169, 2019. https://doi.org/10.2478/foecol-2019-0019. ALBUQUERQUE, Bianca R; HELENO, Sandrina A; OLIVEIRA, M Beatriz P P; BARROS, Lillian; FERREIRA, Isabel C F R. 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Matheus Armelin Nogueira1, Jean Carlos Cardoso2, Gustavo Habermann3 1Programa de Pós-Graduação em Biologia Vegetal, Departamento de Biodiversidade, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Av. 24-A, 1515; 13506-900, Rio Claro, SP, Brazil; 2Laboratório de Fisiologia Vegetal e Cultura de Tecidos, Departamento de Biotecnologia, Produção de Plantas e Animais, Centro de Ciências Agrárias, Universidade Federal de São Carlos, Rodovia Anhanguera, km 174, CEP 13600-970 Araras, SP, Brazil 3Departamento de Biodiversidade, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Av. 24-A, 1515; 13506-900, Rio Claro, SP, Brazil. 31 Abstract Qualea grandiflora and Vochysia tucanorum (Vochysiaceae) are Aluminum- accumulating woody species from the Cerrado vegetation in South America and retain aluminum (Al) in their seeds. Studies suggest Al dependency for these species, but no physiological role has been ascribed to Al in these plants. Due to difficulties in obtaining seedlings of these species (ethylene (Et) seems to enhance their germination), we germinated their seeds in culture medium with increasing Al concentrations and under ethylene (Et) influence to see how important is their seed Al content to germination. Despite showing more than 8,000 (Q. grandiflora) and 32,000 (V. tucanorum) mg Al kg- 1 seeds, both species, but mainly V. tucanorum, germinated better in Al-enriched medium. Et induced germination of V. tucanorum seeds when Al was not present in the medium, suggesting Et could replace Al during germination of this Al-accumulating species. Further research is proposed on the physiological roles of Al and Et in these Al- accumulating species. Key-words: Al3+, Brazilian savanna, culture medium, ethylene, Vochysiaceae. 32 Introduction Aluminum (Al) is the third most abundant element in the Earth's crust, and in acidic soils (pH < 5.0) it is found mainly in the trivalent form (Al3+), which is toxic to most plant species (Matsumoto 2000; Vardar and Ünal 2007). Acidic soils account for 30–40 % of ice-free land (Von Uexküll and Mutert 1995; Kochian et al. 2004). Therefore, acidic soils and Al are potential threats to agriculture with negative impacts for yields. The first symptom of Al toxicity is the inhibition of root growth (Horst et al. 2010; Silva et al. 2019). In sensitive species, approximately 80–90 % absorbed Al is retained in the root system (Vitorello et al. 2005; Silva et al. 2019, 2023). Besides direct effects on the roots, Al can cause ‘indirect’ effects on aboveground organs, such as reduced shoot growth (Jiang et al. 2009), low gas exchange rates and photochemical performance in Coffea arabica (Konrad et al. 2005), Zea mays (Lidon et al. 1999) and Citrus spp. (Jiang et al., 2008, 2009; Banhos et al., 2016; Silva et al., 2018). Some species are tolerant to Al and exude organic acids (OA) (citrate, malate, oxalate, and succinate) through the root cell membranes forming non-toxic Al-OA complexes, avoiding Al uptake in the rhizosphere (Ryan et al. 2011). Other species grow well on acidic soils with high Al saturation (m%) showing no toxicity symptoms (Haridasan 1982; Ma et al. 2001; Bressan et al. 2016). These plants absorb and accumulate Al in their roots (Zaia et al. 2022) and different organs (Timpone and Habermann 2022) but mainly in leaves (Haridasan 1982; Brunner and Sperisen 2013; Bressan et al. 2016; Nogueira et al. 2019). These are considered Al-accumulating species when their leaves show at least 1000 mg Al per kg dry mass (Chenery 1948; Jansen et al. 2002). Some of these Al-accumulating woody species are native to the Cerrado vegetation in South America (Haridasan, 1982) and belong to Melastomataceae (genus 33 Miconia), Rubiaceae, Simplocaceae, and Vochysiaceae (genera Vochysia and Qualea) (Haridasan, 1982; Andrade et al. 2011; Souza et al. 2015; Bressan et al. 2016; Malta et al. 2016). Nevertheless, Cerrado Al-accumulating species show between 4,000 and up to 20,000 mg Al kg-1 dry leaf (Haridasan 1982; Haridasan and De Araújo 1988; Nogueira et al. 2019), demonstrating a large range in Al accumulation. Besides leaves, these plants can also retain Al in seeds (Haridasan et al. 1986; Scalon et al. 2013). Another distribution pattern between these plants involves dependency on Al, as in Vochysia tucanorum (Vochysiaceae). This species does not grow well in calcareous soil, shows necrotic leaves and stunted roots after 45 days in comparison to a perfect development in acidic soil rich in Al (Souza et al. 2017). The same pattern was observed when this species shed their leaves and showed necrotic roots in nutrient solution without Al (Bressan et al. 2021). Qualea grandiflora follows the same response in conditions without Al (Cury et al. 2020). Despite these reports, no physiological role has been ascribed to Al in Al-accumulating and -depending species. Regeneration strategies of Cerrado woody plants involves germination, but most of these woody species show some type of seed dormancy (Zaidan and Carreira 2008). In the case of V. tucanorum seeds, ethylene (Et) was evidenced to break their seed dormancy and induce germination (Pereira et al. 2011), although the physiological reasons for this dormancy break remained unclear. Plant tissue culture is a technique used for plant propagation when regeneration through seed germination is challenging. Given the difficulty in obtaining seedlings of Al-accumulating Cerrado woody species, like Q. grandiflora and V. tucanorum and a possible interaction between these seeds with Et and seed dormancy, we tested germination of seeds of these species in a culture medium with increasing concentrations 34 of Al and under the influence of Et. We hypothesized that their embryos depend on Al to resume growth and development and that Et could enhance the germination performance. Material and methods Study area and plant material The seeds were obtained from fruits collected in two different areas: (i) a forest- type Cerrado vegetation called ‘Cerradão’, which is a more densely treed savanna, whose name is the augmentative of ‘Cerrado’ in Portuguese, and (ii) a remnant of a savanna- type physiognomy named Cerrado sensu stricto (s. str.) broadly known as ‘Brazilian savanna’. The ‘Cerradão’ area was located at the “São José da Conquista” farm (22°15' S, 47°42' W; 770 m of altitude), in the municipality of Itirapina, São Paulo state, southeastern Brazil, and the Cerrado s. str. remnant was located at “Campininha” farm, at the Mogi Guaçu Biological reserve (22°15′ S, 47° 09′ W; 680 m of altitude), an area that has been preserved in its natural conditions since 1950. Five mature plants of Qualea grandiflora Mart. and Vochysia tucanorum Mart. (Vochysiaceae) were randomly selected in both areas between August and September (2020 - 2021), during fruit phase. Soil samples were collected next to the trees at 20–40 cm of depth. Fertility parameters, such as pH, cation exchange capacity (CEC), soil Al saturation (m%) and macro- and micronutrient availabilities were measured (Supplementary material 1). The plants were divided into four quadrants (N, S, E, W), and mature fully expanded green leaves and fruits were collected from each quadrant. Aluminum concentration in donor plants The samples (leaf, fruit, and seed) were taken to a plant nutrition laboratory at the Agronomic Institute of Campinas (IAC, Campinas, Brazil), ground, and digested in a 35 solution of nitric:perchloric acids (3:1, v/v). The concentration of Al was assessed by the inductively coupled plasma optical emission spectroscopy (ICP-OES) method and expressed as mg Al per kg dry mass (Table 1). Experimental strategy Seeds of both species were separated from their fruits, washed, and stored at 25 ± 2 °C. The seeds were immersed for 24 hours in deionized water 240 g L-1 2-chloroethyl phosphonic acid (Ethrel®), an ethylene (Et) -releasing compound. Then, water-treated, and Et-treated seeds had their tegument removed and the embryos were put to germinate (“seeded”) in vitro with culture medium containing 0, 90, 180, 270, and 320 μM Al. The percentage of germination (%G), mean germination time, germination speed index, root length and biomass, shoot length and biomass were assessed over time. To check Et accumulation in the embryos and Et x Al interaction, two groups of seeds (Et-treated and water-treated) were inoculated in vitro with culture medium containing 0, 180, and 320 μM Al and then, Et evolution (production) was quantified in the embryos 14 days after inoculation. Culture medium A MS (Murashige and Skoog 1962) medium at ¼ strength (25% of macronutrients) was used. This reduction in nutrients was necessary to approximate the medium nutrient status to the limited nutrient availability found in Cerrado soils where donor plants grew (Table 1). The medium contained 30 g L-1 sucrose, 0.1 g L-1 myo- inositol, 0.5 mg L-1 6-benzylaminopurine (BAP), and 2 mg L-1 gibberellic acid (GA3) (Duchefa, Haarlem, Netherlands), and it was solidified with 2.7 g L-1 Gelrite®. This medium has been used to successfully germinate seeds of other Cerrado species (Cardoso 36 and da Silva 2013). The medium pH was adjusted to 4.5 ± 0.05, and it was sterilized (with an autoclave) at 121 °C for 25 min. The Al concentrations in the medium (0, 90, 180, 270, and 320 μM Al) were selected in accordance with other studies that tested Al toxicity in Picea glauca (Nosko et al. 1988), Triticum aestivum (Lima and Copeland, 1990), Oryza sativa (Kikui et al. 2005), and Nicotiana tabacum (Vardar et al. 2006). However, there is only one work involving the use of Al in vitro in culture media for Cerrado species (Nogueira et al. 2023). In vitro seed germination essays The seeds were immersed in 70 % ethanol (v/v) for 3 min, followed by a 50 % sodium hypochlorite (containing 1.0–1.25% of active chlorine) solution for 20 min, and rinsed three times with sterilized deionized water. After cleansing, the mature zygotic embryos (MZEs) were retrieved under a laminar flow chamber and inoculated (seeded) in borosilicate test tubes (20 cm high × 2.1 cm wide) containing 65 mL of the culture media and sealed with parafilm®. Test tubes were set in racks under 26 ± 1 °C and 25 μmol photons m-2 s-1 provided with 60W fluorescent bulbs (Phillips, Netherlands) under a 16 h photoperiod. Embryo germination parameters The percentage of germination (%), and root and shoot lengths (cm) were measured at 7, 14, 21, 28, and 35 days after in vitro seeding. Germination speed index (GSI) was calculated according to the method described by Maguire (1962) using the following equation: GSI = G1/N1 + G2/N2 +…+ G5/N5, where Gn/Nn is the number of germinated seeds per number of days in the first (7 days), second (14 days), third (21 days), fourth (28 days), and fifth (35 days) evaluations. 37 We calculated germination time (GT) using the following equation: GT = Σ (Gi x i) / Σ Gi, where “i”, is the number of days between seed inoculation (day 7) and seed germination; Gi, the number of seeds germinated on day i. GT corresponds to the mean germination time of the fraction of seeds that germinated, and does not factor in seeds that failed to germinate (Ellis and Roberts 1980). Ethylene measurements On the 14th day of inoculation, 20 mL of air from test tubes was taken with a gas tithe syringe and analyzed in a gas chromatograph (TRACE 1300 Series, Thermo Fisher Scientific, USA) equipped with a flame ionization detector and 1.5 m column Poropak-Q (80–100 mesh). Chromatograms were generated, Et concentrations calculated and expressed as µL Kg-1 h-1 fresh mass, based on the volume of air in the flasks formed between the culture medium and the sealing, and incubation (for Et production) time. Data analysis The experiments were conducted in randomized blocks, and 10 embryos (replicates) were used per treatment. Each replicate consisted of one testing tube containing one embryo. The embryo germination experiments were repeated twice. Aluminum (0, 90, 180, 270, and 320 μM Al) and Et were studied as the causes of variation. The germination performance (%G), germination time and germination speed index) as well as shoot and root length and biomass were evaluated. The data were submitted to a two-way analysis of variance (two-way ANOVA) to test the Al and Et factors. Tukey test (p < 0.05) was used to compare mean values between treatments. 38 Results Aluminum concentration in leaves, fruits, and seeds Aluminum concentration was significantly higher in the organs of V. tucanorum in comparison to Q. grandiflora. Leaf, fruit, and seed Al concentration was five-, 11- and four-fold higher in V. tucanorum than Q. grandiflora (Table 1). Aluminum and ethylene effect on seed germination Embryos from water-treated seeds of Q. grandiflora germinated well (%G > 70%) (Fig. 1A) when compared with V. tucanorum (%G < 60%) (Fig. 1B), and Al improved its %G only at 180 and 270 μM Al. Ethylene-treated seeds improved %G only at 90 and 320 μM Al (Fig. 1A). Embryos from water-treated seeds of V. tucanorum, however, did not germinate without Al, and only embryos from Et-treated seeds germinated under no Al (Fig. 1B). In the medium with Al, embryos from Et-treated seeds showed increased (+30%) %G (Fig. 1B). Therefore, treating V. tucanorum seeds with Et induced embryo germination when Al was not present in the medium, while Q. grandiflora embryos germinated independent of Al presence in the medium and treating their seeds with Et did not significantly influence embryo germination. Embryos from water-treated seeds of Q. grandiflora showed the highest GSI at 180 μM Al (Fig. 1C), while embryos from water-treated seeds of V. tucanorum showed the highest GSI at 270 μM Al (Fig. 1D). Ethylene-treated seeds improved GSI of Q. grandiflora embryos only when cultivated with Al (Fig. 1C), while V. tucanorum embryos, which did not germinate without Al, showed significantly higher GSI when their seeds were treated with Et and their embryos seeded in media with Al (Fig. 1D). 39 Germination time of embryos from water-treated Q. grandiflora seeds was maximum at 90 μM Al and it was reduced with the increasing Al concentrations in the medium (Fig. 1E). Ethylene-treated seeds did not positively influence Q. grandiflora (Fig. 1E) or V. tucanorum (Fig. 1F) embryo germination. Germination time of embryos from water-treated V. tucanorum seeds was maximum at 90 μM Al (Fig. 1F). Aluminum and ethylene effect on root and shoot growth The root length of germinated embryos from water-treated seeds of Q. grandiflora was the same between Al treatments on each evaluation date (7, 14, 21, 28, and 35 days) (Fig. 2A, B). This parameter did not differ between Al treatments when embryos came from Et-treated seeds (Fig. 2C, D). Vochysia tucanorum embryos from water-treated seeds did not germinate and as expected, did not show root length (Fig. 3A, B). The root length of germinated embryos from water-treated V. tucanorum seeds grew similarly between Al treatments (Fig. 3A, B). Germinated embryos from ethylene-treated V. tucanorum seeds, however, showed higher root length at 90, 180, 270, and 320 μM Al, from 21 days (Fig. 3C, D). Therefore, differences in the root length between Al treatments were only observed in germinated embryos from V. tucanorum Et-treated seeds. Shoot biomass of germinated embryos from Et- and water-treated seeds of Q. grandiflora (Fig. 4A) and V. tucanorum (Fig. 4B) varied considerably between Al treatments. The root biomass of germinated embryos of Q. grandiflora Et-treated seeds was significantly higher than those from water-treated seeds at 0, 90, 180, and 270 μM Al (Fig. 4C). This parameter measured in germinated embryos of V. tucanorum Et-treated seeds was higher than those from water-treated seeds only at 90 μM Al (Fig. 4D). 40 Therefore, the root rather than shoot biomass was improved in the presence of Al, especially for Q. grandiflora. Ethylene production in vitro Although Et was ‘detected’ in germinated embryos from water- and Et-treated seeds of Q. grandiflora and V. tucanorum in media with 0, 180 and 320 μM Al, no significant differences were observed between most treatments. ‘Non-germinated’ embryos from water-treated V. tucanorum seeds produced no Et when compared with germinated embryos from water-treated seeds with 180 and 320 μM Al in the media (Table 2). Therefore, Et was produced in germinated embryos from both species regardless if their seeds were treated with Et or water. Discussion In the present study, we tested the germination of Q. grandiflora and V. tucanorum embryos in culture medium with increasing concentrations of Al under the influence of Et. Embryos from water-treated seeds did not germinate in medium without Al (Fig. 1B). This is intriguing because V. tucanorum seeds showed more than 32,000 mg Al per kg seed (Table 1), meaning that the Al present in their seed, somehow, does not produce the same effect on germination as if provided by the medium in contact with the embryo. The Al concentration in the hypocotyl of germinated embryos from Et-treated V. tucanorum seeds was approximately 20,000 mg kg-1 (data not shown). These results suggest that V. tucanorum seeds require Al in the germination medium to germinate. Soils from the Cerrado, where seeds of these species supposedly lay on (although there is leaf litter before seeds get in contact with the soil) with wind — these are anemochoric seeds 41 (Kawasaki 2007; Ishara and Maimoni-Rodella 2011; Kuhlmann and Ribeiro 2016; Gottsberger and Silberbauer-Gottsberger 2018) — show low pH and m% higher than 70% (Haridasan 1982; Souza et al. 2015; Bressan et al. 2016; Nogueira et al. 2019). On the other hand, treating V. tucanorum seeds with Et induced embryo germination when Al was not present in the medium. This suggests that ethylene may exert a similar effect to Al in promoting germination in this specie. A previous study had already demonstrated Et breaks seed dormancy and improve germination of seeds of V. tucanorum (Pereira et al. 2011), but no further physiological explanation was given by these authors. In Al-sensitive species, it is demonstrated that Al induces overproduction of Et (Massot et al. 2002; Sun et al. 2007), which seems to act as a signal to change auxin distribution in the roots, disrupting AUX1- and PIN2-mediated auxin polar transport, leading to arrest of root elongation (Sun et al. 2010). There are not such refined studies in roots or seeds of Cerrado woody species, but it could be possible that in the case of Al- accumulating species such as V. tucanorum, the Al induces auxin action/transport in these seeds, promoting germination, and Et is able to substitute the Al in this process. Nevertheless, this hypothesis remains to be further studied in seeds of these Cerrado species. Contrary to what is observed in Al-sensitive species, where Al stunts root growth (Horst et al. 2010; Silva et al. 2019, 2023), in the case of Al-accumulating species, but especially V. tucanorum, Al in the medium is critical for seed germination and initial root growth (Fig. 3A, B). In addition, the presence of Et (Et-treated seeds) enhanced even more the root growth (Fig. 3C, D), suggesting a synergistic effect of Et and Al on root growth in this species. In contrast, Q. grandiflora embryos germinated independent of Al presence in the 42 medium and treating their seeds with Et did not significantly influence embryo germination (Fig. 1A) or root elongation (Fig. 2A, B). It was unexpected to observe that Al did not exert a significant effect on these parameters of Q. grandiflora because this is an Al-accumulating species (Haridasan 1982; Bressan et al. 2016; Nogueira et al. 2019) and some studies point out beneficial Al effects on its growth. Saplings of Q. grandiflora supplemented with 150 μM Al in the substrate had healthier appearance, larger shoots, and roots with greater root biomass in comparison with plants not supplemented with Al (Cury et al. 2020). In another study, Q. grandiflora saplings cultivated without Al in nutrient solution show compromised root integrity, reducing the leaf hydration and the Rubisco (Ribulose 1,5-biphosphate carboxylase/oxygenase) in vivo performance, when compared to plants grown with Al (Silva et al. 2023). These studies, however, analyzed saplings (mature plants with some months of age), and it is possible the beneficial Al effects on this species may only become apparent later in development. Our findings suggest that, during in vitro culture, the presence of both Al and ethylene had no significant impact on either germination or initial root growth in seedlings of Q. grandiflora (Fig. 1A; Fig. 2) contrasting with what was observed for V. tucanorum (Fig. 1B; Fig. 3). Despite that, the contrasting response to Al and Et between the two species is also reflected in their root growth. In Q. grandiflora, the root length of germinated embryos remained constant across different Al treatments (Fig. 2), indicating a limited influence of Al on root growth. The root biomass, however, of Q. grandiflora from Et-treated seeds was significantly higher than those from water-treated seeds up to 270 μM Al (Fig. 4C), suggesting a positive interaction between Et and Al in promoting root growth. Root length of V. tucanorum, on the other hand, was higher when seeds were treated with Et and cultivated in media with Al (Fig. 3), but the positive effect of Et was only observed up to 43 90 μM Al in the culture medium (Fig. 4D), indicating the increase in biomass for both species shows a threshold of Al influence. Thus, root elongation and carbon incorporation into biomass are distinct responses and should be evaluated with care. In this study, we demonstrate that Al has a significant effect on the germination and growth of Q. grandiflora and V. tucanorum. 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Sun, P., Q.-Y. Tian, M.-G. Zhao, X.-Y. Dai, J.-H. Huang, L.-H. Li, and W.-H. Zhang. 2007. Aluminum-induced ethylene production is associated with inhibition of root elongation in Lotus japonicus L. Plant and Cell Physiology 48:1229–1235. Oxford University Press. Sun, P., Q. Y. Tian, J. Chen, and W. H. Zhang. 2010. Aluminium-induced inhibition of root elongation in Arabidopsis is mediated by ethylene and auxin. Journal of Experimental Botany 61:347–356. Timpone, L. T., and G. Habermann. 2022. Is aluminum (Al) eliminated by senescent structures of Miconia albicans, an Al-accumulating species from Brazilian savanna? Flora 289:152036. Elsevier. Vardar, F., E. Arican, and N. Gozukirmizi. 2006. Effects of aluminum on in vitro root growth and seed germination of tobacco (Nicotiana tabacum L.). Advances in Food Sciences 28:85–88. Vardar, F., and M. Ünal. 2007. Aluminum toxicity and resistance in higher plants. İstanbul Kültür Üniversitesi. Vitorello, V. A., F. R. Capaldi, and V. A. Stefanuto. 2005. Recent advances in 49 aluminum toxicity and resistance in higher plants. Brazilian Journal of Plant Physiology 17:129–143. Von Uexküll, H. R., and E. Mutert. 1995. Global extent, development and economic impact of acid soils. Plant and soil 171:1–15. Springer. Zaia, M., L. T. Timpone, and G. Habermann. 2022. Do aluminum (Al)-accumulating species from the Brazilian savanna accumulate Al in the roots? Trees 36:1677– 1685. Springer. Zaidan, L. B. P., and R. C. Carreira. 2008. Seed germination in Cerrado species. Brazilian Journal of Plant Physiology 20:167–181. 50 Tables Table 1. Aluminum concentration in leaves, fruit, and seeds of Qualea grandiflora Mart. and Vochysia tucanorum Mart. collected in a cerrado sensu stricto remnant, in the municipalities of Itirapina and Mogi Guaçu, São Paulo state, southeastern Brazil. Mean values (n = 5) ± standard deviation Species Organ mg Al kg-1 dry mass Q. grandiflora Leaf 8,053.66 ± 323.71 Fruit 1,305.88 ± 523.62 Seed 8,406.0 ± 236.66 V. tucanorum Leaf 40,536.66 ± 1,844.59 Fruit 11,036.67 ± 1,294.69 Seed 32,250.0 ± 964.36 51 Table 2. Ethylene concentration (µL/Kg-1 h-1) in germinated embryos from ethylene- and water- treated seeds of Qualea grandiflora Mart. and Vochysia tucanorum Mart., grown for 14 days with 0, 180 and 320 µM Al. Mean values (n = 3) ± standard deviation (-) = water-treated seeds (+) = Ethylene-treated seeds For each Et- or water-treated seeds of the same species, distinct letters represent significant differences between Al treatments by Tukey test (P < 0.05) Species Et µM Al µL kg-1 h-1 Q. grandiflora - 0 0.002 ± 0.00 a 180 0.006 ± 0.10 a 320 0.003 ± 0.02 a Q. grandiflora + 0 0.001 ± 0.01 a 180 0.002 ± 0.03 a 320 0.002 ± 0.01 a V. tucanorum - 0 0 ± 0.00 b 180 0.006 ± 0.04 a 320 0.008 ± 0.02 a V. tucanorum + 0 0.007 ± 0.00 a 180 0.010 ± 0.01 a 320 0.007 ± 0.04 a 52 Figures Figure 1. Mean values (n = 20) of germination percentage (G%) (A, B), time (GT) (C, D), and speed index (GSI) (E, F) of mature zygotic embryos of Q. grandiflora (A, C, E) and V. tucanorum (B, D, F) rescued from water-treated seeds (-Et) and ethylene-treated seeds (+Et) grown on culture 53 medium containing 0, 90, 180, 270 and 320 μM Al. Different lower case and upper case letters indicate significant differences by Tukey test (P < 0.05) between Al-treatments in water-treated and Et-treated seeds, respectively. For each Al treatment, asterisks indicate significant differences by Student t-test (P < 0.05) between water- and Et-treated seeds. Bars = s.e. Significant differences indicated by Tukey test. 54 Figure 2. Mean value (n = 20) of root of mature zygotic embryos of Q. grandiflora rescued from water-treated seeds (-Et) (A) and ethylene-treated seeds (+Et) (C) grown on culture medium containing 0, 90, 180, 270, and 320 μM Al at 14, 21, 28, and 35 days after inoculation. Morphological details of water-treated (B) and Et-treated (D) seedlings at the end of the 35-day evaluation period. No significant differences were detected between treatments as determined by the Tukey and Student’s tests (p > 0.05). Bars = s.e. 55 Figure 3. Mean value (n = 20) of root of mature zygotic embryos of V. tucanorum rescued from water-treated seeds (-Et) (A) and ethylene-treated seeds (+Et) (C) grown on culture medium containing 0, 90, 180, 270, and 320 μM Al at 14, 21, 28, and 35 days after inoculation. Morphological details of water-treated (B) and Et-treated (D) seedlings at the end of the 35-day evaluation period. Different letters indicate significant differences by Tukey test (P < 0.05) between Al-treatments in Et-treated seeds. For each Al treatment, the significant differences between the water- and Et-treated seeds were determined by Student’s t-test (P < 0.05). Bars = s.e. Significant differences indicated by Tukey test. Ellipses indicate statistically similar treatments. 56 Figure 4. Mean values (n = 20) of shoot biomass (A, B) and root biomass (C, D) of mature zygotic embryos of Q. grandiflora (A, C) and V. tucanorum (B, D), rescued from water-treated seeds (- Et) and ethylene-treated seeds (+Et) grown on culture medium containing 0; 90; 180; 270, and 320 μM of Al. Different lower case and upper case letters indicate significant differences by Tukey test (P < 0.05) between Al-treatments in water-treated and Et-treated seeds, respectively. For each Al treatment, asterisks indicate significant differences by Student t-test (P < 0.05) between water- and Et-treated seeds. Bars = s.e. Significant differences indicated by Tukey test. 57 Supplementary material Supplementary material 1. Macro and micronutrient concentrations and fertility parameters in a cerrado sensu stricto remnant, in the municipalities of Itirapina and Mogi Guaçu, São Paulo state, southeastern Brazil. Soil Type pH OM P resin Al3+ H+Al3+ K+ Ca2+ Mg2+ BS CEC (CaCl2) (g dm−3) (mg dm−3) ----------------------------------------mmol charges dm−3---------------------------------------- Itirapina 3.8 ± 0.1 a 9.4 ± 1.9 b 1.8 ± 0.4 a 15.8 ± 2.6 a 41.6 ± 5.7 b 0.4 ± 0.2 a 2.4 ± 0.9 a 1 ± 0.0 a 3.8 ± 1.0 a 45.4 ± 5.3 a Mogi Guaçu 3.8 ± 0.5 a 14.74 ± 3.4 a 0.7 ± 0.0 b 12.6 ± 0.8 a 50.16 ± 6.3 a 0.3± 0.5 b 0.24 ± 0.1 b 0.48 ± 0.04 b 1.04 ± 0.15 b 51.2 ± 6.4 a V% m% B Cu Fe Mn Zn (%) (%) --------------------------------------------------mg dm−3-------------------------------------------------- 8.6 ± 2.8 a 80.4 ± 6.8 b 0.27 ± 0.0 a 0.28 ± 0.0 b 55.2 ± 17.2 a 1.4 ± 0.6 b 0.1 ± 0.0 b 2.0 ± 0.0 b 92.2 ± 0.4 a 0.18 ± 0.0 b 0.34 ± 0.1 a 56.1 ± 15.1 a 1.92 ± 0.5 a 0.2 ± 0.0 a OM = Organic matter; BS = Base saturation (K+ + Ca2+ + Mg2+); CEC = K+ + Ca2+ + Mg2+ + H+ + Al3+ ; V % = Fertility rate [V = 100 ( K+ + Ca2+ + Mg2+ ) CEC− 1], %; Soil Al saturation (m%) = (100 × Al3+)/( BS + Al3+) Mean values (n = 5) ± standard deviation Different letters represent significant difference between soil types by Student t-test 58 CAPÍTULO II In vitro organogenesis, content phenols and antioxidant capacity of two aluminum accumulator plants species from the Cerrado region, Brazil Matheus Armelin Nogueira1*, Vitor Rodrigues Marin2, Gustavo Habermann3, Jean Carlos Cardoso4 1Programa de Pós-Graduação em Biologia Vegetal, Departamento de Biodiversidade, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Av. 24-A, 1515, 13506-900, Rio Claro, SP, Brazil 2Departamento de Biologia Geral e Aplicada, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Av. 24-A, 1515, 13506-900 Rio Claro, SP, Brazil 3Departamento de Biodiversidade, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Av. 24-A, 1515, 13506-900, Rio Claro, SP, Brazil 4 Laboratório de Fisiologia Vegetal e Cultura de Tecidos, Departamento de Biotecnologia, Produção de Plantas e Animais, Centro de Ciências Agrárias, Universidade Federal de São Carlos, Rodovia Anhanguera, km 174, CEP 13600-970 Araras, SP, Brazil * Corresponding author email: matheus.armelin@unesp.br 59 Abstract Aluminum (Al) is the most plentiful metal present in the Earth's crust, and when present in acidic soils (pH < 5.0), it becomes soluble and toxic to most plant species. Species from acidic and aluminum-rich soil regions, such as Brazilian Cerrado, developed mechanisms allowing for their growth in these conditions. Some can accumulate Al in their tissues, especially in the leaves. However, the possible functions of Al in these plants are unknown. This study investigated the impact of Al on the organogenesis and production of phenolic compounds in extracts from cotyledonary leaf segments of two Al- accumulator species, Qualea grandiflora Mart. and Vochysia tucanorum Mart. (Vochysiaceae). The addition of Al at 320 μM in the absence of BAP resulted in the regeneration of both species’ adventitious roots in cotyledonary leaf segments. In contrast, adding BAP without Al did not generate regeneration responses. However, shoot regeneration and development occurred when 0.44 and 0.88 μM BAP was added to the culture medium with 320 μM Al. Both species exhibited a noteworthy phenolic content, further enhanced by adding Al and, or, BAP. The antioxidant capacity of extracts also demonstrated a significant increase from the addition of Al and, or, BAP in both species. These findings have important significance for the cultivation and propagation of these species and demonstrate a close relationship between Al and the evolution of these plants. This study is the first to relate Al with phenolic content and antioxidant activity in these two Cerrado plant species, filling a gap in existing research. Keywords: Aluminum, adventitious shoots, cytokinins, organogenesis, Plant species 60 Introduction The Cerrado biome in Brazil is a diverse and endangered area, covering about 20% of the country and containing high levels of endemism (Colli et al. 2020). It is considered a vital center of plant diversity with approximately 10,000 plant species, many of which are unique to this biome (Ratter et al. 2003; Klink and Machado 2005). The Cerrado soil is acidic (pH < 5.0), dystrophic, has low nutrient availability, and it also contains high levels of available aluminum (m% > 70%) [m%= (100 x Al)/(BS + Al), where BS is the base saturation] (Vitorello et al. 2005; Singh et al. 2017). In acidic soils (pH < 5.0), aluminum minerals undergo solubilization, resulting in the formation of Al3+ ions as well as related oxides and hydroxides. These forms of aluminum are considered toxic to numerous plant species (Kochian et al. 2015). However, despite the toxicity, the flora in this region thrives in these acidic soils (Haridasan 2008; Habermann and Bressan 2011; de Souza et al. 2017; Nogueira et al. 2019). Although some species are capable of growing in soils with high Al saturation (m%), others also developed the capacity to accumulate Al in different tissues and organs without any damage or sign of toxicity that could affect their metabolism or development (Haridasan 1982; Delhaize and Ryan 1995; de Andrade et al. 2011; Bressan et al. 2021). Examples of such species include Fagopyrum esculetum Moench (Polygonaceae), Hydrangea macrophylla L. (Hydrangeaceae), and Camellia sinensis (L.) Kuntze (Theaceae) as reported by Ma et al. (2001) and Watanabe and Osaki (2002). Plants that store more than 1 g of aluminum per kg of dry leaf mass are referred to as Al- accumulators (Chenery 1948). Qualea grandiflora Mart. and Vochysia tucanorum Mart. are native plant species from the Cerrado that accumulate Al in their leaves and roots (Haridasan 1982; Brunner and Sperisen 2013; Nogueira et al. 2019; Bressan et al. 2021; 61 Silva et al. 2023). Previous research has shown that this metal might exhibit a benign response in this group of plants, increasing plant biomass and root elongation in C. sinensis, Melastoma malabathricum L. (Melastomataceae) and Quercus serrata Murray (Fagaceae) (Bojórquez-Quintal et al. 2017). The specific relationships and potential impact of Al on physiological parameters that explain the positive influence of aluminum on plant growth in aluminum-accumulating species are seldom documented or not commonly reported. In the Brazilian Cerrado biome, many plant species produce phenolic compounds and have developed adaptations to resist the oxidative stress caused by extreme environments (Farias et al. 2013; Moreira-Araújo et al. 2019). These adaptations include a higher expression and activity of antioxidant enzymes and an increased synthesis of phytochemicals, particularly phenolic compounds (Arruda et al. 2022). Phenolic acids are the most abundant secondary metabolites in plants and play important roles in diverse physiological processes (Zhang et al. 2022). They play an important role in plant defense mechanisms by being deposited in cell walls after pathogen infection. When they are excreted from plant root systems, they can inhibit growth within the adjacent rhizosphere and affect bacterial flora of the soil. Phenolics can also act as modulators of plant development by regulating indole acetic acid (IAA) catabolism. They are effective in regulating plant growth, cell differentiation and organogenesis (Kumar et al. 2020, 2023). In addition to the adaptations that many plant species in the Brazilian Cerrado biome have developed to resist oxidative stress, such as the production of phenolic compounds, these species also face other challenges, such as the presence of aluminum (Al) in the soil. Studying the effects of Al on these plants can be difficult due to the 62 challenges of propagating and growing them in ex situ conditions that isolate the effects of Al from other elements (Cardoso and Silva 2013; Leite et al. 2021; Lima et al. 2022). In vitro propagation can be a valuable technique for conserving, cultivating, and propagating Cerrado species under controlled conditions, allowing for the isolation and testing of factors, such as Al, in the culture media. This study aimed to test the hypothesis regarding the significance of aluminum (Al) and its potential interaction with the cytokinin 6-benzylaminopurine (BAP) in promoting adventitious shoot and root organogenesis in Q. grandiflora and V. tucanorum. Additionally, this study investigated if the presence of Al and BAP has a relationship between the total phenolics content and antioxidant activity in extracts of these two Cerrado species. Material and Methods Plant Material Seeds of Qualea grandiflora Mart. and Vochysia tucanorum Mart. were obtained from fruits collected during the dehiscence season (between August and December 2021) in reminiscent areas of Cerrado at the municipalities of Itirapina and Mogi Guaçu, São Paulo State, Brazil. The fruits were opened, and the seeds were removed and stored at 25 °C ± 2 in an acclimatized room until their use for in vitro seedling germination. The experiment was conducted at the Center for Agricultural Sciences at the Federal University of São Carlos (CCA/UFSCar) in Araras, SP, Brazil. 63 Aluminum concentration The samples (seeds and cotyledonary leaves) were taken to the Plant Nutrition Laboratory of the Agronomic Institute of Campinas (IAC, Campinas, Brazil), ground, and digested in a solution of sulfuric, nitric, and perchloric acids (1:10:2, v/v/v). After digestion, Al concentrations were measured using atomic absorption spectroscopy (Sarruge and Haag 1974). The values were expressed as mg Al per kg of dry mass (Supplementary material 1). In vitro seed germination and seedling development The modified Murashige and Skoog (MS; Murashige and Skoog 1962) was the basal culture medium, reducing the original salt concentration to ¼ of macronutrients. The medium (¼ MS) was supplemented with 30.0 g L-1 of sucrose (Synth, Diadema, Brazil), and 0.1 g L-1 of myo-inositol (Synth) and solidified with 2.7 g L-1 of Gelrite® Gellan gum (Duchefa, Haarlem, Netherlands). Also, plant growth regulators were added to the culture medium: 0.44 µM 6-benzylaminopurine (BAP), 5.7 µM gibberellic acid (GA3) (Duchefa), and 90.0 µM of aluminum chloride AlCl3 . 6H2O (Synth). This culture medium was successfully used to germinate other Cerrado species (Cardoso and da Silva 2013). The pH of the culture medium was adjusted to 4.5 ± 0.05, and it was sterilized by autoclave at 121 °C for 25 min. The Al range concentration in the medium (0 and 320.0 µM of Al) were selected in accordance with other studies that tested Al toxicity in Picea glauca (Nosko et al. 1988), Triticum aestivum (Lima and Copeland 1990), Oryza sativa (Kikui et al. 2005), and Nicotiana tabacum (Vardar et al. 2006). The asepsis of seeds was realized by immersion of the seeds in 70% ethanol (Synth) (v/v) for 3 min, followed by a solution containing 50% sodium hypochlorite 64 (Candura, Piracicaba, Brazil) (1.0 to 1.25% of active chlorine) for 20 min, and, at last, rinsed three times with sterilized distilled water for 3 min each. After this superficial disinfestation, the mature zygotic embryos (MZEs) were excised under a laminar flow chamber and inoculated in test tubes containing 65 mL of the culture media. After, inoculated seeds were incubated for 35 d in 26±1°C and maintained under a 16-hr photoperiod (60 W fluorescent lamps; Phillips, Amsterdam, Netherlands). Organogenesis Induction from Cotyledonary Leaves Segments of Germinated Seedlings Mature and healthy cotyledonary leaves from 35-d-old in vitro Vochysiacea spec