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UNIVERSIDADE ESTADUAL DE SANTA CRUZ
PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E
BIOLOGIA MOLECULAR
Avaliação das alterações fisiológicas e metabólicas de plantas
cítricas submetidas ao déficit hídrico
DAYSE DRIELLY SOUZA SANTANA VIEIRA
ILHÉUS-BAHIA-BRASIL
Fevereiro de 2016
DAYSE DRIELLY SOUZA SANTANA VIEIRA
Avaliação das alterações fisiológicas e metabólicas de plantas
cítricas submetidas ao déficit hídrico
Tese apresentada à Universidade
Estadual de Santa Cruz como parte das
exigências para a obtenção do título de
Doutora em Genética e Biologia Molecular.
Área de Concentração: Genética e
Melhoramento Vegetal
Orientador: Dr. Abelmon da Silva
Gesteira
ILHÉUS-BAHIA-BRASIL
Fevereiro de 2016
V658 Vieira, Dayse Drielly Souza Santana. Avaliação das alterações fisiológicas e metabólicas de plantas cítricas submetidas ao déficit hídrico / Dayse Drielly Souza Santana Vieira. – Ilhéus, BA: UESC, 2016. xi, 103 f. : il. Orientador: Abelmon da Silva Gesteira. Tese (doutorado) – Universidade Estadual de Santa
Cruz. Programa de Pós-graduação em Genética e Biologia Molecular.
Inclui referências.
1. Cítricos. 2. Porta-enxertos. 3. Stress (Fisiologia). 4. Frutas cítricas – Condições hídricas. 5. Frutas cítricas – Doenças e pragas. 6. Secas. I. Título.
CDD 634.3
DAYSE DRIELLY SOUZA SANTANA VIEIRA
AVALIAÇÃO DAS ALTERAÇÕES FISIOLÓGICAS E
METABÓLICAS DE PLANTAS CÍTRICAS SUBMETIDAS AO
DÉFICIT HÍDRICO
Tese apresentada à Universidade
Estadual de Santa Cruz como parte das
exigências para a obtenção do título de
Doutora em Genética e Biologia Molecular.
Área de Concentração: Genética e
Melhoramento Vegetal
APROVADA: Ilhéus - Bahia, 25 de fevereiro de 2016.
__________________________________ _______________________________
Dr. Alfredo Augusto Cunha Alves Drª. Bruna Carmo Rehem (Embrapa CNPMF) (IFBA)
__________________________________ _______________________________
Drª. Fabienne Florence L. Micheli Drª. Virgínia Lúcia Fontes Soares (CIRAD - UESC) (UESC)
_______________________________
Dr. Abelmon da Silva Gesteira (Embrapa CNPMF - Orientador)
ii
À minha amada e
abençoada família, pelo amor
e apoio incondicional.
OFEREÇO
Aos meus pais, Sidelma e
Sérgio, e ao meu marido Jonathan.
DEDICO
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AGRADECIMENTOS
À DEUS, pela vida, oportunidades, inspiração e força interior.
À Universidade Estadual de Santa Cruz (UESC), em especial ao Programa
de Pós-Graduação em Genética e Biologia Molecular (PPGGBM), pela
oportunidade.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), pela bolsa.
Ao programa Ciência sem Fronteira (CAPES), pela oportunidade e bolsa
do doutorado sanduíche.
Ao meu DEZorientador, Dr. Abelmon da Silva Gesteira, pelo apoio,
confiança, orientação e amizade.
A todos os membros da “família Gesteira” pela ajuda, apoio, abrigo em
Cruz das Almas e amizade, especialmente para Diana Matos, Lucas da Hora,
Diogo Moraes, Liziane Marques, Maria Santos e Naiara Almeira.
A toda a equipe da Embrapa Mandioca e Fruticultura, especialmente ao
Dr. Maurício Coelho pela ajuda na montagem e condução do experimento. Além
disso, gostaria de agradecer a todos os técnicos de campo e de laboratório,
representados aqui por Sr. Santana, Sr. Raimundo, Mabel e Andressa, por toda
ajuda.
Ao grupo do Istituto per la Protezione Sostenibile delle Piante
(CNR/Florença-Itália) e do Instituto Valenciano de Investigaciones Agrarias
(Valencia – Espanha), em especial a Drª Biancaelena Maserti e ao Dr. Raphael
Morillon, pela recepção, atenção e orientação.
Aos amigos “recebidos” no doutorado sanduíche, aqui representados por
minha família italiana - Família Claps -, a Fabiana Zanelato e família, Valdir
Mano, Eliana Tassi, Aansa Rukya, Alireza Khaleghi e Wafa.
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A todo o grupo do Laboratório de Fisiologia do Desenvolvimento Vegetal
(IB/USP), de forma especial ao Dr. Luciano Freschi e a Aline Bertinatto Cruz,
pela recepção, atenção e ajuda.
À Fabrícia, Mara e Kátia pela eficiência e carinho no atendimento da
secretaria do PPGGBM.
À minha amada, abençoada e grande família, pelo amor, confiança e
exemplo de vida.
Aos meus pais, Sidelma e Sérgio, por todo amor e dedicação, pela minha
educação, pelos “puxões de orelha” e por serem meus maiores exemplos!
Ao meu marido Jonathan Vieira, pelo amor, cuidado, apoio, incentivo e
compreensão. E ao meu filho cachorro, o Buddy, pelo amor e carinho, além da
companhia nas madrugadas!
A todos os meus amigos de vida, especialmente para Marília, Matheus,
Aurizangela, Ícaro, Daniela e a família “caminho de casa” - Manu, Keu, Josi e
Louise -, pela verdadeira amizade.
Aos professores, familiares e amigos que contribuíram direta ou
indiretamente para a minha formação profissional e pessoal.
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“Quem decidir se colocar como juiz da Verdade e do
Conhecimento será naufragado pela gargalhada dos deuses.”
Albert Einstein
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ÍNDICE
EXTRATO ........................................................................................................... vii
ABSTRACT .......................................................................................................... ix
LISTA DE FIGURAS .......................................................................................... xi
1. INTRODUÇÃO ................................................................................................ 12
1.1 Objetivos ......................................................................................................... 14
1.1.1 Geral .............................................................................................................. 14
1.1.2 Específicos .................................................................................................... 14
2. REVISÃO DE LITERATURA ....................................................................... 16
2.1 A citricultura .................................................................................................. 16
2.2 Interação copa/porta-enxerto ........................................................................ 18
2.3 Déficit hídrico ................................................................................................. 21
2.3.1 Estratégias de sobrevivência ......................................................................... 23
2.3.2 Hormônios vegetais.......................................................................................24
2.3.3 Carboidratos..................................................................................................26
2.3.4 Compostos orgânicos voláteis.......................................................................27
CAPÍTULO 1: Polyploidization alters constitutive emission of volatile organic compounds (VOC) and improves membrane stability under water deficit in Volkamer lemon (Citrus limonia Osb.) leaves. .................................. 29
CAPÍTULO 2: Survival strategies of citrus rootstocks subjected to drought.47
3. CONCLUSÕES GERAIS ................................................................................ 95
4. REFERÊNCIAS ............................................................................................... 96
vii
EXTRATO
SANTANA-VIEIRA, Dayse Drielly Souza, Universidade Estadual de Santa Cruz,
Ilhéus, Fevereiro de 2016. Avaliação das alterações fisiológicas e metabólicas de
plantas cítricas submetidas ao déficit hídrico. Orientador: Abelmon da Silva
Gesteira.
A produção de citros possui grande importância na economia mundial e
brasileira. Entretanto, as plantas cítricas estão sujeitas a uma série de estresses bióticos e abióticos que são os principais limitantes da produção. Para suportar o período de estresse, as plantas desenvolvem diversos mecanismos em nível fisiológico, molecular e metabólico. Dentre os estresses abióticos, uma atenção especial tem sido dada ao déficit hídrico, especialmente devido às modificações nos regimes de chuvas das regiões produtoras, bem como das possíveis alterações climáticas previstas para os próximos anos. Aliado a isto, a citricultura é cultivada pelo método da enxertia, em que duas plantas são unidas para constituir uma única. O porta-enxerto, que é o responsável pelo desenvolvimento do sistema radicular, é de suma importância para a tolerância às intempéries ambientais. Alguns estudos relatam que plantas poliploides, que podem ocorrer naturalmente em citros, possuem melhor adaptação as alterações do ambiente. Nesse contexto, trabalhos que identifiquem e/ou caracterizem o comportamento dessas plantas, especialmente os porta-enxertos, sob estresse por seca, são de suma importância na busca de genótipos mais tolerantes, que podem ser diretamente aplicados à agricultura. Dessa forma, o objetivo do presente trabalho foi avaliar o comportamento fisiológico e metabólico de porta-enxertos de citros, enxertados ou não enxertados, visando à identificação dos mecanismos desenvolvidos e dos genótipos mais tolerantes, além de avaliar qual a estratégia de sobrevivência adotada durante o déficit. Na tentativa de contribuir e/ou elucidar estas questões, dois experimentos independentes foram desenvolvidos: i) avaliar as trocas gasosas, além do perfil de compostos orgânicos voláteis (VOCs) e da expressão de genes nos porta-enxertos Limão Volkameriano diploide (2xVL) e tetraploide (4xVL) submetidos ao déficit hídrico; e ii) avaliar as trocas gasosas, bem como os perfis dos açúcares e hormônios, dos porta-enxertos Limoeiro Cravo ‘Santa Cruz’
(RL) e Tangerineira Sunki ‘Maravilha’ (SM) em condição não-enxertada (RL e SM), com combinações inversas (SM/RL e RL/SM), além de combinações com duas copas comerciais Laranjeira Valencia (VO – VO/RL e VO/SM) e Lima ácida
viii
Tahiti (TAL – TAL/RL e TAL/SM), também submetidos ao déficit hídrico. Como principais resultados do primeiro experimento, foi constatado que o nível de ploidia das plantas - 2xVL e 4xVL – pode alterar a emissão constitutiva de VOCs, fator importante no desenvolvimento de tolerância ao estresse. Além disso, apesar de não ser constatado diferenças significativas no comportamento fisiológico de 2xVL e 4xVL sob déficit hídrico, as plantas 4xVL demonstraram possuir membranas mais resistentes ao estresse. Já no segundo experimento, foi possível observar que os porta-enxertos RL e SM desenvolvem estratégias diferentes de sobrevivência ao déficit hídrico, sendo ‘evitar a desidratação’ e ‘tolerar a desidratação’, respectivamente. Além disso, o porta-enxerto SM, quando comparado ao RL, apresentou maiores valores de rafinose e trealose, bem como de ABA e SA, na situação de déficit hídrico severo, sendo que tais resultados demonstram que o SM possue um sistema de proteção mais eficiente que o desenvolvido por RL, justificando assim a estratégia adotada por este. Além disso, foi observado que o SM foi capaz de induzir nas copas enxertadas um comportamento semelhante ao desenvolvido por ele quando não exertado. Estes resultados sugerem que, em um estresse prolongado, como os previstos para os próximos anos, as plantas enxertadas em RL podem chegar primeiro ao ponto de murcha permanente que as em SM.
Palavras-chave: citros, estresse abiótico, seca, porta-enxerto, tolerância
ix
ABSTRACT
SANTANA-VIEIRA, Dayse Drielly Souza, Universidade Estadual de Santa Cruz,
Ilhéus, Fevereiro de 2016. Evaluation of physiological and metabolic changes of
citrus plants submitted to drought stress. Supervisor: Abelmon da Silva Gesteira.
The citrus production have been great importance in the world and the Brazilian economy. However, citrus plants are subject to a variety of biotic and abiotic stresses which are the major factor limiting production. To withstand the stress period, the plants developed several mechanisms to physiological, metabolic and molecular levels. Among the abiotic stresses, special attention has been given to water stress, especially due to changes in rainfall regimes in the producing regions as well as the possible climate change expected for the next years. Allied to this, the citrus industry is grown by the method of grafting, in which two plants are joined to form a single. The rootstock, which is responsible for the development of the root system, is of paramount importance for tolerance to environmental weathering. Some studies report that polyploid plants, which may occur naturally in citrus, have better adaptation to environmental changes. In this context, work to identify and / or characterize the behavior of these plants, especially rootstocks, under drought stress, are extremely importance to identify the most tolerant genotypes that can be directly applied to agriculture. Thus, the objective of this study was to evaluate the physiological and metabolic behavior of citrus rootstocks, grafted or not grafted, aiming at the identifying the mechanisms developed and more tolerant genotypes and to evaluate which survival strategy adopted during the deficit. In an attempt to contribute and / or clarify these issues, two independent experiments were developed: i) evaluate the physiological behavior and the volatile profile (VOCs) of rootstock Lemon Volkameriano diploid (2xVL) and tetraploid (4xVL) submitted to water deficit ; and ii) assess physiologically as well as the profiles of sugars and hormones, rootstock Limoeiro Cravo 'Santa Cruz' (RL) and Sunki 'mandarin Wonder' (SM) in non-grafted condition (RL and SM), with inverse combinations (SM / RL and RL / SM), as well as combinations of two commercial tops Orange Valencia (VO - VO / RL and PO / SM) and acid Lima Tahiti (TAL - TAL / RL and TAL / SM), also submitted to deficit. The main results of the first experiment, it was found that the ploidy level of plants - 2xVL and 4xVL - can change the constitutive emission of VOCs, an important factor in the development of tolerance to stress. Further,
x
although not found significant differences in physiological and behavioral for 2xVL and 4xVL plants under drought, the 4xVL plants were found to have more resistant to stress membranes. In the second experiment, it was observed that the rootstocks RL and SM develop survival strategies to different water deficit, and 'avoid dehydration' and 'tolerate dehydration', adopted by them respectively. Furthermore, the rootstock SM when compared to RL, showed higher raffinose and trehalose values, as well as ABA and SA during the severe drought, and these results show that SM had a more efficient protection system than RL, thus justifying the strategy adopted by him. Furthermore, it was observed that the SM was able to induce the scions grafted behavior similar to that developed by him when ungrafted. These results suggest that in a prolonged stress, as envisaged for the coming years, the plants grafted on RL can arrive first at the permanent wilting point than SM. Key words: citros, abiotic estress, drought, rootstock, tolerance
xi
LISTA DE FIGURAS
Figura 1. Ilustração de algumas das principais maneiras em que os porta-enxertos
podem afetar a água nas copas, bem como suas relações e crescimento. Adaptado
de Hamlyn Jones, 2012 (How do rootstocks control shoot water relations? /New
Phytlogist).. ............................................................................................................ 19
Figura 2. Efeitos primários e secundários em plantas causadas pelo déficit
hídrico. Adaptado de Taiz e Zeiger, 2013.. ............................................................ 22
Figura 3. Balanço entre a tolerância ao estresse e manutenção do crescimento.
Adaptado de Claeys e Inzé, 2013.. ......................................................................... 24
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1. INTRODUÇÃO
A produção de citros gera um grande volume de receita, possuindo assim
ampla importância mundial. Nesse contexto, o Brasil se destaca como o segundo
maior produtor, ficando atrás apenas da China, que produz, essencialmente,
tangerina (Franck Curk, 2014). A laranja é a fruta mais produzida no território
nacional, sendo considerada uma commodity bastante expressiva no Produto
Interno Bruto (PIB), e tendo relevância na geração de emprego e renda (Neves et
al., 2010; Neves e Jank, 2006). A produção brasileira de laranja representa cerca
de 30% da safra mundial da fruta (MAPA-Citros, 2016), e valores próximos a
98% da laranja nacional é exportada em forma de suco concentrado para países
como EUA, União Europeia, Japão e China (Lohbauer, 2011). Essa produção
corresponde a cerca de 60% da produção mundial de suco (MAPA-Citros, 2016).
A safra brasileira de laranja 2015/2016 tem previsão de atingir 410
milhões de caixas de 40,8kg cada, representando um aumento de 10 milhões de
caixas, relativa à safra anterior (2014/2015) (Canal Rural, 2016; Revista Globo
Rural, 2016). Este aumento da safra se deve, especialmente, ao volume de chuva
satisfatório no ano de 2015. Entretanto, apesar da expressividade da produção
brasileira, problemas ocasionados por estresses bióticos e abióticos estão
ameaçando a produtividade (Neves e Jank, 2006). Dentre estes problemas podem
ser citados o avanço do greening (Huanglongbing/HLB), causada pelas bacterias
Candidatus Liberibacter asiaticus e Candidatus Liberibacter americanus, que são
transmitidas para as plantas de citros pelo psilídeo Diaphorina citri. O HLB é uma
das doenças mais graves que vem atingindo a cadeia citrícola brasileira desde
2004 (Fundecitrus, 2016). Além desse fator, as alterações climáticas,
13
especialmente o déficit hídrico – compreendendo os períodos de seca prolongados
na época da floração e/ou enchimento dos frutos – preocupam os produtores.
As plantas cítricas precisam de umidade no solo durante praticamente todo
o ano (de 600 a 1300 mm anuais) para que tenham um bom crescimento e uma
boa produção, mantendo assim os níveis de produtividade comercial (Vieira,
1991; Sentelhas et al., 2005). Na grande parte das regiões de produção do Brasil, a
exemplo dos estados de São Paulo, Minas Gerais, Bahia e Paraná, existe um
volume de chuvas adequado a produção, contudo a distribuição pluviométrica é
irregular. Nesse contexto, os períodos prolongados de estiagem prejudicam o
desenvolvimento da cultura, levando a reduções de produção entre 30 a 40%,
como o observado no ano de 2007 (Viana e Braga, 2007). A irrigação é uma
alternativa para mitigar este problema, além de proporcionar um aumento de
produção entre 35 a 75% quando comparado ao não irrigado (Coelho et al, 2004).
Porém, a irrigação possui alto custo de implantação, manutenção e operação
(custos com água e energia), o que interfere na adoção da tecnologia pelos
produtores. Outra alternativa, que por sua vez pode ter um menor custo ao
produtor, é a utilização de variedades que suportem melhor os períodos de
estiagem prolongados, e que também apresentem uma boa capacidade de
recuperação após as primeiras chuvas.
Durante o período de estiagem, as plantas apresentam diversas alterações
em níveis fisiológicos, moleculares e metabólicos, objetivando sobreviver ao
estresse por déficit hídrico (Verslues et al, 2006; Taiz e Zeiger, 2013). Dentre as
modificações apresentadas pelas plantas, pode-se citar a alteração nas trocas
gasosas, como a redução dos valores de condutância estomática (gs), resultando
na diminuição das taxas fotossintéticas (A) e de transpiração (E); além de
diferentes padrões de expressão gênica, bem como na emissão e produção de
compostos voláteis (VOCs), e nos conteúdos de hormônios, açucares e prolina. Os
Citros apresentam uma alta diversidade genética (Fang e Roose, 1997),
abrangendo laranjas (Citrus sinensis), tangerinas (Citrus reticulata e Citrus
deliciosa), limões (Citrus limon), limas ácidas como o Tahiti (Citrus latifolia) e o
Galego (Citrus aurantiifolia), e doces como a lima da Pérsia (Citrus limettioides),
o pomelo (Citrus paradisi), a cidra (Citrus medica), a laranja-azeda (Citrus
aurantium) e as toranjas (Citrus grandis) (Mattos Junior et al., 2005). Além disso,
14
as plantas cítricas possuem uma porcentagem, que gira em torno de 7%, de
formação de plantas poliploides naturais (Barrett e Hutchinson, 1982; Saleh et al.,
2008), incrementando assim a sua diversidade. A composição genética das plantas
pode vir a determinar a sua maior ou menor tolerância ao estresse, bem como qual
será a estratégia de sobrevivência que esta adotará. De forma geral, as plantas
podem adotar a estratégia de evitar a desidratação - objetivando a diminuição da
perda de água, e mantendo a produção; ou a estratégia de tolerar a desidratação -
onde adotam mecanismos para evitar os danos causados pelo estresse na planta, e
reduzem a produção, na tentativa de sobreviver ao estresse (Verslues, 2006). Com
base no exposto, o objetivo do presente trabalho é entender o comportamento
fisiológico e metabólico de plantas cítricas expostas a condição de estresse hídrico
por falta de água, visando identificar genótipos mais tolerantes, bem como obter
um melhor entendimento da tolerância ao estresse hídrico.
1.1 Objetivos
1.1.1 Geral
Avaliar as possíveis modificações fisiológicas e metabólicas promovidas
pelo déficit hídrico em plantas de citros.
1.1.2 Específicos
· Cap. I – Projeto Doutorado Sanduíche (Itália/Espanha)
o Analisar as trocas gasosas do Limão Volkameriano diploide (V2X)
e tetraploide (V4X) sujeito ao déficit hídrico;
o Diferenciar o perfil de compostos voláteis (VOCs) produzidos em
situação de controle, estresse hídrico severo e reidratação com 24h
em folhas de V2X e V4X;
15
o Identificar as diferenças entre V2X e V4X nas três situações
supracitadas referente ao conteúdo de prolina e melondialdeído
(MDA);
o Avaliar a expressão gênica de genes importantes na tolerância ao
déficit hídrico nas plantas de V2X e V4X.
· Cap. II – Projeto Brasil
o Analisar as trocas gasosas e os potenciais hídrico foliar, osmótico e
matricial dos porta-enxertos Sunki Maravilha (SM) e Limão Cravo
Santa Cruz (RL), bem como em suas combinações invertidas
(SM/RL e RL/SM), além desses com duas copas comerciais
Laranjeira Valencia (VO) e Lima ácida Tahiti (TAL), em três
situações: controle, déficit hídrico severo e reidratado com 48h;
o Avaliar as alterações nos perfis de hormônios e açucares nas oito
combinações supracitadas;
o Identificar qual a estratégia de sobrevivência adotada pelos porta-
enxertos em estudo.
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2. REVISÃO DE LITERATURA
2.1 A citricultura no Brasil
A laranja foi introduzida no Brasil por volta de 1540 pelos portugueses,
com sementes provenientes da Espanha (Lima, 2014). As primeiras plantas foram
cultivadas em Salvador – Bahia, onde através de uma mutação natural da
variedade ‘Seleta’, surgiu à variedade ‘Bahia’. Posteriormente, a laranja foi
propagada nos estados de São Paulo e Rio de Janeiro, e em decorrência das
condições climáticas favoráveis para o desenvolvimento das frutas e a
aglomeração de pessoas nesses estados, formou-se o núcleo citrícola do país
(Donadio et al., 2005).
Entretanto, o grande marco do desenvolvimento de citros no Brasil se deu
na década de 60, quando ocorreu a queda na produção de laranja nos Estados
Unidos da América (EUA) - principal produtor na época - dando espaço para o
surgimento de um novo produtor. Além disso, nessa mesma época, empresas
extratoras de suco começaram a se instalar na região de São Paulo, bem como
incentivos governamentais para produção de laranja e suco concentrado foram
desenvolvidos, formando assim o cinturão citrícola do país (Paullilo, 2006). Pode-
se dizer que o cultivo de laranja no Brasil se divide em dois períodos distintos: de
1990 a 1999 - que se caracteriza pelo aumento da produção e conquista da posição
de líder do setor; e a partir de 1999 até os dias atuais – onde ocorreu a
consolidação da capacidade e desempenho produtivo. O Ministério da Agricultura
17
e Pecúaria (MAPA), objetivando manter a liderança do setor, no que diz respeito à
produção de laranja e suco concentrado, vêm investindo no apoio a adoção de
sistemas mais eficientes, como a produção integrada, com medidas para reduzir os
custos, e aperfeiçoar e ampliar a comercialização do produto (MAPA, 2016).
Associado aos incentivos do governo e em decorrência do aumento da
produção e a percepção de que a atividade citrícola era lucrativa, campos
experimentais começaram a ser montados, dando suporte aos produtores
brasileiros, que seguiam os padrões de produção dos EUA, não adequados ao
clima e solo local (Donadio et al., 2005). Desde então, a citricultura nacional
evoluiu bastante, tanto pelo interesse dos produtores, quanto pelo incentivo e
desenvolvimento científico. Nesse contexto, surgiu em 1988 o Programa de
Melhoramento Genético de Citrus na Embrapa Mandioca e Fruticultura
(CNPMF), localizada no município de Cruz das Almas – BA. Como suporte para
desenvolvimento das pesquisas, a instituição conserva um Banco Ativo de
Germoplasma (BAG) com mais de 800 acessos em campo, sendo que grande parte
já está em telados. Além disso, instituições públicas e privadas servem de apoio
aos projetos desenvolvidos, a exemplo da Universidade Estadual de Santa Cruz
(UESC). Dentre os principais objetivos desse programa podem ser citados:
selecionar genótipos, principalmente porta-enxertos, mais tolerantes à seca e ao
alumínio, reduzir o período juvenil, aumentar a longevidade dos pomares, obter
variedades mais resistentes à gomose de Phytophthora spp. e ao complexo do
Vírus da Tristeza dos Citros – CTV (Citrus Tristeza Vírus), além de adaptados a
altas densidades populacionais [Soares-Filho, W. dos S., 2003(a); Soares-Filho,
W. dos S., 2003(b)] .
Os maiores estados produtores de laranja no Brasil são São Paulo, Bahia,
Paraná, Minas Gerais e Sergipe, nessa ordem. O sudeste detém a maior produção,
com cerca de 80%, enquanto Bahia e Sergipe, juntamente, representam em torno
de 9% da produção nacional (IBGE, 2013). As estimativas até 2023 é que a
produção de citros no país cresça de 20,2 (2013) para 23,8 mil toneladas, um
aumento de 17,8%, ao passo que a exportação de suco concentrado deve crescer
de 2,1 (2013) para 2,6 mil toneladas (AGE/Mapa e SGE/Embrapa, 2016).
Contudo, para que tais níveis de produção sejam mantidos e/ou aumentados,
problemas relativos ao controle de pragas e doenças, bem como as variações
18
climáticas e ambientais, especialmente relativas ao regime de chuvas, devem ser
minimizados e/ou superados.
2.2 Interação copa/porta-enxerto
A enxertia é uma técnica milenar utilizadas em diversas culturas (Mudge
et al., 2009). Nessa técnica, duas plantas distintas – a copa e o porta-enxerto – são
unidas para formar um único genótipo, sendo que uma planta será responsável
pelo desenvolvimento do sistema radicular – absorvendo água e os nutrientes do
solo – e outra pela copa – responsável pela realização da fotossíntese, floração e
consequente produção de frutos (Ribeiro et al, 2005). Em diversas culturas, sejam
elas perenes ou anuais, a enxertia é utilizada para unir sistemas radiculares
resilientes (porta-enxerto) com uma copa produtiva, formando assim um
“produto” que gere “frutos” (Warschefsky et al., 2015). A depender da forma da
união dessas duas plantas – seja pela técnica utilizada ou pelas características
intrínsecas da planta –, essa pode ser um fator determinante para limitar o
crescimento da copa, bem como a taxa de sobrevivência da planta enxertada
(Johkan et al., 2009; Martínez-Ballesta et al., 2010).
A utilização adequada da combinação copa/porta-enxerto pode ser de
fundamental importância para o aumento de produtividade, visto que, a utilização
de um único porta-enxerto para diferentes copas, pode subestimar o potencial
produtivo destas. Na planta enxertada, a comunicação entre a parte aérea e o
sistema radicular ocorre como em uma planta não enxertada. Entretanto, o porta-
enxerto, responsável pelo sistema radicular, pode interferir diretamente nas
características da copa, tais como: tamanho, precocidade de produção e
produtividade de frutos, época de maturação, peso do fruto, coloração da casca e
da polpa dos frutos, teor de açúcares e de ácidos, permanência dos frutos na
planta, conservação do fruto após a colheita, transpiração e composição química
das folhas, fertilidade do pólen, capacidade de absorção, síntese e utilização de
nutrientes, tolerância à seca, à salinidade e ao frio, resistência e/ou tolerância a
pragas (Pompeu Junior, 1991).
A disponibilidade de água no solo é um dos fatores mais limitantes da
produção mundial, e nesse contexto, o melhoramento vegetal visa selecionar
19
plantas que sejam mais tolerantes aos períodos secos, e que, consequentemente,
tenham uma maior eficiência do uso da água (EUA) (Condon et al., 2004). Dentro
deste cenário, o porta-enxerto tem fundamental importância para que este objetivo
seja alcançado. Em diversas culturas, já foi comprovada a influência que o porta-
enxerto exerce na característica de tolerância ao déficit hídrico (Soar et al., 2006;
García-Sánchez et al., 2007; Sánchez-Rodríguez et al., 2012; Liu et al., 2012).
Dentre os mecanismos desenvolvidos na interação copa/porta-enxerto, podemos
citar a sinalização química – realizada através de estímulos hormonais para
controlar o consumo e perda de água pela parte área – e a sinalização hidráulica –
regulada, em grande parte, pelo tamanho dos vasos do xilema, que influência na
capacidade de captura da água no solo (Figura 1) (Jones, 2012).
Figura 1. Ilustração de algumas das principais maneiras em que os porta-enxertos
podem afetar a água nas copas, bem como suas relações e crescimento. Adaptado
de Jones, 2012 (How do rootstocks control shoot water relations? /New
Phytlogist).
As plantas desenvolvem diferentes mecanismos para sobreviver ao estresse
por déficit hídrico, e estas respostas vão depender da duração e da intensidade do
período de estresse (Bray, 1997). Vários estudos foram desenvolvidos para
identificar mecanismos e/ou características para facilitar a seleção de genótipos de
porta-enxerto mais tolerantes. Em videiras, Marguerit et al. (2012) demonstraram
20
que a taxa de transpiração e a adaptação ao déficit hídrico em copas, são
influenciados geneticamente pelos porta-enxertos. Na revisão de Aroca et al.
(2012), eles enfatizaram a importância da capacidade de extração de água do solo
do porta-enxerto na determinação da maior tolerância a deficiência hídrica.
Enquanto que no trabalho de Martínez-Ballesta et al. (2010), focada
especialmente para enxertia em Solanaceas e Curcurbitaceas, foi relatada a
importância da correta conexão entre copa e porta-enxerto, pois distúrbios
fisiológicos - como inibição de crescimento e restrição de comunicação – podem
ser decorrentes da descontinuidade da união vascular. Já em citros, Tzarfati et al.,
2013, demonstrou que o enxerto promove alterações no padrão de expressão de
micro-RNAs (miRNAs), e que esta interferência pode estar relacionada com a
redução do período juvenil.
Em citros, e também na maioria das culturas enxertadas, as partes unidas
para originar a nova planta, são propagadas assexuadamente (Warschefsky et al.,
2015). O gênero Citrus possui uma característica chamada poliembrionia, onde
em uma semente são gerados vários embriões, e destes, boa parte são nucelares,
ou seja, idênticos a planta mãe. Os porta-enxertos utilizados, em quase 100% dos
casos, são propagados através dessa característica (Oliveira et al., 2008). Ao passo
que, as copas são obtidas através de gemas e/ou pedaços do caule de uma planta
matriz. A citricultura brasileira, atualmente, ainda é mantida, em sua grande parte,
em um único porta-enxerto, o Limoeiro Cravo Santa Cruz, que ganhou o apreço
dos produtores devido a sua rusticidade, potencial produtivo mesmo em condições
adversas, como a seca, além de tolerância a algumas doenças (Oliveira et al.,
2008). Entretanto, o uso de um único porta-enxerto é um risco eminente, devido à
susceptibilidade dos pomares às pragas e doenças. Na década de 40, cerca de 75%
dos pomares brasileiros, enxertados em laranjeira ‘Azeda’, foram dizimados em
decorrência da alta sensibilidade ao vírus da tristeza dos citros (CTV). Dessa
forma, a diversificação de porta-enxertos – que é um tema bastante discutido nos
últimos tempos por pesquisadores e citricultores – é um ponto chave para
manutenção da produção nacional, especialmente com chegada do HLB.
21
2.3 Déficit hídrico
As plantas cultivadas estão sujeitas a uma série de estresses que podem ser
de origem biótica e/ou abiótica. Uma redução que gira em torno de 50 a 70% da
produtividade nas grandes culturas tem sido atribuída aos estresses abióticos
(Mittler, 2006). Adicionado a isto, os estresses abióticos desenvolvem um papel
determinante na distribuição das espécies de plantas nos mais diversos ambientes
do planeta (Verslues et al., 2006). Dentre os estresses abióticos que mais
preocupam os produtores, o deficit hídrico tem se destacado, especialmente com
as previsões de períodos secos mais prolongados nos próximos anos (Marengo,
2014). O estresse hídrico por seca pode ser definido como o período em que
ocorre baixa precipitação quando comparada ao normal, limitando a produtividade
no sistema natural ou agrícola (Kramer e Boyer, 1995; Taiz e Zeiger, 2013).
Dessa forma, o componente que define a seca é a disponibilidade de água no solo
(Verslues et al., 2006).
Para sobreviver a tais condições de estresses, as plantas respondem com
várias modificações através de uma cascata de sinais (Dos Reis et al., 2012).
Dentre estes mecanismos podemos citar como efeitos primários a redução do
potencial hídrico (Ψw), a desidratação celular e a resistência hidráulica.
Adicionado a estes, efeitos secundários ocorrem, a exemplo: redução da expansão
celular; redução das atividades celulares e metabólicas; fechamento estomático e
consequente inibição da fotossíntese; abscisão foliar; cavitação – que é o processo
de redução da coluna líquida sob tensão no xilema; produção de ROS (espécies
reativas de oxigênio); morte celular; desestabilização de membranas e proteínas,
dentre outros (Figura 2) (Taiz e Zeiger, 2013). São estes mecanismos que ajudam
as plantas a sobreviver aos períodos de estresse hídrico, bem como a sua
recuperação após as primeiras chuvas ou reidratação.
Em citrus, diversos estudos foram desenvolvidos no intuito de caracterizar
o comportamento destas plantas durante o déficit hídrico. Rodríguez-Gamir et al.,
(2010) avaliaram três genótipos de citros sob deficiência hídrica e constataram
que o déficit afeta as trocas gasosas e a relação de água nas folhas, além de
constatarem que a tolerância ao déficit é ligada ao genótipo. Este último fato
também foi observado nos trabalhos de Romero et al., (2006), Brito et al. (2012)
22
Argamasilla et al., (2014), e Pedroso et al. (2014), especialmente quando relata-se
a importância do genótipo do porta-enxerto. Allario et al. (2013) trabalharam com
plantas diploides (2x) e tetraploides (4x) de citrus também submetida ao estresse
por seca, e constataram que as plantas 4x são mais tolerantes que as 2x, além de
desenvolverem mecanismos que possibilitam maior aclimatação, a exemplo da
expressão genética diferenciada para controle da sinalização de longa distância
pelo ácido abscísico (ABA).
Figura 2. Efeitos primários e secundários em plantas causadas pelo déficit
hídrico. Adaptado de Taiz e Zeiger, 2013.
No trabalho de Argamasilla e Gómez-Cadenas (2014), que avaliaram dois
genótipos de citros submetidos ao estresse por seca e alagamento, foi constatado
alterações nos perfis de prolina, ácido jasmônico (AJ), ácido indolacético (AIA) e
ABA durante o déficit hídrico, além de enfatizarem que o genótipo possui
características intrínsecas (metabolismo basal) que podem influenciar na maior ou
menor aclimatação a condição de estresse. Já no trabalho de Pedroso et al. (2014),
onde avaliou-se a capacidade dos porta-enxertos na modulação de carboidratos
não estruturais e a tolerância ao déficit hídrico, foi demonstrado que o genótipo
mais tolerante, incentivou o crescimento de raiz, havendo acúmulo de
carboidratos nas mesmas. Além disso, foi constatado neste trabalho que folhas
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jovens maduras possuem maiores taxas fotossintéticas durante o déficit hídrico
que folhas maduras.
2.3.1 Estratégias de sobrevivência
A alteração climática pode ser definida como a indisponibilidade e/ou
alteração das condições e/ou recursos fundamentais para o desenvolvimento das
plantas (Nicotra et al., 2010). Quando submetidas a tais condições, as plantas
desenvolvem respostas induzidas por estas alterações ambientais, resultando em
um processo denominado de plasticidade fenotípica. A plasticidade fenotípica
pode ser entendida por possuir controle genético e/ou hereditário (epigenética),
sendo considerada fundamental para a evolução das espécies (Nicotra et al., 2010;
Reed et al., 2010). O entendimento de como as plantas se comportam e/ou
adaptam a tais alterações ambientais é de suma importância para o
desenvolvimento de técnicas de manejo visando minimizar os impactos nas
culturas.
Alguns estresses abióticos possuem ao menos um dos seus efeitos
negativos no desenvolvimento da planta relativo ao conteúdo de água disponível.
Esse efeito pode ser causado devido à alteração do teor de íons e absorção de água
decorrente do estresse salino, pela desidratação celular devido a formação de gelo
extracelular em conseqüência do frio, ou pela redução da disponibilidade de água
causada pelo estresse hídrico (Verslues et al., 2006). Dentre as alterações
climáticas, o déficit hídrico tem tido destaque devido às limitações que causam na
produção. Para superar o período com restrição hídrica, as plantas podem adotar
diferentes estratégias de sobrevivência, sendo que as duas principais são: evitar e
tolerar a desidratação (Verslues et al., 2006; McDowell et al., 2008; Claeys and
Inzé, 2013).
Na estratégia de evitar a desidratação o ponto principal é o balanço entre a
captura de água do solo e a perda de água por transpiração (Verslues et al., 2006;
Claeys and Inzé, 2013). Normalmente esta estratégia é adotada em estresses
moderados ou de curta duração, onde são desenvolvidos mecanismos como o
aumento do sistema radicular e o fechamento estomático, visando manter o
crescimento e a produção (Figura 3) (Verslues et al., 2006; McDowell et al., 2008;
Claeys and Inzé, 2013).
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Entretanto, quando o estresse hídrico se torna mais severo e/ou mais
prolongado, os mecanismos desenvolvidos na estratégia de evitar a desidratação,
não são suficientes. Nesse contexto, existe a estratégia de tolerar a desidratação,
cujo objetivo principal é a proteção aos danos celulares ocasionados pelo estresse
hídrico. Alguns dos mecanismos desenvolvidos nessa estratégia incluem a
desintoxicação causada pelas espécies reativas de oxigênio (ROS), a acumulação
de proteínas de proteção, bem como de solutos a exemplo da prolina, glicina
betaína e açúcares, que possuem importância na mantenção da turgescência
celular (Verslues et al., 2006; Claeys and Inzé, 2013). Nessa estratégia o ponto
principal é garantir a sobrevivência da espécie após o estresse (Figura 3).
Figure 3 – Balanço entre a tolerância ao estresse e manutenção do crescimento.
Adaptado de Claeys e Inzé, 2013.
2.3.2 Hormônios vegetais
Os hormônios vegetais são substancias que, em pequenas concentrações,
regulam o desenvolvimento e crescimento das plantas (Taiz e Zeiger, 2013).
Normalmente eles são produzidos em uma parte da planta, e posteriormente
translocado para células visinhas ou para outras partes da planta, onde irão induzir
25
as respostas fisiológicas. Dentre os vários tipos de hormônios vegetais, três serão
enfatizados a seguir: o ácido abscísico (ABA), a auxina (ácido indolacético - AIA)
e o ácido salicílico (AS).
O ABA é um hormônio bastante conhecido devido ao seu importante papel
no crescimento e fechamento estomático, principalmente quando as plantas estão
sujeitas as alterações climáticas, de forma especial ao déficit hídrico. Além disso,
o ABA também é importante na maturação, regulação do crescimento e na
dormência das sementes, dormência de gemas e senescência foliar (Taiz e Zeiger,
2013). Muitos estudos relatam que o ABA seria produzido nas raízes e
posteriormente translocados para a parte aérea, especialmente em condições de
deficiência hídrica, induzindo o fechamento estomático. Entretanto, alguns
estudos sugerem que também existe produção de ABA na parte área, e que esta
pode ser de grande importância na indução de uma maior tolerância ao déficit
hídrico (Christmann et al., 2007; Ikegami et al., 2009; Bauer et al., 2013). O ABA
pode ser transportado tanto pelo xilema quanto pelo floema, sendo este transporte
altamente influenciado pelas condições ambientais, especialmente de déficit
hídrico.
A auxina foi o primeiro hormônio descoberto, sendo a sua forma natural
mais comum o ácido indol-3-acético (AIA). Este hormônio é normalmente
sintetizado em meristemas e/ou tecidos jovens em divisão, sendo movida através
do transporte polar, ou seja, do ápice para a região basal (Friml, 2003; Taiz e
Zeiger, 2013). A auxina promove o crescimento através do alongamento celular,
ou seja, ocorre uma alteração nas paredes celulares, e devido ao armazenamento
de água nos vacúolos, ocorre a expansão celular. Além da função de promoção do
crescimento/alongamento celular, a auxina também é importante no
desenvolvimento dos tropismos vegetais e em diversas etapas do desenvolvimento
da plantas, sendo este dependente da composição genética e/ou das condições que
a plantas estão sujeitas (Sachs, 2005).
Já o AS não é reportado como um dos principais grupos de hormônios,
contudo, nos últimos anos, vários estudos estão relatando sua importância como
molécula sinalizadora. O SA é associado, principalmente, a resposta ao ataque de
patogenos, desenvolvendo o fenômeno denominado de resistência sistêmica
adquirida (RSA), onde sinais são enviados do local infectado para o restante da
26
planta através do floema, e também para plantas vizinhas, estimulando assim o
aumento da tolerância ao ataque (Dempsey et al., 2011; Taiz e Zeiger, 2013).
Recentes estudos, contudo, também estão relatando a importância deste hormônio
no desenvolvimento da tolerância ao déficit hídrico, sendo este associado ao
controle estomático em associação com o ABA (Horváth et al., 2007; Miura e
Tada, 2014). De acordo com a composição genética da planta e as condições de
estresse em que são submetidas, os níveis destes hormônios – ABA, AIA e AS –
podem ser aumentados ou reduzidos durante o déficit hídrico, influenciando
diretamente em uma maior ou menor tolerância ao mesmo.
2.3.3 Carboidratos
Os carboidratos são substâncias que fornecem energia para as plantas. É
por meio da fotossíntese que a planta consegue transformar energia luminosa em
energia química (através de pigmentos fotossintéticos), usando água e CO2, e
produzindo carboidratos (Taiz e Zeiger, 2013). Diversos fatores podem influenciar
na maior ou menor eficiência dessa conversão, dentre eles podemos destacar o
estresse hídrico (Santos e Carlesso, 1998; da Cruz et al., 2015). As plantas
normalmente acumulam carboidratos que servem de estoque de energia para um
período com fornecimento energético limitado e/ou para reforçar uma maior
demanda energética (Krasensky e Jonak, 2012).
O amido é a principal reserva de carboidratos das plantas, sendo
rapidamente convertido em açúcares solúveis quando a planta está submetida a
qualquer alteração ambiental – como seca e salinidade (Basu et al., 2007; Kempa
et al., 2008) –, fornecendo energia para continuidade do metabolismo celular
(Krasensky e Jonak, 2012). Estudos relatam o acumulo de açúcares durante do
déficit hídrico (Krasensky e Jonak, 2012; Pedroso et al., 2014; da Cruz et al.,
2015). Durante muito tempo, os açúcares foram considerados como sinalizadores
para a produção de ROS, ou seja, o aumento da concentração de açúcares
intracelular induzia a produção de ROS, desencadeando assim uma resposta das
plantas ao estresse (Couée et al., 2006). Entretanto, trabalhos mais recentes estão
defendendo que o acumulo de açúcares, de forma especial trealose e rafinose
(Keunen et al., 2013; Lunn et al., 2014), podem ser verdadeiros seqüestradores de
27
ROS, auxiliando assim na desintoxicação celulares, podendo induzir uma maior
tolerância ao déficit hídrico.
Em citros, diversos estudos foram realizados para avaliar o comportamento
dos carboidratos durante o déficit hídrico, realçando a importância destes
componentes no metabolismo celular (Yakushiji et al, 1998; Barry et al., 2004;
Magalhaes Filho et al., 2008; Arbona et al., 2013; Pedroso et al., 2014). Embora o
conceito dos açúcares como reguladores na atividade fotossintetica e metabolismo
celular seja bem difundido, é relativamente nova a ideia que os açúcares são
moleculas sinalizadoras (Rolland et al., 2002). Ao nosso conhecimento, estudos
que quantifiquem trealose e rafinose, potenciais sequestradores de ROS, ainda não
foram desenvolvidos em plantas cítricas. Este estudo poderá ser de grande valia
para a abertura de novas vertentes de estudo, bem como servir de suporte para um
melhor entendimento da influência destes carboidratos na indução da tolerância ao
déficit hídrico.
2.3.4 Compostos Voláteis
Os compostos orgânicos voláteis (VOCs) são metabólitos secundários
vegetais que evaporam sob temperatura ambiente (Taiz e Zeiger, 2013). Os VOCs
possuem diversas funções eco-fisiológicas (Pinto-Zevallos et al., 2013) dentre
elas: defesa de plantas contra agentes de estresse, possibilitando a comunicação
entre planta e o meio ambiente; protegem a planta do calor, reduzindo ROS
produzidos em altas temperaturas (Copolovici, et al., 2005); repelem insetos
(McCallum et al., 2011); atraem polinizadores para as flores e animais dispersores
de sementes; além de serem atração para predadores naturais em plantas atacadas
(Arimura et al., 2010).
Loreto e Schnitzler (2010) enfatizam que os VOCs são moléculas
fundamentais de sinalização do estresse e por consequência ativa a RSA e a
resposta de hipersensibilidade (RH). Esta última desencadeia a morte celular no
local da infecção, privando o patógeno de nutrientes e impedindo a propagação na
planta (Taiz e Zeiger, 2013). Os VOCs podem ser emitidos constitutivamente
pelas plantas (Kesselmier e Staut, 1999) ou ser induzido por estresse abiótico
(Loreto, Schnitzler, 2010) e biótico (Paré, Tumlinson, 1999).
28
Dentre os compostos voláteis, podemos citar os terpenos – que são
hidrocarbonetos formados a partir da união de isoprenos –, os compostos
fenólicos e os alcalóides. Estes são emitidos constitutivamente, entretanto, em
condição de ataque de herbívoros, podem apresentar diferentes padrões de
emissão dependendo da espécie do inseto. Além destes, quando tem-se um dano
mecânico, as plantas podem emitir os voláteis de folhas verdes (VGLs), que são
derivados de lipídeos (Taiz e Zeiger, 2013). Os compostos voláteis são emitidos
em vários tecidos, bem como abaixo do solo, e possuem fundamental importância
na comunicação entre plantas, bem como entre plantas e outros organismos,
exercendo assim diversas funções nos estresses bióticos e abióticos (Loreto,
Schnitzler, 2010; Pinto-Zevallos et al., 2013).
Diversos fatores podem influenciar – reduzindo ou estimulando – na
emissão dos voláteis pelas plantas, a exemplo da luminosidade, umidade do solo e
do ar, temperatura, nutrientes disponíveis, herbivoria, etc (Hare, 2011). Dessa
forma, o estudo dos compostos orgânicos voláteis (COV) liberados pelas plantas
em situações de estresse, seja pela picada de insetos, introdução de bactéria,
excesso de luminosidade, deficiência nutricional e/ou estresse hídrico por falta de
água, é de fundamental importância na tentativa de elucidar o papel destes
componentes na maior ou menor tolerância aos estresses bióticos e abióticos, de
forma especial no déficit hídrico.
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CAPÍTULO 1:
Polyploidization alters constitutive emission of volatile
organic compounds (VOC) and improves membrane stability
under water deficit in Volkamer lemon (Citrus limonia Osb.)
leaves
Artigo submetido à Environmental and Experimental Botany em 07/12/2015
Artigo aceito com menores correções na Environmental and Experimental Botany
em 25/01/2016 e resubmetido em 05/02/2016
Artigo publicado na Environmental and Experimental Botany em 15/02/2016
Polyploidization alters constitutive content of volatile organiccompounds (VOC) and improves membrane stability under waterdeficit in Volkamer lemon (Citrus limonia Osb.) leaves
Dayse Drielly Souza Santana Vieiraa,b, Giovanni Emilianic, Marco Michelozzid,Mauro Centrittoc, François Luroe, Raphaël Morillonf, Francesco Loretog,Abelmon Gesteirah, Biancaelena Masertia,*aCNR-IPSP, Istituto per la Protezione Sostenibile delle Piante, Area della Ricerca,Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), ItalybDepartamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Rodovia Jorge Amado, Km 16 45662-900 Ilhéus, Bahia, BrazilcCNR!IVALSA, Istituto per la Valorizzazione del Legno e delle Specie Arboree, Area della Ricerca, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), ItalydCNR-IBBR, Istituto di Bioscienze e Biorisorse, Area della Ricerca, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), ItalyeUMR AGAP!INRA de Corse, équipe APMV, 20230 San Giuliano, FrancefUMR AGAP!CIRAD, équipé APMV – Station de Roujol, 97170 Petit Bourg, Guadaloupe, FrancegCNR-DISBA!Dipartimento di Scienze Bio-Agroalimentari, Piazzale Aldo Moro 7, 00185 Roma, Italyh EMBRAPA Mandioca Fruticultura, Cruz das Almas, Bahia, Brazil
A R T I C L E I N F O
Article history:
Received 7 December 2015Received in revised form 4 February 2016Accepted 14 February 2016Available online 16 February 2016
Keywords:
DREB2A
Green leaf volatileHPL
MonoterpenoidsOxylipinsPolyploidizationVOC
A B S T R A C T
In Citrus species chromosome doubling naturally occurs in somatic embryos and doubled diploid plantsoften show better adaptation to adverse environmental condition. To understand the moleculardeterminants of stress acclimation, we examined the response to water deficit in diploid (2"VL) anddoubled diploid (4"VL) seedlings of Volkamer lemon (Citrus limonia Osb.) assessing the profile ofconstitutive volatile organic compound (VOC) in control and stressed conditions. Physiologicalparameters and leaf volatile compound profiles were measured during water deficit and 24 h afterrehydration of plants to field capacity. Net photosynthesis and stomatal conductance were reduced inwater stressed leaves, with no significant differences between 2"VL and 4"VL plants. Malondialdehydeconcentration, a marker of lipid peroxidation of cellular membranes, was significantly more higher instressed 2"VL leaves than in 4"VL. The blend of constitutive VOC was different in control leaves beingoxygenated sesquiterpenoids more abundant in 2"VL leaves, and monoterpenoids more abundant in4"VL leaves. Water deficit did not stimulate biosynthesis of terpenoids, whereas accumulation of trans-2 hexenal, a green leaf volatile (GLV) synthesized after membrane denaturation, was observed in stressedleaves of 2"VL leaves, but not in 4"VL leaves. Semiquantitative PCR showed an increase of the expressionof HPL, the gene encoding for hydroperoxidase lyase which catalyzes 2-hexenal formation, only in 2"VLplants. The expression of the putative dehydration transcription factor DREB2A was also observed only in2"VL water stressed plants. This work shows that level of ploidy may alter constitutive content of GLV byCitrus, therefore likely affecting plants capacity of protection and interaction with other organisms.Whereas diploid and double diploid plants showed similar physiological responses to water deficit, abiochemical marker indicated that membranes of double diploid leaves were more resistant to the stress.These results provide intriguing insights into the regulation of terpenoids and oxylipins pathways as afunction of polyploidization in a non-model plant species.
ã 2016 Elsevier B.V. All rights reserved.
1. Introduction
Citrus is one of the most important fruit crop in the world andits production is challenged by many environmental constraints.
Citrus varieties are routinely grown on rootstocks to help themcoping with a range of stressful conditions (Levy and Syvertsen,2004; Tadeo et al., 2008). Citrus rootstocks are propagated throughpolyembryonic seeds and the seedlings regenerated from nucellarembryos are genetically identical to the maternal plant. Themajority of cultivated citrus genotypes has a partially apomicticreproduction with a somatic embryogenesis of nucellar cellsinduced by the gamete fertilization (Aleza et al., 2011). Doubled
* Corresponding author.E-mail address: [email protected] (B. Maserti).
http://dx.doi.org/10.1016/j.envexpbot.2016.02.0100098-8472/ã 2016 Elsevier B.V. All rights reserved.
Environmental and Experimental Botany 126 (2016) 1–9
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diploid seedlings in apomictic genotypes are considered to arisefrom somatic chromosome doubling of nucellar embryo cells andshould be genetically identical, at the qualitative level, to the seedsource tree (Rao et al., 2008). In reality, the phenotype of doubleddiploids is modified in various traits, such as a reduced growthvigor and enlargement of cells and organs (Comai, 2005).Polyploidy has been an important determinant in plant evolution,facilitating plant invasiveness and the capability to successfullycolonize habitats characterized by strong fluctuating environmen-tal conditions (Beest et al., 2012). Furthermore, recent studies alsodemonstrate that genome doubling confers to plants also a betteradaptability to various environmental stresses included a highertolerance to salinity (Mouhaya et al., 2010; Jiang et al., 2013) andwater deficit (Li et al., 2009). In citrus plants, Allario et al. (2013)found that in control conditions 4" rootstocks constitutivelyoverexpressed a set of genes putatively involved in water deficittolerance. Podda et al. (2013) found higher levels of antioxidantenzymes, such as superoxide dismutase and ascorbate peroxidase,in 4" Cleopatra and Willow leaf mandarin than the respective 2"plants challenged with salt stress.
Drought stress is a major environmental factor that affects plantgrowth and development and are expected to increase withclimate change (Dai, 2010; Centritto et al., 2011).
Plants release into the surrounding atmosphere a vast range ofvolatile organic compounds (VOC) which have a role as infochem-icals in biotic interaction (Blée, 2002; Niinemets et al., 2013) andalso in abiotic stress acclimation. VOCs are often considered as amechanism for plants to respond to stresses and alleviating theirnegative consequences (Loreto and Schnitzler, 2010).
The terpenoids, and the green leaf volatiles (GLVs), including 2-hexenal (cis,trans aldehydes C6), are two families of volatile organiccompounds (VOC) abundantly emitted by stressed plants (Feuss-ner and Wasternack, 2002; Dudareva et al., 2004). Green leafvolatiles are well-studied as compounds produced by plants inresponse to wounding (Loreto et al., 2006; Brilli et al., 2011), topathogenic infection (Blée, 2002; Scala et al., 2013) and toherbivory (Maja et al., 2014). However, induction of GLV afterabiotic stress was also observed. Emission of (E)-2-hexenal wasdetected in a photoinhibition sensitive Arabidopsis mutant afterexposure to intense light conditions (Loreto et al., 2006), in tomatounder heat and cold stresses (Copolovici et al., 2012), and intobacco after exposure to ozone (Beauchamp et al., 2005), andunder other photooxidative stress condition (Mano et al., 2010).Recently, it has been suggested that 2-hexenal can act asendogenous signal chemical inducing abiotic-responsive genes(Kramell et al., 2000; Savchenko et al., 2014; Yamauchi et al., 2015).
Profiling of VOC has been largely used to evaluate biotic andabiotic stress response in citrus plants, for example in response toCitrus Tristeza Virus (Cheung et al., 2015), winter flooding andsalinity (Velikova et al., 2012) or to blue light (Pallozzi et al., 2013).However, to our knowledge, no study has examined the impact ofploidy combined with water deficit on VOC profile in citrus plants.Thus, the aim of this work was to compare the VOC content in 2"and 4" Volkamer lemon plants, in control and water stressconditions, to gain new insight on the role of VOC in water stressdeficit and on the association of VOC profile with the geneticstructure of citrus plants.
2. Material and methods
2.1. Plant material and growth conditions
Thirty diploid (2"VL) and thirty doubled diploid (4"VL), six-month-old seedlings of Volkamer lemon (Citrus limonia Osb.)were obtained from seeds of fruit picked in adult trees maintainedin the citrus germplasm collection (INRA/CIRAD CRB Citrus), San
Giuliano, Corsica (France). The ploidy status of 2" and 4" plantswas previously checked and confirmed by flow cytometry (Partec I)according to Froelicher et al. (2007). The clonal propagation bynucellar embryogeneis was verified by genotyping the offspringusing 13 SSR markers according to protocols and markersdevelopped by Luro et al. (2008). Markers are listed in theSupplementary Table S1. Non conform Volkamer lemon plantswere discarded. The selected plants were transplanted in 3 L potscontaining commercial fresh soil, and then transferred in achamber of the Institute for Sustainable Plant Protection inFlorence, which was equipped to grow plants for 6 months underthe following controlled condictions: photoperiod of 16 h, withday/night of 25–32 #C/18–20 #C, and relative humidity varyingdaily between 50 and 80%. The photon flux density at leaf level was300–350 mmol m!2 s!1,supplied mainly with cool lights. Theplants were regularly irrigated during the week and werefertilizated with Bayfolan (Bayer) (NPK 5–7–8 + microelements),every 15 days.
2.2. Water deficit
Water deficit experimental design involved two phases(Supplementary Fig. S1). In the first phase, twenty-four plantsfor each genotype were irrigated at field capacity and the excesswater was allowed to drain overnight. After draining, the pots wereweighed to determine the weight at field capacity (Initialpotweight).Each pot was then enclosed in a plastic bag that was tied aroundthe stem to prevent soil evaporation. Then, twelve plants for eachgenotype were water-stressed by withholding water, while other12 plants for each genotype continued to be watered to fieldcapacity (control plants). Water deficit development was followedand parameterized measuring the pot weight and calculating thefraction of transpirable soil water (FTSW) (Sinclair and Ludlow,1986; Brilli et al., 2013). Every two days, during the water deficitexperiment, all plastic bags were unwrapped to weigh plants(nDaypotweight) and to water control plants compensating waterloss by transpiration. The physiologically lower limit of availablesoil water was defined as the FTSW at which stomatal conductanceapproached zero (Sinclair and Ludlow 1986; Brilli et al., 2013).Once this level was achieved, the water-stressed plants wereweighed to determine the final pot weight (Finalpotweight). Then,the FTSW was calculated for each single pot as: FTSW = (nDaypot-weight ! Finalpotweight)/(Initialpotweight! Finalpotweight) and water wasprovided to all plants to reach the initial pot weight. Leaves formetabolomic and biochemical analysis were harvested at (100% ofFTSW) (T0), 50% of FTSW (T1), 0% of FTSW (T2), and after 24 h fromirrigation of water-stressed plants (R).
2.3. Leaf net photosynthesis and stomatal conductance
During water deficit experiment, net photosynthesis andstomatal conductance were recorded with a portable photosyn-thesis system IRGA equipped with an integrated fluorometer (LI-6400, Li-Cor Inc., Nebraska, USA (model 6400; Li-Cor, Lincoln, NE)).Measurements were performed on the central portion of the firstfully expanded leaf using a photosynthetic photon flux density of600 mmol m!2 s!1, a leaf temperature of 25 #C, a relative humiditynear 40% and a CO2 concentration of 390 mmol mol!1. Measure-ments were repeated on 3–5 plants for each genotypes, at the sametimes when destructive sampling was carried out.
2.4. Proline extraction and determination
Proline was extracted according to Bates et al. (1973). Briefly,20 mg of leaf were crushed in liquid N2 with mortar and pestle andhomogenized in 70:30 ethanol:water at 95 #C for 20 min. The
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resulting mixture was then centrifuged at 14000"g for 5 min. Analiquot of the ethanolic supernatant (0.5 mL) was added to 1 mLreaction mix (ninhydrin 1% (w/v) in acetic acid 60% (v/v), ethanol20% (v/v)) in a screw-cup eppendorf tube. The tube was sealed,mixed and heated at 95 #C for 20 min. After cooling at roomtemperature, the tube was centrifuged quickly (1 min, 9000"g)and the content transferred in a 1.5 mL cuvettes. Proline contentwas measured with a spectrophotometer (EASYSPEC SAFAS, UV–vis spectrophotometer) at 520 nm and calculated against a prolinestandard curve (5–2–1–0.5–0.2 mM of proline in 40:60 ethanol:water, 40:60 v/v). Data are expressed as mmol g!1 dry weight(DW).
2.5. Lipid peroxidation
Lipid peroxidation was measured as malondialdehyde (MDA)concentration (being MDA a product of lipid peroxidation),following the method of Hodges et al. (1999), with somemodifications. Briefly, leaf samples (0.05 g) were homogenizedin 1 mL of 0.1% (w/v) trichloroacetic acid (TCA). The homogenatewas centrifuged at 15,000"g for 5 min. An aliquot of thesupernatant (0.5 mL) was added with 0.5 mL of 0.5% (w/v)thiobarbituric acid (TBA) in 20% (w/v) TCA. The mixture washeated at 95 #C for 30 min and then quickly cooled in an ice bath.After centrifugation at 10,000"g for 10 min, the absorbance of thesupernatant was recorded at 532, 600 and 440 nm. The MDAcontent was calculated using e = 155 mM!1 cm!1 and expressed asnmol MDA g!1 DW.
2.6. Volatile organic compounds
Volatile organic compound analysis was done by HS-SPME–GC-MS (Head Space Solid Phase Micro Extraction sampling coupledwith Gas Chromatography Mass Spectrometry).
For sample preparation, 0.1 g aliquots of citrus leaf, finelyground in liquid nitrogen, were transferred to 2 mL screw capheadspace vials and, for each sample, 0.5 mL of distilled water andapproximately 0.150 g of NaCl were added. The vial ensuredhomogeneous sample mixing and favored the partitioning of VOCinto the head space during SPME extraction. An Agilent 7820 GC-chromatograph equipped with a 5977A MSD mass spectrometerwith EI ionisation operating at 70 eV was used for analysis. A three-phase DVB/Carboxen/PDMS 75-mm SPME fibre (Supelco, Bella-fonte, PA, USA) was exposed in the head space of the vials at 60 #Cfor 30 min for volatile compound sampling after a 5-minequilibration time. A Gerstel MPS2 XL autosampler equipped witha magnetic transportation adapter and a temperature controlledagitator (250 rpm with on/cycles of 10 s) was used for ensuringconsistent SPME extraction conditions. A Chromatographic columnJ&W Innovax 30 m, 0.25 mm, ID 0.5 mm DF was used. The GCinjection temperature was 250 #C, splitless mode, and the oven wasprogrammed at 40# for 1 min, followed by a ramp of 5 #C/min to200 #C, and of 10 #C/min to 260 #C. This high temperature was heldfor 5 min. Mass spectra were acquired within the 29–350 m/zinterval with an Agilent 5977 MSD spectrometer at three scans/sspeed. The identification of VOC was done on the basis of both peakmatching with library spectral database, and matching of thecalculated Kovats retention indexes (KRI) with those retrievedfrom literature. The data are expressed as percent area of eachcompound over the sum of all the identified compounds.
As SPME analysis are more qualitative than quantitative, thegreen leaf volatile 2-hexenal whose levels resulted significantlydifferent between control and stressed plants, was quantitatedtogether with its product cis3—hexenol by extraction with pentaneaccording to the protocol of Raffa and Smalley (1995) modified fora better quantitation of the compounds of interest, and GC–MS
analysis. The pentane solution was supplemented with 5 methylhexanol (at 10 mg L!1) as internal standard (IS) instead ofTridecane, as described in the original method. For extraction,200 mg aliquots of the ground samples were soaked in 5 mL of ISpentane solution at room temperature for 24 h, in 20 mL screw capvials. The extracts were then filtered with 0.45 mm PTFE syringefilters and injected in the GC-MS system (1 ml in 1:10 split mode).Chromatographic conditions were the same as for HS-SPME–GC-MS analysis. Calibration lines, constructed with pure 2-hexenaland cis3-hexenol as standards in the same analytical conditionsand in the range 2–50 mg L!1 allowed the calculation of thecompound concentrations in the samples. The data are expressedas nanogram g!1 DW.
2.7. RNA extraction and RT-PCR analysis
In order to design primers for semi-quantitative analyses, thenucleotide coding sequences (CDS) of citrus DREB2A and HPLproteins were retrieved from the Phytozome database (http://www.phytozome.net/). For DREB2A the Arabidopsis thaliana NCBIsequence AY063972 was used as seed for a Blastn analysis on theCitrus (C. sinensis and C. clementina) genome database. Thesequences showing significant hits were downloaded from thePhytozome database, aligned with Muscle (Edgar 2004) and usedto find high similarity regions for primer design (SupplementaryFig. S3). The same approach was used for HPL using C. sinensis NCBIsequence NM_001288924 (Supplementary Fig. S4). The cyclophilin(gene bank: AB981058.1) was used as reference gene for relativequantifications, after preliminary test to select the most suitablereference gene. The web based Primer3Plus toll (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) was usedto design the primers (Supplementary Table S2). To ensure that theprimer was amplifying the targeted genes, the amplicons wereexcised and purified from agarose gels using the Wizard1 SV Geland PCR Clean-Up System (Promega) following the manufacturer’sprotocol, and then sequenced by Dipartimento Integrato Inter-istituzionale of University of Pisa, Italy (Supplementary Figs. S3 andS4). Total RNA extraction and cDNA synthesis were performed asalready reported in Podda et al. (2013) modifying the protocol ofthe Taqman Gene Expression Cells-to-CT TM Kit (AppliedBiosystems). The following standard thermal profile was usedfor PCR: 94 #C for 5 min; 33 cycles (for CYP, 35 for DREB2A and HPL)of 94 #C for 30 s, 58 #C for 50 s for cyclophilin and 60 #C for DREB2A
and HPL, and 72 #C for 60 s; 72 #C for 7 min as final extension. NoTemplate Controls (NTC) were include in each PCR. Each samplewas amplified in duplicate reactions (technical replicates). Threeindependent biological and two technical replicates were used.PCR products were separated by 1.5% agarose gel electrophoresisand stained with SYBR safe DNA gel stain (Invitrogen). Gel imageswere acquired with a GE DOC XR+ system (BioRad) and bandsquantification was performed with the Immage Lab Software(BioRad). The transcript levels were expressed in arbitrary units asmean $ SE.
2.8. Statistical analysis
For physiological, biological and expression analysis, thestatistical significance of differences between genotypes orbetween experimental conditions was determined by ANOVAand t-test, using STATISTICA software (StatSoft, Italy) at theprobability level <0.05. Data on volatile compounds were notnormally distributed (Kolmogorov–Smirnov one sample test) andthey were analysed by the non-parametric tests of Kruskal–Wallisand Mann–Whithney using SYSTAT 12.0 software (Systat SoftwareInc., Richmond, California, USA). All data are expressed as the meanvalue $ SE.
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3. Results
3.1. Physiological parameters
Net photosynthesis and stomatal conductance showed a signifi-cant decrease along with water deficit progression in bothgenotypes. However both parameters decreased faster (since 70%ofFTSW) in4"VL. Alsonet photosynthesiswassignificant lowerthanthose in 2"VL at 20% of FTSW and at the end of water stressexperiment (Fig. 1a and b), whereas stomatal conductance showedsignificant lower levels in 4"VL than 2"VL since FTSW was reducedof about 50%. At the end of the experiment stomatal conductanceof stressed 4"VL leaves were about 0.008 mmol m!2 s!1,whereas the value in stressed 2"VL leaves was 3-fold higher(0.020 mmol m!2 s!1). After rewatering net photosynthesis andstomatal conductance returned to values around 40% of respectivecontrols, both in 2"VL and 4"VL plants.
3.2. Proline content
At the beginning of the experiment, the concentration ofproline in control 4"VL leaves was slightly higher (99 $ 4.6 mmolg!1 DW) than in control 2"VL leaves (87 $ 2.5 mmol g!1 DW)(Fig. 2A). At the end of the water stress experiment and after 24 h ofrehydration, proline concentration was higher in stressed 2"VLand 4"VL with no significant difference in the levels betweengenotypes.
3.3. Lipid peroxidation
The content of MDA was not different in control 2"VL and 4"VLleaves (about 31.8 $ 0.3nmol g!1 and 32.7 $ 0.2 nmol g !1 DW,
respectively) (Fig. 2B). However, there was a marked increase inMDA concentration during the course of water deficit in bothgenotypes, and especially in 2"VL leaves which reached MDAcontent as high as 44.9 $ 2.8 nmol g!1 DW at the end of theexperiment.
3.4. Volatile organic compounds
3.4.1. Terpenoid profiling in unstressed leaves of the two genotypes
In order to characterize the profile of VOC in 2"VL and 4"VLleaves, SPME coupled with mass chromatography was used.Overall, 23 compounds belonging to the monoterpenoid familywere detected and identified in control leaves of 2"VL and 4"VL.Interestingly, the volatile profile was different in the control of thetwo genotypes (Fig. 3) indicating that some compounds might beused as phenotyping markers for ploidy level. In particular, 4"VLleaves were characterized by significantly higher relative contentsof a-pinene, sabinene, myrcene, limonene, and ocimene, andlower relative contents of citronellol, citral z, citral, and geraniol,with respect to 2"VL leaves.
3.4.2. Terpenoid profiling during and after water deficit
No significant changes in the profile and in the relative amountof monoterpenoids was found during progression of water stress,and after rewatering in the leaves of the two genotypes (Fig. 4A andB).
3.4.3. Green leaf volatile profiling during and after water deficit
Pentane extraction was used to quantify the 2-hexenalconcentration in the two genotypes together with the levels ofcis-hexenol, produced from hexenal after conversion catalyzed byalcohol dehydrogenase (Supplementary Fig. S2). The 2-hexenal
Fig. 1. The effect of water deficit on: Photosynthesis rate and leaf stomatal conductance. White circle: 2"VL, control; black circle: 2"VL, stress condition; white square: 4"VL,control; black square: 4"VL, stress condition. Lower case letters show statistical differences between control and stressed conditions; upper case letters show statisticaldifferences between genotypes (P < 0.05). Bars indicate means $ SE (n = 3–5 plants).
Fig. 2. Proline (A) and MDA (B) concentrations in leaves of 2"VL (grey line) and 4"VL (dark line) plotted against FTSW % during water deficit. Lower case letters showsignificant differences between control and stressed leaves; upper case letter show significant differences between genotypes (P < 0.05). Bars indicate means $ SE (n = 3plants).
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content of control leaves was 1.8-fold higher in 4"VL than in 2"VL(32.9 $ 1.7 and 18.1 $1.5 ng g!1 DW, respectively) (Fig. 5). After 6 dof water deficit (50% FTSW) the level of 2-hexenal of 2"VL leaveswas maximum, about 3-fold higher than in controls (from18.1 $1.5 to 58.8 $ 3.2 ng !1 DW), whereas no variation wasobserved in 4"VL leaves. At progressing water deficit levels 2-hexenal content of 2"VL leaves decreased from the value observedat T1, but remained significantly higher than in controls, andsimilar to the content of 4"VL leaves. No accumulation of cis-hexenol was observed in the two genotypes during and after thewater deficit (Fig. 5).
3.5. Expression profile of HPL and of the putative dehydration
transcription factor DREB2A
Semiquantitative PCR technique was performed to evaluate theexpression profile of HPL, the gene encoding for hydroperoxidaselyase, the enzyme synthetizing 2-hexenal from (9Z,11E,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13-HPOT). As shown inFig. 5a and b the expression of HPL in 2"VL leaves increased withwater deficit, reaching values approximately 3-fold higher than incontrols (from 1.2 to 3 arbitrary units) at T2, and strongly droppedafter rehydration at a value significantly lower than in controls
Fig. 3. Comparison of monoterpenoid profile from unstressed leaves of 2"VL and 4"VL. Data are expressed as percentage of the total monoterpenoid levels. Asterix showsignificant differences between genotypes (P < 0.05). Bars represent means $ SE (n = 3 plants).
Fig. 4. Comparison of the monoterpenoid profile from control and water-stressed leaves of 2"VL (A) and 4"VL (B). White rectangle: T0 100% of FTSW; light grey: T1, 50% ofFTSW, dark grey: T2, 0% of FTSW, black: R, 24 h rehydrated plants. Data are expressed as percentage of the total monoterpenoid levels. Bars represent means $ SE (n = 3 plants).
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(0.5 arbitrary units). The expression of HPL of 4"VL control leaveswas similar as control 2"VL leaves. However, HPL expression didnot change during water deficit in 4"VL leaves, and dropped afterrehydration (Fig. 6).
In order to evaluate the potential role of 2-hexenal as signalingcompound, the expression of a putative DREB2A, a gene encoding adehydration transcription factor, was measured in the twogenotypes in control and in water deficit conditions (Fig. 6). Theexpression profile of DREB2A was very similar to that of HPL,
showing a significant increase during water deficit, and a strongdecrease at control levels after rehydration of 2"VL leaves, but nosignificant changes throughout the water deficit experiment in4"VL leaves (Fig. 6).
4. Discussion
The aim of the study was to detect whether polyploidy has aneffect on VOC signature during and after a water deficit episode inVolkamer lemon seedlings. Tetraploidization related to chromo-some doubling is a spontaneous phenomenon in citrus. It leads toanatomical differences when compared to diploid plants, and mayalso lead to specific changes in the phenotype such as dwarfism,reduced number but increased size of the stomata with higher leafwater content (Allario et al., 2011) and enhanced constitutive rootabscisic acid production leading to better tolerance to water deficitconstraint (Allario et al., 2013).
The relative proportion of constitutive monoterpenoid n (themonoterpenoid profile) has been shown to be under strong geneticcontroland isgenerallyunaffectedbyabiotic factors(Hanover,1992;Plomion et al.,1996; Loreto et al., 2009). Therefore, monoterpenoidshave been largely used as interspecific and intraspecific chemo-taxonomical markers, mainly in forest genetics (Müller-Starck et al.,1992; Loreto et al., 2009), and in aromatic plants and tree crops suchas Citrus (Hosni et al., 2010; Lota et al., 2000, 2001; Lin et al., 2010).Thesignificantdifferencesrelatedtoploidyfoundinourexperiment,as 2"VL plants synthesized prevalently aldehydes and alcoholcompounds (namely z-citral, e-citral and e-geraniol) whereas 4"VLplants synthesized more non-oxygenated monoterpenoids
Fig. 5. Levels of 2- hexenal and cis-hexenol in 2"VL and 4"VL during the waterdeficit experiment. 2"C control; 2"T1: 50% of FTSW; 2"T2: 0% of FTSW; 2"R: 24 hafter rehydration. 4"C: control; 4"T1 50% of FTSW; 4"T2: 0% of FTSW; 4"R: 24 hafter rehydration. Bars represent means $ SE (n = 3 plants). Lower case letters showdifferences between control and stressed conditions; upper case letters showdifferences between genotypes (P < 0.05).
Fig. 6. Expression levels of hydrogen peroxide lyase (HPL) and the putative dehydration transcription factor DREB2A in 2"VL and 4"VL during water deficit experiment.Cyclophiline (CYP) was used as housekeeping gene. 2"C control, 2"T150% of FTSW, 2"T2 0% of FTSW; 2"R:24 h after rehydration. 4"C: control; 4"T1: 50% of FTSW; 4"T2: 0%of FTSW, 4"R: 24 h after rehydration. Bars represent means $ SE (n = 3 plants). Different letters indicate significant differences between control and stressed leaves.
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(limonene, a-pinene, mircene, sabinene and ocimene), are intrigu-ing. Several authors suggested that complex forms of geneticdominance determine the biosynthesis pathways of volatilecompounds in allotetraploid citrus hybrids (Gancel et al., 2003)andinsomatichybrids(cybrids)(Fanciullinoetal.,2005).Howeveritis not clear how chromosome doubling might have affected VOCbiosynthesis in unstressed 2"VL and 4"VL opening the way tofurther investigations on the nuclear- cytoplasmic interactions in4"VL plants and whether these differences might be important instressful environments or forcommunicating with otherorganisms.as VOC have prominent roles in plant interactions with biotic andabiotic stresses (Dicke and Loreto 2010).
To study the impact of drought stress on VOC content, 2"VL and4"VL were subjected to a water deficit followed by rehydration.Several reports suggest that photosystems of doubled diploidplants were stressed by water deficit less than in diploid plants (Liet al., 2009; Ruiz et al., 2015). In our work, the photosynthesisparameters decreased along with the water deficit in 2"VL and4"VL leaves with a similar profile. However, stomatal conductancedecreased faster (since 70% of FTSW) in stressed 4"VL respect to2"VL. This behavior might be a strategy for a better adaptation towater deficit trading water saving for carbon assimilation (Chaveset al., 2002; Centritto et al., 2011; Marino et al., 2014).
Drought and salinity may lead to the development of osmoticstress (Bartels and Sunkara, 2005) and lipid peroxidation (Blokinaet al., 2003). Proline is a well-known osmolyte (Ashraf and Foolad,2007) acting as mediator of osmotic adjustment under stressconditions. The similar profile accumulation of proline in bothgenotypes suggested that diploid and doubled diploid Volkamerlemon respond in the same way through osmotic adjustment forsurvival under water deficit condition. Conversely, MDA, asmarker of lipid peroxidation, showed lower concentrations in4"VL than 2"VL plants during and after water deficit. This mayindicate activation of others mechanisms protecting the photo-synthetic membranes regardless of the observed inhibition ofphotosynthesis under water deficit conditions. A strong antioxi-dant system was reported by Zhang et al. (2010) in 4" Dioscorea
plants exposed to heat stress, and by Podda et al. (2013) in 4"citrus plants under salt stress. Enhanced functionality of anti-oxidants might therefore be common to polyploid plants, helpingthem cope with all forms of oxidative stress. Volatile isoprenoidsmight also be a component of the antioxidant system protectingphotosynthetic membranes in 4"VL trees, as HS-SPME analysisshowed that the VOC profile was characterized by high proportionof monoterpenoids in 4"VL plants and of oxygenated terpenoidsin 2"VL plants. Monoterpenoids are believed to cooperate withnon-volatile isoprenoids in scavenging ROS in leaves (Loreto andSchnitzler, 2010). However, water deficit did not cause anymodification in the profiles of VOCs emitted by the leaves from2"VL and 4"VL plants in the condition applied in this work. Thismay indicate that VOC profile is under a strong systematic controland that VOC accumulation of 2"VL and 4"VL plants areconstitutively different. Variation in monoterpenoid profilesclassified into different chemotypes has been reported to affectinsects and fungal pathogens (Michelozzi et al., 1995; Taft et al.,2015). Further investigations might clarify whether differences inmonoterpenoid profiles also have a role in the chemical defense ofdiploid and doubled diploids citrus plants.
The higher levels of lipid peroxidation observed in stressed andrehydrated 2"VL might explain the increased concentration of theGLV 2-hexenal also detected in the same plants. Savchenko andDehesh (2014) found that drought stress finely tuned the oxylipinspathway leading to increased amount of hexenal, without howeverconcurrently increasing hexenol, as typically observed afterwounding. We also found no changes in hexenol concentrationsboth in 2"VL and in 4"VL under water deficit. Hexenal originates
in the hydroperoxide lyase branch of the oxylipin pathway and isformed from fatty acids, mainly as consequence of membraneperoxidation (Matsui, 2006). Up-regulation of HPL, the geneencoding for hydroperoxide lyase, in stressed diploid plants but notin 4"VL plants supports the idea that 2-hexenal is an indicator ofmembrane denaturation in response to water deficit in 2"VLplants. However, gene expression study in rice in response todehydration revealed significant enhancement of the HPL geneexpression also in tolerant lines (Lenka et al., 2011). De Domenicoet al. (2012) reported that during acclimation of sensitive andtolerant pea varieties to water deficiency conditions, the tolerantplants activated components of the lipoxygenase pathwayincluding lipoxygenase, two HPLs, and AOS, faster and strongerthan the sensitive variety. The first enzyme in the pathway, HPL, isencoded by one or more HPL genes, differing in their subcellularlocalization, including microsomes (Pérez et al., 1999), lipid bodies(Mita et al., 2005), the outer envelope of chloroplasts (Froehlichet al., 2001), and in some cases, with no specific localization in aparticular organelle (Nordermeer et al., 2000). Thus, the differentHPL induction in 2"VL and 4"VL plants might depend by adifferential regulation of this pathway, perhaps in response todifferent water deficit effects in the two genotypes.
The GLV 2-hexenal and cis-hexenol should be rapidly emittedafter biotic (Bleé, 2002; Ninemeets et al., 2013) and abioticstresses (Loreto et al., 2006; Loreto and Schnitzler, 2010;Savchenko et al., 2014). Nevertheless, these compounds are moresoluble than non-oxygenated terpenoids, and can be stored insolution, especially when stomata are closed, in response tostresses (Ormeno et al., 2011). Indeed, Mano et al. (2010) foundincreased endogenous levels of (E)-2-pentenal and (E)-2-hexenalin leaves of photo-sensitive and photo-tolerant tobacco plantsunder photo-inhibitory illumination, suggesting that lipid per-oxidation had occurred, and Catola et al. (2015) found higherlevels of GLV in drought stressed than in control pomegranates. Itshould be noted that, according to some authors, 2-hexenalconcentration in leaves might also have a signaling function.Yamauchi et al. (2015) reported that vaporizing trans-2- hexenalrapidly but transiently increased the internal 2-hexenal concen-tration in Arabidopsis plants, and concluded that 2-hexenal mayact as a signaling molecule that induces stress-related genes suchas HSFA and DREB2A. In our experiment, the expression of aputative drought-related transcription factor DREB2A showed aresponse to the stress similar to 2-hexenal and HPL expression.Thus, it cannot be excluded that 2-hexenal also acts as signalingcompound triggering adaptation of the 2"VL genotype to waterdeficit.
5. Conclusion
In conclusion, we reported that VOC content may be underconstitutive, genetic control in citrus, and that the VOC relativeproportion may change with ploidy level. Whereas both 2"VL and4"VL plants were similarly negatively affected by a water deficit,the 4"VL genotype seemed to have less damage to thephotosynthetic membranes, that we also attribute to the mono-terpenoids characterizing its VOC profile. Our data provide newinsights in the understanding of the relationships between VOCand ploidy level in plants and the role of GLV in the response towater deficit in a non-model plant.
Acknowledgements
Dayse Drielly Souza Santana Vieira spent nine months at CNR-IPSP and one month at CIRAD-IVIA laboratories in the framework ofa doctorate sandwich financed by CAPES, http://www.capes.gov.br/(grant: CSF SDW-0268/13-5), Ciências Sem Fronteiras.
D.D.S.S. Vieira et al. / Environmental and Experimental Botany 126 (2016) 1–9 7
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.envexpbot.2016.02.010.
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39
Supplementary material
Metabolomic analysis reveals involvement of 2-hexenal in the response to water deficit of diploid Citrus volkameriana but not in the
respective doubled diploid plants.
Fig. S1. Water deficit experimental strategy for 2xVL and 4xVL. (A): Each plant is one biological replicate. The experiment consisted of
two treatments, control and water-stress and two genotypes (2xVL and 4xVL). (B): The physiological parameters were collected every two
40
days from 12 different plants (3 - 6 for each experimental condition and genotype). Volatile organic compounds were extracted from 3 plants at
T0- 100% FTSW T1- (about 51% FTSW), T2- (0% FTSW) and R- 24 hours after rehydration .
Fig.S2. GC-MS analysis of volatile compounds extracted by pentane from leaves of Volkamer lemon.
41
Table S1. Name, primer sequence of the SSR markers and size of amplified DNA fragments in Volkamer lemon with these markers
Forward primer (5'-3') Reverse primer (5'-3') cDNA ID
Allele size
(in bases)
MEST 308 CCTCTTCATTTTTCTCTGAAACTAA TTGCAACATCGTTCCTCTTG FC883472.1 253 - 262
MEST 356 CAAAATTCCATGGCTTGCTT GGCTTGGGAATTTGATTTGA FC909938.1 129 - 153
MEST 391 TGAAGTCCCTCCAAGAAAGC AGTCAGAGCCAGAGCCAGAG FC897601.1 160 - 166
MEST 502 TCAGCAGAAGAAGGAGACTCG CGGACATGGATGTAATCAGG FC893337.1 180 - 189
MEST 775 AGCTCCGTCTCCAGCATAAA CCACACACCCTTTTAACGAGA FC885563.1 148 - 150
MEST 910 TCCAAAACGACTCCCTTAACA GAAGCTGTGAGAGGCTTTGG FC913597.1 132 - 136
MEST 914 CAGCCTCCTCTCCCTTTCTT CAGCAATTCCGAGTGAGTGA FC896872.1 166 - 176
MEST 1047 AAACAAAATCAATGGCCGAG TGGGTTTATTGTTGGGCTGT FC881048.1 176 - 188
MEST 1344 GAAGCCAAGAAATGCATCGT AAAGGAGGGATGGTATTGCC FC896899.1 204 - 210
MEST 1361 GGAGATGTGCCATGGAAGTAA AAGATTACCAACAGGAGTTTATATGAG FC887186.1 140 - 156
MEST 1047 AAACAAAATCAATGGCCGAG TGGGTTTATTGTTGGGCTGT FC881048.1 176 - 188
MEST 1344 GAAGCCAAGAAATGCATCGT AAAGGAGGGATGGTATTGCC FC896899.1 204 - 210
MEST 1361 GGAGATGTGCCATGGAAGTAA AAGATTACCAACAGGAGTTTATATGAG FC887186.1 140 - 156
42
Table S2. Primers used for semi-quantitative gene expression analyses in Volkamer lemon. Primers were designed using the web-based
Primer3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Constitutivelyexpressed cyclophilin (CYP) was amplified
as a reference gene; HPL, hydroperoxidelyase; DREB2a, dehydration-responsive element-binding protein 2A
Genename PCR product
size Forward primer sequence (5’–3’) Reverse primersequence (5’–3’)
DREB2A 239 GAAGAGTTCCGGCCAAGG ATTAAGCCGTGCACAAGGAC
HPL 204 TTCTCCATGCTCGACAAGTG TGTTAGCCCAAACTCGTCCT
CYP 111 CGGATCTCAGTTCTTCGTCTG ACTTTCTCGATGGCCTTGAC
43
AY063972.1_A.thaliana_putative_DREB2A_protein ATGGCAGTTTATGATCAGAGTGGAGATAGAAACAGAACACAAATTGATACATCGAGGAA-
XM006486183.1_C.sinensis_dehydration-responsive_element ATGGCTATT------CAAAGCAAAGATTCTTCTCCGTCTCTTA-TGATGCCACTGAGTAG
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
AY063972.1_A.thaliana_putative_DREB2A_protein ---AAGGAAATCTAGAAGTAGAGGTGACGGTACTACTGTGGCTGAGAGATTAAAGAGATG
XM006486183.1_C.sinensis_dehydration-responsive_element TGACAGGAAAAGGAAACGAAGAGATGGCGT---TAATGTTGCTGAGACTCTTGAACGGTG
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
AY063972.1_A.thaliana_putative_DREB2A_protein GAAAG-------AGTATAAC--GAGACCG---TAGAAGAAGTTTCTACCAAGAAGAGGAA
XM006486183.1_C.sinensis_dehydration-responsive_element GAGGCGATATAATGAATCGCTTGAATCTGGCAATGGCGAG-GATAAACCA--ATGAGAAG
C.volkameriana_sequenced_amplicon --------------------------------------------------------GAAG
*.*.
AY063972.1_A.thaliana_putative_DREB2A_protein AGTACCTGCGAAAGGGTCGAAGAAGGGTTGTATGAAAGGTAAAGGAGGACCAGAGAATAG
XM006486183.1_C.sinensis_dehydration-responsive_element AGTTCCGGCCAAGGGTTCGAAAAAGGGTTGTATGAAAGGTAAAGGAGGACCGGAGAATGG
C.volkameriana_sequenced_ampliconAGTTCCGGCCAAGGGTTCGAAAAAGGGTTGTATGAAAGGTAAAGGAGGACCGGAGAATGG
***:** ** **.** *****.*****************************.******.*
AY063972.1_A.thaliana_putative_DREB2A_protein CCGATGTAGTTTCAGAGGAGTTAGGCAAAGGATTTGGGGTAAATGGGTTGCTGAGATCAG
XM006486183.1_C.sinensis_dehydration-responsive_element ACGGTGTGATTATCGAGGTGTGAGGCAGAGGACCTGGGGTAAGTGGGTTGCGGAGATAAG
C.volkameriana_sequenced_ampliconACGGTGTGATTATCGAGGTGTGAGGCAGAGGACCTGGGGTAAGTGGGTTGCGGAGATAAG
.**.***..**: .****:** *****.**** ********.******** *****.**
AY063972.1_A.thaliana_putative_DREB2A_protein AGAGCCTAATCGAGGTAGCAGGCTTTGGCTTGGTACTTTCCCTACTGCTCAAGAAGCTGC
XM006486183.1_C.sinensis_dehydration-responsive_element GGAGCCAAACAGGGGAAATAGGCTATGGCTTGGTACTTTTCCAAGTGCTGTTGAGGCTGC
C.volkameriana_sequenced_ampliconGGAGCCAAACAGGGGAAATAGGCTATGGCTTGGTACTTTTCCAAGTGCTGTTGAGGCTGC
.*****:** .*.**:*. *****:************** **:* **** ::**.*****
AY063972.1_A.thaliana_putative_DREB2A_protein TTCTGCTTATGATGAGGCTGCTAAAGCTATGTATGGTCCTTTGGCTCGTCTTAATTTCCC
44
XM006486183.1_C.sinensis_dehydration-responsive_element CCTTGCTTATGATCATGCTGCTAGGGCTATGTATGGTCCTTGTGCACGGCTTAATTTGCC
C.volkameriana_sequenced_ampliconCCTTGCTTATGATCATGCTGCTAGGGCTATGTATGGTCCTTGTGCACGGCTTAAT-----
********** * *******..**************** **:** ******
AY063972.1_A.thaliana_putative_DREB2A_protein T-CGGTC-----------TGATGCGTCTGAGG---------TTACGAGTACCTCAAGTCA
XM006486183.1_C.sinensis_dehydration-responsive_element CGATGTTTCAAGATTGAATGAGTCTTCGAAGGATTCTGATTCAACGACGTCATCAAACCA
C.volkameriana_sequenced_amplicon------------------------------------------------------------
AY063972.1_A.thaliana_putative_DREB2A_protein GTCTGAGGTGTG---TACTGTTGAGACTCCTGGTTGTGTTCATGTGAAAACAGAGGATCC
XM006486183.1_C.sinensis_dehydration-responsive_element GTCTGAGATTGAGGATGCTAAAGTGAAGA--------------ATGACGCAAGAGAAGCC
C.volkameriana_sequenced_amplicon------------------------------------------------------------
AY063972.1_A.thaliana_putative_DREB2A_protein AGATTGTGAATCTAAACCCTTCTCCGGTGGAGTGGAGCCGAT---GTATTGTCTGG----
XM006486183.1_C.sinensis_dehydration-responsive_element GAATCTAAAATAATTGCCCAAC--CTGAGGCCGAGTTACTAAGTAGTCCAGTCAAACCAA
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
AY063972.1_A.thaliana_putative_DREB2A_protein ---AGAATGGTGCGGAAGAGATGAAGAGAGGTGTTAAAGCGGATAAGCATTGGCTGAGCG
XM006486183.1_C.sinensis_dehydration-responsive_element AAGCTAAAGATGAGGCTGAGGA--------------------------------------
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
AY063972.1_A.thaliana_putative_DREB2A_protein AGTTTGAACATAACTATTGGAGTGATATTCTGAAAGAGAAAGAGAAACAGAAGGAGCAAG
XM006486183.1_C.sinensis_dehydration-responsive_element -TAATGAAAAGTACTACTGGGGTGAACAGCTAGAT-----TGCAAGCCGG---AAGCAAG
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
Fig. S3. Alignment of Arabidopsis thaliana DREB2A gene mRNA, Citrus sinensis dehydration responsive element, and Citrus x
volkameriana DREB2 fragment sequenced in this work (green) and used for mRNA accumulation analyses. Specific DREB2A motif, bold;
EREB/AP2 domain, underline; primer annealing regions (red).
45
DQ866816_C_aurantium_HPL GACACACTGTTCGACACCGTCGAGAAGGAACTCTCCGAAAAGAACAGCATAAGCTACATG
NM_001288924_C_sinensis_HPL GACACACTGTTCGACACCGTCGAGAAAGAGCTCTCCGAAAAGAACAGCATAAGCTACATG
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
DQ866816_C_aurantium_HPL GTCCCTTTACAAAAATGCGTCTTTAACTTCCTCTCAAAATCGATCGTGGGAGCCGACCCA
NM_001288924_C_sinensis_HPL GTCCCTTTACAAAAATGCGTCTTTAACTTCCTCTCAAAATCGATCGTGGGAGCCGACCCA
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
DQ866816_C_aurantium_HPL AAAGCCGACGCCGAAATCGCCGAGAACGGCTTCTCCATGCTCGACAAGTGGCTGGCCTTG
NM_001288924_C_sinensis_HPL AAAGCCGACGCCGAAATCGCCGAGAACGGCTTCTCCATGCTCGACAAGTGGCTGGCCTTG
C.volkameriana_sequenced_amplicon ------------------------------TTCTCCATGCTCGACAAGTGGCTGGCCTTG
******************************
DQ866816_C_aurantium_HPL CAGATCCTGCCCACAGTCAGCATAAACATCTTGCAGCCCCTTGAAGAGATCTTTCTTCAC
NM_001288924_C_sinensis_HPL CAGATCCTGCCCACAGTCAGCATAAACATCTTGCAGCCCCTTGAAGAGATCTTTCTTCAC
C.volkameriana_sequenced_ampliconCAGATCCTGCCCACAGTcaGCATAAACATCTTGCAGCCCCTTGAAGAGATCTTTCTTCAC
************************************************************
DQ866816_C_aurantium_HPL TCTTTTGCTTACCCTTTTGCCCTTGTCAGTGGAGACTACAACAAGCTCCACAACTTCGTT
NM_001288924_C_sinensis_HPL TCTTTTGCTTACCCTTTTGCCCTTGTCAGTGGAGACTACAACAAGCTCCACAACTTCGTT
C.volkameriana_sequenced_ampliconTCTTTTGCTTACCCTTTTGCCCTTGTCAGTGGAGACTACAACAAGCTCCACAACTTCGTT
************************************************************
DQ866816_C_aurantium_HPL GAAAAGGAAGGCAAAGAAGTTGTGCAGCGGGGGCAGGACGAGTTTGGGCTAACAAAAGAA
NM_001288924_C_sinensis_HPL GAAAAGGAAGGCAAAGAAGTTGTGCAGCGGGGGCAGGACGAGTTTGGGCTAACAAAAGAA
C.volkameriana_sequenced_amplicon GAAAAGGAAGGCAAAGAAGTTGTGCAGCGGGGGCAGGACGAGTTTGGGCTAACA------
******************************************************
46
DQ866816_C_aurantium_HPL GAAGCCATTCACAACTTGCTGTTCATTCTAGGGTTTAATGCTTTCGGTGGGTTCTCTATT
NM_001288924_C_sinensis_HPL GAGGCCATTCACAACTTGCTGTTCATTCTAGGGTTTAATGCTTTCGGTGGGTTCTCTATT
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
DQ866816_C_aurantium_HPL TTTTTGCCAAAGCTGATTAATGCAATTGCTAGTGACACAACTGGGTTGCAGGCAGAGTTA
NM_001288924_C_sinensis_HPL TTGTTGCCAAAGCTGATTAATGCAATTGCTAGTGACACAACTGGGTTGCAGGCAAAGTTA
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
DQ866816_C_aurantium_HPL AGAAGTGAAGTGAAAGAGAAATGTGGGACATCCGCCTTGACTTTTGAGTCAGTCAAGAGT
NM_001288924_C_sinensis_HPL AGAAGTGAAGTGAAAGAGAAATGTGGGACATCCGCCTTGACTTTTGAGTCAGTCAAGAGT
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
DQ866816_C_aurantium_HPL CTAGAGTTGGTTCAGTCTGTGGTTTGCGAAACTCTGAGACTTAACCCACCGGTTCCTCTC
NM_001288924_C_sinensis_HPL CTAGAGTTGGTTCAGTCTGTGGTTTACGAAACTCTGAGACTTAACCCACCGGTTCCTCTC
C.volkameriana_sequenced_amplicon ------------------------------------------------------------
Fig.S4. Alignment of Citrus sinensis and Citrus aurantium, hydroperoxidelyase (HPL) gene mRNA and Citrus volkameriana fragment
sequenced in this work (green) and used for mRNA accumulation analyses. Primer annealing regions are highlighted (red).
47
CAPÍTULO 2:
Survival strategies of citrus rootstocks subjected to drought
(Artigo submetido à Plant & Cell Physiology em 15/01/2016)
48
Survival strategies of citrus rootstocks subjected to drought
Dayse Drielly Souza Santana-Vieira1, Luciano Freschi2, Lucas Aragão da
Hora Almeida1, Diana Matos Neves1, Diogo Henrique Santos de Moraes1, Liziane
Marques dos Santos3,5, Fabiana Zanelato Bertolde4, Walter dos Santos Soares
Filho5, Maurício Antônio Coelho Filho5, Abelmon da Silva Gesteira1,5
1 – Departamento de Biologia, Centro de Genética and Biologia Molecular,
Universidade Estadual de Santa Cruz, Ilhéus – Bahia, 45662-900, Brazil 2 – Departamento de Botânica, Instituto de Biociências, Universidade de
São Paulo, São Paulo 05508-090, Brazil 3 – Departamento de Ciências Agrárias, Universidade Federal do
Recôncavo da Bahia, Cruz das Almas - Bahia, 44380-000, Brazil 4 – Departamento de Ensino, Instituto Federal da Bahia – Campus
Eunápolis, Eunápolis - Bahia, 45823-431, Brazil 5 – Embrapa – Mandioca e Fruticultura, Cruz das Almas - Bahia, 44380-
000, Brazil
Corresponding author:
Abelmon da Silva Gesteira - [email protected]
Telephone: +55(75)3312-8046 / Fax: +55(75)3312-8097
Abbreviations: Sunki Maravilha mandarin - SM
Rangpur lime - RL
Valencia orange - VO
Tahiti acid lime - TAL
reactive oxygen species - ROS
systemic acquired acclimation - SAA
leaf water potential - ΨL
49
leaf osmotic potential - Ψπ
soil matric potentials - soil Ψ
photosynthetic rate - A
stomatal conductance - Gs
transpiration rate - E
intrinsic water use efficiency - A/Gs
water use efficiency - A/E
salicylic acid – SA
50
MANUSCRIPT TITLE: Survival strategies of citrus rootstocks subjected to
drought
Abstract
In the present study, Citrus limonia Osb. (Rangpur lime - RL) and Citrus sunki
(Sunki Maravilha mandarin - SM) rootstocks were tested, either ungrafted or
grafted with their reciprocal graft combinations (SM/RL and RL/SM) or with
shoot scions of two commercial citrus varieties: Citrus sinensis (L.) Osb.
(Valencia orange - VO) and Citrus latifolia Tanaka (Tahiti acid lime - TAL). All
the combinations were subjected to drought through gradual decreases in soil
water content. RL adopted a dehydration avoidance strategy and maintained
growth, whereas SM adopted a dehydration tolerance strategy focused on plant
survival. Compared with RL, the leaves and roots of SM exhibited higher
concentrations of abscisic acid (ABA) and salicylic acid (SA), which induce
drought tolerance, and of carbohydrates such as trehalose and raffinose, which are
reported to be important reactive oxygen species (ROS) scavengers. SM
rootstocks adopted a dehydration tolerance strategy in response to drought when
ungrafted, and the same strategy was induced in RL, VO and TAL shoot scions.
RL rootstocks are widely used by Brazilian citrus producers, and SM and its
derivatives may be an alternative for genetic diversification. However, because of
their different survival strategies, RL reaches the permanent wilting point more
quickly than SM, and SM may recover from prolonged droughts, such as those
predicted for upcoming years, more efficiently than RL. The present study is one
of the most complete studies of drought tolerance mechanisms in citrus crops and
is the first to use reciprocal grafting to clarify scion/rootstock interactions.
51
Keywords: Citrus plants, drought stress, interaction scion/rootstock, reciprocal
grafting, rootstock, survival strategy.
Introduction
Citrus crops are widespread and have great importance worldwide (Wu et
al., 2013). Because they are perennial crops grown in orchards with long
production periods (Molinari et al., 2004), citrus crops are subjected to several
biotic and abiotic stresses. Drought is one of the most threatening abiotic factors
for citrus cultivation, causing decreased plant growth, development and
productivity (Osakabe et al., 2014). In addition, drought severity and/or intensity
is increasing worldwide (Shukla et al., 2012).
Rootstock selection and improvement with the goal of increasing plant
water use efficiency is an efficient strategy to minimize the effects of climate
changes on plant production (Berdeja et. al, 2015). Grafting is a millenary
technique widely used in several different cultures (Mudge et al., 2009). A recent
study showed the importance of rootstocks for food security by increasing the
efficiency of natural (water and soil) resource utilization and decreasing the use of
chemical inputs (Albacete et al., 2015). Rootstocks increase the resistance of
citrus crops to biotic and abiotic factors (Garcia-Sanchez et al., 2007; Rodrígues-
Gamir et al., 2010; Machado et al., 2013). In the case of drought, this resistance is
due to physiological changes that lead to changes in leaf water potential, stomatal
conductance and hydraulic conductance (Romero el al., 2006; Garcia-Sanchez et
al., 2007; Rodrígues-Gamir et al., 2011).
Drought-associated dehydration results from an imbalance between root
water uptake and water loss via transpiration; the result is a decrease in gas
52
exchange and, consequently, in photosynthetic rates (Arbona et al., 2005; Garcia-
Sanchez et al., 2007; Aroca et al., 2012; Mittler and Blumwald, 2015). Perception
and signal transduction of abiotic stresses, including drought, is initiated by a
signaling cascade termed systemic acquired acclimation (SAA) (Mittler and
Blumwald, 2015). SAA results from plant physiological responses that may
include changes at the transcriptional, proteomic and post-transcriptional
modification levels, as well as metabolic changes and/or metabolite accumulation
(Verslues et al., 2006). These responses to abiotic stresses may be, for example,
hormonal changes (Koshita and Takahara, 2004; Arbona et al., 2008; Pérez-
Clemente et al., 2012; Ollas et al., 2012) or the accumulation of solutes and/or
carbohydrates (Verslues et al., 2006; Peters et al., 2007; Tardieu et al., 2011).
Depending on their intrinsic characteristics, plants may adopt different
strategies to cope with drought periods. These different strategies can be divided
into two major categories, dehydration avoidance and dehydration tolerance,
which involve different physiological processes (Levit, 1972; Verslues et al.,
2006; Lawlor, 2013). Dehydration avoidance comprises multiple strategies to
prevent water loss, such as solute accumulation and cell wall hardening.
Dehydration tolerance involves mechanisms to avoid cell damage caused by water
loss, such as synthesis of osmoprotectant proteins and solutes, metabolic changes,
and detoxification of reactive oxygen species (ROS) (McDowell et al., 2008;
Claeys and Inzé, 2013). Whereas dehydration avoidance is focused on the
maintenance of plant growth and productivity, dehydration tolerance is focused on
plant survival, especially during prolonged drought (Verslues et al., 2006). Zhoa
et al. (2015) evaluated different cassava cultivars under drought conditions and
reported that the plants that adopted survival strategies exhibited stomatal closure,
53
decreased shoot growth, the diversion of carbon and energy to the storage and
biosynthesis of protective compounds. Plants that adopted growth maintenance
strategies showed reprogramming of energy metabolism, osmotic adjustment, and
maintenance of cell wall flexibility (Claeys and Inzé, 2013).
Rangpur lime (RL) and Sunki Maravilha mandarin (SM) rootstocks
exhibit different patterns of soil water uptake and protein profiles under drought
conditions (Neves et al., 2013; Oliveira et al., 2015), indicating different drought
survival strategies. This discrepancy prompted us to investigate the influence of
scions on rootstocks, and vice versa. These interactions were investigated using
reciprocal grafting between these two citrus crops (SM/RL and RL/SM). In
addition, the behavior of the reciprocal grafts was compared with that of the
commercial scions Valencia orange (VO) and Tahiti acid lime (TAL) grafted onto
RL and SM rootstocks.
The gas exchange measurements and hormonal and carbohydrate profiles
obtained in the present study confirmed that RL and SM rootstocks adopt different
strategies to cope with drought stress. Whereas RL tried to maintain growth and
productivity by adopting a dehydration avoidance strategy, SM exhibited
dehydration tolerance mechanisms, which help plants to survive, especially during
longer drought periods. To our knowledge, the present study is the first to use
reciprocal grafting to clarify scion/rootstock interactions. Furthermore, the present
results showed that the rootstocks tested, especially SM, influenced the VO, TAL,
and reciprocal scions, which exhibited similar behavior to the ungrafted
rootstocks. The present study is one of the most complete studies of drought
tolerance mechanisms in citrus crops.
54
Results
Morphological and physiological responses of eight different
scion/rootstock combinations subjected to drought
Drought was induced by withholding water supply to plants. Plants
reached severe stress, as indicated by the leaf water potential (ΨL ≤ -2.0 MPa), on
different days. During the experiment, the air relative humidity decreased slightly;
however, no sudden changes in climate conditions were observed (Fig. 1).
Leaf area values of drought-exposed plants before and after drought
application, for all the scion/rootstock combinations tested, indicated no
significant interactions between plant combination and water availability
conditions, except for comparison group 2 (Fig. 2). In comparison group 2, the
RL/SM combination exhibited a 54.5% decrease in leaf area following drought
compared to the control, which was significantly different from SM/RL under the
same conditions (Fig. 2B). Significant differences in leaf area between the
beginning and end of the experiment were observed for the TAL/RL combination
only, with a 31.5% decrease (Fig. 2D).
All plants were harvested at soil Ψ values lower than -1.5 MPa (theoretical
wilting point) and at leaf water potential (ΨL) values ≤ -2.0 MPa (Figs. 3A-D and
4), which indicates severe drought stress and was adopted as the threshold ΨL
value for plant harvest in the present study (Fig. 3A-D). All control plants,
independent of the combination, exhibited ΨL ≥ -0.5 MPa. Similar values were
observed following 48-h rehydration.
A significant interaction between plant combination and drought treatment
was observed for leaf osmotic potential (Ψπ) exclusively in comparison groups 1
(Fig. 3E) and 2 (Fig. 3F). Ψπ was significantly lower for SM than for RL
55
following rehydration. RL exhibited significantly different Ψπ values under severe
drought, under control conditions, and following rehydration. For SM, the Ψπ
values under control conditions were significantly different from the Ψπ values
under the remaining water availability conditions. Regarding the reciprocal graft
combinations (group 2), SM/RL exhibited significantly lower Ψπ under severe
drought than under control conditions and following rehydration, whereas no
significant differences were observed for RL/SM. Except for ungrafted SM, no
significant differences between different water availability conditions were
observed for plants with SM roots (RL/SM, VO/SM and TAL/SM) for any of the
tested genotypes. This result indicates that SM roots can determined plant
behavior during severe drought stress. All the combinations with RL roots,
including ungrafted RL, exhibited significantly lower Ψπ under severe drought
than under control conditions or following rehydration. This finding indicates that
plants with RL roots exhibited osmotic adjustment compared to plants with SM
roots.
Net photosynthetic rate (A), stomatal conductance (Gs), transpiration rate
(E) decreased under severe drought compared to control plants or following 24-h
rehydration for all tested plant combinations (Table 1). Significant differences in
A were observed only for the comparison between RL and SM (Table 1), with SM
exhibiting higher A following 24-h rehydration. Significant differences in A
between different water availability conditions were observed for all plant
combinations tested, except for the comparison between TAL/RL and TAL/SM,
which revealed significant differences between severe drought and following
rehydration, with A being higher for TAL/SM. It should be noted that no
significant differences in A were observed between plants with RL and SM roots
56
under control conditions; however, plants with RL or SM roots exhibited
pronounced decreases (78-100%) in A under severe drought compared to control
conditions. Following rehydration, A increased 30.2- (RL), 71.6- (SM/RL), 6.9-
(VO/RL) and 299.8-fold (TAL/RL) compared to plants under severe drought
stress for plants with RL roots, and 4.6- (SM), 6.1- (RL/SM), 4.0- (VO/SM) and
2.6-fold (TAL/SM) for plants with SM roots.
SM exhibited Gs values almost twice as high as RL following 24-h
rehydration and no significant differences were observed among the remaining
genotypes (Table 1). However, significant differences between different water
availability conditions were observed for all plant combinations. Significant
differences in E between different water availability conditions were observed for
each plant combination (Table 1). For groups 1 (RL and SM), 2 (SM/RL and
RL/SM), and 4 (TAL/RL and TAL/SM), the highest E values were observed
under control conditions, followed by rehydration and then severe drought. For
group 3 (VO/RL and VO/SM), E under severe drought was significantly different
from the control treatment and following 24-h rehydration, but not between the
latter two treatments. Two observations regarding transpiration rates should be
highlighted. First, plants with RL roots exhibited a stronger recovery of
transpiration rates following 24-h rehydration than plants with SM roots, with E
values 7.4- (RL), 7.2- (SM/RL), 4.1- (VO/RL) and 4.9-fold (TAL/RL) higher than
under severe drought stress. Plants with SM roots exhibited E values 2.8- (SM),
3.6- (RL/SM), 3.4- (VO/SM) and 1.9-fold (TAL/SM) higher following
rehydration than under severe drought stress. Second, the TAL/SM combinations
exhibited the highest transpiration rates during severe drought, which were
significantly different from those of TAL/RL. This finding is consistent with the
57
photosynthetic rates and stomatal conductance results for these plant combinations
(Table 1).
Significant interactions for intrinsic water use efficiency (A/Gs) were
observed for all the comparison groups (Table 1). It should be noted that A and Gs
were markedly higher for plants with SM roots than for plants with RL roots
under severe drought. No significant differences were observed between plants
with RL and SM roots under control conditions or following 24-h rehydration. For
instant efficiency of water use (A/E), significant differences were observed
between RL and SM only, with higher levels for SM following rehydration and
under severe drought. For group 2 (SM/RL and RL/SM), only SM/RL exhibited
significant differences in A/E between severe drought stress and under control
conditions or following rehydration. For groups 3 (VO/RL and VO/SM) and 4
(TAL/RL and TAL/SM), and also for VO/RL and TAL/RL significant differences
between all the tested water availability conditions were observed for, whereas
VO/SM and TAL/SM exhibited significant differences between the control
treatment and severe drought or following rehydration, but not between the latter
two conditions.
Drought-induced changes in hormone levels
Plant hormones are regulated by environmental changes and by simulated
stress conditions, such as drought. Overall, drought, especially severe drought,
affected hormone concentrations in all the plant combinations, resulting in
decreased or increased concentrations, depending on the hormone (Fig. 5). In
addition, hormone concentrations were observed to be higher in leaves than in
roots, especially under control conditions, in all plant combinations. Except for
the leaf IAA and SA concentrations in SM/RL, plants with SM roots (ungrafted or
58
grafted) exhibited significantly higher or similar concentrations of all tested
hormones compared to plants with RL roots (ungrafted or grafted) under control
conditions. In addition, rootstocks were observed to influence scion behavior, and
vice versa. This finding was particularly evident for the comparisons between the
reciprocal graft combinations SM/RL and RL/SM (Fig. 5B, F, J, N, R and V),
confirming the importance of scion/rootstock interactions for plant development
and survival in different environments and/or stress conditions.
Both leaf and root ABA concentrations increased under severe drought for
all plant combinations (Fig. 5A-H). This result is in agreement with the decreased
stomatal conductance observed under the same conditions. The only exception
was RL, which exhibited no significant differences in leaf ABA content between
the different water availability conditions. Notably, plants with SM roots
(ungrafted or grafted) exhibited higher ABA concentration than plants with RL
roots (ungrafted or grafted) under both severe drought stress and control
conditions, which may indicate an influence of rootstocks on scion behavior.
Furthermore, the influence of scions on rootstock behavior was confirmed by the
finding that the reciprocal graft combination SM/RL exhibited pronouncedly
higher ABA levels than RL (ungrafted); the difference was not as pronounced for
the remaining tested scions grafted onto RL. Similar ABA levels were observed
for RL/SM and the ungrafted SM.
Overall, IAA content decreased under severe drought stress compared to
control conditions (Fig. 5I-P). This finding is in agreement with the increases
observed in ABA concentrations (Fig. 5A-H) because ABA and IAA have been
demonstrated to antagonistically interact during drought-induced responses
(Dunlap and Binzel, 1996; Niculcea et al., 2013). It should be noted that under
59
control conditions, plants with SM roots (ungrafted or grafted) exhibited higher
leaf and/or root IAA levels than plants with RL roots. However, SM/RL exhibited
higher leaf IAA content under control conditions than the reciprocal graft
combination RL/SM (Fig. 5J), demonstrating the influence of scions on rootstock
behavior.
SA levels increased under severe drought in all plant combinations (Fig.
5Q-Z), except for RL (Fig. 5Q) and RL/SM (Fig. 5R) in leaves, and SM/RL (Fig.
5V), VO/RL (Fig. 5X) and TAL/RL (Fig. 5Z) in roots. It should be highlighted
that the plants with SM roots (ungrafted or grafted) exhibited higher root SA
concentrations than those with RL roots (ungrafted or grafted) under severe
drought stress (Fig. 5U-Z). Higher leaf SA under severe drought were also
observed for SM (Fig. 5Q) and TAL/SM (Fig. 5T) compared to RL (Fig. 5Q) and
TAL/RL (Fig. 5T), respectively. This finding is interesting because SA is very
important for plant defense signaling in response to biotic and abiotic stresses,
such as drought (Horváth et al., 2007; Miura et al., 2013; Okuma et al., 2014).
Drought-induced changes in carbohydrate profile
Osmoprotection is essential to the maintenance of cell activity during
drought stress. Carbohydrates play an important role as osmoprotectants in the
maintenance of cell turgor (Salerno and Curatti, 2003; Peshev and Van den Ende,
2013). The leaf and root concentrations of raffinose, trehalose, galactose, fructose,
glucose and sucrose for the eight plant combinations and three water availability
conditions evaluated are presented in Table 2. Overall, drought resulted in
increased carbohydrate concentrations. Raffinose, trehalose and galactose
concentrations were higher in roots than in leaves, whereas fructose, glucose and
sucrose concentrations were similar in leaves and roots.
60
Except for RL, SM/RL and TAL/RL, all plant combinations exhibited
increased leaf raffinose under severe drought, which was particularly evident in
plants with SM roots (Table 2). Except for TAL/SM, all plant combinations with
SM roots exhibited significantly higher raffinose content under severe drought;
these concentrations decreased to levels similar to the control treatment following
48-h rehydration.
Fluctuations in trehalose levels resembled those observed for raffinose
levels. In all plants with SM roots (ungrafted or grafted), leaf and root trehalose
levels increased under severe drought and then decreased following 48-h
rehydration (Table 2). No significant differences in leaf trehalose content between
the three water availability conditions were observed for plants with RL roots
(ungrafted or grafted). Root trehalose content under different water availability
conditions was significantly higher following 48-h rehydration for SM/RL, and
under severe drought stress for VO/RL.
For leaf galactose, significant differences were observed only between RL
and SM and between their reciprocal grafts, SM/RL and RL/SM, whereas
significant differences in root galactose were observed between VO/RL and
VO/SM and between TAL/RL and TAL/SM (Table 2). Sucrose is formed by the
combination of fructose and glucose, and overall, the concentrations of these three
carbohydrates followed similar tendencies. Interestingly, in plants with SM roots,
the leaf concentrations of fructose, glucose and sucrose increased under severe
stress and decreased again following 48-h rehydration (Table 2). Root fructose
and sucrose levels exhibited a tendency similar to that observed for leaves, with
increased concentrations under severe drought stress. However, root glucose
increased following 48-h rehydration in plants with SM roots.
61
Discussion
In the present work, drought caused marked changes in leaf water
potential, leaf osmotic potential, gas exchange, and hormone and carbohydrate
profiles in the tested plant combinations. RL and SM rootstocks were selected
based on previous studies indicating their different behaviors when subjected to
drought stress (Neves et al., 2013; Oliveira et al., 2015). In the present study,
reciprocal graft combinations (SM/RL and RL/SM) were used to further
investigate scion/rootstock interactions. In addition, two commercial scions were
used, Valencia orange (VO) and Tahiti acid lime (TAL), which were grafted onto
SM and RL rootstocks to compare the behavior of the two rootstocks under the
same scion.
Changes and interactions between gas exchange and hormone and
carbohydrate profiles under drought
Among the first drought-induced responses in plants, stomatal closure (to
avoid water loss) and an increased root:shoot ratio (Claeys and Inzé, 2013) are
considered critically important for plant survival. However, during longer periods
of drought, plants lose the ability to balance water uptake and loss, and leaf water
potential (ΨW) decreases. Two strategies may then be adopted: (i) dehydration
avoidance or (ii) dehydration tolerance. Dehydration avoidance is characterized
by solute accumulation and cell wall hardening to decrease water loss.
Dehydration tolerance involves the production of protective solutes and proteins,
metabolic changes and ROS detoxification to avoid damages caused by cellular
water loss (Verslues et al., 2006). In the present study, the tested rootstocks, RL
and SM, were observed to exhibit different strategies to tolerate/survive drought.
62
Both tested rootstocks, RL and SM, and their different graft combinations
exhibited decreased leaf water potential (ΨW; Fig. 3A-D) and soil matric potential
(Fig. 4A-D), indicating that they were affected by the severe drought treatment. In
addition, A, Gs and E were observed to decrease under severe drought in all plant
combinations (Table 1). Similar findings have been previously reported for
different citrus plants under drought conditions (Romero et al., 2006; Allario et
al., 2011). It should be highlighted that no significant differences in A, Gs or E
were observed between RL and SM rootstocks (grafted or ungrafted) under
control conditions, indicating that RL and SM possess similar capacities for CO2
absorption and control of water loss by transpiration under high soil water
availability conditions. Significant differences were observed between the
ungrafted rootstocks (RL and SM) following 24-h rehydration; SM exhibited
higher A and Gs, indicating that SM has a greater capacity to recover from severe
drought. In addition, plants with SM roots exhibited higher A and Gs levels under
severe drought than plants with RL roots, indicating that even under field
conditions, where RL was observed to maintain production levels (data not
shown), SM decreases production and uses the available water more efficiently to
maintain metabolic processes and survive prolonged drought periods. A/Gs is
very important for plant development under water limited conditions, such as in
semi-arid regions. Studies have identified citrus plants with high A/Gs, such as
those exhibited by SM, as suitable for semi-arid regions and/or regions with
partial irrigation methods (García-Sánchez et al., 2007; Tejero et al., 2011; Hutton
and Loveys, 2011; Pérez-Péres et al., 2012).
ABA is considered the primary hormone involved in regulation of plant
responses to abiotic stresses, especially drought (Fang et al., 2010; Gómez-
63
Cadenas et al., 2015). ABA levels were observed to increase in both roots (all
tested plant combinations) and leaves (except for RL) (Fig. 5A-H). One of the
main functions of ABA is the induction of stomatal closure, which occurs through
the inhibition of the flow of potassium ions into guard cells to control water loss
due to transpiration (Shinozaki and Shinozaki, 2006; Peleg and Blumwald, 2011).
This phenomenon also results in decreased photosynthetic rates. An interaction
between increased leaf and root ABA contents and decreased gas exchange was
observed under severe drought stress for all combinations tested.
Except for the reciprocal grafts SM/RL and RL/SM, plants with SM roots
exhibited significantly higher ABA content than plants with RL roots under
control conditions. It was long believed that endogenous ABA under high water
availability could decrease shoot growth. However, normal levels of endogenous
ABA have been reported to be required to maintain shoot growth (Sharp and
LeNobel, 2002). It should be highlighted that no significant differences in Gs, A
or E were observed under control conditions between plants with SM roots, which
exhibited higher ABA levels, and plants with RL roots. However, under optimal
environmental conditions, SM exhibited slower growth than RL but eventually
reached the same growth and production pattern (data not shown). In addition, in
TAL/RL, the TAL scions, which are tall and exhibit vigorous growth and
abundant foliage, induced the RL rootstocks to produce ABA levels under control
and severe drought conditions (Fig. 5H) that were at least twice the levels of the
remaining combinations with RL rootstocks (RL, SM/RL and VO/RL; Fig. 5E-G).
This finding indicates that due to their characteristics, TAL scions demanded
more ABA from rootstocks, which resulted in higher production of ABA by RL
roots in an attempt to control water loss by TAL shoots.
64
Interestingly, under severe drought, all plants with SM roots (grafted and
ungrafted) exhibited higher leaf ABA (Fig. 5A-D) than plants with RL roots
(grafted or ungrafted). It should also be noted that under severe drought, SM
scions grafted onto RL exhibited leaf ABA levels approximately 2.5-fold higher
than ungrafted RL. ABA has been frequently reported to be produced in roots and
to translocate into leaves, especially under drought conditions (Ollas et al., 2012;
Neves et al., 2013). However, other studies have shown that ABA can also be
produced in shoots and/or guard cells, protecting plants from water loss by
inducing stomatal closure (Zeevaart, 1977; Holbrook et al., 2002; Christmann et
al., 2007; Ikegami et al., 2009; Bauer et al., 2013; Hu et al., 2013; Boursiac et al.,
2013). Therefore, these data indicate that ABA production can be stimulated in
SM leaves under severe drought conditions, which, in turn, leads to increased
ABA production in grafted scions as well.
Auxins, particularly IAA, play critical roles in numerous plant growth and
development responses (Wang et al., 2001). IAA is mainly produced in young
shoot regions and is redistributed via both cell-to-cell polar transport an non-polar
transport in phloem to other plants regions, such as the the roots, whose growth
and development is strongly modulated by this hormone (Aloni et al., 2010).
Although IAA is not a significant component of the xylem sap, it controls xylem
morphological changes under stress conditions (Pérez-Alfocea et al., 2011).
Except for RL/SM, leaf IAA levels under control conditions were higher for
plants with SM roots than with RL roots (Fig. 5M-P). It should also be noted that
SM/RL exhibited higher IAA content than the reciprocal combination under
control conditions, with shoots exhibiting a prevalence of SM characteristics
(ungrafted), whereas the root IAA concentration was more similar to those
65
observed for ungrafted RL. The decreased IAA levels observed under severe
drought may result from the effect of water deficit on IAA polar transportation
(Wang et al., 2009). Studies with different crops have shown increased ABA and
decreased IAA levels under osmotic stress due to drought or salinity stress
(Niculcea et al., 2013; Dunlap and Binzel, 1996). In the present study, increased
ABA and decreased IAA concentrations were observed under severe drought for
all plant combinations. This change may have caused the decreased leaf and root
growth rates observed during drought for both genotypes. It should also be
highlighted that IAA affects the stomatal aperture (Peleg and Blumwald, 2011),
therefore, the reductions in IAA content observed in drought-exposed plants may
facilitate the promotive action of ABA on stomatal closure and, consequently, on
the minimization of water loss due to transpiration.
SA has generally been reported to be critical for tolerance to biotic
stresses, usually being produced at sites of infection (Halim et al., 2006; Dempsey
et al., 2011). However, SA has also been observed to play significant roles in
tolerance to abiotic stresses (Horváth et al., 2007), plant growth and development,
and stomatal movements (Raskin, 1992; Miura and Tada, 2014). Recent studies
based on exogenous applications of SA (Horváth et al., 2007; Kang et al., 2012)
or using SA-deficient or overproducer mutants (Miura et al., 2013; Okuma et al.,
2014) have also shown that SA increases drought tolerance in plants. This
increased tolerance is directly related to the induction of ROS production
mediated by peroxidases and the promotion of stomatal closure (Okuma et al.,
2014). Stomatal closure induced by SA is also related to defense against
pathogens by preventing pathogen entry through the stomata (Lee et al., 2007). In
the present study, except for leaf SA content in RL, SM/RL and RL/SM (Fig. 5Q
66
and R) and root SA levels in SM/RL VO/RL and TAL/RL (Fig. 5V, X and Z),
plants exhibited increased SA concentrations during severe drought (Fig. 5Q-Z).
Therefore, these increased levels of SA in drought-exposed citrus plants may also
have facilitated stomatal closure under these environmental circumstances (Table
1), very likely acting in conjunction with the increased ABA levels also detected
under drought conditions (Fig. 5A-H). It should be highlighted that all plant
combinations that did not exhibit increased SA under severe drought had RL roots
and/or shoots, and this hormone can be very important for inducing an increased
tolerance to stress. During drought, RL adopts a strategy of maintenance of plant
growth/production, whereas SM adopts a strategy focused on plant survival, i.e.,
ensuring that plants survive the stress period.
Interestingly, SA supplementation before stress was observed to increase
ABA contents (Bandurska and Stroinski, 2005) and both hormones are known to
induce ROS production and stomatal closure. An SA increase can directly result
in higher drought tolerance for both analyzed genotypes and for the different plant
combinations. However, the increase in SA was more pronounced in plants with
SM roots or scions (SM/RL) than in those with RL roots. Induction of SA
accumulation may play a protective role during water stress (Miura and Tada,
2014). SM may therefore possess a more efficient system to protect against
damages caused by drought than RL, i.e., a protective mechanism to ensure plant
survival under drought conditions.
A large proportion of the evaluated plant combinations exhibited increased
carbohydrate concentrations under severe drought stress in both leaves and roots,
with only a few plant combinations exhibiting decreased carbohydrate
concentrations (Table 2). Carbohydrates, especially sucrose, the main product of
67
photosynthesis, play a central role in plant life by affecting plant development,
growth, storage, signaling, and acclimatization to biotic and abiotic stresses
(Salerno and Curatti, 2003). In general, drought increases the concentrations of
soluble carbohydrates, which are synthesized in response to osmotic stress and act
as osmoprotectants by stabilizing cell membranes and maintaining plant turgor
(Peshev and Van den Ende, 2013). Some of the main functions of carbohydrates,
such as sucrose, raffinose and trehalose, are replacing the water lost under drought
conditions, binding to the polar ends of membrane phospholipids and maintaining
cell turgor. In addition, carbohydrate accumulation during osmotic stress affects
ROS production. ROS are mainly produced in chloroplasts through excitation or
partial reduction of molecular O2, mitochondria and peroxisomes (Keunen et al.,
2013). This relation between carbohydrates and ROS may be mediated by both
increases and decreases in carbohydrate cell contents (Couée et al., 2006). It
should be highlighted that excess ROS causes oxidative damage to cells (Blokhina
et al., 2003); therefore, the production of ROS scavengers is of great importance.
Soluble carbohydrates produced during stress were previously thought to
be direct or indirect signals for the production of ROS scavengers and/or repair
enzymes. However, recent studies have suggested that soluble carbohydrates,
namely raffinose and trehalose, may act as true ROS scavengers (Keunen et al.,
2013; Lunn et al., 2014). In addition to being osmoprotectants, the raffinose
family of oligosaccharides (RFOs) has been recently described to have antioxidant
action by acting as ROS scavengers and inducing higher tolerance in plants under
osmotic stress (Nishizawa et al., 2008; ElSayed et al., 2014). Raffinose levels
were observed to increase in leaves and roots under severe drought stress,
especially in plants with SM roots, with more pronounced increases in root tissues
68
(Table 2). Roots are the first plant organs to perceive water stress, initiating a
series of reactions that help plants prevent or tolerate stress. The pronounced
increase in raffinose in leaves of drought-exposed citrus plants may be associated
with its roles both in signaling and as ROS scavengers. The finding that this
increase was more pronounced in plants with SM roots again indicates that SM
has more efficient protective mechanisms than RL under severe drought stress to
ensure plant survival to the stress.
Trehalose can be maintained at high levels without damaging cell
metabolism because of its high solubility and non-reducing nature (Lunn et al.,
2014). In addition, recent studies using Arabidopsis thaliana mutants have shown
that trehalose metabolism plays an important role in the response of guard cells to
ABA by controlling stomatal conductance (Avonce et al., 2004; Houttle et al.,
2013; Lunn et al., 2014). This finding is in agreement with the present study, in
which high levels of ABA (Fig. 5A-H) and trehalose (Table 2) were observed to
coincide with low stomatal conductance (Table 1). This is the first report of
increased trehalose and raffinose concentrations in citrus plants during drought.
This finding is of great importance for the understanding of drought stress
tolerance in citrus plants, and it suggests that further, more specific, studies of
these carbohydrates would be valuable.
The evaluated citrus plants exhibit different survival strategies to drought
Drought causes several physiological, morphological and
metabolic/biochemical changes in plants, namely decreased gas exchange,
resulting in decreased growth (Readdy et al., 2004; Arbona et al., 2013), leaf
abscission (Gómez-Cadenas et al., 1996), and changes in hormone (Sharp and
LeNoble, 2002; Argamassila et al., 2014) and/or carbohydrate profiles (Peters et
69
al., 2007; Keunem et al., 2013). In addition, plants may develop different
strategies for surviving and/or coping with drought period, greatly depending on
its intensity and duration (Zhao et al., 2015). These strategies may focus on
maintaining plant growth and competitiveness or on plant survival (Claeys and
Inzé, 2013). However, plants may develop characteristics to cope with drought
that are not advantageous for the maintenance of plant production (Lopes et al.,
2011).
Neves et al. (2013) and Oliveira et al. (2015) measured physiological
parameters, ABA concentrations, gene expression of proteins associated with the
ABA biosynthetic pathway, and protein profiles of RL and SM rootstocks grown
in pots under drought conditions, and they observed that RL and SM exhibited
different responses to drought stress. SM, compared with RL, exhibited higher
ABA concentrations and greater numbers of differentially expressed proteins in
response to drought. In addition, proteins found exclusively in SM were involved
with DNA repair and processing (Oliveira et al., 2015), whereas RL exhibited up-
regulation of proteins responsible for transportation, protein metabolism, stress
response and proteolysis. These studies, together with the field trials (Muriti
Farm, São Paulo), are in agreement with the present results. Overall, plants with
SM roots exhibited high levels of hormones important for the induction of drought
tolerance, such as ABA and SA (Fig. 5A-H and 5Q-Z), and high levels of
carbohydrates, such as sucrose, raffinose and trehalose (Table 2), that help prevent
the cell damage caused by drought stress and maintain cell metabolism and turgor.
It should be highlighted that decreased water availability under field
conditions is intensified by evaporation; when these decreases are systematic and
continuous, even genotypes such as SM, which possess drought survival
70
mechanisms based on water saving, will also suffer from drought due to
evaporation. It is therefore believed that under severe and long periods of drought,
RL will reach the permanent wilting point sooner than SM because the drought
survival mechanism of SM is more effective under prolonged drought stress.
In addition, a detailed analysis of the behavior of RL and SM, especially
their hormone and carbohydrate profiles, indicates that the two exhibit different
survival strategies (Fig. 6). This conclusion was mainly based on the lower or
similar hormone and carbohydrate concentrations observed for RL, ungrafted or
grafted with SM, VO and TAL scions, than for SM. SM generally exhibited
higher hormone and carbohydrate concentrations than RL. This finding indicates
that even under control conditions, SM has stronger protective mechanisms than
RL, which may confer an advantage to this rootstock under conditions of
prolonged drought. In addition, the lower concentrations observed for RL than for
SM indicate that the main focus of RL is maintaining plant growth and, therefore,
plant production. This finding was confirmed in the field (data not shown),
indicating that RL adopts a strategy of dehydration avoidance. Because SM has
more robust mechanisms of stress protection, it develops a strategy of dehydration
tolerance, which may decrease plant production. It’s important to highlight that a
selection of rootstocks that guarantee plant survival and maintain plant growth and
production rates under prolonged drought conditions is essential for sustainable
citrus production. Hybridization between genotypes with these characteristics may
therefore be the key to obtaining these varieties.
The present study is, to our knowledge, the first study conducted in citrus
plants using reciprocal grafting to clarify scion/rootstock interactions. It is
therefore one of the most complete studies of drought tolerance in citrus plants
71
performed to date. The results show that the rootstocks tested, especially SM, tend
to modify scion behavior (VO, TAL, and reciprocal grafts) into behavior similar
to that exhibited by the ungrafted rootstock.
Materials and methods
Plant material and drought treatment
Two rootstocks, Citrus limonia Osb. (Rangpur lime) and Citrus sunki
(Sunki Maravilha mandarin), which exhibit different responses to drought (Neves
et al., 2013; Oliveria et al., 2015), and two commercial scions, Citrus sinensis (L.)
Osb. (Valencia orange) and Citrus latifolia Tanaka (Tahiti acid lime), which are
economically important worldwide and locally, respectively, were used.
Rootstocks were obtained by seed germination, and scions were obtained from
buds of healthy mother plants from the Citrus Germplasm Bank (Banco de
Germoplasma de Citros) of Embrapa Cassava and Fruit Crops (Embrapa
Mandioca e Fruticultura). Bud grafting was performed when the rootstocks were 6
months old. Eight different combinations were tested: Rangpur lime (RL) and
Sunki Maravilha mandarin (SM) ungrafted rootstocks and RL and SM rootstocks
grafted with different scions – Sunki Maravilha mandarin/Rangpur lime (SM/RL),
Rangpur lime/Sunki Maravilha mandarin (RL/SM), Valencia orange/Rangpur
lime (VO/RL), Valencia orange/Sunki Maravilha mandarin (VO/SM), Tahiti acid
lime/Rangpur lime (TAL/RL), and Tahiti acid lime/Sunki Maravilha mandarin
(TAL/SM). Following grafting, plants were transferred into 45-L pots containing
Plantmax®, washed sand and clay (2:1:1). The plants were maintained under an
anti-aphid screen with daily irrigation. NPK and micronutrient fertilizers were
applied every two weeks until the plants were 2 years old.
72
Following this period, plants were homogenized by pruning the scions.
After one month, the plants were divided into two groups: (i) control treatment:
plants grown in soil kept at field capacity with constant irrigation and (ii) drought
treatment: plants grown without irrigation. The pots were covered with transparent
plastic and aluminum foil to avoid water loss due to evaporation. The experiment
lasted 17 days, with drought developing gradually as the soil water content
decreased. Soil moisture was monitored daily using a time domain reflectometry
(TDR) probe. When the leaf water potential of the plants became lower than -2.0
MPa, they were harvested, and rehydration of these plants was started. The plants
reached water deficit on different days. Plants subjected to rehydrationwere
harvested 48-h after rewatering.
Leaf water potential, leaf osmotic potential and matric potential
Leaf water potential (ΨL) was determined before dawn using a Scholander
pressure chamber (m670, PMS Instrument Co., Albany, OR, USA). ΨL was
measured every other day after confirming that photosynthetic parameters were
decreasing via measurements using an infrared gas analyzer (IRGA). Leaves were
detached from the plants using a stylus and were immediately used for ΨL
measurements. Leaves and roots of plants under severe drought were harvested
when they reached ΨL ≤ -2.0 MPa. ΨL was also measured for control plants and
following 48-h rehydration, after which roots and leaves were harvested.
Leaf osmotic potential (Ψπ) was measured using a Vapro 5520 vapor
pressure osmometer (Wescor, Inc., Logan, USA) calibrated using NaCl standards
with known concentrations (mmol kg-1). Fresh leaves were collected from the
middle third of the plants and were macerated, pressed, and filtered to extract the
sap. A 10-µL aliquot was used to determine tissue osmolarity. Ψπ values were
73
obtained in mmol kg-1 and converted into osmotic potential using the Van’t Hoff
equation:
Ψπ=R×T×C
where R is the ideal gas constant (0.00813), T is temperature (K),
and C is the leaf extract concentration (mmol kg-1, converted to moles).
Soil matric potential (soil Ψ) was determined using the soil moisture
values (measured daily with a TDR probe) and the matric potential (measured
with a WP4 Dewpoint PotentiaMeter dew-point mirror psychrometer).
Leaf area
The leaf area of drought-exposed plants was measured at the beginning
and end (following stress application) of the experiment. The total leaf area was
obtained by measuring the length and width once every five leaf of each plant.
The leaves were measured from the stem to the branches, starting at the mark
made during the previous measurement. Leaf area was determined by multiplying
the leaf length by the leaf width with a correction factor of 0.72 (Neves et al.,
2013). The total leaf area was determined by summing all the leaf areas obtained
for each plant and multiplying the total by five. The total leaf area was obtained in
cm2 and converted into m2.
Photosynthetic parameters
The net photosynthetic rate (A), stomatal conductance (Gs) and
transpiration (E) were measured every other day in fully expanded mature leaves
that had previously been selected and marked. Gas exchange measurements (A,
Gs and E) were performed using an LCpro-SD portable IRGA (ADC
BioScientific Limited, UK) at 1000 µmol photons m-2 s-1 photosynthetically active
radiation (PAR) and ambient leaf temperature, air humidity and CO2
74
concentration. Measurements were performed between 8 AM and 11 AM in two
leaves of each plant once the readings had stabilized, i.e., exhibited similar values.
Gas exchange measurements in rehydrated plants were performed following 24-h
rehydration.
Hormonal measurements
Endogenous indoleacetic acid (IAA), salicylic acid (SA) and abscisic acid
(ABA) levels were determined by gas chromatography tandem mass
spectrometry-selected ion monitoring (GC-MS-SIM). Leaf and root samples (50-
100 mg FW) were extracted as described in Rigui et al. (2015). Approximately
0.25 μg of the labeled standards [2H6]ABA (OlChemIm Ltd.), [13C6]IAA
(Cambridge Isotopes, Inc.) and [2H6]SA (Cambridge Isotopes, Inc.) was added to
each sample as internal standards. For ABA quantification, aliquots of the extract
were methylated as described in Rigui et al. (2015). For IAA and SA
quantifications, aliquots were evaporated and resuspended in 50 mL of pyridine,
followed by a 60-min derivatization at 92°C using 50 mL of N-tert-
butyldimethylsilyl-N-methyltrifluoroacetamide (with 1% tert-
butyldimethylchlorosilane). Analysis was performed on a gas chromatograph
coupled to a mass spectrometer (model GCMS-QP2010 SE, Shimadzu) in
selected ion monitoring mode. The chromatograph was equipped with a fused-
silica capillary column (30 m, 0.25 mm ID, 0.50-mm-thick internal film) DB-5 MS
stationary phase using helium as the carrier gas at a flow rate of 4.5 mL min–1 in
the following program: 2 min at 100°C, followed by gradients of 10°C min–1 to
140°C, 25°C min–1 to 160°C, 35°C min–1 to 250°C, 20°C min–1 to 270°C and
30°C min–1 to 300°C. The injector temperature was 250°C, and the following MS
operating parameters were used: ionization voltage, 70 eV (electron impact
75
ionization); ion source temperature, 230°C; and interface temperature, 260°C.
Ions with a mass ratio/charge (m/z) of 244, 202 and 130 (corresponding to
endogenous IAA); 250, 208 and 136 (corresponding to [13C6]-IAA); 309, 195 and
209 (corresponding to endogenous SA); 315, 201 and 215 (corresponding to
[2H6]SA); 190, 162 and 134 (corresponding to endogenous ABA); and 194, 166
and 138 (corresponding to [2H6]ABA) were monitored. Endogenous
concentrations were calculated based on extracted chromatograms at m/z 244 and
250 for IAA, 309 and 315 for SA, and 190 and 194 for ABA.
Sugar profiling
For soluble carbohydrate profiling, leaf and root samples (50-100 mg FW)
were extracted as described in Freschi et al. (2010). The supernatant residue was
resuspended in 50 μL of pyridine and derivatized for 40 min at 60°C with 20 µL
of N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane.
The trimethylsilylated extracts were analyzed by GC-MS (model GCMS-QP2010
SE, Shimadzu), and the total ion current spectra were recorded in the mass range
of 50–700 atomic mass units in scanning mode. The chromatograph was equipped
with a fused-silica capillary column DB-5 MS stationary phase using helium as
the carrier gas at a flow rate of 4.5 mL min–1. The initial running conditions were
100°C for 5 min, followed by a gradient up to 320°C at 8°C min-1. Endogenous
metabolite concentrations were obtained by comparing the peak areas of the
chromatograms against commercial standards.
Statistical analysis
A completely randomized experimental design (CRD) was used, with 3
replicates for the control group and 3 replicates for the groups subjected to
drought (for each of the eight tested combinations), for a total of 48 plants. The
76
eight combinations were divided into four comparison groups, each with two
combinations, according to the similarity between them. Comparison group 1
consisted of the two ungrafted rootstocks (RL and SM), group 2 was the
reciprocal grafts of the two rootstocks (SM/RL and RL/SM), group 3 was
Valencia orange scions grafted onto the two rootstocks (VO/RL and VO/SM), and
group 4 was Tahiti acid lime scions grafted onto the two rootstocks (TAL/RL and
TAL/SM). Analysis of variance (ANOVA) followed by the Scott-Knott test was
performed for each comparison group to test for significant differences between
combinations within each comparison group, significant differences between
different water availability treatments for each combination, and significant
interactions between plant combination and drought treatment, with significance
defined as p≤0.05. Six replicates were performed for the photosynthetic
parameters (n=6), three each for leaf water potential, leaf osmotic potential, soil
matric potential, and leaf area (n=3) and five for hormone contents and
carbohydrate profiles (n=5).
Funding information: National Council for Scientific and Technological
Development - CNPq (grant: 301356/2012-2 and 472733/2013-3). Scholarship of
Dayse Drielly Souza Santana Vieira financed by CAPES in Brazil and also by
CAPES/Ciências Sem Fronteiras (grant: CSF SDW-0268/13-5) of a doctorate
sandwich.
Acknowledgements: The trainee Victor Pereira Brito for the illustration of figure
6.
77
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Tables
Table 1 – Physiological parameters of eight different scion/rootstock combinations under three different water availability conditions:
Control: ΨL ≤ 0.5; severe drought stress: ΨL ≥ 2.0; and 48-h rehydration. Treatments were divided into four comparison groups. A:
photosynthetic rate (µmol m2 s-1); Gs: stomatal conductance (mol m2 s-1); E: transpiration rate (mmol m2 s-1); A/Gs: intrinsic water use
efficiency; A/E: water use efficiency. RL: Rangpur lime; SM: Sunki Maravilha mandarin; VO: Valencia orange; TAL: Tahiti acid lime. Values
are averages ± standard errors (n=6). Different uppercase letters indicate significant differences between combinations, and different lowercase
letters indicate significant differences within the same combination, according to the Scott-Knott test (p<0.05).
88
Comparison group
Combinations Condictions A
µmol m-2s-1 Gs
µmol m-2s-1 E
µmol m-2s-1
A/Gs A/E
1
RL C 8.28 ± 0.61 cA 0.17 ± 0.01 cA 2.16 ± 0.16 cA 48.45 ± 1.36 bA 3.84 ± 0.05 cA S 0.12 ± 0.13 aA 0.00 ± 0.00 aA 0.18 ± 0.09 aA 13.83 ± 8.91 aA 0.93 ± 0.37 aA R 3.57 ± 0.51 bA 0.04 ± 0.01 bA 1.35 ± 0.17 bA 82.19 ± 2.27 cA 2.65 ± 0.30 bA
SM C 7.71 ± 0.58 cA 0.15 ± 0.02 cA 1.98 ± 0.12 cA 51.97 ± 2.70 aA 3.90 ± 0.22 bA S 1.22 ± 0.18 aA 0.01 ± 0.00 aA 0.52 ± 0.06 aA 99.25 ± 14.04 bB 2.47 ± 0.34 aB R 5.59 ± 0.19 bB 0.08 ± 0.00 bB 1.42 ± 0.06 bA 72.00 ± 2.70 aA 3.94 ± 0.11 bB
2
SM/RL C 5.38 ± 0.98 cA 0.12 ± 0.02 cA 1.69 ± 0.19 cA 43.38 ± 2.56 bA 3.08 ± 0.31 aA S 0.05 ± 0.18 aA 0.00 ± 0.00 aA 0.19 ± 0.02 aA 0.00 ± 0.00 aA 1.29 ± 0.48 bA R 3.46 ± 0.31 bA 0.04 ± 0.00 bA 1.39 ± 0.11 bA 84.66 ± 10.63 cA 2.57 ± 0.29 aA
RL/SM C 6.60 ± 0.41 cA 0.16 ± 0.01 cB 1.97 ± 0.06 cA 41.69 ± 2.65 aA 3.36 ± 0.19 aA S 0.66 ± 0.27 aA 0.01 ± 0.00 aA 0.42 ± 0.08 aA 63.03 ± 11.20 bB 2.18 ± 0.62 aA R 4.03 ± 0.68 bA 0.06 ± 0.01 bA 1.49 ± 0.28 bA 66.06 ± 5.34 bA 2.89 ± 0.37 aA
3
VO/RL C 5.17 ± 0.38 cA 0.11 ± 0.01 cA 1.60 ± 0.10 bA 46.38 ± 2.00 aA 3.24 ± 0.14 cA S 0.54 ± 0.20 aA 0.01 ± 0.01 aA 0.39 ± 0.12 aA 31.97 ± 7.95 aA 1.10 ± 0.28 aA R 3.74 ± 0.54 bA 0.05 ± 0.00 bA 1.60 ± 0.24 bA 70.24 ± 8.71 aA 2.37 ± 0.11 bA
VO/SM C 5.62 ± 0.33 cA 0.12 ± 0.01 cA 1.67 ± 0.08 bA 48.44 ± 2.36 aA 3.40 ± 0.23 bA S 1.07 ± 0.23 aA 0.01 ± 0.00 aA 0.48 ± 0.03 aA 107.33 ± 23.37 bB 2.21 ± 0.46 aA R 4.26 ± 0.71 bA 0.06 ± 0.01 bA 1.62 ± 0.26 bA 75.56 ± 6.34 bA 2.62 ± 0.18 aA
4
TAL/RL C 7.34 ± 0.30 cA 0.17 ± 0.01 cA 2.02 ± 0.08 cA 44.81 ± 2.43 aA 3.65 ± 0.13 cA S 0.01 ± 0.11 aA 0.00 ± 0.00 aA 0.25 ± 0.05 aA 9.64 ± 4.00 aA 0.66 ± 0.43 aA R 2.50 ± 0.30 bA 0.03 ± 0.00 bA 1.22 ± 0.02 bA 84.35 ± 6.89 bA 2.06 ± 0.25 bA
TAL/SM C 7.69 ± 0.20 cA 0.17 ± 0.01 cA 2.11 ± 0.17 cA 47.30 ± 3.04 aA 3.75 ± 0.27 bA S 1.67 ± 0.30 aB 0.02 ± 0.00 aA 0.79 ± 0.13 aB 102.24 ± 35.55 aB 2.23 ± 0.42 aB R 4.32 ± 0.56 bB 0.06 ± 0.01 bB 1.54 ± 0.17 bA 69.62 ± 4.36 aA 2.79 ± 0.13 aA
89
Table 2 – Raffinose, trehalose, galactose, fructose, glucose and sucrose concentrations in the leaves and roots of eight different scion/rootstock
combinations under three different water availability conditions. Conditions: control: ΨL ≤ 0.5; severe drought stress: ΨL ≥ 2.0; and 48-h
rehydration. RL: Rangpur lime; SM: Sunki Maravilha mandarin; VO: Valencia orange; TAL: Tahiti acid lime. Values are averages ± standard
errors (n=5). Different uppercase letters indicate significant differences between combinations, and different lowercase letters indicate
significant differences within the same combination, according to the Scott-Knott test (p<0.05).
90
91
Figure legends
Figure 1 – Microclimate conditions under the screen where the experiment was
performed. The daily maximum and minimum temperatures (°C) and air relative humidity
(%) for the days of the experiment are represented as lines and bars, respectively.
Figure 2 – Plant leaf areas of eight scion/rootstock combinations before (control –
gray bars) and after drought stress (after stress – black bars). RL: Rangpur lime; SM: Sunki
Maravilha mandarin; VO: Valencia orange; TAL: Tahiti acid lime. Values are averages ±
standard errors (n=6). Different uppercase letters indicate significant differences between
combinations, and different lowercase letters indicate significant differences within the same
combination, according to the Scott-Knott test (p<0.05).
Figure 3 – Pre-dawn leaf water potential (A-D; ΨL; MPa), and leaf osmotic potential
(E-H; Ψπ; MPa) of eight scion/rootstock combinations (RL: Rangpur lime; SM: Sunki
Maravilha mandarin; VO: Valencia orange; TAL: Tahiti acid lime), under three different
water availability conditions: control: ΨL ≤ 0.5 (white bars); severe drought stress: ΨL ≥ 2.0
(black bars); and 48-h rehydration (gray bars). Values are averages ± standard errors (n=3).
Different uppercase letters indicate significant differences between combinations, and
different lowercase letters indicate significant differences within the same combination,
according to the Scott-Knott test (p<0.05).
Figure 4 – Soil matric potential (soil Ψ; MPa) under severe drought stress (ΨL ≥ 2.0
MPa – black bars) for eight scion/rootstock combinations. RL: Rangpur lime; SM: Sunki
Maravilha mandarin; VO: Valencia orange; TAL: Tahiti acid lime. Values are averages (n=3).
Error bars indicate standard errors.
Figure 5 – ABA, IAA and SA concentrations in the leaves (A-D, I-L, and Q-T,
respectively) and roots (E-H, M-P, and U-Z, respectively) of eight different scion/rootstock
combinations under three different water availability conditions: control: ΨL ≤ 0.5 (white
92
bars); severe drought stress: ΨL ≥ 2.0 (black bars); and 48-h rehydration (gray bars). RL:
Rangpur lime; SM: Sunki Maravilha mandarin; VO: Valencia orange; TAL: Tahiti acid lime.
Values are averages ± standard errors (n=5). Different uppercase letters indicate significant
differences between combinations, and different lowercase letters indicate significant
differences within the same combination, according to the Scott-Knott test (p<0.05).
Figure 6 – Strategies of RL and SM rootstocks during drought. The physiological
responses of drought avoidance (RL) and drought tolerance (SM) are described in the lateral
boxes.
Figures
Figure 1
Figure 2
93
Figure 3
Figure 4
Figure 5
94
Figure 6
95
3. CONCLUSÕES GERAIS
· A ploidia influência na emissão de VOCs;
· Plantas tetraploides de limão Volkameriano apresentaram membranas mais resistentes
ao déficit hídrico;
· Porta-enxertos de citros adotam diferentes estratégias de sobrevivência quando
submetidos ao déficit hídrico;
· O porta-enxerto tangerineira Sunki ‘Maravilha’ é capaz de induzir às copas enxertadas
sobre ele, a mesma estratégia de sobrevivência adotada quando em condição não enxertado;
96
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