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UNIVERSIDADE ESTADUAL DE SANTA CRUZ
PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E
BIOLOGIA MOLECULAR
CARACTERIZAÇÃO MOLECULAR E FUNCIONAL DE GENES
DAS FAMÍLIAS MIP (PROTEÍNA INTRÍNSECA PRINCIPAL) E
GolS (GALACTINOL SINTASE) ENVOLVIDOS NA RESPOSTA
A ESTRESSES BIÓTICOS E ABIÓTICOS EM CITROS
CRISTINA DE PAULA SANTOS MARTINS
ILHÉUS – BAHIA – BRASIL
Março de 2016
CRISTINA DE PAULA SANTOS MARTINS
CARACTERIZAÇÃO MOLECULAR E FUNCIONAL DE GENES
DAS FAMÍLIAS MIP (PROTEÍNA INTRÍNSECA PRINCIPAL) E
GolS (GALACTINOL SINTASE) ENVOLVIDOS NA RESPOSTA
A ESTRESSES BIÓTICOS E ABIÓTICOS EM CITROS
Tese apresentada à
Universidade Estadual de Santa Cruz,
como parte das exigências para
obtenção do título de Doutor em
Genética e Biologia Molecular.
Orientador: Prof. Dr. Marcio Gilberto
Cardoso Costa
ILHÉUS – BAHIA – BRASIL
Março de 2016
M379 Martins, Cristina de Paula Santos. Caracterização molecular e funcional de genes das famílias MIP (Proteína Intrínseca Principal) e GoIS (Galactinol Sintase) envolvidos na resposta a estres- ses bióticos e abióticos em citros / Cristina de Paula Santos Martins. – Ilhéus, BA: UESC, 2016. 142 f. : il. Orientador: Marcio Gilberto Cardoso Costa. Tese (Doutorado) – Universidade Estadual de Santa Cruz. Programa de Pós-Graduação em Genética e Biologia Molecular. Inclui referências.
1. Frutas cítricas – Cultivo. 2. Biotecnologia. 3.
Estresse hídrico. 4. Frutas – Melhoramento genético.
I. Título.
CDD 634.3
CRISTINA DE PAULA SANTOS MARTINS
CARACTERIZAÇÃO MOLECULAR E FUNCIONAL DE GENES DAS FAMÍLIAS
MIP (Proteína Intrínseca Principal) E GolS (Galactinol Sintase) ENVOLVIDOS
NA RESPOSTA A ESTRESSES BIÓTICOS E ABIÓTICOS EM CITROS
Tese apresentada à
Universidade Estadual de Santa Cruz,
como parte das exigências para
obtenção do título de Doutor em
Genética e Biologia Molecular.
APROVADA em 04 de março de 2016
_____ _____
Prof. Dr. Luciano Freschi
USP
Prof. Dr. Raúl René Meléndez Valle
CEPLAC
_____
Prof. Dr. Fabienne Florence Luciene Micheli
UESC
Prof. Dr. Fátima Cerqueira Alvim
UESC
__________
Prof. Dr. Alex-Alan Furtado de Almeida
UESC
“Esperar não significa inércia, muito menos desinteresse;
Renunciar não quer dizer que não ame; Abrir mão não
quer dizer que não queira; O tempo ensina, mas não cura.”
Martha Medeiros
Aos meus queridos pais, Maria Lúcia e Valmir.
DEDICO
AGRADECIMENTOS
A Deus, por me proteger e iluminar o meu caminho em todos os dias da minha
vida.
A toda a minha família, em particular aos meus pais (Maria Lúcia e Valmir), por
colocarem sempre os meus interesses em primeiro lugar e por todos os
sacrifícios que fizeram e fazem por mim, o meu profundo agradecimento. Amo
vocês!
Ao meu orientador, Prof. Dr. Marcio Gilberto Cardoso Costa pelo apoio,
paciência, incentivo e disponibilidade, tornando possível a realização deste
trabalho.
Aos funcionários e professores do PPGGBM, pelo apoio.
A Coordenação de Aperfeiçoamento de Pessoal de Nível superior (CAPES),
pela concessão da bolsa de estudo.
Aos colegas da University of Florida pela recepção e acolhimento durante o
período da bolsa sanduíche, em especial ao Dr. Frederick G. Gmitter Jr. pela
orientação.
Pela amizade, risadas e contribuições da família CBG (Monique, Luciana,
Diana, Jamilly, Lívia, Laís, Andressa, Jam, Juliano, Jacque e Carol).
Aos meus preciosos e verdadeiros amigos de Rio Acima e da UFMG, pelo
companheirismo, compreessão e paciência durante todos esses anos de
convívio.
A todos aqueles que por diferentes razões possibilitaram a realização deste
trabalho, muito obrigada!
ÍNDICE
EXTRATO ............................................................................................................... i ABSTRACT ........................................................................................................... iii
2.1 Objetivo geral ............................................................................................ 5 2.2 Objetivos específicos ................................................................................ 5
3 REVISÃO DE LITERATURA .......................................................................... 6 3.1 Aspectos gerais e econômicos da citricultura ........................................... 6 3.2 Respostas moleculares e fisiológicas à deficiência hídrica em citros ....... 8 3.3 Huanglongbing ........................................................................................ 14 3.4 Major Intrinsic Proteins (MIPs) ................................................................ 10 3.5 Galactinol sintase ................................................................................... 12
4. REFERENCIAS ............................................................................................ 17 CAPÍTULO 1 ................................................................................................ 29
ABSTRACT ...................................................................................................... 30 1 INTRODUCTION ........................................................................................... 31 2 MATERIALS AND METHODS ...................................................................... 33
2.1 Plant materials and stress treatments ..................................................... 33 2.2 Identification and classification of CsMIPs .............................................. 34 2.3 Analysis of CsMIP protein properties and conserved amino acid residues ...................................................................................................................... 35 2.4 Analysis of promoter sequences and chromosomal locations of CsMIPs 36 2.5 Expression analysis of CsMIPs ............................................................... 36 3 Results and Discussion.............................................................................. 37 3.1 MIP encoding genes in the sweet orange genome ................................. 37 3.2 CsMIP protein properties and conserved amino acid residues ............... 41 3.3 Genomic organization of CsMIPs ........................................................... 43 3.4 Expression patterns of CsMIP genes in different tissues ........................ 45 3.5 Expression patterns of CsMIP genes under abiotic and biotic stresses .. 47
4 CONCLUSION .............................................................................................. 51 5 ACKNOWLEDGEMENTS ............................................................................. 52 6 REFERENCES .............................................................................................. 52 7 SUPPORTING INFORMATION .................................................................... 59
CAPÍTULO 2 ................................................................................................ 66 ABSTRACT ...................................................................................................... 67 1. INTRODUCTION .......................................................................................... 67 2. MATERIALS AND METHODS ..................................................................... 69
2.1 Plant materials and stress treatments ..................................................... 70 2.2 RNA extraction and quantitative real-time RT-PCR (qPCR) analysis ..... 70 2.3 CsTIP2;1 cloning and generation of transgenic tobacco ......................... 69 2.4 Analysis of H2O2 accumulation ............................................................... 72 2.5 Leaf anatomy .......................................................................................... 72 2.6 H2O2 sensitivity assay of leaves .............................................................. 72 2.7 Physiological and growth analyses ......................................................... 73 2.8 Statistical analysis .................................................................................. 74
3 RESULTS ...................................................................................................... 74
3.1 Overexpression of CsTIP2;1 in transgenic tobacco ................................ 74 3.2 CsTIP2;1-overexpressing tobacco plants show enhanced tolerance to dehydration ................................................................................................... 74 3.3 Dehydration tolerance of CsTIP2;1-overexpressing transgenic lines correlates with the inhibition of H2O2 accumulation ....................................... 75 3.4 CsTIP2;1-overexpressing tobacco plants show improved tolerance to water and salt stresses ................................................................................. 76 3.5 CsTIP2;1 overexpression in tobacco improves their growth, photosynthetic capacity and WUE under drought stress .............................. 78 3.6 CsTIP2;1 overexpression promotes mesophyll cell expansion and aquiferous parenchyma differentiation .......................................................... 82 3.7 CsTIP2;1 expression increases the resistance of transgenic tobacco to exogenous H2O2 application ......................................................................... 84
4 DISCUSSION ................................................................................................ 85 5 CONCLUSIONS ............................................................................................ 88 6 ACKNOWLEDGMENTS ............................................................................... 88 7 REFERENCES .............................................................................................. 88 8 SUPPORTING INFORMATION .................................................................... 96
CAPÍTULO 3 ................................................................................................ 98 1 INTRODUCTION ........................................................................................... 99 2 MATERIALS AND METHODS .................................................................... 101
2.1 Identification and sequences analysis .................................................. 101 2.2 Plant materials and stress treatments ................................................... 102 2.3 RNA extraction and expression analysis of CsGolS in citrus ................ 104 2.4 CsGolS6 cloning and generation of transgenic tobacco ....................... 105 2.5 Leaf anatomy ........................................................................................ 106 2.6 Metabolic profile primary ....................................................................... 106 2.7 Statistical analysis ................................................................................ 107
3 RESULTS AND DISCUSSION .................................................................... 107 3.1 Identification and sequence analysis of the CsGolS ............................. 107 3.2 Expression of CsGolS genes in leaf and root under abiotic and biotic stresses conditions ..................................................................................... 109 3.3 CsGolS6-overexpressing transgenic tobacco shows enhanced tolerance to dehydration and salt stresses ................................................................. 112 3.4 Physiologyand growth analysis of CsGolS6-overexpressing transgenic plants under drought stress ........................................................................ 113 3.5 Mesophyll anatomy change in the CsGolS6-overexpressing transgenic plants under drought stress ........................................................................ 116 3.6 Metabolomic changes in CsGolS6-overexpressing transgenic plants .. 118 4 conclusion ................................................................................................ 120
5 REFERENCE .............................................................................................. 120 6 SUPPORTING INFORMATION .................................................................. 124 APÊNDICE .................................................................................................... 128 1 INTRODUÇÃO ............................................................................................ 129 2 OBJETIVO GERAL ..................................................................................... 132
2.1 Objetivos específicos ............................................................................ 132 3 MATERIAL E MÉTODOS ........................................................................... 132
3.1 Material vegetal e estirpe de Agrobacterium ......................................... 132 3.2 Análise de expressão gênica ................................................................ 132 3.3 Clonagem Gateway .............................................................................. 133
3.4 Ensaios de coinfiltração de folhas de Nicotiana benthamiana e análise de fluorescência ............................................................................................... 134
4 RESULTADOS ............................................................................................ 134 5 CONCLUSÃO ............................................................................................. 138 6 REFERÊNCIAS ........................................................................................... 138
CONCLUSÃO GERAL ............................................................................... 142
i
EXTRATO
MARTINS, Cristina de Paula Santos, Ms., Universidade Estadual de Santa
Cruz, Ilhéus, marçode 2016. Caracterização molecular e funcional de genes
das famílias MIP (Proteína Intrínseca Principal) e GolS (Galactinol Sintase)
envolvidos na resposta a estresses bióticos e abióticos em citros. Orientador:
Dr. Marcio Gilberto Cardoso Costa. Co-orientadores: Dr. Alex-Alan Furtado de
Almeida e Dr. Abelmon da Silva Gesteira
Na agricultura mundial, as frutas cítricas ocupam o primeiro lugar em volume
de produção em fruticultura, sendo a laranja a principal fruta cítrica produzida.
O Brasil se destaca como o maior produtor mundial de laranja e exportador de
suco de laranja concentrado e congelado. A citricultura brasileira encontra-se
sustentada principalmente no porta enxerto limoeiro ‘Cravo’ (Citrus limonia
Osb.), cuja suscetibilidade a doenças vem provocando expressiva diminuição
na produção de frutos, tornando necessário uma diversificação dos porta
enxertos. Os principais porta enxertos alternativos ao limoeiro ‘Cravo’
apresentam algumas limitações, como suscetibilidade à seca. A biotecnologia
tem oferecido novas alternativas ao melhoramento genético de citros,
permitindo a introdução de genes de interesse sem alterar o padrão varietal.
Com a recente disponibilização da sequência do genoma da laranja doce
(Citrus sinensis), tornou-se possível acelerar o processo de identificação e
caracterização de genes candidatos de resistência/tolerância a estresses
bióticos e abióticos para exploração em programas de melhoramento genético
de citros empregando-se estratégias biotecnológicas. O presente trabalho teve
como objetivo caracterizar molecular e funcionalmente genes das famílias MIP
(proteína intrínseca principal) e GolS (galactinol sintase) envolvidos na resposta
a estresses bióticos e abióticos em citros. Para isso, ferramentas de análises
de sequências de DNA e proteínas, bem como de expressão gênica e função
gênica em sistemas-modelo foram empregadas, explorando-se as informações
do genoma de referência de C. sinensis disponíveis na base de dados do
Phytozome. Um total de 34 genes que codificam MIPs de C. sinensis (CsMIPs)
ii
foram identificados e divididos em cinco subfamílias (CsPIPs, CsTIPs, CsNIPs,
CsSIPs e CsXIPs), que se encontram distribuídas em todos os cromossomos
de laranja doce. A expressão gênica de CsMIPs foi analisada em diferentes
tecidos e sob estresses por deficiência hídrica e salino, bem como com
infecção por 'Candidatus Liberibacter asiaticus', agente causador do HLB
(huanglongbing). Um papel especial na regulação do fluxo de água e nutrientes
é proposto para CsTIPs e CsXIPs durante estresse por deficiência hídrica, e
para a maioria de CsMIPs durante estresse salino e o desenvolvimento da
doença HLB. Plantas transgênicas de tabaco superexpressando CsTIP2;1
apresentaram maior crescimento e adaptação a estresses por deficiência
hídrica e salino devido ao maior conteúdo de água nas células, resultando na
maior dissolução do sal e auxiliando na expansão celular. Outros fatores que
contribuíram para amaior tolerância das plantas transgênicas foram a
atenuação dos danos oxidativos, pela detoxificação do H2O2, e indução da
abertura estomática, que contribuiu para a maior capacidade fotossintética,
taxa de transpiração e eficiência do uso da águasob condições de deficiência
hídrica. No genoma de laranja doce foram identificados oito genes da família
CsGolS. Análises de expressão gênica em plantas submetidas a estresses por
deficiência hídrica e salino, bem como com infecção por 'Candidatus
Liberibacter asiaticus', demonstraram que CsGolS foram diferencialmente
regulados pelos diferentes estresses, com maiores níveis de expressão
observado em folhas e raiz. Um papel fisiológico na tolerância a estresses por
deficiência hídrica e salino é proposto para CsGolS6, e para CsGolS5 na
tolerância ao HLB. CsGolS6 foi superexpresso em plantas de tabaco e as
linhagens transgênicas apresentaram maior capacidade fotossintética, porém
mantendo a taxa de transpiração e condutância estomática similares àquelas
da linhagem selvagem. Diante desses resultados, pode-se concluir que os
genes das famílias MIP e GolS desempenham papéis importantes na resposta
e tolerância de plantas cítricas a estresses bióticos e abióticos, representando
novos alvos para o melhoramento genético de citros.
Palavras–chave: Biotecnologia, citricultura, estresse hídrico, estresse osmótico
e HLB
iii
ABSTRACT
MARTINS, Cristina de Paula Santos, Ms., University of Santa Cruz, Ilheus,
February 2016.Molecular and functional characterization of MIP (Major Intrinsic
Protein) and GolS (Galactinol Synthase) gene families involved in the response
to biotic and abiotic stresses in citrus. Advisor: Dr. Marcio Cardoso Gilberto
Costa. Co-advisors: Dr. Alex-Alan Furtado de Almeida and Dr. Abelmon da
Silva Gesteira
In the world agriculture, citrus fruits are top ranked in volume of production
among all the fruit crops. Brazil stands out as the world's largest orange
producer and exporter of frozen concentrated orange juice. The Brazilian citrus
industry is supported mainly in the rootstock Rangpur lime (Citrus limonia Osb.),
whose susceptibility to diseases has caused a significant decrease in the fruit
production. The major alternative rootstocks to Rangpur lime have some
limitations, such as susceptibility to drought stress. Biotechnology has offered
new alternatives to the genetic improvement of citrus, allowing the introduction
of genes of interest without changing the varietal background.With the recent
availability of the genome sequence of sweet orange (Citrus sinensis), it
became possible to accelerate the process of identification and characterization
of candidate genes for resistance/tolerance to biotic and abiotic stresses for
exploitation in citrus breeding programs using biotechnological strategies.The
objective of this study was to characterize molecular and functionally genes of
MIP (major intrinsic protein) and GolS (galactinol synthase) families involved in
the response to biotic and abiotic stresses in citrus. For this, DNA and protein
sequences analyses, as well as analyses of gene expression and gene function
in model systems, have been employed by exploiting the information of the
reference genome of C. sinensis available in the Phytozome database. A total
of 34 genes encoding C. sinensis MIPs (CsMIPs) were identified and divided
into five subfamilies (CsPIPs, CsTIPs, CsNIPs,CsSIPs and CsXIPs), which are
distributed over all the chromosomes of sweet orange. The CsMIPs gene
expression was analyzed in different tissues and under water deficit and salt
iv
stresses, as well as to infection with 'Candidatus Liberibacter asiaticus', the
causalagent of the HLB (huanglongbing) disease. A special role in regulating
the water and nutrient flow is proposed to CsTIPs and CsXIPs during water
deficit, and for most CsMIPs during salt stress and the development of HLB
disease. Transgenic tobacco plants overexpressing CsTIP2;1 showed a higher
growth and a better adaptation to stresses by water deficit and salinity due to
the higher water content in their cells, resulting in increased salt dissolution and
contributing to the cell expansion. Other factors contributing to the increased
tolerance of the transgenic plants were the attenuation of oxidative damage by
detoxifying the H2O2, and stomatal opening, which contributed to the higher
photosynthetic capacity, transpiration rate and water use efficiency under water
stress conditions. Eight genes belonging to the CsGolS family were identified in
the sweet orange genome. Analysis of gene expression in plants subjected to
water deficit and salt stresses, as well as to infection with 'Ca. Liberibacter
asiaticus', showed that these genes were differentially regulated by the different
stresses, with the higher expression levels observed in leaves and roots. A
physiological role in the stress tolerance towater deficit and salinity is proposed
for CsGolS6, and for CsGolS5 in the tolerance to HLB. CsGolS6 was
overexpressed in tobacco plants and the transgenic lines showed a higher
photosynthetic capacity while maintaining the transpiration rate and stomatal
conductance similar to those of the wild-type. Thus, it was possible to observe
that this gene plays an important role in the response and tolerance of plants to
abiotic stresses. From these results, it can be concluded that the genes of the
MIP and GolS families play important roles in the response and tolerance of
citrus plants to biotic and abiotic stresses, representing new targets for genetic
improvement of citrus.
Keywords: Biotechnology, citriculture, water stress, osmotic stress and HLB
1
1 INTRODUÇÃO GERAL
Os citros pertencem à família Rutaceae e os principais gêneros
comerciaissão Fortunella, Poncirus e Citrus. O gênero Citrus é o de maior
relevância comercial, sendo que as principais espécies cultivadas sãoa laranja
doce (Citrus sinensis L. Osb.), tangerinas (C. reticulata, C. deliciosae C.
clementina), limão (C. limon), limas ácidas (C. latifolia e C. aurantifolia) e
pomelo (C. paradisi Macf.) (SIMÃO, 1998; PIO et al.,2005). A cultura dos citros
desempenha um papel de acentuada importância sócio-econômica onde, neste
contexto, o Brasil se destaca comoo maior produtor mundial de laranja, com
produção de cerca de 30% da produção mundial (MAPA, 2015). O Brasil
também ocupa uma posição de destaque no cenário internacional como o
maior exportador de suco de laranja concentrado congelado, com produção de
810,7 mil toneladas, representando 60% da produção mundial de suco de
laranja (MAPA,2015). A agroindústria citrícola brasileira está concentrada no
Estado de São Paulo, sendo responsável por 68% da produção nacional de
laranja e pelo processamento de praticamente toda a safra nacional de laranja
(IBGE, 2015). O Nordeste brasileiro detém, após o Estado de São Paulo, a
citricultura de maior expressão, graças à liderança nesse setor dos Estados da
Bahia e Sergipe (IBGE, 2015).
A principal forma de propagação do citros é por enxertia, sendo que os
porta enxertos influenciam em mais de 20 características hortícolas e
patológicas dos citros, destacando-se: a absorção, síntese e utilização de
nutrientes; transpiração e composição química das folhas; resposta aos
produtos de abscisão de folhas e de frutos; porte, precocidade de produção e
longevidade das plantas; maturação, peso e permanência de frutos na planta;
coloração da casca e do suco; teores de açúcares, ácidos e de outros
componentes do suco; tolerância aos insetos-praga, doenças e fatores
abióticos, como frio, salinidade e seca; conservação pós-colheita; produtividade
e qualidade da frutas (POMPEU JUNIOR, 1991; 2005).
Possível híbrido natural entre o limoeiro verdadeiro [C. limon (L.) Burm.
f.] e a tangerineira (C. reticulata sensu Swingle), o limoeiro ‘Cravo’(Citrus
2
limonia Osbeck) possui diversas características que o qualificam como porta
enxerto de preferência na citricultura brasileira por apresentar: tolerância à
seca e a tristeza dos citros, facilidade de obtenção de sementes,
compatibilidade adequada com a maioria das variedades copas, indução de
crescimento, produção precoce e alta produtividade às copas nele enxertadas
(POMPEU JUNIOR, 2005). Porém a suscetibilidade de ‘Cravo’ à Morte Súbita
dos Citros (MSC) se tornou um sério problema na atualidade, tendo como
consequência a expressiva diminuição na produção de frutos (CANTAGALLO
et al., 2005). A diversificação de portaenxertos na citricultura é fundamental,
com ênfase na busca de variedades melhores adaptadas às condições de
déficit hídrico, já que a citricultura brasileira é conduzida praticamente sem
irrigação (SANTOS et al., 1999). O desenvolvimento de variedades tolerantes
ao déficit hídrico associado à utilização racional da água é indicado para a
manutenção da produção agrícola em situações de escassez hídrica (CHAVES
et al., 2003).
O desenvolvimento de novas variedades portaenxerto capazes de
substituir o ‘Cravo’ é uma tarefa difícil, devidoaos problemas associados a
biologia reprodutiva dos citros que limitam a execução de programas
convencionais de melhoramento genético desse grupo de plantas. Por outro
lado, a biotecnologia, por meio das técnicas de cultura de tecidos e
transformação genética, tem oferecido novas alternativas para o melhoramento
dos citros, permitindo a introdução de genes de interesse sem alterar o padrão
varietal. Em citros a transformação genética já é um processo bem
estabelecido (COSTA et al., 2007).No entanto, a principal limitação para o uso
dessa ferramenta no melhoramento de citros é a escassez de genes
identificados e caracterizados governando caracteres de importância
agronômica em plantas cítricas. Com a recente disponibilização da sequência
do genoma da laranja doce (WU et al., 2014), esse processo passou a ser
facilitado consideravelmente.
MIPs constituem uma superfamília de proteínas de membrana, as quais
têm sido caracterizadas por atuarem como componentes centrais das relações
hídricas em plantas. MIPs que transportam especificamente água são
chamadas de aquaporinas (JOHANSON etal., 2001). A descoberta de
aquaporinas trouxe uma nova visão do mecanismo de transporte de água
3
transmembrana, fornecendo a base molecular para uma rápida e sólida
regulação reversível de transporte de água em determinados processos
fisiológicos (MAUREL, 1997; MAUREL e CHRISPEELS, 2001). A abundância e
a atividade dessas proteínas na membrana plasmática e tonoplasto podem ser
reguladas, permitindo que a planta controle o fluxo de água dentro e fora das
células (MAUREL, 2007; KALDENHOFF; FISCHER, 2006; JOHANSSON et al.,
2000; JAVOT; MAUREL, 2002). Estudos têm demonstrado que a expressão de
genes codificando paraaquaporinas é induzida em resposta a
estressesabióticos, como seca e salinidade, melhorando a eficiencia das
plantas com relação à utilização da água (LU; NEUMANN, 1999; SIEFRITZ et
al., 2002; JANG et al., 2004; ALEXANDERSSON et al., 2010).
Membros da família GolS codificam para a enzima-chave na biossíntese
de oligossacarídeos de rafinose, catalizando a formação de galactinol e UDP a
partir de UDP-gal e mio-inositol. GolS possui papel chave como regulador na
distribuição de carbono entre sacarose e oligossacarídeos de rafinose
(HANDLEY et al., 1983; SARAVITZ et al., 1987; LOWELL; KUO, 1989).
Oligossacarídeos de rafinose são sintetizados a partir da sacarose, por meioda
subsequente adição de ativadores de Gal doado por galactinol (PETERBAUER;
RICHTER, 2001). A expressão de enzimas relacionadas com a biossíntese de
galactinol e oligossacarídeos de rafinose e sua acumulação intracelular em
células vegetais estão intimamente associados com as respostas de estresse
ambiente, contribuindo para a distribuição de solutos compatíveis para
proteção das células (TAJI et al., 2002; DOWNIE et al., 2003; ZUTHER et al.,
2004; KAPLAN et al., 2004; PANIKULANGARA et al., 2004; PETERS et al.,
2007).
O presente estudo partiu da hipótese de que genes das famílias MIP e
GolS desempenham importantes funções na tolerância de plantas cítricas a
estresses bióticos e abióticos. No Capítulo 1 é apresentado um estudo sobre a
identificação da série completa de genes MIPs presentes no genoma de laranja
doce, bem como a caracterização quanto às suas sequências, relações
filogenéticas, organização genômicae expressão gênica em diferentes tecidos
e em resposta a diferentes estresses bióticos e abióticos. O Capítulo 2 trata da
caracterização funcional de um gene da família MIP, CsTIP2;1, por meio da
sua superexpressão em plantas de tabaco e análise das plantas transgênicas.
4
O Capítulo 3 apresenta um estudo sobre a identificação e caracterização de
genes da família GolS presentes no genoma de laranja doce, bem como a
caracterização funcional de um de seus membros, CsGolS6, por meio da
superexpressão e análise de plantas transgênicas de tabaco. Coletivamente,
os resultados obtidos no presente estudo permitiram ampliar a base de
conhecimento potencialmente aplicável ao melhoramento, bem como facilitar o
desenvolvimento de novas variedades porta enxerto tolerantes a estresses
bióticos e abióticos e potencialmente úteis para a citricultura brasileira.
5
2 OBJETIVOS
2.1 Objetivo geral
Caracterizar molecular e funcionalmente genes das famílias MIP e GolS
envolvidos na resposta a estresses bióticos e abióticos em citros.
2.2 Objetivos específicos
1) Identificar genes das famílias MIP e GolS presentes no genoma de
referência de C. sinensis;
2) Analisar as relações filogenéticas dos membros de cada família com
homólogos de outras espécies vegetais;
3) Caracterizar as sequências de DNA e proteínas dos membros de cada
família;
4) Caracterizar a expressão gênica dos membros de cada família em
plantas submetidas a estresses bióticos e abióticos;
5) Caracterizar a função dos genes candidatos de tolerância a estresses
por meio da expressão transgênica em sistemas-modelo.
6
3 REVISÃO DE LITERATURA
3.1 Aspectos gerais e econômicos da citricultura
Os citros são originários de regiões com clima tropical e subtropical do
sul e sudeste Asiático (MOORE, 2001; NICOLOSI, 2007). A classificação das
espécies de Citrus é complexa, sendo variável principalmente pelo número de
espécies. Os sistemas taxonômicos que mais se destacam são os de Swingle e
Reece (1967) e o de Tanaka (1977). O gênero Citrus pertence à ordem
Geraniales, família Rutaceae, subfamília Aurantioideae, tribo Citreae, subtribo
Citrineae, sendo composto por seis gêneros Poncirus, Fortunella e Microcitrus,
Clymenia, Eremocitrus e Citrus (SWINGLE, REECE, 1967; TANAKA, 1977;
NICOLOSI, 2007), que são coletivamente chamadas de citros.
Horticulturamente, os citros podem ser agrupados em laranjeiras doce
[Citrus sinensis (L.) Osbeck], tangerineiras (Citrus reticulata Blanco), limoeiros
[Citrus limon (L.) Buen. F.], limeiras ácidas [Citrus aurantifolia (Christm.)
Swingle e Citrus latifolia (Yu. Tanaka)], pomeleiros (Citrus paradisi Macf.), entre
outras (SIMÃO, 1998; PIOet al.,2005). Algumas espécies e seus híbridos são
utilizadas principalmente como porta-enxertos, como o limoeiro ‘Cravo’ (C.
limonia Osbeck), limoeiro ‘Volkameriano’ (C. volkameriana V. Ten. & Pasq.),
tangerineira ‘Sunki’ (C. sunki hort. ex Tanaka), tangerineira ‘Cleopatra’ (C.
reshni hort. ex Tanaka), laranjeira azeda (C. aurantium L.) e citrumeleiro
‘Swingle’ [C. paradisi Macf. cv. Duncan x P. trifoliata (L.) Raf.] (POMPEU
JUNIOR, 2005).
As plantas cítricas podem ser multiplicadas por sementes (via sexual),
por alporquia, estaquia e enxertia (via assexual), sendo este último método
mais utilizado comercialmente por apresentar algumas vantagens, entre as
quais se destacam a uniformidade das mudas, precocidade no início de
produção e aumento na produtividade, além de se obter mudas idênticas à
planta-mãe e maior resistência ou tolerância a condições desfavoráveis de
clima, solo, pragas e doenças de acordo com o porta enxerto ou combinação
utilizada (POMPEU JUNIOR, 2005).
7
As características apresentadas pelo limoeiro ‘Cravo’ o tornaram o
preferido pelos citricultores, de modo que a partir da década de 60 passou a
ser praticamente o único portaenxerto da citricultura paulista (POMPEU
JÚNIOR, 2005). O limoeiro ‘Cravo’ é considerado um híbrido natural entre
limoeiro e tangerineira, originado na região de Canton, no sul da China.
Apresenta média resistência às gomoses de P. parasitica e P. citrophthora, é
suscetível a nematóides (Tylenchulu semipenetrans e Pratylenchus jaehni),
tolerante ao vírus da tristeza dos citros, suscetível ao declínio dos citros e
tolerante à seca. Apresenta melhor desempenho quando plantado em solos
arenosos e profundos e pode apresentar produtividade inferior à das
tangerineiras ‘Cleópatra’ e ‘Sunki’ quando plantado em solos argilosos. Em
2014, os porta enxertos mais empregados na citricultura paulista foram o
limoeiro ‘Cravo’(53%), o citrumeleiro ‘Swingle’ (32%), a tangerineira ‘Sunki’
(11%), a tangerineira ‘Cleópatra’ (1,6%), o Poncirus trifoliata (1,4%) e o limoeiro
‘Volkameriano’ (0,7%) (IAC, 2014).
Cultivado em mais de 130 países, os citros constituem uma das mais
importantes fruteiras cultivadas no mundo, com produção de aproximadamente
131 milhões de toneladas por ano. China, Brasil e Estados Unidos são os
principais países produtores de citros, respondendo por mais de 50% da
produção mundial (FAO, 2014). A citricultura brasileira ocupa uma posição de
destaque no cenário internacional, sendo o maior produtor de laranja
(aproximadamente 30% da produção mundial) e o maior exportador de suco de
laranja concentrado e congelado, onde representa 60% da produção mundial
de suco de laranja (MAPA, 2015). Levantamentos destacam que o Estado de
São Paulo é o líder na produção de laranja em território nacional, participando
com aproximadamente 68% da produção e pelo processamento de
praticamente toda a safra nacional de laranja. Minas Gerais é o segundo maior
produtor de laranja doce seguidos pelo estado de Bahia e Sergipe, que
praticamente se igualam na produção de citros (IBGE, 2015).
As mudanças com relação à migração do local de produção de laranja
estão relacionadas principalmente com os problemas fitossanitários que vêm
atingindo os pomares. Esse fato ocorre em razão da base genética dos
pomares estarem concentradas em copas de laranja ‘Pera’ e no uso de porta
enxerto limão ‘Cravo’ (FERREIRA et al., 2013). Dentre as doenças que causam
8
expressivos danos na produção pode-se destacar a gomose de Phytophthora
(Phytophthora spp.), pinta preta (Guignardia citricarpa Kiely), tristeza dos citros
(Citrus tristeza virus), cancro cítrico (Xanthomonas citri pv. citri), a morte súbita
dos citros (MSC) e o huanglongbing (HLB), que está associado a bactéria
Candidatus Liberibacter spp. (MATTOS-JUNIOR, 2005). Há necessidade de
diversificação das variedades de porta-enxerto, porém essas devem apresentar
características de tolerância à seca como o limoeiro ‘Cravo’ e a doenças
agressivas como o HLB.
3.2 Respostas moleculares e fisiológicas à deficiência hídrica em citros
As plantas que são tolerantes à seca apresentam uma série de
mecanismos para que, durante o estresse, as respostas sejam rapidamente
ativadas. Cada planta possui uma percepção do sinal, assim como a regulação
molecular e fisiológica, que se reflete nas diferenças de tolerância à seca inter
e intra-específica (BARTELS; SUNKAR, 2005). Secas tendem a promover
alterações no crescimento e desenvolvimento das plantas de citros
promovendo a queda das folhas e aumentando a massa do sistema radicular
para promover a aquisição de água nos solos mais profundos (RODRIGUEZ-
GAMIR et al., 2010). Diferenças entre indivíduos da mesma espécie foram
relatadas para Citrus (GARCÍA-SÁNCHEZ et al., 2007) em relação às variáveis
fisiológicas, como eficiência do uso da água (KNIGHT et al., 2006;
KLAMKOWSKI;TREDER, 2008) e ao estágio de desenvolvimento e à atividade
de diversas enzimas (HONG et al., 2005). O balanço hídrico dos portaenxertos
limão Volkameriana (Citrus volkameriana P.), limão Cravo (Citrus limonia O.),
limão Rugoso (Citrus jambhiri L.) e tangerina Cleópatra (Citrus reshni Hort. Ex
Tan.) foi analisado em condições de campo, sendo a laranja Pêra (Citrus
sinensis L.) utilizada como copa. De acordo com os resultados, o porta enxerto
menos adaptados ao estresse hídrico foi à tangerina Cleópatra e o limão Cravo
foi o que melhor se adaptou por apresentar menor taxa de evapotranspiração
(CINTRA et al., 2000). O limão 'Cravo' se destaca como variedade tolerante à
seca, enquanto tangerina 'Sunki Maravilha' mostra maior suscetibilidade ao
estresse hídrico em comparação a primeira, confirmada pela desidratação foliar
acentuada apresentada por esta tangerina durante o estresse hídrico e análise
9
de parâmetros fisiológicos, como razão de área foliar, potencial de água foliar,
resistência estomática, e análise molecular como níveis de ABA e perfil de
expressão gênica da família NCED (NEVES et al., 2013). Outros porta enxertos
de plantas cítricas, como tangerineira Cleópatra e Poncirus trifoliata, também
respondem à seca por meio do envio de sinais para as folhas, induzindo o
fechamento dos estômatos, reduzindo assim a perda de água por transpiração
(RODRIGUEZ-GAMIR et al., 2010).
As plantas necessitam de gás carbônico (CO2) para realizar
fotossíntese, porém muitas vezes elas têm que se adaptar entre a perda de
água (H2O) e a absorção de CO2, que é modulado pela regulação estomática.
Este mecanismo depende do vapor de água, do CO2 e das variações da
pressão de turgescência das células guarda. Em situação de seca, a regulação
da abertura estomática é controlada pelo ácido abcísico (ABA), o principal
hormônio envolvido no fechamento estomático, assim como pelas citocininas,
para a abertura dos estômatos (TAIZ; ZEIGER, 2003). É comum o fechamento
dos estômatos em resposta à seca. Assim, com os estômatos fechados, o CO2
deixa de entrar na folha, o que leva a uma limitação do substrato disponível
para a enzima RUBISCO, reduzindo a taxa fotossintética e a assimilação de
CO2(TAIZ; ZEIGER, 2003). A entrada de CO2 nas plantas é regulada em
função da disponibilidade deste gás, assim como pela abertura dos estômatos,
como demonstram as diferenças na resposta da fotossíntese à concentração
interna (Ci) de CO2 (TALLMAN, 2004).
Pesquisas sobre a genética das respostas à deficiência hídrica em
plantas cítricas têm sidodesenvolvidas por alguns autores. Citros em condição
de deficit hídrico, ocorreu aumento da regulação específica de NCED3 em
tecidos fotossinteticamente ativos, como folhas e ovários em desenvolvimento
(AUGUSTI et al., 2007). A resposta do transcriptoma da tangerina 'Clementina'
(C. clementina Ex Tanaka) enxertada em tangerina 'Cleópatra' (C. hort reshni
Ex Tanaka) foi analisada em condições de déficit hídrico (GIMENO et al.,
2009). Genes que codificam para proteínas envolvidas na síntese de lisina,
prolina e catabolismo da rafinose, redução de peróxido de hidrogênio,
transporte de malato vacuolar e defesa (incluindo osmótica, desidratação e
chaperones) foram induzidos sob a condição de estresse (GIMENO et al.,
2009). Análise da família NAC de fatores de transcrição resultou na
10
identificação de um membro, CsNAC1, que foi induzido por déficit hídrico nas
folhas de tangerina 'Cleópatra' e limão Cravo e pelo estresse salino, frio e ácido
abscísico (ABA) em folhas e raízes de tangerina 'Cleópatra' (OLIVEIRA et al.,
2011). A expressão dos genes PIP1 e PIP2, foi investigada em raízes de
Poncirus trifoliata (L.) Raf. e tangerina ‘Cleópatra’ (C. reshni Hort ex Tan.)
submetidos ao déficit hídrico moderado (RODRÍGUEZ-GAMIR et al., 2011), e
em raízes de P. trifoliata, tangerina ‘Cleópatra’ e citrange ‘Carrizo’[C. sinensis
(L.) Osb. × P. trifoliata (L.) Raf.], submetido a tratamento de estresse salino
(RODRÍGUEZ-GAMIR et al., 2012). A diferença de expressão de PIP1 e PIP2
foi correlacionada com alterações na condutividade hidráulica da raiz e a
exclusão de cloreto de sódio das folhas e, consequentemente, com a tolerância
à estresses hídrico e salino, respectivamente. Da mesma forma, a
condutividade hidráulica da raiz e transpiração em citros foram reduzidas em
condição de estresse salino (RODRIGUEZ-GAMIR et al., 2012).
3.3 Proteínas Intrínsecas Principais (MIPs)
As MIPs constituem uma superfamília de proteínas transmembranas,
com peso molecular entre 23-31 kDa, que se encontram presentes em todos os
organismos. As MIPs, também conhecidas como aquaporinas, possuem uma
estrutura tetramérica, composta por quatro monômeros, cada um com um poro
ativo (MAUREL et al., 2008).
Esta família com membros em animais, plantas e micróbios, é composta
por proteínas integrais de membrana que facilitam a transferência bidirecional
de água e pequenos solutos em membranas biológicas (JOHANSON et al.,
2001). A função das aquaporinas, como reguladores do transporte de água, foi
descoberta através da expressão de uma aquaporina humana em oócitos de
Xenopus, em que se obteve um aumento da permeabilidade à água
(TYERMAN et al., 2002). A primeira aquaporina descoberta em plantas foi da
subfamilia TIP (tonoplast intrinsic protein), presente no tonoplasto, o que
potencializou a pesquisa de mais genes desta família (TYERMAN et al., 2002).
Inicialmente, as aquaporinas foram identificadas como canais de
transporte seletivo de água (MAUREL et al., 1993). Porém, novos estudos
evidenciaram que esses canais também podem transportar glicerol (GERBEAU
11
et al., 1999.; LI et al., 2008), uréia (KLEBL et al., 2003; GERBEAU et al, 1999;
DYNOWSKI et al, 2008), peróxido de hidrogênio (H2O2) (BIENERT et al., 2007;
DYNOWSKI et al., 2008) e compostos gasosos como a amônia (NH3) (JAHN et
al., 2004; LOQUE et al., 2005; DYNOWSKI et al., 2008) e dióxido de carbono
(CO2) (WU; BEITZ, 2007).
As aquaporinas possuem um papel importante em situações de
estresses abióticos, como seca e salinidade. Vários estudos demonstraram
que, em casos de estresse hídrico, a transcrição de genes das aquaporinas é
alterada, sendo que o padrão de expressão destes genes é coincidente com
um aumento da tolerância das plantas (MARTRE et al., 2002; MAUREL et al.,
2008). Em mudas de arroz submetidas ao estresse hídrico, ocorreu o aumento
no funcionamento fisiológico dos canais de água pelo aumento da atividade de
água do canal da raiz ou pelo aumento do número de aquaporinas em
comparação com mudas de plantascontrole não submetidas ao estresse (LU;
NEUMANN, 1999). A diminuição da expressão do gene aquaporinI, que está
presente na membrana plasmática em plantas de Arabidopsis thaliana,reduziu
a condutividade hidráulica dessa planta, levando ao decréscimo na tolerância à
seca (SIEFRITZ et al., 2002).
A regulação transcricional das MIPs é realizada de diferentes formas.
Podem ser induzidas por diferentes hormônios, como ABA e giberelinas, por
diferentes fatores de estresse abiótico e por modificações pós-traducionais,
através de fosforilação (MAUREL et al., 2002). A fosforilação das aquaporinas
leva à abertura do poro, em oposição ao mecanismo de fechamento, que passa
pela percepção de protóns (MAUREL et al., 2008).
Em seres humanos (Homo sapiens) foram descritos 13 MIPs (MAGNI et
al., 2006), enquanto que em plantaso número total de MIPs é particularmente
elevado, em comparação com os animais, com 35 membros identificados no
genoma de Arabidopsis (JOHANSON et al., 2001), 36 em milho (Zea mays)
(CHAUMONT et al., 2001), 33 em arroz (Oryza sativa) (SAKURAI et al., 2005),
28 em videira (Vitis vinifera) (FOUQUET et al., 2008), 56 em Populus
trichocarpa (GUPTA; SANKARARAMAKRISHNAN, 2009).Por homologia, os
genes de Arabidopsis thaliana foram agrupados em quatro classes ou
subfamílias diferentes. O maior subgrupo é formado pelas proteínas intrínsecas
da membrana plasmática (PIPs), contendo treze genes, seguido pelas
12
proteínas intrísecas do tonoplasto (TIPs), que possuem dez genes homólogos.
Proteínas intrínsecas do tipo Nodulin-26 (NIPs) foram inicialmente
caracterizadas como exclusivamente expressas em membranas
peribacteróides fixadoras de nitrogênio em raizes simbióticas, em nódulos de
leguminosas, e em Arabidopsis thaliana, em que nove membros estão
presentes. A quarta categoria inclui as proteínas intrínsecas pequenas (SIPs)
(LUU; MAUREL, 2005). Pesquisas atuais mostram a existência de outros
membros das aquaporinas (GUSTAVSSON et al., 2005; DANIELSON;
JOHANSON, 2008), porém os quatro grupos descritos acima ainda são
considerados os principais.
As aquaporinas possuem diversas funções de transporte em vários
compartimentos subcelulares. Isoformas das proteínas intrínsecas de
membrana (PIP1 e PIP2) estão relacionadas com a via secretora, que promove
o transporte de partículas a partir do retículo endoplasmático (ER). PIPs
também passam por ciclos repetidos de endocitose e reciclagem, através de
compartimentos endossomais, antes de eventualmente voltarem para o
vacúolo lítico através dos corpos multivesiculares (ROBINSON et al., 1996). As
proteínas intrínsecas do tonoplasto TIP1s (GAMA-TIPs) são encontradas na
membrana do vacúolo lítico, já TIP2s (DELTA-TIPs) e TIP3s (BETA-TIPs) estão
preferencialmente associadas a vacúolos que acumulam proteínas de reserva
em células vegetativas e em sementes, respectivamente. As proteínas
intrínsecas do tipo Nodulin-26 (NIP) mostram um amplo espectro no padrão de
localização subcelular. AtNIP2;1 está localizada no RE e na membrana
plasmática (CHOI; ROBERTS, 2007; MIZUTANI, et al., 2006). Em Oryza sativa,
OsNIP2;1 (transportador de silício) e em Arabidopsis thaliana, AtNIP5;1
(transportador de ácido bórico), estão localizadas na membrana plasmática,
enquanto que em soja (Glycine max), GmNOD26 é exclusivamente expressa
na membrana peribacteroide (MAUREL et al., 2008).
3.4 Galactinol sintase
A família dos oligossacarídeos da rafinose (RFO) possui múltiplas
funções em plantas e é encontrada nas folhas de várias famílias de plantas
superiores (LEE et al., 1970; KUO et al., 1988; CASTILLO et al.,1990). Os
13
oligossacarídeos da família dos RFOs estão relacionados com a proteção
contra estresses abióticos (BACHMANN et al., 1994; HARITATOS et al.,1996).
A biossíntese de RFOs inicia-sea partir da sacarose através da adição de
unidades de galactose doados por galactinol (O-α-D-galactopiranosil- (1 → 1) -
L-mio-inositol). Galactinol sintase (GolS, CE 2.4.1.123) pertence a uma família
de glicosiltransferases envolvidas na primeira etapa da biossíntese RFOs. GolS
catalisa a transferência de UDP-D-galactose para mio-inositol (SCHNEIDER;
KELLER, 2009) e é considerado o principal regulador da via biossintética
(PETERBAUER; RICHTER, 2001). Outros oligossacarídeos desta via
(estaquiose, verbascose e ajugose) são formados sequencialmente pela ação
de galactosil-transferases específicas usando galactinol como o doador de
galactosil. Vários estudos têm demonstrado que a expressão dos genes
relacionados com a biossíntese e acumulação intracelular de galactinol e
rafinose está associadaa respostas de estresse ambientais (TAJI et al., 2002;
PANIKULANGARA et al., 2004; PETERS et al., 2007).
Dez genes GolS foram identificados no genoma de A. thaliana
(NISHIZAWA et al., 2008), três dos quais (AtGolS1, AtGolS2 eAtGolS3) foram
induzidos por estresses abióticos (TAJI et al., 2002). Os genes AtGolS1 e 2
foram induzidos em condições de estresse hídrico e salino, enquanto que o
AtGolS3 foi induzido em baixas temperaturas. As plantas de A. thaliana
submetidas às condições de estresse acumularam grande quantidade de
galactinol e rafinose, sendo que nas plantas não estressadas esses açúcares
não foram detectados. Isto sugere que rafinose e galactinol estão envolvidos na
tolerância a estresses abióticos, atuando como osmoprotetores (TAJI et
al.,2002). Foi observada relação entre a expressão de AtGolS1 nas folhas de
A. thaliana e a síntese de RFOs em plantas submetidas aseca, sugerindoum
papel importante deste gene na expressão do estresse induzido pelo calor
(PANIKULANGARA et al., 2004).
Plantas de tabaco superexpressando constitutivamente o gene CsGolS
de Corynespora cassiicola aumentaram a quantidade de galactinol em raízes e
apresentaram resistência contra os patógenos Botrytis cinerea e Erwinia
carotovora. Tanto as plantas de tabaco transgênicas como o controle, tratadas
com galactinol em aplicação exógena, também demonstraram um aumentona
tolerância a seca e ao estresse salino (KIM et al., 2008). Transcritos de
14
BhGolS1 da planta Boea hygrometrica foram induzidos por ABA e a
superexpressão desse gene em plantas de tabaco aumentou a tolerância à
desidratação (WANG et al., 2009). A regulação diferencial de três isoformas de
GolS em Coffea arabica (CaGolS1, CaGolS2, CaGolS3) sob déficit hídrico, alto
teor salino e condições de choque térmico, ressaltaram a formação de rafinose
e estaquiose durante esses estresses (dos SANTOS et al., 2011). Entre estes
genes, CaGolS1 foi expressa em plantas sob condições normais de
crescimento e também foi a mais responsiva a estresse quando as plantas
foram submetidas à deficiência hídrica.
3.5 Huanglongbing
O huanglongbing (HLB), também conhecido como ‘greening’, é
considerado uma das doenças mais destrutivas de citros, causando a morte de
milhões de plantas cítricas em todo o mundo (BOVE 2006; GOTTWALD et al.
2007; GOTTWALD, 2010). Foi relatada pela primeira vez em 1919, no sul da
China, como a doença do ramo amarelo (“yellow shoot disease”) (REINKING,
1919; CHEN, 1943; LIN, 1956). Na África, em 1937, uma doença com sintomas
similares, porém associada a uma espécie diferente de Liberibacter, foi descrita
como “greening” (VAN DE MERWE; ANDERSON, 1937). Atualmente o HLB
está disseminado nos cinco continentes do mundo (BOVE, 2006). No Brasil a
doença foi relatada pela primeira vez em 2004 na região de Araraquara, Estado
de São Paulo (COLLETA-FILHO et al., 2004; TEIXEIRA et al., 2005).
Atualmente a doença também já foi relatada em outras regiões citrícolas do
estado de São Paulo, Minas Gerais e Paraná. Nos Estados Unidos, a primeira
constatação ocorreu no estado da Flórida, em 2005 (TEIXEIRA et al., 2005). O
HLB foi relatado em Cuba em 2007 (LLAUGER et al., 2008, PANTOJA et al.,
2008, LUIS et al., 2009), na República Dominicana (MATOS et al., 2009) e no
México (NAPPO - www.pestalert.org) em 2008, em Belize, Jamaica e Porto
Rico em 2009 (International Society for Infectious Diseases -
www.promedmail.org).
O HLB está associado a três espécies de bactérias gram-negativas que
são denominadas de Candidatus Liberibacter asiaticus, Candidatus Liberibacter
africanus e Candidatus Liberibacter americanus (GARNIER et al., 1984;
15
JAGOUEIX et al., 1994; TEIXEIRA et al., 2005). O prefixo Candidatus é uma
denominação temporária (MURRAY; SCHLEIFER, 1994), já que a classificação
taxonômica definitiva ainda não foi realizada devido à dificuldade de cultivo do
Liberibacter, mesmo após algumas tentativas (SECHLER et al., 2009). A
identificação e classificação destas espécies foram obtidas com base em
estudos de microscopia eletrônica (GARNIER; DANEL; BOVÉ, 1984) e de
comparações de sequências da região 16S do DNA ribossomal (JAGOUEIX;
BOVÉ; GARNIER, 1994). As bactérias são disseminadas no campo por inseto,
por meio de borbulhas infectadas (LOPES; FRARE, 2008; COLETTA-FILHO et
al., 2010) ou da planta parasita Cuscuta spp. (GARNIER; BOVÉ, 1983). O
psilídeo asiático dos citros, Diaphorina citri Kuwayama (Hemiptera: Liviidae) é
descrito como o vetor de Ca. L. asiaticus e Ca. L. americanus (CAPOOR et
al.,1967; CATLING, 1970; PANDE, 1971; YAMAMOTO et al., 2006), e o
psilídeo africano Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) o vetor
de Ca. L. africanus (McCLEAN et al.,1965). As bactérias associadas a esta
enfermidade mantém uma relação persistente com o inseto vetor (XU et al.,
1988; HUNG et al., 2004).
A identificação de plantas doentes é feita por meio da observação dos
sintomas, que se iniciam como manchas amareladas assimétricas no limbo
foliar que contrastam com a cor verde normal das folhas. Outros sintomas
relacionados à doença são sistema vascular de coloração alaranjada, folhas
com tamanhos reduzidos e cloróticas similar a deficiência de manganês, ferro e
zinco (BOVÉ, 2006). Floradas fora de época podem ocorrer, intensa desfolha
dos ramos afetados, progredindo por toda a planta, causando seca e morte dos
ponteiros (HALBERT; MANJUNATH, 2004). Os frutos de ramos sintomáticos
apresentam-se de tamanho reduzido, assimétricos, com maturação irregular,
escurecidos ou abortadas (BOVE, 2006). O surgimento dos sintomas varia de
acordo com a transmissão da bactéria, sendo que infecção via enxertia o
tempo necessário é em torno de quatro meses (LOPES; FRARE, 2008). Caso a
infecção ocorra via inseto vetor, o tempo é em geral de oito meses (HUNG et
al., 2001). Ainda não há variedades de citros resistentes ao HLB e nem
tratamento fitossanitário capaz de reverter os sintomas da doença (LOPES et
al., 2007). Assim impedir a infecção das plantas é fundamental para o controle
do HLB que atualmente se baseia no plantio de mudas sadias, eliminação de
16
plantas com sintomas, controle químico do inseto vetor e a eliminação das
plantas de murta (Murraya exotica), pois é considerada hospedeira de
Liberibacter (LOPES et al., 2010; GASPAROTO, 2010).
17
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29
CAPÍTULO 1
GENOME-WIDE CHARACTERIZATION AND
EXPRESSION ANALYSIS OF MAJOR INTRINSIC
PROTEINS DURING ABIOTIC AND BIOTIC STRESSES
IN SWEET ORANGE (Citrus sinensis L. Osb.)
Artigo publicado: PLoS ONE
Citation: de Paula Santos Martins C, Pedrosa AM, Du D, Gonçalves LP, Yu Q,
Gmitter FG Jr, et al. (2015) Genome-Wide Characterization and Expression
Analysis of Major Intrinsic Proteins during Abiotic and Biotic Stresses in Sweet
Orange (Citrus sinensis L. Osb.). PLoS ONE 10(9): e0138786.
doi:10.1371/journal.pone.0138786
30
ABSTRACT
The family of aquaporins (AQPs), or major intrinsic proteins (MIPs), includes
integral membrane proteins that function as transmembrane channels for water
and other small molecules of physiological significance. MIPs are classified into
five subfamilies in higher plants, including plasma membrane (PIPs), tonoplast
(TIPs), NOD26-like (NIPs), small basic (SIPs) and unclassified X (XIPs) intrinsic
proteins. This study reports a genome-wide survey of MIP encoding genes in
sweet orange (Citrus sinensis L. Osb.), the most widely cultivated Citrus spp. A
total of 34 different genes encoding C. sinensis MIPs (CsMIPs) were identified
and assigned into five subfamilies (CsPIPs, CsTIPs, CsNIPs, CsSIPs and
CsXIPs) based on sequence analysis and also on their phylogenetic
relationships with clearly classified MIPs of Arabidopsis thaliana. Analysis of key
amino acid residues allowed the assessment of the substrate specificity of each
CsMIP. Gene structure analysis revealed that the CsMIPs possess an exon-
intron organization that is highly conserved within each subfamily. CsMIP loci
were precisely mapped on every sweet orange chromosome, indicating a wide
distribution of the gene family in the sweet orange genome. Investigation of their
expression patterns in different tissues and upon drought and salt stress
treatments, as well as with ‘Candidatus Liberibacter asiaticus’ infection,
revealed a tissue-specific and coordinated regulation of the
different CsMIP isoforms, consistent with the organization of the stress-
responsive cis-acting regulatory elements observed in their promoter regions. A
special role in regulating the flow of water and nutrients is proposed
for CsTIPs and CsXIPs during drought stress, and for most CsMIPs during salt
stress and the development of HLB disease. These results provide a valuable
reference for further exploration of the CsMIPs functions and applications to the
genetic improvement of both abiotic and biotic stress tolerance in citrus.
31
1 INTRODUCTION
Aquaporins (AQPs) are integral membrane proteins that assist the rapid
movement of water as well as other low molecular weight molecules across
cellular membranes [1-3]. AQPs belong to the ancient family of major intrinsic
proteins (MIPs) found in microorganisms, plants and animals. While a small
number of different AQPs have been identified in E. coli (2), S. cerevisiae (9), C.
elegans (11) and mammals (13) [4], a surprisingly large number of MIP
homologues have been found in plants; for example, 35 AQPs were found in
Arabidopsis [5], 36 in Zea mays [6], 33 in Oryza sativa [7], 28 in Vitis vinifera [8],
55 in Populus trichocarpa [9], 71 in Gossypium hirsutum [10], 47 in Solanum
lycopersicum [11] and 66 in Glycine max [12]. These observations highlight a
major role for plant MIPs as key regulators of the intricate flows of water and
solutes required for growth and adaptive responses to the ever-changing
environment.
Plant MIPs were originally categorized into four subfamilies on the basis
of sequence homologies and subcellular localization: plasma membrane (PIP),
tonoplast (TIP), nodulin-like (NIP) and small basic (SIP) intrinsic proteins [2,13].
More recently, studies in the moss Physcomitrella patens revealed the presence
of novel AQP isoforms in addition to the four conserved plant AQP subfamilies:
a homologue of the Escherichia coli intrinsic protein GlpF (GIPs), intrinsic hybrid
proteins (HIPs) and unclassified X intrinsic proteins (XIPs) [9,13-17]. XIP
homologues have also been identified in some higher plants, such as Solanum
lycopersicum, Populus trichocarpa and Glycine max [9,11,12,15]. These
findings suggest that the family of plant MIPs is larger and much more complex
than previously anticipated and, hence, may play critical roles in a wide range of
biological processes that go far beyond the current knowledge.
AQP-mediated water transport in plants has been implicated to play a
central regulatory step in diverse biological processes, including cell elongation,
seed germination and osmoregulation [18]. In addition, AQPs facilitate the
transport of small uncharged molecules of physiological significance like
glycerol, urea, boric acid, silicic acid, hydrogen peroxide (H2O2), ammonia (NH3)
and carbon dioxide (CO2) through the plant cell membranes [1,2] and also
32
regulate phloem sap loading and unloading, stomatal and leaf movement, and
cytoplasmic homeostasis [1,2,13,19]. Therefore, it is not surprising that their
expression and biological activities have been shown to be developmentally and
differentially regulated in a cell-specific manner, via phytohormones such as
abscisic acid (ABA), gibberellins and possibly brassinosteroids, and by
environmental signals such as light, water stress, nematode infection, low
temperature, and salinity [4]. However, a general expression pattern among the
distinct MIP isoforms cannot be distinguished, as they are either up- or
downregulated depending on the stimulus and/or the cell-type studied [4,19,20].
This difference in transcriptional regulation suggests that each MIP isoform may
play distinct roles in plant growth, development and stress response [4].
As a major horticultural crop, the cultivated Citrus spp. face constant
biotic and abiotic constraints in the main regions of production, including
drought, salinity, extreme temperatures and serious diseases like
Huanglongbing (HLB, or citrus greening), which are predicted to increase in
intensity, frequency, and geographic extent as a consequence of global climate
change. Despite the highlighted importance of AQPs, there are only a few
studies to date on citrus MIPs and their predicted role in the transport of water
and solutes required for plant growth, development and adaptive responses to
the environment. The expression of two MIP genes, PIP1 and PIP2, has been
investigated in roots of Poncirus trifoliata (L.) Raf., Cleopatra mandarin (C.
reshni Hort exTan.) and one of their hybrid, subjected to moderate water deficit
[21], and in roots of P. trifoliata, Cleopatra mandarin and Carrizo citrange (C.
sinensis [L.] Osb.×P. trifoliata [L.] Raf.), subjected to salt treatment [22]. PIP1
and PIP2 gene expression differences were correlated with alterations in root
hydraulic conductance (Kr) and chloride (Cl-) exclusion from leaves and, hence,
plant tolerance to water and salt stresses, respectively. With the recent
completion and publication of the draft genome sequences of sweet orange [23-
25], it is now possible to identify and characterize the complete repertoire of
MIPs in citrus, as well as to carry out comparative genome analysis in order to
understand their evolutionary history. Therefore, the objective of the present
study was to identify sweet orange MIP genes through a genome-wide analysis
and to characterize their sequences, evolutionary relationships, putative
functions and expression patterns in various tissues and in response to abiotic
33
stresses and ‘Candidatus Liberibacterasiaticus’ infection. This is the first
comprehensive study of the MIP gene family in sweet orange, providing
valuable information for further exploration of the functions of this important
gene family in citrus.
2 MATERIALS AND METHODS
2.1 Plant materials and stress treatments
Two-year-old sweet orange plants grafted on Rangpur lime
(C. limonia Osbeck), a rootstock highly resistant to drought, were used in the
drought stress experiment. Plants were first pruned and acclimatized to
greenhouse conditions (25±4°C, 16 h of light and relative humidity oscillating
between 80 and 95%) during 90 days to obtain adequate root development and
uniform leaf flushes. During acclimatization, plants were grown in plastic pots of
45-L, containing a mixture of soil and sand (ratio 3:1) and micronutrient mix FTE
(50g per pot), irrigated with tap water twice a week, and fertilized weekly with 1
liter of the following nutrient solution: 1.0g l-1 Ca(NO3)2, 0.4g l-1 KNO3, 0.6g l-
1 MgSO4 and 0.4g l-1 NH4H2P04 (MAP). Thereafter, the pots were closed with
aluminum foil to prevent water loss by evaporation, and a set of 10 plants was
randomized over the experimental area and subjected to the following
treatments: (i) 5 plants in control, in which plants were maintained at leaf
predawn water potential values of -0.2 to -0.4 MPa by daily irrigation and (ii) 5
plants in drought, in which the plants were exposed to a progressive soil water
deficit until their leaves reach predawn water potential values of -1.5 MPa. The
leaf predawn water potential was recorded on the third fully expanded mature
leaf from the apex of each plant, between 2 AM and 4 AM, using a Scholander-
type pressure pump (m670, Pms Instrument Co., Albany, USA).
For salt treatment, sweet orange seeds were germinated in vitro as
described by de Oliveira et al. [26]. Twenty-day-old seedlings of nucellar origin
were selected based on their uniformity, and transferred to MS medium alone
(control) or containing 150 mM NaCl (Merck, Darmstadt, Germany). Each
treatment consisted of 15 nucellar plants (biological replicates). Leaves and
34
roots were harvested 20 days after the treatments and immediately frozen in
liquid nitrogen and stored at -80°C.
Plants were infected with ‘Candidatus Liberibacter asiaticus’ as described
in Fan et al. [27]. Briefly, two-year-old seedlings of rough lemon (C. jambhiri
Lush.) and sweet orange (C.sinensis L. Osbeck) were graft-inoculated with bud
wood from ‘Ca. L. asiaticus’ infected ‘Carrizo’ citrange trees kept under
greenhouse conditions. For controls, the plants were grafted with bud wood
from healthy Carrizo trees. All these plants were kept in a United States
Department of Agriculture Animal and Plant Health Inspection Service and
Center for Disease Control-approved and secured greenhouse at the University
of Florida, Citrus Research and Education Center, Lake Alfred. Three biological
replicates were produced for each citrus species in each treatment. Quantitative
real-time PCR was performed to confirm the presence of ‘Ca. L. asiaticus’ in the
inoculum source and inoculated plants as described in Li et al. [28]. Four fully
expanded leaves were sampled separately from ‘Ca. L. asiaticus’ inoculated
plants and mock-inoculated plants (used as controls) of each citrus species at
0, 7, 17, and 34 weeks after inoculation (WAI). Leaves were immediately frozen
in liquid nitrogen and stored at -80°C until use. Three biological replicates were
produced for each condition. In total, 12 plants with 48 leaf samples were
collected (2 species x 2 treatments x 3 replicates x 4 time points).
2.2 Identification and classification of CsMIPs
The HMM (Hidden Markov Model) profile of the PFAM
(http://pfam.sanger.ac.uk/) [29] motif PF00230 (major intrinsic protein) was used
as a keyword to search the sweet orange genome sequence database
(http://www.phytozome.org/citrus)[25]. The KEGG Orthology (KO) terms
K09872 (aquaporin PIP), K09873 (aquaporin TIP), K09874 (aquaporin NIP) and
K09875 (aquaporin SIP) were also used as keywords to search the sweet
orange genome sequence at Phytozome. To avoid the deficiencies of the
automatic annotation, the 35 Arabidopsis thaliana MIP protein sequences were
retrieved from TAIR (http://www.arabidopsis.org/), according to previous reports
[5,30], and also used to align the sweet orange genome sequence assembly
available at Phytozome using the TBLASTN tool. After merging the results from
35
all these strategies, unique entries (with unique locus ID) were identified to
remove the redundancy. The resulting sequences were manually inspected for
the presence of characteristic and functionally important MIP amino acids and
motifs.
The sweet orange MIPs were classified in different isoforms based on
sequence analysis of the multiple alignments and on their phylogenetic
relationship with those clearly classified MIPs of Arabidopsis thaliana, Ricinus
communis and Nicotiana benthamiana, downloaded from the TAIR and NCBI
databases. Multiple sequence alignments of the deduced amino acid
sequences of CsMIPs and those of A. thaliana, R. communis and N.
benthamiana were performed using the default parameters of ClustalW [31].
The dendrogram was generated by the MEGA 6 program [32] using the
Neighbor-Joining (NJ) method [33] and bootstrap analysis (1,000 replications).
2.3Analysis of CsMIP protein properties and conserved amino acid
residues
Information about coding sequence (CDS), full-length sequence and
predicted amino acid sequence was obtained for each sweet orange MIP gene
from the Phytozome database. The GRAVY (grand average of hydropathy),
molecular weight and isoelectric point (pI) of the deduced amino acid
sequences were predicted by the PROTPARAM tool available on the Expert
Protein Analysis System (ExPASy) proteomics server
(www.expasy.ch/tools/protparam.html). The subcellular localization of MIP
proteins was predicted using the WoLF PSORT tool available at
http://www.genscript.com/psort/wolf_psort.html. Careful visual inspection of
amino acid sequence alignments were performed in order to identify the
characteristic MIP amino acids and motifs and the residues in seven key
positions that have been reported to be specific for each functional subgroup
[12,30,34,35].
36
2.4 Analysis of promoter sequences and chromosomal locations of
CsMIPs
One kb upstream region from the translation start site was extracted from
all the sweet orange MIP genes and subsequently analyzed in the PLACE
database (http://www.dna.affrc.go.jp/PLACE/signalscan.html) to identify the
presence of the stress-responsive cis-acting regulatory elements ABRE (ABA-
responsive element; ACGTG), DRE/CRT (dehydration responsive element/C-
repeat; G/ACCGAC), MYBS (MYB binding site; TAACTG) and LTRE (low-
temperature-responsive element; CCGAC) in their promoters. The physical
locations of CsMIPs were determined by confirming the starting position of all
genes on each chromosome, using BLASTN searching against the local
database of the Citrus sinensis Annotation Project (CAP;
http://citrus.hzau.edu.cn/orange/). MapChart software was used to plot the gene
loci on the sweet orange chromosomes [36].
2.5 Expression analysis of CsMIPs
Total RNA isolation, cDNA synthesis and quantitative real-time RT-PCR
(qPCR) analysis were performed as described previously [26]. qPCR primers
were designed in order to avoid the conserved regions. Primer sequences are
shown in detail in S1 Table. Glyceraldehyde-3-phosphate dehydrogenase C2
(GAPC2) was used as an internal reference gene to normalize expression
among the different samples [37]. Data were obtained from a pool of three
biological replicates that were individually validated.
RNA-Seq data were downloaded from CAP [24] and used to analyze the
expression patterns of CsMIPs in different tissues, namely callus (C), flower
(Fl), leaf (L), fruit (Fr), and mixed tissues from fruit at three developmental
stages (Mix.1, Mix.2, and Mix.3). The heatmap was generated using R 3.1.0
software.
37
3 Results and Discussion
3.1 MIP encoding genes in the sweet orange genome
Searches in the sweet orange genome sequence database at
Phytozome using annotation information, as well as the 35 protein sequences of
the complete set of A. thaliana MIPs (AtMIPs) as query sequences, resulted in
the identification of 34 different genes encoding C. sinensis MIPs (CsMIPs)
(Table 1). The retrieved sequences were manually inspected for the presence
of characteristic and functionally important MIP domains and motifs, such as the
highly conserved NPA (Asn-Pro-Ala) motifs, and considered to be correct. The
number of MIP genes described in this study is similar to that found in the
genomes of Arabidopsis [5], maize [6], rice [7] and grape [8], but significantly
lower than that identified in the genomes of poplar [9], cotton [10], tomato [11]
and soybean [12]. The absence of recent whole-genome duplication (WGD)
events in the sweet orange genome, as described by Xu et al. [23], could
account for the relatively small size of the MIP family in the citrus genome.
38
1
Table 1. Genes and encoded MIP proteins in sweet orange. 2
Gene name
Locus
Chromosome location
Group
Kegg Orth
ID
Polypeptide
length
(MW)
pI
GRAVY
Predicted subcellular
localization
CsPIP1;1 orange1.1g018895m chr7:
31,253,722…31,256,103
PIP K09872 349
(37.56kDa)
9.39 0.397 plasma membrane
CsPIP1;2 orange1.1g023021m chr7:
31,247,369…31,248,902
288
(30.67kDa)
7.71 0.414 plasma membrane
CsPIP1;3 orange1.1g023107m chr5:
1,804,896…1,807,677
287
(30.70kDa)
8.97 0.344 plasma membrane
CsPIP1;4 orange1.1g023069m chr6:
9,907,825…9,909,541
287
(30.82kDa)
8.96 0.343 plasma membrane
CsPIP2;1 orange1.1g023108m chr6:
12,833,884…12,835,042
287
(30.67kDa)
8.74 0.376 plasma membrane
CsPIP2;2 orange1.1g022966m chr8:
19,657,287…19,659,497
289
(31.05kDa)
7.62 0.392 plasma membrane
CsPIP2;3 orange1.1g019681m chr7:
26,202,220…26,205,546
337
(36.34KDa)
9.77 0.403 plasma membrane
CsPIP2;4 orange1.1g023370m chr8:
981,841…983,571
283
(30.14kDa)
8.99 0.431 plasma membrane
CsTIP1;1 orange1.1g025548m chrUn:
46,663,407…46,665,011
TIP K09873 251
(26.06kDa)
6.12 0.675 vacuole
CsTIP1;2 orange1.1g025600m chr8:
20,659,157…20,660,437
250
(25.65kDa)
5.32 0.841 cytosol
CsTIP1;3 orange1.1g037978m chr8:
20,659,157…20,660,437
124
(12.92kDa)
4.37 0.734
cytosol
CsTIP1;4 orange1.1g025464m chr7:
29,135,182…29,136,531
252
(26.01kDa)
5,69 0,786 vacuole
CsTIP2;1 orange1.1g025817m chr1:
18,627,617…18,629,472
247
(25.15kDa)
5.59 0.894 vacuole
CsTIP2;2 orange1.1g025865m chr1:
18,627,617…18,629,472
247
(25.10kDa)
5.59 0.902 vacuole
CsTIP2;3 orange1.1g038895m chr5:
5,749,487…5,750,939
206
(20.55kDa)
4.72 0.979 vacuole
CsTIP3;1 orange1.1g025197m chr5:
16,938,542…16,940,192
256
(26.99kDa)
7.07 0.626 cytosol
CsTIP4;1 orange1.1g025864m chr4: 247 6.27 0.825 vacuole
39
19,032,254…19,033,990 (16.27kDa)
CsTIP5;1 orange1.1g046726m chr9:
14,144,215…14,145,199
161
(16.86kDa)
9.00 0.770 cytosol
CsTIP6;1 orange1.1g042738m chr9:
14,144,215…14,145,199
107
(11.11kDa)
4.54 0.636 secreted
CsNIP1;1 orange1.1g023184m chr2:
2,151,220...2,153,387
NIP K09874 286
(30.45kDa)
8.64 0.434 vacuole
CsNIP2;1 orange1.1g036721m chr6:
18,134,848…18,136,228
223
(23.70kDa)
9.69 0.889 cytosol
CsNIP2;2 orange1.1g040981m chr6:
18,134,848…18,136,228
211
(22.32kDa)
9.39 0.967 cytosol
CsNIP2;3 orange1.1g040755m chr2:
13,464,261…13,465,771
275
(29.39kDa)
8.88 0.628 plasma membrane
CsNIP3;1 orange1.1g023102m chr6:
20,482,599…20,486,038
287
(30.30kDa)
8.40 0.387 plasma membrane
CsNIP4;1 orange1.1g046511m chr3:
23,770,831...23,774,009
282
(29.28kDa)
8.84 0.372 vacuole
CsNIP5;1 orange1.1g035030m chr1:
13,680,675…13,682,992
75
(7.75kDa)
8.98 0.291 chloroplast
CsNIP5;2 orange1.1g027840m chr1:
13,678,241…13,681,090
218
(22.51kDa)
7.75 0.462 plasma membrane
CsNIP6;1 orange1.1g039196m chr9:
3,798,017…3,800,780
288
(30.20kDa)
7.53 0.718 plasma membrane
CsSIP1;1 orange1.1g026039m chr5:
28,968,880…28,972,401
SIP K09875 244
(25.92kDa)
9.35 0.727 plasma membrane
CsSIP1;2 orange1.1g026082m chr3:
1,234,876…1,236,556
244
(26.17kDa)
9.83 0.749 plasma membrane
CsSIP2;1 orange1.1g026600m chr6:
17,078,102…17,081,323
236
(25.57kDa)
9.70 0.600 chloroplast
CsXIP1;1 orange1.1g036381m chr8:
7,139,938…7,141,114
XIP - 235
(25.09kDa)
8.70 0.821 plasma membrane
CsXIP1;2 orange1.1g040654m chr8:
7,131,064…7,132,799
302
(32.68kDa)
8.74 0.573 plasma membrane
CsXIP2;1 orange1.1g045670m chr8:
7,128,184...7,129,448
319
(34.58kDa)
8.32 0.681 plasma membrane
3
40
The CsMIPs were classified in five different subfamilies, PIPs, TIPs, NIPs, SIPs
and XIPs, based on analysis of the amino acid residues located in seven key
positions (P1 to P7) that were previously proposed [12,34,35] to discriminate
the different subfamilies (S2 Table), as well as on their phylogenetic
relationships with the well classified MIPs of A. thaliana and XIPs of R.
communis and N. benthamiana (S1 Fig). Our analysis revealed the presence of
8 PIPs, 11 TIPs, 9 NIPs, 3 SIPs and 3 XIPs in the sweet orange genome (Table
1). The CsMIPs were named according to the nomenclature proposed in
classification of the MIPs of A. thaliana. This nomenclature was based on
phylogenetic analyses and where the names in a systematic way reflect distinct
clades that are evolutionarily stable [5]. PIPs, TIPs, NIPs and SIPs from sweet
orange grouped with their respective Arabidopsis counterparts, indicating the
large extent of conservation between the sweet orange and ArabidopsisMIP
gene families (S1 Fig). The only exception was CsTIP6;1, which was found to
encode a N- and C-terminally truncated TIP protein compared to the rest of the
subfamily.
To examine whether the number of MIP genes found in the diploid sweet
orange is comparable to that of the dihaploid sweet orange and haploid
Clementine mandarin, we also performed homology searches against the
dihaploid sweet orange draft genome available at the Citrus sinensis Annotation
Project (CAP) and the reference haploid Clementine mandarin (C.clementina)
genome available at Phytozome (S3 Table). Although the total number of MIP
genes was roughly similar among the different citrus genomes, significant
differences were observed in the number of members within the subfamilies
(Table 2). BLAST similarity analysis revealed that the dihaploid sweet orange
and haploid Clementine contained two additional PIPisoforms closely related to
the CsPIP2;1 (S3 Table). Clementine also contained one additional PIP isoform
closely related to the CsPIP1;2 and one PIP, TIP and NIP isoform without
homology to any MIP sequence from the diploid sweet orange (S3
Table). CsTIP1;2 andCsTIP1;3, CsTIP2;1 and CsTIP2;2, CsTIP5;1 and CsTIP6;
1, and CsNIP2;1 and CsNIP2;2 exhibited significant hits to the
same MIP isoforms of the dihaploid sweet orange and haploid Clementine (S3
Table). These observed variations in the size of the MIP subfamilies may be a
41
consequence of the different sequencing depth and assembly quality between
the diploid and dihaploid sweet orange genomes [23,25] and the evolutionary
origin of Clementine, which is a hybrid of Willowleaf mandarin and sweet-
orange [25] and, thereby, it contains more C.reticulata haplotype regions than
found in sweet orange.
Table 2. Comparison of the number of the different MIP family members in
diploid (Phytozome) and dihaploid (CAP) sweet oranges (C. sinensis) and
haploid Clementine (C. clementina).
Subfamily Diploid
C. sinensis
Dihaploid
C. sinensis
C. clementina
PIP 8 10 11
TIP 11 8 9
NIP 9 8 8
SIP 3 3 3
XIP 3 3 2
Total 34 32 33
3.2 CsMIP protein properties and conserved amino acid residues
The CsMIPs encode proteins ranging from 75 (7.7 kDa) to 349 (37.6
kDa) amino acids in length, and pI values ranging from 4.37 to 9.77 (Table 1).
The average protein length of PIPs, TIPs, NIPs, SIPs and XIPs were 300.8
(32.2 kDa), 213.4 (21.1 kDa), 238.3 (25.1 kDa), 241.3 (25.9 kDa) and 285.3
(30.8 kDa) amino acids, respectively. The average pI of PIPs, TIPs, NIPs, SIPs
and XIPs were 8.77, 5.86, 8.68, 9.63, and 8.59, respectively. These data reveal
that CsTIPs are not only smaller, but most of them are also much more acidic
than the other CsMIPs, as reported for Arabidopsis MIPs [38]. The cause of
these large differences in TIPs has been attributed to the smaller amount of
basic residues found at the carboxyl termini of TIPs compared with the other
MIPs [5]. All the CsMIPs had a positive grand average hydropathy (GRAVY)
score (Table 1), suggesting that they are hydrophobic proteins, which is a
necessary character for AQPs [1]. Analysis of the predicted subcellular
localization showed that all CsPIPs and CsXIPs were localized to plasma
membrane (Table 1). The predicted localization of CsTIPs, CsNIPs and CsSIPs
was more diverse, including vacuole (CsTIP1;1, CsTIP1;4, CsTIP2;1, CsTIP2;2,
CsTIP2;3, CsTIP4;1, CsNIP1;1 and CsNIP4;1), cytosol (CsTIP1;2, CsTIP1;3,
42
CsTIP3;1, CsTIP5;1, CsNIP2;1 and CsNIP2;2), plasma membrane (CsNIP2;3,
CsNIP3;1, CsNIP5;2, CsNIP6;1, CsSIP1;1 and CsSIP1;2), chloroplast
(CsNIP5;1 and CsSIP2;1) and secreted (CsTIP6;1) (Table 1). These results
seem to be in agreement with the experimentally determined localizations of
MIPs reported in the literature [13,39].
MIP folding is characterized by six transmembrane a-helices (H1 to H6)
that are connected by five loops (loops A-E), forming an aqueous
transmembrane pore that constitutes the functional core of MIPs [35]. Loops B
(LB) and E (LE) contain two highly conserved NPA (Asn-Pro-Ala) motifs that are
considered to be critical for the substrate selectivity of MIPs [40,41]. Another set
of four conserved residues forms the aromatic/Arginine selectivity filter (ar/R
filter), which has been proposed to act as a size exclusion barrier for substrate
molecules [42]. The first two residues are located in H2 and H5, while the latter
two are found in LE (LE1 and LE2). Finally, seven key amino acid residues
(named positions P1 to P7) have been proposed to discriminate the five
subfamilies [12,34]. P1 is located in the terminal part of H3, while P2 and P3 are
located in LE, just behind the second NPA motif (2nd and 6th residues after 2nd
NPA, respectively). P4 and P5 correspond to two consecutive amino acids
located in H6, while P6 and P7 also correspond to two consecutive amino acids
located in H3. The multiple sequence alignments were carefully analyzed and
all these conserved motifs and amino acid residues were identified in most
CsMIPs, indicating that they are functional channels for water and other solutes
(S2 Table). All the CsPIPs showed the dual typical NPA motifs and an ar/R filter
configuration characteristic for a water-transporting MIP (F,H,T,R). Additional
presence of the S-A-F-W residues at P2-P5 positions, as observed in all
CsPIPs, except for CsPIP1;1, has been interpreted as a signature of CO2
transporters PIPs [35]. All the CsTIPs also had the two canonical NPA motifs,
except CsTIP1;3, CsTIP5;1 and CsTIP6;1, which were found to encode
truncated proteins lacking either the first (CsTIP1;3) or the second NPA motif
(CsTIP5;1 and CsTIP6;1), as well as other conserved amino acid residues of
the ar/R filter region (S2 Table). TIPs containing the H-I-A-V or H-I-G-R
residues in the ar/R filter and T-A-A-Y-W or T-S-A-Y-W residues at P1-P5
positions, like CsTIP1s, CsTIP2s and CsTIP3, have been shown to transport
urea and H2O2 [35]. The CsNIP1;1, CsNIP2;1, CsNIP2;2 and CsNIP2;3 showed
43
an ar/R filter configuration identical to that of soybean Nodulin 26, indicating that
they are also able to facilitate water and solute transport capability [30]. The
residue at the H5 position of the ar/R filter of AtNIP5;1 was shown to play a key
role in the membrane permeability to water, silicic acid (Si) and boric acid (B)
[42]. AtNIP5;1 with AIGR residues for the ar/R filter was shown to transport
water, B and arsenite (As), but not Si [42]. CsNIP3;1, with GSGR residues for
ar/R filter, can be expected to transport water, Si and B, while CsNIP4;1 (AIGR
residues) may transport water, B and As. The CsSIPs showed a less conserved
first NPA motif, while the second NPA motif was perfectly conserved in all
members (S2 Table). AtSIP1 isoforms, but not AtSIP2;1, were shown to be
functional water channels [43], suggesting that the latter may be involved in the
transport of solutes. However, SIP transport function and structural organization
still await biochemical characterization. The CsXIPs also showed a modified
first NPA motif, NPL (CsXIP1s) or SPV (CsXIP2), and a conserved second NPA
motif (S2 Table). The four positions in the ar/R filter region contained amino
acid residues that were strictly conserved among the CsXIPs. CsXIP1;1 was
observed to contain an internal deletion of 13 amino acid residues in the H2
region that abolished the conserved amino acid V at position H2 of the ar/R
selective filter. Since the first three amino acid of the ar/R filter have rather
hydrophobic residues (VVAR or VVVR), the CsXIPs might be involved in the
transport of molecules other than water [35]. In fact, a recent study has
indicated that the Solanaceae XIPs are plasma membrane aquaporins involved
in the transport of many uncharged substrates, such as urea, H2O2 and B [44].
3.3 Genomic organization of CsMIPs
The exon-intron structure of all 34 CsMIP genes was analyzed using the
sweet orange gene models annotated in Phytozome. With a few exceptions, the
number and size of the exons, but not of the introns, were observed to be
conserved within each CsMIP subfamily (S2 Fig). All the CsPIPs presented
three introns and four exons, as reported for all Arabidopsis [5], poplar [9],
tomato [11] and soybean PIPs [12]. The majority of CsTIPs contained two
introns and three exons, with exception of CsTIP1;1, CsTIP2;3 and CsTIP6;1,
which showed one intron and two exons, and the truncated CsTIP1;3 (one
44
exon) gene. Such a more varied pattern of exon-intron structure has been also
observed in Arabidopsis [5], poplar [9], tomato [11] and soybean TIPs [12]. Most
CsNIPs contained four introns and five exons, like all Arabidopsis [5] and most
poplar [9], tomato [11] and soybean NIPs [12]. The exceptions were CsNIP2;2
(three introns and four exons), CsNIP5;2 (two introns and three exons) and the
truncated CsNIP5;1 (one intron and two exons) genes. The CsSIPs featured
two introns and three exons, except CsSIP1;2 (one exon and one intron in the
3’-UTR region). Similar patterns of exon-intron structure were also reported for
tomato SIPs [11], while all the Arabidopsis [5] and poplar SIPs [9] had two
introns and three exons. The gene structure of CsXIPs varied among all
members, which contained two introns and three exons (CsXIP1;2), either one
intron and two exons (CsXIP2;1) or only one exon (CsXIP1;1). The pattern of
two introns and three exons has been reported for most poplar [9] and tomato
XIPs [11], while all soybean XIPs contained a single intron and two exons [12].
The positions of all 34 CsMIPs were mapped on the sweet orange
chromosomes by homology searches against the full-length sweet orange
genome assembly available at the CAP database (Table 1 and S3 Table).
Except for CsTIP1;1, which was not exactly located on any chromosome
because of an incomplete physical map for sweet orange, all the CsMIP loci
were precisely mapped on every sweet orange chromosome, indicating a wide
distribution of the gene family in the sweet orange genome (Table 1 and S3
Fig). The closely related CsMIP isoforms CsTIP1;2 and CsTIP1;3, CsTIP2;1
and CsTIP2;2, CsTIP5;1 and CsTIP6;1, and CsNIP2;1 and CsNIP2;2 were
respectively mapped on identical chromosome positions since they showed
significant hits to the same genes in the CAP database (S3 Table). Seven
CsMIPs were found to be tandem duplicated genes according to the criteria of
Hanada et al. [45], which defined tandem duplicates as genes in any gene pair,
T1 and T2, that (1) belong to the same gene family, (2) are located within 100 kb
each other, and (3) are separated by 10 or fewer nonhomologous spacer
genes. These were CsNIP5;1 and CsNIP5;2 on chromosome 1, CsPIP1;1 and
CsPIP1;2 on chromosome 7, and CsXIP1;1, CsXIP1;2 and CsXIP2;1 on
chromosome 8 (Table 1 and S3 Fig). These results suggest that all these
CsMIPs may have evolved from tandem duplication events, as also recently
proposed for the tomato XIPs [11].
45
Analysis of previously mapped traits revealed that the 282-kb region
surrounding the Citrus Tristeza Virus resistance (Ctv) locus is physically linked
(~40-kb) to the CsNIP5;1 and CsNIP5;2 genes,on the chromosome 1 [46]. This
region was also reported to contain Tyr1, the major locus controlling citrus
nematode (Tylenchulus semipenetrans) resistance [47,48].
3.4 Expression patterns of CsMIP genes in different tissues
To investigate the expression patterns of CsMIPs in different tissues,
RNA-seq data were downloaded from CAP [24]. The heatmap generated
showed a differential transcript abundance of the 34 CsMIPs in four major
tissues, namely callus, flower, leaf, fruit, and mixed fruit tissues at three
developmental stages (S4 Fig). Some of the CsMIPs (CsPIP1;1, CsPIP1;3,
CsPIP2;2, CsPIP2;3 and CsSIP1;1) showed higher expression in all the seven
tissues, indicating a role in constitutive transport processes throughout the
plant. Others genes were found to have a low expression in all the tissues
(CsTIP1;1, CsTIP1;2, CsTIP1;3, CsTIP2;3, CsTIP5;1, CsTIP6;1, CsNIP2;1,
CsNIP2;2, CsNIP2;3, CsNIP5;1, CsNIP5;2, CsXIP1;1 and CsXIP2;1). The
putative tandem duplicated CsMIP genes were observed to have divergent
expression profiles, which probably has contributed to their maintenance
through regulatory subfunctionalization and neofunctionalization [49]. CsPIP1;1
showed a higher expression than CsPIP1;2 in all the seven tissues analyzed.
CsXIP1;1 and CsXIP2;1 showed low expression in all the seven tissues
analyzed, while CsXIP1;2 exhibited a high expression in flower, leaf and mixed
fruit tissue (Mix.3).
The cell type localization of aquaporin expression can also provide clues
about their physiological roles. For instance, expression of PIP aquaporins is
generally localized in organs and tissues characterized by large fluxes of water,
such as vascular tissues, guard cells, flowers and fruits [4]. Their expression in
roots and leaves has been also correlated with the presence of apoplastic
barriers, the exodermis and endodermis in roots or in suberized bundle sheath
cells in leaves, suggesting their essential role in the transmembrane water
diffusion when its movement is hindered [19,20,50-55]. Except for CsPIP2;1, all
the CsPIPs were found to be highly expressed in flower, leaf, fruit and mixed
46
fruit tissues (S4 Fig), supporting their active role in the transport of water and
solutes across these tissues. By contrast, the expression of TIP isoforms has
been more related to developmental stages and/or organ specificity [56]. For
instance, the expression of AtTIP2;1 is especially high in the vascular system of
the shoot but is barely detectable in the root [57]. AtTIP3;1 is highly expressed
in cotyledons and associated with the membrane of protein storage vacuoles
[4]. TIPs are also differentially expressed during fruit maturation, e.g., the
TIP1;1 homolog in pear is highly expressed in the young fruit, whereas TIP
proteins levels in grape gradually increase along with ripening [58,59]. Vacuoles
participate in cell expansion and, thus, their enlargement by water
compartmentation is essential to provoke the rapid fruit growth that is
characteristic of the ripening process. The lack of specific regulation observed
along fruit ripening for PIPs isoforms points out the essential role of TIPs in this
process [60]. The differential expression of CsTIP1;3, CsTIP1;4, CsTIP2;1,
CsTIP2;2, CsTIP3;1, CsTIP4;1 and CsTIP5;1 highlights the functional
importance of these CsMIPs on each tissue and stage of fruit development
analyzed (S4 Fig). The overall level of NIP expression is usually lower than the
expression of PIPs and TIPs, and it is usually associated with specialized
organs and cells [10]. For instance, AtNIP2;1 is specifically expressed in the
endoplasmic reticulum (ER) of roots, whereas AtNIP5;1 is a plasma membrane
MIP mainly expressed in root elongation zones [39,61]. Our analysis showed
that the CsNIPs had preferential expression either in flower (CsNIP6;1), leaf
(CsNIP2;2 and CsNIP3;1) or mixed fruit tissues at different developmental
stages (CsNIP1;1, CsNIP2;2, CsNIP3;1, CsNIP4;1 and CsNIP6;1) (S4 Fig).
SIPs seem to be expressed in a range of tissues in Arabidopsis, including
young roots, flowers and pollen [62]. It is remarkable that SIPs are also strongly
expressed in suspension cultured cells compared to other MIPs [62]. CsSIP1;1
was observed to be constitutively expressed in all the seven tissues analyzes,
while the others were preferentially expressed in flower and fruit tissues (S4
Fig). XIPs were reported to be expressed in almost all the poplar tissues [9]. By
contrast, CsXIPs showed a low expression in all tissues analyzed, except
CsXIP1;2, which show a relatively high expression in flower, leaf and mixed fruit
tissues at third developmental stage (Mix.3) (S4 Fig).
47
3.5 Expression patterns of CsMIP genes under abiotic and biotic stresses
To identify CsMIPs with a potential role in abiotic and biotic stress
response of sweet orange, the expression patterns of all the 34 sweet orange
MIPs were investigated in plants exposed to drought, high salinity and ‘Ca. L.
asiaticus’ (HLB) infection, by qPCR. Considering the log2 fold change (LFC) of
≥1.00 or ≤-1.00 as cutoff threshold between stressed and control plants, the
qPCR analyses showed that all the CsMIPs were differentially expressed in at
least one stress condition and tissue analyzed (Figs.1-3). Twelve CsMIPs
(CsPIP1;1, CsPIP2;4, CsTIP1;3, CsTIP2;1, CsTIP2;2, CsTIP3;1, CsTIP4;1,
CsNIP1;1, CsSIP1;2, CsXIP1;1, CsXIP1;2 and CsXIP2;1) were observed to be
differentially expressed in response to all the three stress conditions analyzed,
in at least one tissue studied. Interestingly, only CsTIP1;1 showed differential
expression exclusively in response to the abiotic stress treatments, while six
CsMIPs (CsPIP1;2, CsPIP2;2, CsNIP2;2, CsNIP5;2, CsNIP6;1 and CsSIP1;1)
were differentially expressed exclusively under the biotic stress treatment.
These results seem to be consistent with the respective organization of the
stress-responsive cis-acting regulatory elements observed in the CsMIPs
promoters (S5 Fig). CsTIP1;1 was observed to contain the highest number of
ABRE copies in the promotor region among the sweet orange MIP genes, while
no or a low number of ABRE (less than 2 copies) and other stress-responsive
cis-acting regulatory elements was detected in the promoter regions of the
CsMIPs that were not induced by the abiotic stress treatments (S5 Fig). A single
copy of DRE/CRT has been observed to be sufficient for ABA-independent
stress-responsive gene expression, while more than two ABRE sequences are
usually required for the ABA-responsive transcription [63].
48
Fig 1. Expression analysis of the complete set of sweet orange MIPs in
response to drought treatment. Ratios (log2) of relative mRNA levels between
stressed and control plants for all 34 CsMIPs in leaves and roots, as measured
by qPCR. GAPC2 was used as an endogenous control. The bars show means
± SE from three biological replicates.
49
Fig 2. Expression analysis of the complete set of sweet orange MIPs in
response to salt treatment. Ratios (log2) of relative mRNA levels between
stressed and control plants for all 34 CsMIPs in leaves and roots, as measured
by qPCR. GAPC2 was used as an endogenous control. The bars show means
± SE from three biological replicates.
Fig 3. Expression analysis of the complete set of sweet orange MIPs in
response to ‘Ca. L. asiaticus’ infection in rough lemon (LEM) and sweet
orange (SWO). Ratios (log2) of relative mRNA levels between infected and
control plants at 0, 7, 17, and 34 WAI for all 34 CsMIPs, as measured by qPCR.
GAPC2 was used as an endogenous control.
All the CsTIPs and CsXIPs were upregulated in leaves but
downregulated in roots by drought treatment, while only two CsPIPs (CsPIP1;1
and CsPIP2;4) and one CsNIP (CsNIP1;1) and CsSIP (CsSIP1;2) were
differentially upregulated by drought treatment in leaves (Fig 1). Most CsMIPs
were upregulated by salt treatment in roots, and either upregulated or
downregulated by this treatment in leaves, depending on the isoform (Fig2). A
coordinated up- and downregulation, depending on the MIP gene and organ
examined, has been described as a general pattern of MIP regulation during
drought and salt stresses in Arabidopsis [64-67], soybean [12] and rice [68]. It
has been proposed that a general downregulation of MIPs might be a way for
50
the plant to minimize water loss and the hence loss of turgor in specific organs,
and that the upregulation of individual MIPs could be a way for the plant to
direct water flow to certain organs or cells that are crucial for plant survival
during stress, or necessary for its fast recovery upon rehydration [65]. Thus, the
concomitant upregulation in leaves and downregulation in roots of the CsTIPs
and CsXIPs by drought treatment likely reveal a coordinated regulation of these
MIP isoforms to direct the water flow through the leaf plasma membrane and
tonoplast, while avoiding the water loss in roots. On the other hand, the
upregulation of most CsMIPs in roots by salt treatment suggests their
coordinated regulation to increase the overall water flow into this organ, since it
is well known that salt stress reduces the hydraulic conductivity in roots,
resulting in decreases of water flow from root to shoot. Those CsMIPs that were
upregulated in leaves by salt treatment (i.e., CsPIP1;4, CsTIP1;1, CsTIP2;1,
CsTIP2;2, CsTIP3;1, CsNIP2;3, CsNIP4;1, CsXIP1;1 and CsXIP1;2) may also
contribute to increase the water flow into this organ. The time-course
transcriptional analysis in response to ‘Ca. L. asiaticus’ infection showed that 32
out of 34 CsMIPs were strongly downregulated at the early stage (7 weeks) in
the highly susceptible sweet orange, but not in the tolerant rough lemon, whose
expression levels of all CsMIPs were either upregulated or unchanged at this
stage (Fig3). At the later time points (17 and 34 weeks), significantly differential
expression levels between the susceptible and tolerant citrus species were
essentially limited to CsPIP2;2, CsTIP1;2, CsTIP2;1, CsTIP2;2 and CsNIP5;1.
‘Ca. L. asiaticus’ is the most widely distributed of the three species of the
phloem-limited a-proteobacterium, designated as Candidatus Liberibacter (i.e.,
‘Ca. L. asiaticus’, ‘Ca. L. africanus’ and ‘Ca. L. americanus’), which has been
associated with the most destructive disease affecting citrus worldwide, the
Huanglongbing (HLB) disease [69]. Leaf symptoms of HLB disease have been
well documented [69]. These include vein yellowing and blotchy mottle, reduced
leaf size and premature leaf abscission.Anatomical alterations caused by the
disease in leaves include the excessive accumulation of starch, callose
depositions, phloem plugging, necrosis and collapse, swelling of sieve elements
and companion cell walls, and the disruption of chloroplast inner grana
structures [27,69-74]. Significant differences on phloem ultrastructure and
phloem loading activity were also observed between HLB-infected sweet
51
orange and rough lemon plants, highlighting some underlying differences
between tolerance and susceptibility mechanisms to HLB disease [27]. While
the phloem transport activity in the midribs of leaves was considerably impaired
in diseased sweet orange, it was much less significantly affected in infected
rough lemon [27]. The downregulation of most CsMIPs as observed 7 WAI in
the susceptible sweet orange, but not in the tolerant rough lemon, correlates
with the onset of disease symptoms, such as blotchy leaf mottle and yellowing
[27]. The continued contrasting expression patterns of CsPIP2;2, CsTIP1;2,
CsTIP2;1, CsTIP2;2 and CsNIP5;1 between the tolerant and susceptible citrus
species throughout the 17 and/or 34 WAI suggests an involvement of these
CsMIP genes in further symptom development, such as growth inhibition and
impaired phloem loading [27]. Taken together, these results indicate that MIP
genes may play an active role in the pathogenesis of HLB disease by regulating
the flow of water and nutrients required for the normal growth and development
of citrus plants. The recent finding in a microarray experiment that CsPIPs were
observed to be downregulated in stems of HLB-symptomatic sweet orange
trees, 16 months after inoculation of ‘Ca. L. asiaticus’, is consistent with this
interpretation [74].
4 CONCLUSION
This study presented a genome-wide survey of the MIP gene family in
sweet orange. A total of 34 open reading frames (ORFs) encoding MIP proteins
were identified and characterized as to their sequences, phylogenetic
relationships, genomic organization, tissue-specific gene expression and
expression profiles upon abiotic and biotic stresses. Our results allow us to
assess the relative contribution of each CsMIP member to water and solute
transport in different tissues and in response to drought, salinity and ‘Ca. L.
asiaticus’ infection. These results suggest a special role for CsTIPs and CsXIPs
in delivering water to the leaves while preventing root tissue dehydration under
drought stress, and for most CsMIPs in increasing the overall water flow into the
roots during salt stress and also regulating the flow of water and nutrients
during the development of HLB disease. Taken together, our results support the
52
idea that these CsMIP genes represent an important genetic resource for
improving citrus tolerance or resistance to both abiotic and biotic stresses.
5 ACKNOWLEDGEMENTS
The authors are grateful to Dr. Eduardo A. Girardi (Embrapa Cassava & Fruits,
Cruz das Almas, Bahia, Brazil) for kindly providing the grafted citrus plant
material used in this study. We gratefully acknowledge the Ph.D. scholarship to
C.P.S.M. by the CAPES Foundation (Brasília, Brazil).
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7 SUPPORTING INFORMATION
S1 Fig. Phylogenetic relationships of the complete set of 34 sweet orange
MIPs with members of Arabidopsis and other plants. The deduced amino
acid sequences were aligned using ClustalW2 and the phylogenetic tree was
generated using Bootstrap N-J tree (1,000 resamplings) method and MEGA
program (v6.0.5). Numbers at internal nodes denotes the results of
bootstrapping analysis (n = 1000). Black diamonds indicate MIP gene from
sweet orange. Cs, Citrus sinensis; At, Arabidopsis thaliana; Rc, Ricinus
communis; Nb, Nicotiana benthamiana.
60
S2 Fig. Analysis of exon-intron structures of the 34 sweet orange MIP
genes.NOI denotes the number of introns, E the exon and I the intron.
S3 Fig. Chromosomal locations of CsMIPs. The chromosomal position of
each CsMIP was mapped according to the Citrus sinensis Annotation Project
(CAP). The scale is in Mb. CsSIP (circle), CsPIP (star), CsNIP (square), CsTIP
(triangle), CsXIP (diamond).
61
S4 Fig. Heatmap of the expression of CsMIPs in different tissues of sweet
orange. Mix.1, Mix.2 and Mix.3 indicate mixed fruit tissues from different
developmental stages. The heatmap was generated using R 3.1.0 software.
The color scale shown represents RPKM-normalized log2-transformed counts.
S5 Fig. Analysis of the stress-responsive cis-elements ABRE (ACGTG),
DRE/CRT (G/ACCGAC), MYBS (TAACTG) and LTRE (CCGAC) in promoters
of sweet orange MIP genes. The cis-elements were analyzed in the 1 kb
upstream promoter region of translation start site of all CsMIPs using the
PLACE database.
62
S1Table. Primers used in the qPCR analysis.
Name Locus ID Primer Size product
(bp)
CsPIP1;1 orange1.1g018895 F: 5’-CATTCTCATCACAACATCAAACG-3’
R: 5’-CTGCTAGTCCCTCAAAAACACAA-3’
84
CsPIP1;2 orange1.1g023021 F: 5’-TCACTCCCGTAGCAAGATCA-3’
R: 5’-TTTCGCTCGCTCTTCTTCA-3’
90
CsPIP1;3 orange1.1g023107 F: 5’-CCTCAACTTTCTCGCTACGC-3’
R: 5’-TGATGAACCTCTCTCTCGCTCT-3’
85
CsPIP1;4 orange1.1g023069 F: 5’-TCTGCTGTATATGTACAACCCTTCG-3’
R: 5’-ATAGGAATCGGCCATGAACA-3’
80
CsPIP2;1 orange1.1g023108 F: 5’-TAGGCGGCAATGCTAAGTTT-3’
R: 5’-ATGATGAAGAAGGGCGAAGA-3’
89
CsPIP2;2 orange1.1g022966
F: 5’-GCAAACACAACAGTCGTAGCTCT-3’
R: 5’-CTTCAACATCCTTCCCCATTT-3’
90
CsPIP2;3 orange1.1g019681 F: 5’-TGTTGTCATTTTGCTACTCGTTTC-3’
R: 5’-GGCGTGCCATATTGCTTTTA-3’
85
CsPIP2;4 orange1.1g023370 F: 5’-TTTCTGTTATTTGTTCGCTTGTGT-3’
R: 5’-AATGGAAAAATAAAGAGAAGGGTCA-3’
81
CsTIP1;1 orange1.1g025548 F: 5’-AAGCTCCTAGTTGAGAAGTGGAGA-3’
R: 5’-ACGATGAAGATTGAACCTTTGG-3’
81
CsTIP1;2 orange1.1g025600 F: 5’-AACGGTCGGTTAAGCAGATT-3’
R: 5’-GAGGGCGGAGAAAATATTGA-3’
82
CsTIP1;3 orange1.1g037978 F: 5’-GAGGCTCCAAAACCACAAAT-3’
R: 5’-TTTCTTAGCGCGATGGGTAT-3’
90
CsTIP1;4 orange1.1g025464 F: 5’-AACGGAGCACAAAACAGAGC-3’
R: 5’-CGAAAATTTTGTCAAAGAAAAACG-3’
83
CsTIP2;1 orange1.1g025817 F: 5’-TCATTTCTTTTGGAGGTTGTAAAAA-3’
R: 5’-TCCCAATCCATCCATTATCG-3’
80
CsTIP2;2 orange1.1g025865
F: 5’-TGTGGTGGGGTGTATGAAAA-3’
R: 5’-TTATCTGACGAAACCCCATT-3’
83
CsTIP2;3 orange1.1g038895
F: 5’-GATCTGTTTGGGCTTTTTGG-3’
R: 5’-TTAAACATGACGAGGCACAA-3’
83
CsTIP3;1 orange1.1g025197
F: 5’-CGCAGCATCATCCATTAACA-3’
R: 5’-AAAGCTGCTTCTGCTTCTGC-3’
83
CsTIP4;1 orange1.1g025864 F: 5’-AAGCTGCTGTTTCTCTCTTGATG-3’
R: 5’-CAAAATGACAGCAGCCAAAAA-3’
88
CsTIP5;1 orange1.1g046726
F: 5’-TCTTGCGGAATTCATCTCAAC-3’
R: 5’-GCTGCGTCTGGACTCAATTT-3’
87
CsTIP6;1 orange1.1g042738 F: 5’-CTTGAAATCCACGAACCTCA-3’
R: 5’-AGCCCACCAATGGAAATAAA-3’
85
CsNIP1;1 orange1.1g023184 F: 5’-CCAAGAGGAGGACGCTGTT-3’
R: 5’-CCAGTACATGCCATTCACACA-3’
86
CsNIP2;1 orange1.1g036721 F: 5’-TTATTGGAACGGTGACAGGA-3’
R: 5’-AGGAATTGATGTGCAGTTGG-3’
81
CsNIP2;2 orange1.1g040981 F: 5’-CCATCATTCAAAAGGCCAGT-3’
R: 5’-AACCGGTTGTGCCAATAAAT-3’
82
CsNIP2;3 orange1.1g040755 F: 5’-CTCCTGCCTCAACAAAATGC-3’ 84
63
R: 5’-TTGCCTTTTGGAGGAGTTGA-3’
CsNIP3;1 orange1.1g023102
F: 5’-AAGAATTCGGCTGTGTCTGT-3’
R: 5’-CACGGAATTGAAACCGTGTA-3’
82
CsNIP4;1 orange1.1g046511 F: 5’-AGGCGAAGTCAGGATTCAAGT-3’
R: 5’-AACTGGAATATCCGGGAAGC-3’
89
CsNIP5;1 orange1.1g035030 F: 5’-ATGAATCTGGCATTGTGCAG-3’
R: 5’-CCCGGCTATGAGTATGTTGA-3’
83
CsNIP5;2 orange1.1g027840 F: 5’-CATGTGTGCGTAGCCATATGTAG-3’
R: 5’-AAATTAACGTGAAAATGCAGCAG-3’
87
CsNIP6;1 orange1.1g039196
F: 5’-GTTTCTGCCTTCTGGGTTGA-3’
R: 5’-GCCAAAGCATTGAGCTTCAC-3'
87
CsSIP1;1 orange1.1g026039 F: 5’-AAGAAATACACGTGGCTAAAATTCA-3’
R: 5’-TTTGGTTGGGTGCCAATAAC-3’
82
CsSIP1;2 orange1.1g026082 F: 5’-TCCTTCAACTCCAACAACCA-3’
R: 5’-ATGTTGCCCCAAAGTGAAAG-3’
84
CsSIP2;1 orange1.1g026600 F: 5’-ATCAGCTCAGATGAAGGCAAAT-3’
R: 5’-ATTCCAAAGGGATTCATCTACAAA-3’
90
CsXIP1;1 orange1.1g036381 F: 5’-ATCGACACTGGGTTTTCTGG-3’
R: 5’-TGGAGATGCTGACTTGGAAT-3’
88
CsXIP1;2 orange1.1g040654 F: 5’-TTACTGTTTGTTGGGCTTGG-3’
R: 5’-TTGAAGCATACCCATCCACA-3’
80
CsXIP2;1 orange1.1g045670
F: 5’-TCGCAACTATCACAGCCTTT-3’
R: 5’-GGCCTTTTTGCTAACCCTTT-3’
86
GAPC2
Glyceraldehyde-3-phosphate
dehydrigenase C2
F: 5’-TCTTGCCTGCTTTGAATGGA -3’
R: 5’-TGTGAGGTCAACCACTGCGACAT-3’
80
S2Table. Conserved specificity-determining amino acid residues in sweet
orange MIPs.
NPA ar/R Filter SDP1
Subfa
mily
1st 2nd H2 H5 LE
1
LE
2
P1 P2 P
3
P4 P5 P6 P7
CsPIP NPA NPA F H T R Q/E/
M
S A F/
D
W/
F
C/S G
CsTIP NPA NPA H/N I A/
G
R/
V
T/A/
V
S/
A
A Y W V/A/
M
A
CsNIP NPA/S
/G
NPA
/V
W/A/
G/T
V/I/
S
A/
G
R F/L/
V
S/
T
A Y/
F
I/L L/C/
S
A/
G
CsSIP NPT/S
/L
NPA V/A/S I/V/
H
P/
G
N/
S
M/F A/
V
A Y W G/A/
T
G
CsXIP N/SPL
/V
NPA V V A/
V
R V/M C A F W F/V G
1Specificity determining positions according to Froger et al. [34] and Zhang et al [12].
64
S3 Table. Similarity analysis of MIP genes from diploid (Phytozome) and
dihaploid (CAP) sweet oranges (C. sinensis) and haploid Clementine (C.
clementina).
DiploidSweet Orange Dihaploid Sweet Orange Haploid Clementine
Gene
name
ID
(Phytozome)
ID
(CAP)
Identity
(%)
E-value
Score
ID
(Phytozome)
Identity
(%)
E-value
Score
CsPIP1;1 orange1.1g018895 Cs7g31420 98 0 2858 Ciclev10032308m 98 0 2874
CsPIP1;2 orange1.1g023021 Cs7g31410 100 0 2607 Ciclev10032308m
Ciclev10032298m
99
99
0
0
2607
2621
CsPIP1;3 orange1.1g023107 Cs5g03460 100 0 3947 Ciclev10021502m 98 0 3864
CsPIP1;4 orange1.1g023069 Cs6g07970 100 0 2587 Ciclev10012384m 99 0 2565
CsPIP2;1 orange1.1g023108 Cs6g11660 Cs6g11690
Cs6g11700
97 96
94
0 0
0
1915 1905
1832
Ciclev10012375m Ciclev10012379m
Ciclev10012633m
99 92
92
0 0
0
2223 1880
1847
CsPIP2;2 orange1.1g022966 Cs8g16640 100 0 3713 Ciclev10028975m 99 0 3916
CsPIP2;3 orange1.1g019681 Cs7g25610 98 0 4703 Ciclev10032302m 98 0 5012
CsPIP2;4 orange1.1g023370 Cs8g02530 99 0 2968 Ciclev10029003m 99 0 3000
- No Homology No Homology - - - Ciclev10003297m - - -
CsTIP1;1 orange1.1g025548 orange1.1t03005 100 0 2558 Ciclev10012553m 99 0 2558
CsTIP1;2 orange1.1g025600 Cs8g17900 99 0 1976 Ciclev10029134m 99 0 2713
CsTIP1;3 orange1.1g037978 Cs8g17900 97 0 1062 Ciclev10029134m 97 4e-180 632
CsTIP1;4 orange1.1g025464 Cs7g28650 99 0 2351 Ciclev10032534m 99 0 2365
CsTIP2;1 orange1.1g025817 Cs1g15440 97 0 2888 Ciclev10026351m 100 0 3086
CsTIP2;2 orange1.1g025865 Cs1g15440 100 0 3081 Ciclev10026351m 97 0 2879
CsTIP2;3 orange1.1g038895 Cs5g08710 99 0 1293 Ciclev10021867m 99 0 1290
CsTIP3;1 orange1.1g025197 Cs5g17210 100 0 2403 Ciclev10021799m 97 0 2262
CsTIP4;1 orange1.1g025864 Cs4g19580 99 0 2378 Ciclev10009294m 99 0 2392
CsTIP5;1 orange1.1g046726 Cs9g14770 99 0 1237 Ciclev10006865m 99 0 1263
CsTIP6;1 orange1.1g042738 Cs9g14770 100 5E-114 412 Ciclev10006865m 97 0 1346
- No Homology No Homology - - - Ciclev10023306m - - -
CsNIP1;1 orange1.1g023184 Cs2g04370 100 0 3500 Ciclev10016171m 96 0 3231
CsNIP2;1 orange1.1g036721 Cs6g17690 100 0 2016 Ciclev10013768m 97 0 1887
CsNIP2;2 orange1.1g040981 Cs6g17690 98 0 1852 Ciclev10013768m 99 0 1945
CsNIP2;3 orange1.1g040755 Cs2g16610 98 0 2580 Ciclev10017700m 98 0 2580
CsNIP3;1 orange1.1g023102 Cs6g21290 100 0 2017 Ciclev10012382m 97 0 5222
CsNIP4;1 orange1.1g046511 Cs3g20790 100 2.00E-
156
1423 Ciclev10001994m 99 0 3113
CsNIP5;1 orange1.1g035030 Cs1g11150 98 0 1136 Ciclev10026151m 99 0 1409
CsNIP5;2 orange1.1g027840 Cs1g11140 97 0 2396 Ciclev10026151m 99 0 2533
CsNIP6;1 orange1.1g039196 Cs9g06260 100 0 2598 Ciclev10005554m 97 0 2495
- No Homology No Homology - Ciclev10003256m - - -
CsSIP1;1 orange1.1g026039 Cs5g26100 99 0 6557 Ciclev10021916m 99 0 6482
CsSIP1;2 orange1.1g026082 Cs3g01900 100 0 1579 Ciclev10005734m 99 0 1553
CsSIP2;1 orange1.1g026600 Cs6g16190 100 0 1789 Ciclev10012628m 98 0 4933
65
CsXIP1;1 orange1.1g036381 Cs8g08830 99 0 1170 Ciclev10029916m 99 0 1272
CsXIP1;2 orange1.1g040654 Cs8g08820 100 0 2099 Ciclev10028106m 99 0 2049
CsXIP2;1 orange1.1g045670 Cs8g08810 100 0 2283 Ciclev10028106m 99 0 2208
66
CAPÍTULO 2
EXPRESSION OF A CITRUS TONOPLAST INTRINSIC
PROTEIN (CsTIP2;1) GENE IMPROVES TOBACCO
PLANT GROWTH, ANTIOXIDANT CAPACITY AND
PHYSIOLOGICAL ADAPTATION UNDER STRESS
CONDITIONS
67
ABSTRACT
Tonoplast intrinsic proteins (TIPs) are a subfamily of aquaporins (AQPs),
belonging to the major intrinsic protein (MIPs) family. In a previous study,we
have shown thatthe CsTIP2;1expression is upregulated in citrus leaves and in
response to salt and drought stresses, suggesting its potential association
withthe leaf water status in both normal and stress conditions.Here, we show
that the CsTIP2;1 overexpression in transgenic tobacco increases plant growth
under optimal and water- and salt-stress conditions and also significantly
improves the water and oxidative status, photosynthetic capacity, transpiration
rate and water use efficiency of plants subjected to a progressive soil drying.
These results correlated with the enhanced mesophyll cell expansion, midrib
aquiferous parenchyma abundance, H2O2 detoxification and stomatal
conductance observed in the transgenic plants. Taken together, our results
indicate that CsTIP2;1 plays an active role in regulating the water and oxidative
statusrequired for plant growth and adaptation to stressful environmental
conditions and may be potentially useful for engineering tolerance to salinity
and drought stressesin citrus and other crop plants.
Key words: abiotic stress, aquaporin (AQP), major intrinsic protein (MIP),
orange, photosynthesis, reactive oxygen species (ROS).
1. INTRODUCTION
Crop plants are frequently exposed to a range of environmental factors
that adversely affect their growth and productivity. These include physical
factors, such as heat, chilling, freezing, drought and salinity, that collectively
result in water-deficit stress, leading ultimately to the production of reactive
oxygen species (ROS) and the generation of oxidative stress (Slater et al.,
2008). Understanding the basic mechanisms underlying the responses of plants
to water-deficit stress is therefore essential for the development of multistress-
tolerant crop plants.
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It has been shown that the plant water status is tightly regulated by
aquaporins (AQPs), which are membrane proteins that act as channels for
water and other small uncharged molecules of great physiological significance,
such as glycerol, urea, boric acid, silicilic acid, hydrogen peroxide (H2O2),
ammonia (NH3) and carbon dioxide (CO2) (Maurel et al., 2008, 2009). AQPs are
part of the large family of major intrinsic proteins (MIPs), found in
microorganisms, plants and animals, which is further subdivided in seven
subfamilies according to their sequence similarity and subcellular localization:
plasma membrane (PIP), tonoplast (TIP), nodulin-like (NIP), small basic (SIP),
GlpF-like (GIP), hybrid (HIP) and X (XIPs) intrinsic proteins (Johanson et al.,
2001; Maurel et al., 2009; Wudick et al., 2009). In plants, the total number of
MIPs is particularly high as compared with animals, with 35 members identified
in the genome of Arabidopsis (Johanson et al., 2001), 36 in maize (Zea mays)
(Chaumont et al., 2001), 33 in rice (Oryza sativa) (Sakurai et al., 2005), 28 in
Vitis vinifera (Fouquet et al., 2008), 56 in Populus trichocarpa (Gupta and
Sankararamakrishnan, 2009), 34in Citrus sinensis and 33 in Citrus clementina
(Martins et al., 2015). Such observation reinforces their critical role in the
regulation of the intrincate flows of water and solutes required for plant growth,
development and adaptive responses to the ever-changing environment.
TIPs represent a subfamily of plant AQPs that are predominantly located
at the tonoplast, although some members are found in membranes of
specialised organelles such as protein storage vacuoles, lytic vacuoles and
small vacuoles (Fleurat-Lessard et al., 2005; Jauh et al., 1998). TIP subfamily
consists of five subgroups regarding their sequence homologies: TIP1 (the
former γTIP), TIP2 (the former dTIP), TIP3 (the former aTIP and bTIP), TIP4
(the former eTIP) and TIP5 (the former zTIP) (Johanson et al., 2001). TIP1
isoforms are preferentially associated with the large lytic/central vacuole,
whereas TIP2s and TIP3s are preferentially associated with vegetative protein
storage vacuoles and seed protein storage vacuoles, respectively (Maurel et al.,
2008). Besides water, members of almost all TIP subclasses are also able to
transport urea and glycerol (Gomes et al., 2009; Wudick et al., 2009). Members
of some TIP subclasses of Arabidopsis have been also demonstrated to
transport gaseous NH3 (AtTIP2;1, AtTIP2;3) (Loqué et al., 2005) and H2O2
(AtTIP1;1, AtTIP1;2 and AtTIP2;3) (Bienert et al.,2007; Dynowski et al., 2008).
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TIPs are the most abundant proteins found in the tonoplast, accounting
for up to 40% of total tonoplast protein content (Higuchi et al., 1998). Thus, they
are believed to be responsible for the typically higher water permeability of the
tonoplast in comparison with the plasma membrane. For instance, water
transport in tonoplast vesicles from tobacco suspension cells was 100-fold
higher than that of plasma membrane vesicles (Maurel et al., 1997). It would
allow a rapid movement of water and other small molecules into the vacuole in
order to maintain the osmolality and turgor pressure required for cell expansion
and plant growth in a changing environment. However, reports demonstrating a
casual relationship among TIPs, water status and plant tolerance to abiotic
stresses remain limited and controversial (Peng et al., 2007; Sade et al., 2009;
Wang et al., 2011).
In our previous study, we characterized the complete set of Citrus
species MIPs (CsMIPs) by exploiting the available reference genomes of Citrus
sinensis and C. clementina and identified a TIP2;1 isoform, named CsTIP2;1,
which was highly expressed in citrus leaves and in response to drought and salt
stresses (Martins et al., 2015). Based on these data, we have proposed
CsTIP2;1 as a candidate gene involved in the maintanance of the leaf water
status during normal and stress conditions.Thus, the aim of the present study
was to address the functional characterization of this gene in order to test the
hypothesis of its involvement in the modulation of the leaf water status and
stress tolerance.
2. MATERIALS AND METHODS
2.1CsTIP2;1 cloning and generation of transgenic tobacco
The coding sequence of CsTIP2;1 was amplified from roots of drought-
stressed ‘Rangpur’ lime by RT-PCR, using the primers 5’-
TCCCTATTCTCTAGCTCTTTCTTGA-3’ and 5’-
CAAAGACTTTTTACAACCTCCAAAA-3’, cloned in pGEM-T, and then under
cloned in sense orientation into the SalI/NotI sites of pUC118 under the control
of the CaMV 35S promoter (35S-P) and terminator (35S-T) sequences. The
resulting 35S-P::CsTIP2;1::35S-T expression cassette was then removed from
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the pUC118 vector by digestion with PstI and inserted into the same restriction
site of the pCAMBIA2301 vector (Cambia, Australia). This vector contains the
neomycin phosphotransferase II (nptII) selectable marker gene and the beta-
glucuronidase (GUS) uidA scorable marker gene driven by the 35S-P. It was
introduced into the EHA105 Agrobacterium tumefaciens strain by direct DNA
uptake, and used for Agrobacterium-mediated genetic transformation of tobacco
as described previously (Cidade et al., 2012). Several independent transgenic
lines (T0 generation) derived from distinct transformation events were
transferred to soil and grown under standardized greenhouse conditions to
generate seeds by self-pollination. T1 seeds were collected individually from
each T0 plant and screened on plates containing MS medium + 50 mg L-1
kanamycin for analysis of antibiotic-resistant:-susceptible segregation ratio.
GUS histochemical assays (Jefferson et al., 1987) and PCR screening (Cidade
et al., 2012) were used to distinguish between transgenic and nontransgenic
plants.
2.2 RNA extraction and quantitative real-time RT-PCR (qPCR) analysis
Isolation of total RNAs, cDNA synthesis and qPCR analysis of the
transgenic and wild-type (WT) plants were performed as described previously
(Cidade et al., 2012). CsTIP2;1 primers 5’-CGCAGTTGCAATTGGTGCTA-3’
and 5’-GATGCCAGTGAGGATGGTGAT-3’ were used for qPCR analysis. The
actin primers 5’-CATCCCTCAGCACCTTCC-3’ and 5’-
CCAACCTTAGCACTTCTCC-3’ were used for the normalization of qPCR
analysis. Data were obtained from a pool of three biological replicates that were
individually validated.
2.3 Stress treatments
In vitro-grown plants of tobacco (Nicotiana tabacum cv. Havana) were
used in the experiments of Agrobacterium-mediated genetic transformation. The
stress tolerance of WT and transgenic lines was examined in three sets of
experiments. First, the WT and transgenic (T0 generation) plants were
transferred to soil and grown under standardized greenhouse conditions for
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three months. Then, five leaf disks (1-cm diameter) were collected from the first
fully expanded leaf from the apex of each three biological replicates and
subjected to dehydration for up to 180 min at room temperature (~22oC). The
fresh weight (FW) of the leaf disks was measured every 30 min using an
analytical balance, and the dehydration rate was determined as the percentage
FW loss relative to the initial weight. At the end of dehydration, the leaf disks
were sampled for H2O2 staining. In the second set of experiments, seven-day-
old WT and transgenic (T1 generation) seedlings were removed from Murashige
and Skoog (MS) germination medium for the WT and MS medium + 50 mg L-1
kanamycin (Sigma, St. Louis, USA) for the transgenic lines and transferred to
MS medium only (control treatment) or MS medium containing 200 mM
mannitol (Vetec, Rio de Janeiro, Brazil) or 150 mM NaCl (Merck, Darmstadt,
Germany). Each treatment contained three replicates composed of fifteen
seedlings for each line. The FW of individual seedlings was measured 20 days
after the treatments. In the third set of experiments, the 30 day-old WT and
transgenic (T2 generation) plants, with an average of 10 to 15 leaves, were
transplanted to 20 L pots, containing soil and washed sand (2:1 ratio), and
maintained in greenhouse under controlled humidity and air temperatures (70 to
80% relative humidity and 25 to 30ºC) for 35 days before the beginning of
drought stress experiment. Thereafter, the pots were closed with aluminum foil
to prevent water loss by evaporation, and a set of 80 plants (4 lines x 2
treatments x 10 biological replicates) subjected to the following treatments: (i)
10 plants in control of each line, in which plants were maintained at leaf
predawn water potential values of -0.2 to -0.4 MPa by irrigation; (ii) 10 plants in
drought of each line, in which the plants were exposed to a progressive soil
water deficit until their leaves reach predawn water potential values of -1.0 to -
1.5 MPa. The leaf predawn water potential was recorded on the third fully
expanded mature leaf from the apex of each plant, between 2 AM and 4 AM,
using a Scholander-type pressure pump (m670, Pms Instrument Co., Albany,
USA). Physiological and growth parameters were measured in individual plants
35 days after the beginning of the experiment (soil matric potentials of -0.1 MPa
and -1.3 MPa for control and drought stress treatments, respectively, as
estimated from the soil water retention curve).
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2.4 Analysis of H2O2 accumulation
In situ accumulation of H2O2 was examined based on histochemical 3,3′-
diaminobenzidine (DAB) staining as described (Thordal-Christensen et al.,
1997). Five leaf disks from each three biological replicates of the WT and
transgenic plants were immersed in 1 mg ml-1 DAB solution (pH 7.5) and
infiltrated using vacuum for 4 h. Leaf disks of control treatments were vacuum
infiltrated with water alone or H2O2 plus DAB. After incubation in room
temperature, samples were boiled in a solution containing 96% ethanol for 20
min and rinsed twice with 50% ethanol. Observations and photographic
documentation were done using a magnifying glassLeica EZ4D (Leica
Microsystems, Wetzlar, Germany).
2.5 Leaf anatomy
Leaf samples were collected from the third leaf from the apex of WT and
transgenic plants maintained for three months under standardized greenhouse
conditions. Samples of the median portion of the leaf blade were fixed in FAA
70 (Johansen, 1940) and stored in 70% (v/v) etanol. Samples were then
dehydrated in a graded ethanol series and embedded in methacrylate
(HistoResin, Leica), as described by Feder and O'Brien (1968). The material
was sectioned at 10 μm using a rotary microtome and sections were stained
with toluidine blue (Feder and O'Brien, 1968). Anatomical featureswere
measured, including adaxial epidermis, abaxial epidermis, palisade parenchyma
and spongy parenchyma. Observations and photographic documentation were
done using a photonic microscope Leica DM 300 (Leica Microsystems, Wetzlar,
Germany). Three biological replicates were used, each containing three slides.
2.6 H2O2 sensitivity assay of leaves
Photosynthetically active leaves from WT and transgenic plants were
excised and the petioles were immediately dipped in 5%, 10% and 20% H2O2
solutions and subjected to vacuum infiltration for 20 min. Leaves were then
incubated in a growth chamber for 48 h at 26±2oC under 16-h photoperiod. The
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extent of necrosis was recorded 24 and 48 h after the treatments. Each
treatment was composed of five replicates and the experiment was repeated
twice.
2.7 Physiological and growth analyses
Control (irrigated) and drought-stressed WT and transgenic (T2
generation) plants were subjected to physiological and growth analyses, which
included net CO2 assimilation rate (A), stomatal conductance to water vapor
(gs), leaf transpiration rate (E), instantaneous water use efficiency (WUE; A/E),
leaf relative water content (RWC), plant height, root collar diameter, root
volume, root length and root fresh and dry biomass. A, gs and E were measured
in the morning (08:00 - 09:00 AM) using a portable photosynthesis system LI-
6400 (Li-Cor) equipped with an artificial light resource (6400-02B RedBlue). All
measurements were made on the third, fully expanded mature leaf from the
apex of five biological replicates of each WT and transgenic lines. The artificial
light source was adjusted to provide a photosynthetic photon flux density of
1000 μmol m-2 s-1, above the saturation irradiance of N. tabacum. CO2 flux and
temperature were adjusted to maintain a concentration of 380 μmol mol-1 CO2
and 26°C inside the leaf chamber. The minimum pre-established time for
reading stabilization was set at 60 s and the maximum to save each reading, at
120 s. The maximum admitted coefficient of variation to save each reading was
0.3%. The parameters net CO2 assimilation rate (A) and transpiration rate (E)
were used to calculate the instantaneous WUE, the amount of CO2 fixed per
unit amount water lost by transpiration. The leaf relative water content (RWC)
was measured in six 0.5 cm diameter discs collected from the third, fully
expanded mature leaf from the apex of three biological replicates of each WT
and transgenic lines. The leaf discs were immediately weighed, floated on
distilled water at 28°C in the dark for 24 h, blotted, weighed again and finally
dried at 60°C for 48 h for dry weight determination. The leaf RWC was
determined as follows: RWC = (fresh weight – dry weight)/(turgid weight – dry
weight) ´ 100. Plant height and root length were measured in five biological
replicates using a ruler, while the root collar diameter was measured using a
calliper. Root volume was measured using the intact root system to displace the
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water column in a graduated cylinder. Roots were weighed to obtain fresh
biomass and dried at 60 °C for 48 h to obtain dry biomass.
2.8 Statistical analysis
Statistical analysis was carried out with the software BIOESTAT
(Universidade Federal do Pará, Brazil), which tested the experiments as a
completely randomized design. Statistical differences were assessed based on
the analysis of variance (ANOVA) and means were separated by Dunnett test,
with a critical value of p≤ 0.05, p≤ 0.01 and p≤ 0.001. Segregation patterns were
analyzed using the Chi-square test (χ 2) against the expected Mendelian ratios
of 3:1 or 15:1 for single- or double-locus insertion, respectively.
3 RESULTS
3.1 Overexpression of CsTIP2;1 in transgenic tobacco
To analyze the biological functions of CsTIP2;1, transgenic tobacco
plants overexpressing CsTIP2;1 were produced. Several independent
regenerants on kanamycin-containing medium, representing distinct
transformation events, were screened for the presence of the transgene by
GUS histochemical assay (S1a Fig.) and PCR (S1b Fig.). All the PCR-positive
transgenic lines expressed CsTIP2;1 at relatively high and varying levels, as
revealed by the qPCR analysis (S1c Fig.). The transgenic plants exhibited a
normal phenotype and were fertile. Segregation analysis of the nptII gene in the
T1 generation, based on the observed frequencies of kanamycin-resistant and -
susceptible T1 seeds, demonstrated that the transgene was integrated into the
genome of transgenic lines as a single locus (S1 Table). Transgenic lines
overexpressing CsTIP2;1 at varying levels were selected for further analysis.
3.2CsTIP2;1-overexpressing tobacco plants show enhanced tolerance to
dehydration
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WT and transgenic lines were subjected to short-term dehydration to
investigate whether the CsTIP2;1 overexpression correlates with stress
tolerance. Leaf disks of both WT and transgenic lines showed a steady
decrease in FW when they were dehydrated in an ambient environment.
However, at any time point after 30 min (L8) or 60 min (L1, L9 and L12) of
dehydration, the transgenic lines lost remarkably less water than the WT (Fig.
1a).At the end of 180 min of dehydration, the turgor differences between the WT
and transgenic lines were of significantly greater magnitude. These findings
sugests that the transgenic lines were more tolerant to dehydration.
3.3 Dehydration tolerance of CsTIP2;1-overexpressing transgenic lines
correlates with the inhibition of H2O2 accumulation
Since water leakage may also reflect the extent of membrane damage
caused by oxidative stress, it was of interest to determine the ROS
accumulation in the WT and transgenic lines after dehydration stress.
Histochemical staining by DAB was used to reveal in situ accumulation of H2O2
(Fig. 1b). Before dehydration, leaf disks of WT plants were stained less
intensely and mainly at the periphery, as a result of wounding. The intensity of
staining has increased when the WT leaf disks were treated with H2O2. After
180 min dehydration, the leaf disks of WT plants were more heavily stained
throughout and clearly distinct from the transgenic lines, which exhibited
significantly less intense DAB staining. Taken together, these data suggest that
there is a strict relationship between the inhibition of H2O2 accumulation and the
tolerance of CsTIP2;1-overexpressing transgenic lines to dehydration stress.
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Fig.1.Overexpression of CsTIP2;1 enhances the dehydration tolerance in
transgenic tobacco. (a) Relative dehydration rate in the WT and transgenic
lines (L1, L8, L9 and L12) under dehydration for 180 min in an ambient
environment, as measured by the reduction of fresh weight (FW) every 30 min.
Statistically significant differences at p≤0.05 (*) and p≤0.01 (**) between WT
and transgenic lines, at the same time point, are indicated. (b) In situ
accumulation of H2O2 in leaf disks from the WT and transgenic lines under
dehydration stress, as measured by histochemical staining with DAB.
Representative photographs showing staining of H2O2 in leaf disks of WT (180
min) and transgenic lines (L1, L8, L9 and L12) after 180 min dehydration. WT
leaf disks before (0 min) dehydration and treated with H2O2 or only H2O (without
DAB) were used as control treatments.
3.4CsTIP2;1-overexpressing tobacco plants show improved tolerance to
water and salt stresses
The WT and transgenic lines were also subjected to long-term water- and
salt-stress treatments to assess the effect of CsTIP2;1 overexpression on stress
tolerance. Seven-day-old seedlings of WT and transgenic lines were
transplanted onto MS medium containing NaCl or mannitol, or MS medium
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alone as a control, and maintained for 20 days. After the treatments, the
transgenic lines developed significantly higher fresh weights than the WT under
both normal (control) and stress growth conditions (Fig. 2).Under control
conditions, the seedling biomass of the transgenic lines was 1.7- to 2-´ higher
than the WT, except for L9. However, the biomass differences between all
transgenic lines tested and WT drastically increased from ~3- to 5-´ under
stress conditions. These results indicate that the overexpression of CsTIP2;1
had a significant effect on the improvement of plant growth and water- and salt-
stress tolerance.
Fig.2.Performance of WT and CsTIP2;1-overexpressing transgenic lines
under normal (control) and stress (salt or mannitol) growth conditions.
WT and transgenic (L1-L12) seedlings were transplanted onto MS medium
containing 200 mM mannitol, 150 mM NaCl, or MS medium only (control) and
the fresh weight of individual seedlings was measured 20 days after the
treatments. The data are means ± SE of three technical replicates composed of
five seedlings for each line. Statistically significant differences at P≤0.01 (**)
between WT and transgenic lines, at the respective stress treatment, are
indicated.
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3.5CsTIP2;1 overexpression improves tobacco growth, photosynthetic
capacity and WUE under drought stress
To dissect the physiological mechanisms of drought stress tolerance
induced by the CsTIP2;1 overexpression, WT and transgenic lines were also
grown in pots under greenhouse conditions and then exposed to a progressive
water deficit for 35 days, until reach leaf water potentials of ~-1.5 MPa, or
watered to soil field capacity daily as a control. After the treatments, the
CsTIP2;1 transgenic lines displayed distinctive drought resistance as compared
with the WT, which showed leaf rolling, wilting and drying symptoms (Fig. 3A).
Interestingly, the transgenic lines also exhibited a more developed root system
than the WT under drought stress (Fig. 3B). Analyses of growth confirmed that
drought-stressed CsTIP2;1 transgenic plants had significantly higher height,
root collar diameter, root volume, root length and root fresh and dry
biomass(Fig. 4).
Analysis of the leaf RWC revealed that drought-stressed CsTIP2;1
transgenic lines were able to maintain 80-90% of the leaf RWC of their control
counterparts. However, the leaf RWC of drought-stressed WT plants dropped to
more than 50% of that of the non-stressed (control) WT (Fig. 5). These results
seem to be consistent with theincreased size of theirpalisade parenchyma cells
as compared with the WT (see Fig. 8).
Photosynthetic gas exchange parameters showed that the drought-
stressed CsTIP2;1 transgenic lines were also able to maintain significantly
higher rates of A and E compared with the drought-stressed WT (Fig. 5). These
data correlated with the increased gs values found in drought-stressed CsTIP2;1
transgenic lines (Fig. 5). Higher rates of photosynthesis associated with the
non-corresponding increase in transpiration levels resulted in a significant 40-
70% increment in the instantaneous WUE (A/E) in two out of three drought-
stressed CsTIP2;1 transgenic lines (Fig. 5). Taken together, these results
indicate thatCsTIP2;1 overexpression improves growth, photosynthetic capacity
and WUE in the transgenic plants by maintaining a better water status of roots
and leaves and an increased stomatal conductance under drought stress
conditions.
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Fig.3.Phenotype of drought-stressed WT (left) and CsTIP2;1-
overexpressing transgenic lines (right). Pictures of the aerial part (A) and
roots (B) were taken 35 days after the beginning of drought stress experiment.
See materials and methods for further details.
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Fig.4.Growth analyses of WT and CsTIP2;1-overexpressing transgenic
lines under control (irrigated) and drought-stress conditions. WT and
transgenic (L1-L9) plants grown under greenhouse conditions were exposed to
a progressive soil water deficit until reach leaf water potentials of ~-1.5 MPa, or
maintained at leaf water potentials of -0.2 to -0.4 MPa as the control (irrigated)
treatment. Growth parameters were measured in individual plants 35 days after
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the treatments. The data are means ± SE of five biological replicates.
*Significantly different from drought-stressed WT at P£0.05.
Fig.5.Physiological responses of WT and CsTIP2;1-overexpressing
transgenic lines. WT and transgenic (L1-L9) plants were exposed to control
(well-watered) and drought-stress conditions as depicted in the legend of Fig. 4.
Physiological parameters were measured in individual plants 35 days after the
treatments. The data are means ± SE of five biological replicates.
*,**Significantly different from drought-stressed WT at P£0.05 or P£0.01,
respectively.
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3.6CsTIP2;1 overexpression promotes mesophyll cell expansion and
aquiferous parenchyma differentiation
To analyze the effects of CsTIP2;1 overexpression at the anatomical level,
leaf cross-sections of the WT and transgenic plants were collected and
visualized under a photonic microscope. The leaf thickness and anatomy were
significantly altered by the overexpression of CsTIP2;1 (Figs.6 and 7). The
transgenic plants showed a significant increase in the size of palisade (and also
spongy for L1) parenchyma cells as compared with the WT (Fig. 8). The
transgenic plants also exhibited, in comparison with WT, epidermis cells of
altered shape in the adaxial and/or abaxial face of leaves (Fig. 6). More
interestingly, the transgenic plants displayed the presence of abundant
aquiferous parenchyma cells over the midrib (Fig. 7), an anatomical
modification never reported before in AQP-overexpressing transgenic plants.
Fig.6.Leaf cross-section of WT (A) and the CsTIP2;1-overexpressing
transgenic lines L1 (B), L8 (C) and L9 (D), observed under photonic
microscope. ep, epidermis cells; st, stomata; pp, palisade parenchyma; sp,
spongy parenchyma; ci, crystal idioblasts. Arrow indicates the cuticle.
Magnification bars represent 100 µm.
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Fig.7.Cross-section of the leaf midrib vein of WT (A) and the CsTIP2;1-
overexpressing transgenic lines L1 (B), L8 (C) and L9 (D), observed under
photonic microscope. pa, aquiferous parenchyma; ci, crystal idioblasts; xy,
xylem cells. Magnification bars represent 200 µm.
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Fig.8.Quantitative analysis of the anatomy of leaf cross-section of WT and
the transgenic lines L1, L8 and L9; epidermis; palisade parenchyma;
spongy parenchyma; mesophyll. The data are means ± SE of three technical
replicates, each containing three slides composed for each line. Statistically
significant differences at P≤0.05 (*) and P≤0.01 (**) between WT and transgenic
lines.
3.7 CsTIP2;1 expression increases the resistance of transgenic tobacco to
exogenous H2O2 application
Since the WT and transgenic lines differed in their resistance toward
oxidative stress, we considered the possibility that CsTIP2;1 could permeate
H2O2 across the tonoplast for further detoxification. Thus, we tested the effects
of the exogenous application of different H2O2 concentrations in
photosynthetically active leaves of WT and transgenic plants. After 24 h, H2O2-
treated leaves of the transgenic plants did not exhibit the typical symptoms of
necrosis, starting near the base and then proceeding to the apex of the leaves,
as did the WT plants (Fig. 9). Leaves of the transgenic plants were more
resistant to H2O2 even 48 h after treatment, when WT leaves turned necrosed in
a great extent (Fig. 9). These results suggest that CsTIP2;1 contributes to
detoxification of excess amounts of H2O2 in the cytosol.
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Fig.9. Sensitivity of tobacco plants to H2O2-induced necrosis.
Photosynthetically active leaves of WT and CsTIP2;1-overexpressing
transgenic plants were treated with 5%, 10% and 20% H2O2 solutions.
Symptoms were recorded 24 h and 48 h after the treatments. Representative
results of experiments performed in five replicates are shown.
4 DISCUSSION
Transgenic lines overexpressing CsTIP2;1 at high and varying levels
(Fig. S1) exhibited significantly improved tolerance to either short- (dehydration)
(Fig. 1a) or long-term (water-deficit and salt) (Fig. 2) stresses as compared with
the WT.
The transgenic seedlings were able to accumulate larger biomass than
the WT under both favorable (control) and stress (mannitol, salt) conditions (Fig.
2). Such an observation can be explained by the enhanced mesophyll cell
expansion found in the CsTIP2;1-overexpressing transgenic lines (Fig. 6). The
central vacuole occupies as much as 90% of the volume in fully elongated cells
and its enlargement depends on the transport of osmotically active substances
and water influx across the tonoplast (Wudick et al., 2009). This influx
generates turgor pressure that drives cell expansion. Thus, the increased ratio
of vacuole per cytoplasm is believed to be the cause of the enhanced mesophyll
cell expansion, plant growth and beneficial adaptation of CsTIP2;1-
overexpressing transgenic plants to salt and water-deficit stresses, since their
larger cells can also be able to sequester greater amounts of toxic compounds
in relatively larger vacuolar compartments (Peng et al., 2007). Consistent with
this interpretation are the emerging studies reporting a causal relationship
between TIP expression and cell elongation. For instance, the overexpression
of a cauliflower TIP1-GFP fusion in tobacco suspension cells led to a two-fold
increase in cell surface (Reisen et al., 2003), while the overexpression of an
NtTIP1;1-GFP construct in tobacco BY-2 protoplasts increased 24% their
expansion rate (Okubo-Kurihara et al., 2009). A related result has been
obtained at the whole-plant level in Arabidopsis, which showed significantly
increased growth of vegetative (roots, leaves) and reproductive (seeds) organs
by overexpressing the PgTIP1;1 from Panax ginseng (Lin et al., 2007). These
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observations suggest that TIP activity has direct effects on vacuolar and cellular
expansion.
The CsTIP2;1-overexpressingtransgenic lines were observed to retain
their growth advantage when exposed to an adverse situation that they would
face in the field, of progressive soil drying (Figs. 3 and 4). Comparisons of the
root fresh and dry biomass (Fig. 4), as well as of the leaf RWC (Fig. 5), between
drought-stressed WT and CsTIP2;1-transgenic lines further confirm that the
improved growth of the transgenic plants under drought was due to the better
water status of their roots and leaves. Consistent with these findings was the
unusual observation that CsTIP2;1 overexpression drastically increased the
presence of midrib aquiferous parenchyma in the transgenic plants (Fig. 7). To
the best of our knowledge, such AQP-induced anatomical modification has
never been reported before, although the preferential expression of some AQPs
in vascular-associated tissues and cells types has been described in several
plant species (see Prado and Maurel, 2013). AQP expression in these sites has
been interpreted as crucial for the radial cell-to-cell water movement during exit
from the xylem vessels (Prado et al., 2013) and for the osmotically driven water
loading in xylem vessels during embolism refilling (Sakr et al., 2003; Secchi and
Zwieniecki, 2010). Altogether, our data provide functional evidence about the
role of CsTIP2;1 in water compartmentation, which is essential for turgor-driven
cell expansion and cell water status maintenance that ultimately promote plant
growth and drought resistance.
Our results also show that CsTIP2;1 overexpression has significantly
increased A, E, gs and WUE in the transgenic plants under soil drying (Fig. 5).
Since the stomatal conductance regulates A, E and WUE, improvement in these
components were caused by the changed stomatal behavior in the transgenic
plants. A role of aquaporins in the regulation of stomata movement has long
been suggested based on observations that the expression of specific AQPs,
including TIPs, was high or water stress-induced in guard cells (Fraysse et al.,
2005; Kaldenhoff et al., 1995; Otto and Kaldenhoff, 2000; Sarda et al., 1997;
Sun et al., 2007). However, only recently that a direct relationship between AQP
expression and stomatal movement has emerged. The initial studies showed
increased gs in NtAQP1-overexpressing transgenic tobacco and decreased gs in
NtAQP1-antisense lines growing under favorable conditions (Flexas et al.,
87
2006; Uehlein et al., 2003). A similar result has been obtained in rice
overexpressing the barley aquaporin HvPIP2;1 (Hanba et al., 2004). Tomato
plants overexpressing the tobacco aquaporin NtAQP1 also showed significantly
increased gs, transpiration and stomatal aperture under both normal and salt-
stress conditions (Sade et al.,2010). More recently, tobacco plants
overexpressing the ice plant aquaporin McMIPB were observed to show less
significant decreases in the photosynthetic rate and stomatal conductance than
the WT plants under soil water deficit conditions (Kawase et al., 2013). The
impact of AQPs on guard cells has been related to their direct effects in
transporting CO2 (Flexas et al., 2006; Uehlein et al., 2003) and water (Sade et
al., 2010), with the latter affecting stomatal aperture via changes in turgor
pressure of guard cells.
Interestingly, the enhanced dehydration tolerance in the transgenic lines
was correlated with the inhibition of H2O2 accumulation, as revealed by the
histochemical DAB staining analysis (Fig. 1b). We then postulated that
CsTIP2;1 might be involved in H2O2 detoxification, since functional studies
based on in vitro yeast-survival and -fluorescence transport assays have
recently shown that some plant TIPs are able to specifically conduct H2O2 (Azad
et al., 2012; Bienert et al., 2007; Dynowski et al., 2008). Our in vivo H2O2
sensitivity assay revealed that the transgenic lines were in fact more resistant to
H2O2 than the WT (Fig. 9), suggesting that CsTIP2;1 plays a function in the
subcellular partitioning of H2O2. Such a role is further supported by the fact that
CsTIP2;1 contains the typical signature sequences of H2O2 transporter MIPs
(Hove and Bhave, 2011), such as H-I-G-R in the ar/R filter and T-S-A-Y-W at
the P1-P5 positions (Martins et al., 2015).Although some TIPs from plants have
been recently shown to permeate H2O2 (Azad et al., 2012; Bienert et al., 2007;
Dynowski et al., 2008), the physiological role of H2O2 permeability across the
tonoplast still remains unclear (Dynowski et al., 2008). Yet, it is well known that
stressed plants must maintain their ROS pools at low levels in order to minimize
the cellular damage caused by oxidative stress (Harb et al., 2010; Foyer and
Shigeoka, 2011; Miller et al., 2010). It is also well documented that ROS
accumulation depends greatly on the balance between production and
concurrent scavenging by non-enzymatic (i.e., ascorbate, glutathione, a-
tocopherol, carotenoids and flavonoids) as well as enzymatic [i.e., superoxide
88
dismutases (SOD), catalases (CAT), ascorbate peroxidases (APX), guaiacol
peroxidases (POX), glutathione peroxidases (GPX), glutathione reductases
(GR) and glutathione-S-transferases (GST)] antioxidants (Buchanan and
Balmer, 2005). Based on our findings, we propose that TIPs may provide
another important mechanism of H2O2 detoxification by translocating this
reactive oxygen species into vacuoles, which possess a powerful scavenging
system involving the activities of peroxidases and flavonoids (Bienert et al.,
2006).
5 CONCLUSIONS
In conclusion, the results from this study suggest that CsTIP2;1 plays a
role in plant growth and adaptation to salt and drought stresses by a
combination of three mechanisms: (i) water permeation, resulting in increased
water and salt compartmentation and turgor-driven cell expansion; (ii) H2O2
detoxification, thus attenuating the stress-induced oxidative damage; and, (iii)
stomatal opening, contributing to higher photosynthetic carbon assimilation,
transpiration and water use efficiency. Although these mechanisms cannot work
under very severe drought scenarios, they will probably provide some degree of
homeostasis required for plant growth and productivity in naturally occurring dry
scenarios compatible with agriculture.
6 ACKNOWLEDGMENTS
This work was supported by research grants from Embrapa
(Macroprograma 2), CNPq (Brasília, Brazil) and FAPESP (São Paulo, Brazil).
We gratefully acknowledge the Ph.D. Degree scholarship to C.P.S. Martins by
CAPES (Brasília, Brazil).
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8 SUPPORTING INFORMATION
S1 Fig.Overexpression of CsTIP2;1 in transgenic tobacco plants. (a) GUS
activity detected in tissues of CsTIP2;1-overexpressing transgenic tobacco
plants. (b) PCR amplification of nptII gene in genomic DNA from leaves of WT
and transgenic tobacco plants. Lanes:M molecular weight marker, C- WT
tobacco, C+ pCAMBIA 2301/CsTIP2;1 vector, L1–L21 independent transgenic
lines. (c) qPCR analysis of CsTIP2;1 expression in transgenic tobacco plants.
The levels of mRNA expression were normalized to the corresponding value of
the b-actin signal. Normalized values of mRNAs accumulation are represented
using the signal value of L13 as a reference (1). The data are means ± SE of
three biological replicates and three technical replicates per plant. L1-L21
independent transgenic lines.
S1 Table.Segregation of the kanamycin resistance in the T1 generation of
CsTIP2;1-overexpressing transgenic tobacco plants.
97
Transgenic
line
Number of T1 seeds Number loci estimated
from Chi-square value
Chi-square
value
P value
Kanamycin
resistant
Kanamycin
sensitive
L1 119.3 37.7 1 0.43 (3:1) 0.93
L8 123.5 34.5 1 0.86 (3:1) 0.83
L9 114.0 35.0 1 1.38 (3:1) 0.71
L10 119.8 40.8 1 0.32 (3:1) 0.96
L12 125.8 41.5 1 0.08 (3:1) 0.99
98
CAPÍTULO 3
NOVEL CONNECTIONS BETWEEN GALACTINOL
SYNTHASE (GolS) EXPRESSION AND STRESS
TOLERANCE REVEALED BY THE MOLECULAR AND
FUNCTIONAL CHARACTERIZATION OF CITRUS GolS
GENES
99
ABSTRACT Genes encoding for Galactinol Synthase (GolS) are found in diverse plant
species and play important roles in the physiology of plant stress resistance,
photosynthate translocation and seed development. In this study we have
identified and characterized the complete set of GolS genes encoded by the
sweet orange genome. By homology searches against the Arabidopsis thaliana
GolS (AtGolS) sequences, eight CsGolS related genes were identified in the
sweet orange genome.Phylogenetic analysis showed that only three CsGolS
genes (CsGolS6, 7 and 8) have close orthologs in Arabidopsis and the others
seem to have evolved later from gene duplication events. The CsGolS genes
are distributed throughout four sweet orange chromosomes and exhibit anexon-
intron structureconserved with the Arabidopis orthologs, as well as conserved
domains at the amino acid level. Several stress-related cis-acting regulatory
elements were found in the promoter region of the CsGolS genes, including
ABRE, DRE/CRT, MYBS and LTRE. Analysis of gene expression in plants
subjected to drought and salt stresses, as well as to infection with 'Candidatus
Liberibacter asiaticus', showed that the CsGolS genes were differentially
regulated by the different stresses, suggesting a role for these genes in stress
tolerance.CsGolS6-overexpressing transgenic tobacco plants showed higher
photosynthetic capacityand growth under drought conditionsin comparison with
the wild-type (WT) plants. These results correlated with the increased levels of
ascorbate and dehydroascorbate produced by the transgenic plants.
Key words: abiotic stress, biotic stresses, Galactinol Synthase,orange,
physiological
1 INTRODUCTION
Environmental stresses are factors that limit the development or cause
injury to an organism. An important protection mechanism against
environmental stresses in plants is the accumulation of osmoprotectants
(Ronteinet al., 2002). Raffinose family of oligosaccharides (RFOs) have been
proposed to play important roles in stress protection in plants under biotic and
100
abiotic stress (Smirnoff and Cumbes 1989; Morelli et al., 2003; Morsy et
al.,2007; Nishizawa et al.,2008; Van den Ende and Valluru, 2009; Van den
Ende et al., 2011; Peshev et al., 2013). The principal metabolites of the RFOs
pathway are galactinol and myo-inositol. Galactinol is formed from UDP-
galactose and myo-inositol via the activity of galactinol synthase (GolS), which
is considered a key regulatoryenzyme in the pathway (Keller and Pharr 1996;
Peterbauer et al., 2001). GolS genes are found in diverse plant species and
associated with multiple developmental and environmental responses.
Evidences indicate that GolS is important in the physiology of plant stress
resistance, photosynthate translocation and seed development (Zhou et al.,
2012).
In Arabidopsis thaliana, there are seven members belonging to the GolS
gene family, of which AtGolS1, AtGolS2 and AtGolS3 mRNAs were detected in
mature seeds and were also induced by drought, salt and low temperature
stresses (Taji et al., 2002). The overexpression of GolS (AtGolS1, AtGolS2,
AtGolS4) and a raffinose synthase gene in transgenic Arabidopsis increased
the galactinol and raffinose concentrations and resulted in effective ROS
scavenging capacity andoxidative stress tolerance (Nishizawa et al.,2008;
Bolouri-Moghaddam et al., 2010). In leaves of tomato (Solanum lycopersicum)
and Arabidopsis seedlings, the transcript levels of GolS increased under
environmental stress, such as cold and desiccation (Downie et al., 2003; Zuther
et al., 2004). In Coffea arabica, the accumulationof raffinose, stachyose and the
differential transcriptional regulation of three GolS isoforms were observed in
response to abiotic stresses (dos Santos et al., 2011). In citrus,accumulation of
raffinose concomitant with the induction of a raffinose synthase gene was
reported in roots of drought-stressed mandarin, suggesting that raffinose is an
important osmolyte accumulating during drought stress in citrus (Gimeno et al.,
2009).
In biotic stress studies, transgenic tobacco expressing the cucumber
gene CsGolS showed increased galactinol levels in the roots and constitutive
resistance against the pathogens Botrytis cinerea and Erwinia carotovora (Kim
et al., 2008). Galactinol acts as an endogenous molecular signal for induction of
defence responses in plants (Kim et al., 2008). Transcripts encoding enzymes
related to the metabolism of raffinosewere identifiedin citrus plants infected with
101
Candidatus Liberibacter americanus (Calam) (Mafra et al., 2012). Among them,
agalactinol synthase isoform (named here as CsGolS8) increased by seven-fold
in infected plants. RFOs function as potent antioxidants to minimize the
oxidative stress that occurs near to the necrotic sieve elements formed during
infection.
The identification and understanding of the physiological and molecular
mechanisms of plant adaptation to biotic and abiotic stresses is of both
agronomic and economic importance. In the present study, we report the
identification and characterization of the complete set of the Citrus species GolS
(CsGolS) genes through a genome-wide analysis of the sweet orange reference
genome and characterization oftheir sequences, evolutionary relationships,
putative functions and expression patterns in various tissues and in response to
abiotic stresses and ‘Candidatus Liberibacter asiaticus’ infection. Transgenic
tobacco plants overexpressing the CsGolS6 were also generated and analyzed.
This study provides several evidences that the GolS gene family plays
important roles in the response and tolerance of citrus plants to both biotic and
abiotic stresses.
2 MATERIALS AND METHODS
2.1 Identification and sequences analysis
The HMM (Hidden Markov Model) profile of the PFAM
(http://pfam.sanger.ac.uk/) (Punta et al., 2012) motif 01501 (Glycosyl
transferase family 8) was used as a keyword to search the sweet orange
genome sequence database at Phytozome (http://www.phytozome.org/citrus/)
(Wu et al., 2014). The 10 Arabidopsis thaliana GolS protein sequences were
retrieved from TAIR (http://www.arabidopsis.org/), according to previous reports
(Nishizawa et al. 2008), and also used to align the sweet orange genome
sequence assembly available at Phytozome (v9.1) using the TBLASTN tool.
After merging the results from all these strategies, unique entries (with unique
locus ID) were identified to remove the redundancy. The resulting sequences
were manually inspected for the presence of characteristic and functionally
important GolS amino acids and motifs. Multiple sequence alignments of the
102
deduced amino acid sequences of the CsGolS and those of A. thaliana were
performed using the default parameters of ClustalW (Thompson et al., 1994).
The dendrogram was generated by the MEGA 6 program (Tamura et al., 2013)
using the Neighbor-Joining (NJ) method (Saitou; Nei,1987) and bootstrap
analysis (1,000 replications).
Information about coding sequence (CDS), full-length sequence and
predicted amino acid sequence was obtained for each sweet orange GolS gene
from the Phytozome database. The GRAVY (grand average of hydropathy) and
molecular weight of the deduced amino acid sequences were predicted by the
PROTPARAM tool available on the Expert Protein Analysis System (ExPASy)
proteomics server (www.expasy.ch/tools/protparam.html). The subcellular
localization of the CsGolS proteins were predicted using the WoLF PSORT toll
available at http://www.genscript.com/psort/wolf_psort.html.
One kb upstream region from the translation start site was obtained from
all the sweet orange GolS genes and subsequently analysed in the PLACE
database (http://www.dna.affrc.go.jp/PLACE/signalscan.html) to identify the
presence of the stress-responsive cis-acting regulatory elements ABRE (ABA-
responsive element; ACGTG), DRE/CRT (dehydrationresponsiveelement/C-
repeat; G/ACCGAC), MYBS (MYB binding site; TAACTG) and LTRE (low-
temperature-responsiveelement; CCGAC) in their promoters.
The physical locations of CsMIPs were determined by confirming the
starting position of all genes on each chromosome, using BLASTN searching
against the local database of the Citrus sinensis Annotation Project (CAP;
http://citrus.hzau.edu.cn/orange/). MapChart software was used to plot the gene
loci on the sweet orange chromosomes (Voorrips, 2002).
2.2 Plant materials and stress treatments
Two-years-old sweet orange [Citrus sinensis (L.) Osbeck] plants grafted
on Rangpur lime (Citrus limonia Osbeck), a rootstock highly resistant to
drought, were used in the drought stress experiment. The drought stress
experiment was carried out as described by Martins et al. (2015).For salt
treatments, sweet orange [C. sinensis (L.) Osbeck] seeds were in vitro
germinated as described by de Oliveira et al. (2011). Twenty-days-old seedlings
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of nucellar origin were selected, based on their uniformity, and transferred to
MS medium alone (control), MS medium containing 20% Polyethylene Glycol
6000 (Carbowax 6000, Union Carbide Corporation) or 150 mMNaCl (Merck,
Darmstadt, Germany). Each treatment consisted of fifteen biological replicates.
Leaves and roots were harvested 20 days after the treatments and immediately
frozen in liquid nitrogen and stored at -80oC. Plants were infected with
‘Candidatus Liberibacter asiaticus’ as described in Fan et al. (2012). Briefly,
two-year-old seedlings of rough lemon (C. jambhiri Lush.) and sweet orange (C.
sinensis L. Osbeck) were graft-inoculated with bud wood from ‘Ca. L.asiaticus’
infected ‘Carrizo’ citrange (C. sinensis L. Osbeck × P. trifoliata L. Raf.) trees
kept under greenhouse conditions. Plants were grafted with bud wood from
healthy Carrizo trees. All these plants were kept in a United States Department
of Agriculture Animal and Plant Health Inspection Service and Center for
Disease Control-approved and secured greenhouse at the University of Florida,
Citrus Research and Education Center, Lake Alfred. Three biological replicates
were produced for each citrus species in each treatment. Quantitative real-time
PCR was performed to confirm the presence of ‘Ca. L. asiaticus’ in the inoculum
source and inoculated plants as described in Li et al. (2006). Four fully
expanded leaves were sampled separately from ‘Ca. L.asiaticus’ inoculated
plant and mock-inoculated plants (used as controls) of each citrus species at 0,
7, 17, and 34 weeks after inoculation (WAI). Leaves were immediately frozen in
liquid nitrogen and stored at -80°C until use. Three biological replicates were
produced for each condition. In total, 12 plants with 48 leaf samples were
collected (2 species x 2 treatments x 3 replicates x 4 time points).
The stress tolerance of wild-type (WT) and CsGolS6-overexpressing
transgenic plants of tobacco (Nicotiana tabacum cv. Havana) was examined in
vitro and in greenhouse conditions. The WT and transgenic (T0 generation)
plants were grown in plastic pots of 15L, containing a mixture of soil and
washed sand (ratio 3:1),in agreenhouse for three months. The seeds were
collected and germinated on MS medium [Murashigeand Skoog (MS)] medium
for the WT and MS medium + 50 mg L-1 kanamycin (Sigma, St. Louis, USA) for
the transgenic lines.Ten-day-old WT and transgenic (T1 generation) seedlings
were removed from germination medium and transferred to MS medium only
(control treatment) or MS medium containing 20% PEG 6000 (Carbowax 6000,
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Union Carbide Corporation) or 150 mMNaCl (Merck, Darmstadt, Germany).
Each treatment contained three replicates composed of fifteen seedlings for
each WT and transgenic lines. The FW of individual seedlings was measured
20 days after the treatments.
Uniform plants of WT and transgenic lines (T2 generation) containing an
average of 10 to 15 leaves were used in the drought stress experiment. Plants
were acclimatized to greenhouse conditions (25±4°C, 16 h of light and RH
oscillating between 80 and 95%) during 45 days. The plants were grown in
plastic pots of 20L, containing a mixture of soil and washed sand (ratio 3:1).
Thereafter, the pots were closed with aluminum foil to prevent water loss by
evaporation and a set of 300 plants (5 lines x 4 treatments x 15 biological
replicates) were subjected to the following treatments: (i) 15 plants in control of
each line, in which plants were maintained at leaf predawn water potential
values of -0.2 to -0.4 MPa by daily irrigation; (ii) 15 plants in drought of each
line, in which the plants were exposed to a progressive soil water deficit until
their leaves reach predawn water potential values of -1.0 to -1.5 MPa; (iii) 15
plants in drought of each line, in which the plants were exposed to a
progressive soil water deficit until their leaves reach predawn water potential
values of -1.6 to -2.0 MPa and (iv) 15 plants in re-irrigation of each line, in which
plants recovery their leaf predawn water potential values to-0.2 to -0.4 MPa by
irrigation after drought. The leaf predawn water potential was recorded on the
third fully expanded mature leaf from the apex of each plant, between 2 AM and
4 AM, using a Scholander-type pressure pump (m670, Pms Instrument Co.,
Albany, USA).
2.3 RNA extraction and expression analysis of CsGolS in citrus
Total RNA extraction, cDNA synthesis and quantitative real-time RT-PCR
(qPCR) analysis were performed as described previously (de Oliveira et al.,
2011). qPCR primers were designed in order to avoid the conserved regions.
Primersequences are shown in Table S1. Glyceraldehyde-3-phosphate
dehydrogenase C2 (GAPC2) was used as an internal reference gene to
normalize expression among the different samples (Mafra et al., 2012). Data
were obtained from a pool of three biological replicates that were individually
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validated. The heatmap of expression of plants infected with ‘Candidatus
Liberibacter asiaticus’was generated using R 3.1.0 software.
Table S1. Primers used in the qPCR analysis.
Name Locus ID Primer Size product (bp)
CsGolS1 orange1.1g006648m F: 5’- CCCTGCAAGGAACTCGTGAC-3’ R: 5’- CAAGAACCAACCGGAAGCTG-3’
98
CsGolS2 orange1.1g043696m F: 5’- TCAAGAGATGTGGCCCGTTT-3’ R: 5’- CCAAAAGCACATGCACTGGA-3’
91
CsGolS3 orange1.1g007705m F: 5’- AGCCTTGGGCGTGTTACAGA-3’ R: 5’- AGCTTCCACCACCTCTCGTG-3’
97
CsGolS4 orange1.1g037463m F: 5’- GGGCGATGGTCTTTATGCAA-3’ R: 5’- GCGACTCACCATGTCCCAAT-3’
91
CsGolS5 orange1.1g041855m F: 5’- AAAACTCGAGCAGGGATCTCG-3’ R: 5’- GTTTTCCATCCGGCAGCTCT-3’
88
CsGolS6 orange1.1g024367m F: 5’- AGCACCAAGTCTGCCAAAGC-3’ R: 5’- AGCCCTTAACCAAGCCAACG-3’
89
CsGolS7 orange1.1g020230m F: 5’- GGCGACGACAAACAGTGAAA-3’ R: 5’- CAGCAGATGGGGCATTTCTT-3’
95
CsGolS8 orange1.1g019647m F: 5’-GCCTTCTGCTGATGGTAATGC-3’ R: 5’-CACAAACTGAACGGCAGCAG-3’
85
2.4 CsGolS6 cloning and generation of transgenic tobacco
The coding sequence of the CsGolS6was amplified from leaves of
drought-stressed‘Rangpur’ lime by RT-PCR, using the primers 5’-
ATGGCCCCTGATATCACCCCC-3’ and 5’-CTAAGCAGCAGACGGGGCGG-
3’,cloned in pGEM-T, and then cloned in sense orientation into the SalI/NotI
sites of pUC118 under the control of the CaMV 35S promoter (35S-P) and
terminator (35S-T) sequences. The resulting 35S-P::CsGolS6::35S-T
expression cassette was then removed from the pUC118 vector by digestion
with PstI and inserted into the same restriction site of the pCAMBIA2301 vector.
The pCAMBIA2301 vector contains the neomycin phosphotransferase II (nptII)
selectable marker gene and the beta-glucuronidase (GUS) uidAscorable marker
gene driven by the 35S-P. This vector was introduced into the EHA105
Agrobacterium tumefaciens strain by direct DNA uptake, and used for
Agrobacterium-mediated genetic transformation of tobacco as described
previously (Cidade et al., 2012).
Several independent transgenic lines (T0 generation) derived from
distinct transformation events were transferred to soil and grown under
standardized greenhouse conditions to generate seeds by self-pollination. T1
seeds were collected individually from each T0 plant and screened on plates
containing MS medium + 50 mg L-1 kanamycin for analysis of antibiotic-
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resistant:-susceptible segregation ratio. PCR screening (Cidade et al., 2012)
was used to distinguish between transgenic and nontransgenic plants. qPCR
analysis to assess the CsGolS6 expression levels in the different transgenic
lines was performed as described above.
2.5 Leaf anatomy
Leaf cross-sections of WT and the CsGolS6-overexpressing transgenic
lineswere collected from the third leaf from the apex of plants maintained for 45
days under greenhouse conditions. Samples of the median portion of the leaf
blade were fixed in FAA 70 (Johansen, 1940) and stored in 70% (v/v) etanol.
Samples were then dehydrated in a graded ethanol series and embedded in
methacrylate (HistoResin, Leica), as described by Feder and O'Brien (1968).
The material was sectioned at 10 μm using a rotary microtome and sections
were stained with toluidine blue (Feder and O'Brien, 1968). Anatomical
featureswere measured, including the adaxial epidermis, abaxial epidermis,
palisade parenchyma and spongy parenchyma. Observations and photographic
documentation were done using a photonic microscope Leica DM 300 (Leica
Microsystems, Wetzlar, Germany). Three biological replicates were used, each
containing three slides.
2.6 Primary metabolomics profiling
The extraction of primary metabolites, derivation standard addition and
sample injection for GC-MS analyzes were performed according to Lisec et al.
(2006) with modifications.The extraction of primary metabolites was performed
in 10 mg of lyophilized leaf tissue with the addition of the extracting solution
containing methanol: chloroform: water ( 2.5:1:1 v/v/v) 60 uL per sample and
ribitol internal standard (0.2 mg ml-1 in H2O). The volume of 1.5 ml of the
extraction mix was added to the plant material and homogenized by vortexing
for 10 seconds and incubated at 4°C for 30 min. The incubated was centrifuged
(14,000 rpm, 5 min) and 0.75 mL of ultra pure H2O was added to the
supernatant (1 ml).The mixture was homogenized by vortexing (15 seconds)
and centrifuged at 14,000 rpm for 15 minutes. The polar top phase containing
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the primary polar metabolites were collected and dried in SpeedVac without
heating.
In samples were added 40 uL methoxyamine hydrochloride (20 mg ml-1
in pyridine) for 2 h incubation at 37°C, under agitation of 950 rpm.We added 70
uL of a solution containing 1 ml of N-methyl-N-trimethylsilyltrifluoroacetamide
(MSTFA) and 20 uL of FAME and again incubated for 30 minutes at 37°C under
agitation of 950 rpm. The volume of 100 uL was transferred to vials to be
applied to GC-MS.
The GC-MS TruTOF system was composed by autosampler Split /
Splitless, Agilent 7890A gas chromatograph and mass spectrometer LECO
TruTof. The chromatograms and mass spectra were evaluated using the
Chroma TOF software 4.4 (Leco) and TagFinder 4.0 (Luedemann et al., 2008),
supplied with the library containing the retention time and fragmentation pattern
of the primary metabolites of Golm metabolome database. After identification,
the relative values of the metabolites were normalized by ribitol internal
standard, sample weight and the average of the relative value of the control
treatment (irrigated wild-type plant).
2.7 Statistical analysis
Statistical analysis was carried out with the software BIOESTAT
(Universidade Federal do Pará, Brazil), which tested the experiments as a
completely randomized design. Statistical differences were assessed based on
the analysis of variance (ANOVA) and means were separated by Dunnett test,
with a critical value of p≤ 0.05 andp≤ 0.01.
3 RESULTS AND DISCUSSION
3.1 Identification and sequence analysis of the CsGolS
Eight CsGolS related genes were identified from the sweet orange
genome sequence database at Phytozome (http://www.phytozome.org/citrus/)
by a homology search against the arabidopsis (http://www.arabidopsis.org/),
GolS according to previous report (Nishizawa et al., 2008), using the TBLASTN
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tool. Phylogenetic analysis revealed that CSGolSare highly homologous with
each other (Figure S1) and also share homology to the 10 AtGolS (Nishizawa et
al.,2008).CsGolS1, CSGolS2, CSGolS3, CSGolS4 and CSGolS5 have no
'close' orthologs with Arabidopsis genes, seeming to have evolved from gene
duplication events.CsGolS6,7 are orthologs of AtGolS2,3, while CsGolS8 is
ortholog of AtGolS1.
The physicochemical properties of CsGolS were examined to further
understand their molecular characteristics. The CsGolS genes encode proteins
ranging from 203 (23.68kDa) to 637 (73.62kDa) amino acids in length (Table
S1).All the CsGolS had a negative grand average hydropathy (GRAVY) score
(Table S1), suggesting that they are hydrophilic proteins. Analysis of the
predicted subcellular localization showed that all CsGolS are located in
cytoplasm (Table S1). Galactinol synthase (EC 2.4.1.123; GolS) is involved in
the first step of RFOs biosynthesis (Keller and Pharr, 1996) and issupposed to
act in the cytosol (Schneider and Keller, 2009).The deduced amino acid
sequence of all CsGolS isoforms contain common features of plant GolSs. They
have a domain of family 8 proteins (Glycosyltransferase 8), which is
characteristic of galactinol synthases (Sprenger and Keller 2000;Cantarel et al.,
2009).The disparate functions of the Glycosyltransferase 8 family as proven and
putative plant cell wall polysaccharide biosynthetic α-
galacturonosyltransferases, the eukaryotic galactinol synthases (GolS) as α-
galactosyltransferases that synthesize the first step in the synthesis of the
oligosaccharides stachyose and raffinose.The putative starch initiation protein
α-glucosyltransferases, and the large bacterial Glycosyltransferase 8 family of
diverse α-glucosyltransferases and α-galactosyltransferases involved in
lipopolysaccharide and lipooligosaccharide synthesis. Glycosyltransferase 8
family members are involved in several unique types of glycoconjugate and
glycan biosynthetic processes (Yin et al., 2010).
The exon-intron structure of all eight CsGolS genes was analyzed
usingthe sweet orange gene models annotated in the Phytozome.
CsGolS2,CsGolS3 and CsGolS6 had a single intron and two exons (Fig. S2).
On the other hand,CsGolS4,CsGolS5 and CsGolS8 had two introns and three
exons. CsGolS1 and CsGolS7 contained three introns and four exons. A similar
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number of exons and introns has been observed in the respective Arabidopsis
orthologs (Taji et al., 2002; Nishizawa et al., 2008).
Analysis of the 1 kb upstream promotor region of all the sweet orange
GolS genes revealed the presence ofstress-related cis-acting regulatory
elements, including ABRE, DRE/CRT, MYBS and LTRE. Our analysis showed
that six CsGolS containing the ABRE core motif, three the MYBS motif, three
the LTRE motif and two the DRE/CRT motif (Fig. S3). Four CsGolS genes
contained at least two of the four different cis-regulatory elements analyzed in
their promoterregions, with one gene containing all the four core motifs
(CsGolS7), one gene containing all the three core motifs (CsGolS4), two genes
containing two core motifs (CsGolS5 and CsGolS6), three genes containing
only one of the core motifs (CsGolS2, CsGolS3 and CsGolS8) and CsGolS1
with no one of the core motifs analyzed.
The positions of all eight CsGolS were mapped on the sweet orange
chromosomes by homology searches against the sweet orange genome
assembly available at the CAP database (Fig. S4). The closely related CsGolS
isoforms CsGolS4, CsGolS5 and CsGolS6 were respectively mapped on close
positions in the chromosome 3, suggesting that they evolved from duplication
events (Fig. S4).CsGolS8 was not exactly located on any chromosome probably
because of an incomplete physical map for sweet orange.
3.2 Expression of CsGolS genes upon abiotic and biotic stress conditions
To identify CsGolS genes potentially involved in abiotic and biotic stress
response and tolerance, qPCR analysis was performed in citrus plants
subjected to drought, PEG, high salinity (NaCl) and 'Ca. L. asiaticus' (HLB)
infection.CsGolS3, 5, 6 and 8 were strongly induced by drought in leaves, while
only CsGolS6 was induced by drought stress in roots (Fig. 1). CsGolS6 was
also the only CsGolS gene induced by salt stress in leaves, while CsGolS1, 2
and 5 were induced by this treatment in roots (Fig. 2). PEG treatment induced
the expression of CsGolS1, 2, 3 and 6 in leaves and CsGolS1 and 6 in roots
(Fig. 3).
The time-course transcriptional analysis in response to ‘Ca. L. asiaticus’
infection showed that all theCsGolS were downregulated at the early stage (7
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weeks) of disease in the highly susceptible sweet orange, but not in the tolerant
rough lemon, whose expression levels of all CsGolS did not significantly
changed at this stage (Fig. 4).At the later time points (17 and 34 WAI), the
major difference observed between tolerant and susceptible citrus genotypes
was the expression of CsGolS4 and CsGolS5, suggesting a role for these
genes in the tolerance to HLB.
Ortholog GolS in other species have been shown to be induced by
various stress treatments and hormones (Liu et al., 1998; Sprenger and Keller,
2000; Taji et al., 2002; Zhao et al., 2003; Wang et al., 2009; dos Santos et al.,
2011; Unda et al., 2012). For example, the PtGolS genes were recently shown
to be highly responsive to biotic or abiotic stresses in hybrid poplarand showed
differential induction with gene-specific patterns in source and sink leaves
(Philippe et al., 2010). Similarly, three of the seven A. thaliana GolS genes
(AtGolS1, AtGolS2, and AtGolS3) were shown to be stress-responsive genes
(Taji et al., 2002). It has been hypothesised that galactinol and RFOs function
as signals that modulate stress responses (Valluru and Van den Ende 2011;
Peshev et al. 2013). There is evidence that galactinol can act as a signal during
pathogen-induced systemic resistance, supporting its role in defence against
biotic stresses (Kim et al., 2008; Valluru and Van den Ende, 2011; ElSayed et
al., 2014; Peshev et al., 2013).
Fig.1. Expression analysis of CsGolS genes in response to drought. Ratios
(log2) of relative mRNA levels between stressed and control plants for all eight
CsGolS in leaves and roots, as measured byq PCR. GAPC2 was used as an
endogenous control. The bars show means ± SE from three biological
replicates.
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Fig. 2. Expression analysis of CsGolS genes in response to NaCl stress.
Ratios (log2) of relative mRNA levels between stressed and control plants for all
eight CsGolS in leaves and roots, as measured byq PCR. GAPC2 was used as
an endogenous control. The bars show means ± SE from three biological
replicates.
Fig. 3. Expression analysis of CsGolS genes in response to PEG. Ratios
(log2) of relative mRNA levels between stressed and control plants for all eight
CsGolS in leaves and roots, as measured byq PCR. GAPC2 was used as an
endogenous control. The bars show means ± SE from three biological
replicates.
Fig. 4. Expression analysis of CsGolS genes in response to ‘Ca. L.
asiaticus’ infection in rough lemon and sweet orange.Ratios (log2) of
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relative mRNA levels between infected and control plants at 0, 7, 17, and 34
WAI for all eight CsGolS, as measured by qPCR. GAPC2 was used as an
endogenous control.
3.3 CsGolS6-overexpressing transgenic tobacco shows enhanced
tolerance to dehydration and salt stresses
The CsGolS6 was further selected for functional characterization
because of its potential involvement in stress tolerance based on the results of
gene expression analysis (Figs. 1, 2 and 3). Independent transgenic lines on
kanamycin-containing medium, representing distinct transformation events,
were screened for the presence of the transgene by PCR (Fig. S5A).The PCR-
positive transgenic lines expressed CsGolS6 at relatively high and varying
levels, as revealed by qPCR analysis (Fig. S5B). The transgenic plants
exhibited a normal phenotype and were fertile.
The biomass accumulation was compared between WT and CsGolS6-
overexpressing transgenic plants subjectedto PEG or salt stress treatments. In
controlled conditions the growth between transgenic plants and WT showed
significant difference.WT plants showed yellow color and low growth compared
to transgenic plants in MS medium supplemented with NaCl or PEG6000. The
fresh weights of the transgenic plants were significantly higher than thoseof WT
upon PEG and salt treatments (Figure 5B). In Arabidopsis overexpression
AtGolS2, at 15 days after osmotic stress treatment, transgenic plant showed
stronger stress tolerance to the WTplants (Taji et al., 2002). These results
indicate that the orthologs CsGolS6 and AtGolS2 have similar function.
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Fig. 5.Biomass accumulation of WT and CsGolS6-overexpressing
transgenic lines under normal (control) and stress (salt and PEG) growth
conditions. A)Representative picture of the experiment, taken at 20 days after
the treatments. B) Quantitative analysis. WT and transgenic (L3-L12) seedlings
were transplanted in MS medium containing 20% PEG-6000, 150 mM NaCl, or
MS medium only (control). Statistically significant differences by Dunnett test at
p≤0.05 (*) between WT and transgenic lines, at the respective stress treatment,
are indicated.
3.4 Physiology and growth analysis ofCsGolS6-overexpressing transgenic
plants under drought stress
The CsGolS6-overexpressing transgenic (L3, L6, L10 and L12) and WT
plants were grown under greenhouse conditions and then exposed to drought
stress by decreasing water supply gradually during 45 days. Physiological
aspects such as leaf transpiration rate, stomatal conductance, photosynthetic
rate and leaf relative water content were measured during a dehydration and
rehydration cycle. CO2 assimilation rate (A) analysis showed no difference
between transgenic and WT plantsin control condition (Fig. 6). However, the
CsGolS6 transgenic plants were able to maintain significantly higher rate of
Athan the WT under mild and/or moderate drought stresses. In contrast with the
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WT, the transgenic plants showed photosynthetic rates similar, or even higher,
to those of the control plants after rehydration (Fig. 6), indicating that the
drought stress did not significantly affect their photosynthetic apparatus. Only
L3 and L12 showed stomatal conductance and leaf transpiration rate higher
than the WT in the control treatment (Fig. 6). However, all the transgenic lines
showed significantly higher gs and E than the WT in at least one drought
condition tested. No significant differences among the plants on these
parameters were observed after rehydration, except for L3 and L6, which
showed significantly higher E than the WT upon such treatment. L6 and L12
exhibited increased water use efficiency (WUE) under moderate drought stress,
while L3 and L10 showed decreased WUE in such treatment (Fig. 6).The
transpiration rate of the AtGolS2 transgenic plants was lower than that of
WTplants. When plants were rehydrated, all the transgenic plants recovered,
but none of the control plants survived (Taji et al., 2002).These results suggest
that CsGolS6 has a different physiological function than that performed by the
AtGolS2 ortholog, since the citrus gene did not decrease the transpiration rate
of the plants under drought stress, and even improved the CO2 assimilation
rateof the plants under such conditions. Thus, CsGolS6 plays a physiological
role in enhancing the photosynthetic rate under of the plants under drought
stress. Analysis of the leaf water content relative (RWC) revealed that the
transgenic plants were able to maintain higher RWC levels than the WT under
moderate drought stress (Fig. 7).
All the transgenic plants were significantly taller than the WT, irrespective
of the water treatment (Fig. 8). Transgenic plants exhibited significantly higher
values than WT under control conditions for many growth parameters. In the dry
leaf mass, the only L10 line was not significantly higher compared with WT in
moderate stress. The basal stem diameter in the L3 line not differ statistically
from the WT. The root length and volume of L3, L6 and L12 were significantly
higher than the WT under drought conditions (Fig. 8). The fresh root weight in
lines L3 and L10 was not significantly different from the WT in moderate stress.
Collectively, these results indicate that the transgenic lines showed a better
development in control and drought stress conditions than the WT.
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Fig. 6.Physiological analysis of WT and CsGolS6-overexpressing
transgenic plants under different water regimes. WT and T2transgenic (L3-
L12) plants were exposed to four water regimes: (1) control (leaf water potential
at -0.3 a -0.5 MPa); (2) mild stress 1 (leaf water potential at -1.0 a -1.5 MPa);
(3)moderate stress 2 (leaf water potential at -1.5 a -2.0 MPa);(4) rehydration
(leaf water potential at -0.3 a -0.5 MPa).The data are means ± SE of ten
biological replicates. *,**Significantly different from WT by Dunnett test at p£0.05
or p£0.01, respectively.
Fig. 7. Relative water content of WT and CsGolS6-overexpressing
transgenic lines under control (irrigated) and moderate drought-stress
conditions. WT and T2transgenic (L3-L12) plants. The data are means ± SE of
ten biological replicates. *Significantly different from drought-stressed WT by
Dunnett test at p£0.05.
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Fig. 8.Morphological analyses of WT and CsGolS6-overexpressing
transgenic lines under control (irrigated) and moderate drought-stress
conditions. WT and T2transgenic (L3-L12) plantsgrown under greenhouse
conditions were exposed to a progressive soil water deficit until reach leaf water
potentials of ~-2.0 MPa, or maintained at leaf water potentials of -0.3 to -0.5
MPa as the control treatment. The data are means ± SE of ten biological
replicates. *Significantly different from drought-stressed WT by Dunnett test at
p£0.05.
3.5 Mesophyll anatomy change in the CsGolS6-overexpressing transgenic
plants under drought stress
N. tabacum shows amphistomatic leaves with a dorsiventral mesophyll
type. In control conditions, there was a reduction palisade parenchyma in two
transgenic lines (L3 and L12), but an increase in L6 which, along with the
increase of the spongy parenchyma, contributed to the greater thickness of
itsmesophyll (Fig. 9). In drought conditions, all thetransgenic plants showed a
thicker spongy parenchyma than the WT, which contributed to the increased
thickness of the mesophyll in three (L3, L10 and L12) out of the four transgenic
lines (Fig. 9). The greater thickness of the spongy parenchyma is an adaptation
of some plants that exhibit drought resistance (Ennajeh et al., 2010), since it
increases the abundance of intercellular spaces, thus increasing the internal
117
diffusion of CO2, which contributes to the higher rate of CO2 assimilation under
stress by water deficit.
Fig. 9.Anatomical analysis of WT and transgenic lines overexpressing
CsGolS6. A - Leaf cross-section of WT and the CsGolS6-overexpressing
transgenic lines L3, L6, L10 and L12, observed under photonic microscope
under moderate drought stress, showing the mesophyll region. Magnification
bars represent 100 µm. Adaxialside (adx), abaxial side (adx), palisade
parenchyma (pp), spongy parenchyma (pe), glandular trichomes (tg), stomata
(*), the vascular tissue (arrow), idioblatos (i).B - Quantitative analysis of the
anatomy of leaf cross-section of WT and the transgenic lines L3, L6, L10 and
L12; epidermis cells; palisade parenchyma; spongy parenchyma; mesophyll.
The data are means ± SE of three technical replicates, each containing three
slides composed for each plant line. Statistically significant differences at
P≤0.05 (*) between WT and transgenic plants.
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3.6 Metabolomic changes in CsGolS6-overexpressing transgenic plants
In the control condition, the transgenic plants showed an increase in
ascorbate (1.00 to 7.4), dehydroascorbate (1.00 to 2.91) and fructose (1.00 to
1.69). In stress condition, ascorbate and dehydroascorbate levels were also
higher (1.55 to 4.29 and 0.78 to 3.68, respectively) in most transgenic plants,
while no differences were observed in the fructose levels between transgenic
and WT plants. Surprisingly, the transgenic plants exhibited reduced raffinose
levels (0.32 to 1.00) under control conditions compared to the WT. However,in
contrast with the WT, there was a lightly induction (0.53 to 1.19) in the synthesis
of this sugar in most transgenic plants under stress conditions.The connection
between increased levels of ascorbate and increased CO2 assimilation was
observed in transgenic tomato plants for the gene GME2 (Liu et al., 2011). The
role of ascorbate in photosynthesis is supported by the observation that
transgenic rice plants downregulated for L-galactono-1,4-lactone
dehydrogenase (GLDH), the gene encoding the last stepenzyme for ascorbate
synthesis, resulted in a loss of chlorophyll, a lowerribulose 1,5-bisphosphate
carboxylase/oxygenase protein content, and a lowerrate of CO2 assimilation. As
a consequence, a slower rate of plant growth and lower seed set were
observed. Conversely, increasing GLDH expression maintained high levels of
chlorophyll, rubisco protein, and a higher rate of netphotosynthesis, resulting in
higher seed set (Liu et al., 2011). These data indicate thatthe ascorbate level
and/or GLDH enzyme is closely associated with plant photosynthesis and
growth.
119
Fig.10.Metabolomic analysisof WT and CsGolS6-overexpressing
transgenic lines under control (irrigated) and moderate drought-stress
(non-irrigated) conditions.WT and T2transgenic (L3-L12) plants.
120
4 Conclusion
CsGolS genes act in response to abiotic and biotic stresses in citrus and
CsGolS6 has a physiological role in the tolerance to abiotic stress, increasing
the photosynthetic rate and growth under drought conditions presumably due to
the increased synthesis of ascorbate and dehydroascorbate.
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6 SUPPORTING INFORMATION
Table S1. Characteristics of genes encoding sweet orange GolS proteins.
Name ID phytozome ID CAP Location Polypeptide length (MW)
pI GRAVY Predicted subcellular localization
CsGolS1 orange1.1g006648m Cs1g05500 chr1:5,229,069..5,232,119
637 (73.62kDa)
6.61 -0.353 Cytoplasm
CsGolS2 orange1.1g043696m Cs7g19860 chr7:15,999,350..16,002,178
521 (60.54kDa)
5.95 -0.358 Cytoplasm
CsGolS3 orange1.1g007705m Cs3g23600 chr3:25,840,038..25,843,105
592 (69.36kDa)
9.04 -0.348 Cytoplasm
CsGolS4 orange1.1g037463m Cs3g13670 chr3:18,022,540..18,025,263
344 (40.74kDa)
6.54 -0.497 Cytoplasm
CsGolS5 orange1.1g041855m Cs3g13720 chr3:18,115,719..18,118,535
203 (23.68kDa)
9.54 -0.393 Cytoplasm
CsGolS6 orange1.1g024367m Cs3g15460 chr3:19,579,954..19,581,767
268 (30.85kDa)
5.71
-0.103 Cytoplasm
CsGolS7 orange1.1g020230m Cs9g01710 chr9:468,115..470,386
329 (37.94kDa)
4.78 -0.188 Cytoplasm
CsGolS8 orange1.1g019647m orange1.1t01729
chrUn:27,794,711..27,796,299
337 (38.27kDa)
5.18 -0.193
Cytoplasm
125
Fig.S1. Phylogenetic relationships of sweet orange GolS with those of
Arabidopsis thaliana. The deduced amino acid sequences were aligned using
ClustalW2 and the phylogenetic tree was generated using Bootstrap N-J tree
(1,000 resamplings) method and MEGA program (v6.0.5). Numbers at internal
nodes denotes the results of bootstrapping analysis (n = 1000). Cs, Citrus
sinensis; At, Arabidopsis thaliana.
Fig.S2.Analysis of exon-intronstructures of the eight sweet orange GolS
genes.NOI denotes the number of introns, E the exon and I the intron.
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Fig.S3. Analysis of the stress-responsive cis-elements ABRE (ACGTG),
DRE/CRT (G/ACCGAC), MYBS (TAACTG) and LTRE (CCGAC) in promoters
of sweet orange GolS genes.Thecis-elements were analyzed in the 1 kb
upstream promoter regionof translation start site ofallCsGolS using PLACE
database.
Fig.S4. Chromosomal locations of CsGolS.The chromosomal position of
each CsGolS was mapped according to the Citrus sinensis Annotation Project
(CAP).The scale is in Mb.
127
Fig. S5.Overexpression of CsGolS6 in transgenic tobacco plants. (A) PCR
amplification of nptII gene in genomic DNA from leaves of WT and transgenic
tobacco plants. Lanes: M molecular weight marker, C- WT tobacco, C+
pCAMBIA 2301/CsGolS6 vector, L3–L12 independent transgenic lines. (B)
qPCR analysis of CsGolS6 expression in transgenic tobacco plants.
Normalizedvalues of mRNAs accumulation are represented using the signal
value of WT as a reference. The data are means ± SE of three biological
replicates and three technical replicates per plant.
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APÊNDICE
ESTUDO DA INTERAÇÃO ENTRE FATORES DE
TRANSCRIÇÃO MYBs E GENES MIPs ENVOLVIDOS
NA RESPOSTA DE CITROS A DEFICIÊNCIA HÍDRICA
E A INFECÇÃO POR Candidatus Liberibacter spp.,
BACTÉRIAS CAUSADORAS DO HLB
129
1 INTRODUÇÃO
O Brasil é o segundo maior produtor de citros e o maior produtor de
laranja em nível mundial, sendo responsável por 22% da produção total de
laranja e por 52% da produção de suco de laranja concentrado e congelado
(AGRIANUAL, 2014). Apesar do grande volume de produção dos citros no
mundo, a citricultura tem sofrido com perdas, causada por pragas, doenças e
estresses abióticos, que podem influenciar de maneira determinante na
produtividade (GMITTER et al., 2007).
Dentre os estresses bióticos, destaca-se o Huanglongbing (HLB),
popularmente conhecido como ‘greening’. O HLB é considerado a doença mais
destrutiva da citricultura, sendo relatada nas principais áreas produtoras do
mundo (SUTTON et al., 2005; BOVE, 2006; GOTTWALD, 2010). Sua presença
nos pomares tem gerado significativas perdas de produtividade e considerável
aumento nos custos de produção (BOVE, 2006; GOTTWALD, 2010).Os
sintomas de HLB incluem folha com clorose variegada, crescimento atrofiado,
pouco crescimento das raízes, frutos verdes e malformados e, finalmente, a
morte. Os sintomas iniciais de HLB são muito semelhantes aos causados por
deficiência de nutrientes, tais como zinco, magnésio, ou ferro.
HLB é causada por uma bactéria do floema do gênero Candidatus
Liberibacter. Três espécies deste agente patogênico estão associadas com a
doença em citros. A forma asiática, Candidatus Liberibacter asiaticus (Calas), é
encontradaem todos os países afetados por HLB excluindo-seos países do
continente Africano. O tipo Africano, Candidatus Liberibacter africanus (Calaf) e
o Americano, Candidatus Liberibacter americanus (Callan), são encontrados
atualmente somente na África e no Brasil, respectivamente (BOVE, 2006).
Calas é transmitida por Diaphorina citri kuwayama, o psilídeo asiático do citros.
No Brasil, o HLB foi descrito pela primeira vez na região de Araraquara, em
Março de 2004 (COLLETTA FILHO et al., 2004). Atualmente, a doença ocorre
nos estados do Paraná e Minas Gerais e está presente em todas as áreas de
citros do Estado de São Paulo. Em 2010, sua incidência chegou a 24% dos
pomares em São Paulo, com tendência de crescimento observada em anos
anteriores (BELASQUE Jr. et al., 2010).
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Dentre os estresses abióticos, o que mais influencia negativamente a
produtividade da citricultura no Brasil é a seca. As plantas cítricas necessitam
de água para manter um crescimento contínuo e apresentar boa produção de
frutos, porém a distribuição pluviométrica no Brasil é bastante irregular e
apesar da incidência de déficits temporários nas regiões citrícolas, o cultivo de
citros é realizado praticamente sem irrigação (SANTOS et al., 1999). Diante
dessa situação, a utilização de porta enxertos com tolerância à deficiência
hídrica se faz necessária.
A adaptação de plantas à seca é um fenômeno complexo que envolve
diversas vias de sinalização e alteração do perfil de expressão gênica
(IKEGAMIet al., 2009).
Em plantas superiores, a expressão dos genes geralmente é regulada
por interações entre proteínasregulatórias e promotores. Um número
significativo de fatores de transcrição tem sido identificado em angiospermas,
capazes de influenciarno controle de expressão de vários genes-alvo em vias
de transdução de sinais (VANDEPOELE et al., 2009). MYB compreende uma
família de fatores de transcrição que está presente em todos os eucariontes.
Em animais e fungos observa-se um número restrito de genes que codificam
para esses fatores, enquanto que em plantas há uma grande variabilidade de
fatores MYB. Em Arabidopsis foram identificados 85 genes MYB, 42 em cana-
de-açúcar, 71 em eucalipto (ROMERO et al., 1998; SOARES-CAVALCANTI et
al., 2009) e177 em laranja doce (HOU et al., 2014). Um estudo prévio sobre a
análise do transcriptomade folhas de limão rugoso (Citrus jambhiri; tolerante ao
HLB) e laranja doce (C. sinensis; suscetível ao HLB) em resposta à infecção
por Candidatus Liberibacter asiaticus, empregando-se a tecnologia de
microarranjos, evidenciou a expressão diferencial de MYBs, sendo umgene
induzido(CsMYB250) e três genes(CsMYB130, CsMYB135 eCsMYB69)
reprimidos em limão rugoso e 12 genes induzidos (CsMYB135, CsMYB163,
CsMYB250, CsMYB130, CsMYB260, CsMYB74, CsMYB67, CsMYB66,
CsMYB330, CsMYB81, CsMYB38 e CsMYB156) e quatro genes reprimidos
(CsMYB79, CsMYB480, CsMYB950 e CsMYB70) em laranja doce, na 27ª
semana após a inoculação (FAN et al., 2012).
As MIPs (‘Major Intrinsic Proteins’) ou aquaporinas constituem uma
superfamília de proteínas de membrana que atuam como componentes
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centrais das relações hídricas em plantas (JOHANSON et al., 2001; JAVOT;
MAUREL, 2002). As MIPs vegetais são importantes não somente para relações
planta-água, mas também para diversos aspectos fisiológicos, como resposta a
estresses abióticos, bióticos e de sinalização, já que são capazes de
transportar pequenas moléculas não-carregadas de significância biológica,
incluindo glicerol, ureia, amônia (NH3), CO2, ácido bórico, ácido silícico e
peróxido de hidrogênio (H2O2), etambém de regular o carregamento e
descarregamento do floema, fechamento estomático, movimento foliar e a
homeostase citoplasmática (MAUREL et al., 2008).
Recentementea expressão diferencial de MIPs foi demonstrada entre os
genótipos tolerante (limão rugoso) e suscetível (laranja doce) ao HLB, em
resposta à infecção por Candidatus Liberibacter asiaticus (MARTINS et al.,
2015). Foi observado uma regulação negativa da maioria das CsMIPs em
laranja doce na sétima semana após a inoculação (SAI), porém o mesmo não
foi observado em limão rugoso, podendoesse resultado estar correlacionado
com o aparecimento dos sintomas dadoença no genótipo suscetível, tais como
manchas foliar e amarelecimento (FAN et al., 2012). Os padrões de expressão
de CsPIP2;2, CsTIP1;2, CsTIP2;1, CsTIP2;2 e CsNIP5;1 foram contrastantes
entre as espécies de citros tolerantes e suscetíveis ao longo da 17ªSAI e/ou
34ªSAI, sugerindo envolvimento destes genes no desenvolvimento dos
sintomas mais tardios da doença, tais como a inibição do crescimento e
comprometimento da distribuição do floema (FAN et al., 2012). Em conjunto,
esses resultados sugeremque os genes MIPs podem desempenhar um papel
ativo na patogênese de HLB e que a expressão gênica dessa família pode ser
regulada por MYBs.
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2 OBJETIVO GERAL
Investigar a indução da expressão de genes MIPs por fatores de transcrição
MYBs.
2.1 Objetivos específicos
· Validar, via qPCR, os dados de co-expressão de MYBs e MIPs em
amostras de folhas de limão rugoso e laranja doce infectadas por
Candidatus Liberibacter asiaticus.
· Realizar ensaios de coinfiltração (dual GFP transient assay) em folhas de
Nicotiana benthamiana para comprovar a transativação da transcrição de
MIPs por MYBs;
3 MATERIAL E MÉTODOS
3.1 Material vegetal e estirpe de Agrobacterium
Nicotiana benthamiana e Agrobacterium tumefaciens EHA101 foram
utilizadas para a realização dos ensaios de coinfiltração.
3.2 Análise de expressão gênica
As amostras de cDNA que foram utilizadas nas analises de qPCR foram
cedidas pela Universidade de Florida. As plantas foram infectadas com
Candidatus Liberibacter asiaticus, tal como descrito em Fan et al. (2012) e
Martins et al. (2015).
Os procedimentos de qPCR, incluindo testes, validações e
experimentos, foram conduzidos no aparelho ABI 7500 (AppliedBiosystems,
EUA), seguindo as instruções do fabricante. Gliceraldeído-3-fosfato
desidrogenaseC2 (GAPC2) foi usado como um gene de referência interno para
normalizar expressão entre as diferentes amostras (MAFRA et al., 2012).
Os dados foram obtidos a partir de um pool de três réplicas biológicas
que foram validados individualmente e duas réplicas experimentais, em volume
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de reação de 22 μl, 10 μl contendo 75-100 ng de cDNA, 1 μl de
oligonucleotídeo(F+R) na concentração de 10 nM, 11 μl de Maxima® SYBR
Green/ROX qPCR Master Mix (2X) (Fermentas, EUA). As condições de
amplificação foram realizadas utilizando-se as seguintes etapas: (1) ativação
da Taq DNA polimerase a 50ºC por 2 min, (2) desnaturação inicial a 95ºC por
10 min, (3) desnaturação a 94ºC por 15s, (4) anelamento a 60ºC por 30s e (5)
extensão a 60ºC por 1 min. As etapas 3 a 5 foram repetidas por 40 ciclos.
Para quantificação da expressão gênica foi utilizado o método
comparativo de Ct:2-ΔΔCt. Reações controle desprovidas de cDNA (NTC)
também foram utilizadas em todos os experimentos. O programa Dissociation
Curve 1.0 (AppliedBiosystems, EUA) foi usado para verificar que somente um
único produto de PCR foi gerado pela amplificação dos transcritos. O heatmap
foi gerado para comparação dos resultados,utilizando o softwareR3.1.0.
3.3 Clonagem Gateway
A partir da análise de qPCR foram selecionados alguns genes para
validar a interação deMYBs com asregiões promotoras de MIPs. As
construções MIPpromotor::GFP e 35S::MYB que foram utilizadas para
coinfiltração em folhas de Nicotianabenthamianaforam obtidas utilizando o
sistema de clonagem Gateway (Invitrogen).
Os oligonucleotídeos desenhados para amplificação dos genes contêm
a sequência correspondente do DNA, a sequência relativa aos sítios de
recombinação (attb1 e attb2), a sequência responsável pela ligação ao
ribossomo (Shine-Dalgarno) e o fragmento Kozac. As sequências das regiões
promotoras das diferentes MIPs e região codante dos genes MYBs foram
obtidas por PCR e purificadas a partir de géis de agarose. Estas sequências
foram clonadas no vetor de entrada pENTR/D-TOPO (Gateway, Invitrogen),
utilizando o pENTR Direcional TOPO Cloning Kit (Gateway, Invitrogen),
seguindo as instruções do fabricante.
Escherichia coli da linhagem DH5a (Clontech, Heidelberg, Germany)
competentes foram transformadas com o vetor recombinante por choque
térmico. Os clones positivos foram selecionados por meiode PCR de colônias.
Todos os vetores obtidos foram sequenciados utilizando o oligonucleotídeo de
134
iniciação universal M13 “Forward” em conjunto com o oligonucleotídeo reverso
utilizado para amplificação de cada uma das sequências por PCR, de modo a
confirmar a presença do fragmento de inserção no local e na orientação
corretos.
O vetor TOPO recombinante foi linearizado e dosado para ser utilizado
nas reações de recombinação BP. Colônias transformadas foram plaqueadas
em meio LB-canamicina líquido, o DNA e cDNA foi quantificado para ser
utilizado nas reações de recombinação LR. Para a reação de recombinação
foram usados 300ng do vetor pDest15™ e de 100 a 300ng do vetor
pEntry/pKGWFS7 e pEntry/pH2GW7.
3.4 Ensaios de coinfiltração de folhas de Nicotiana benthamiana e análise
de fluorescência
Folhas de N. benthamiana foram coinfiltradas com suspensão de
Agrobacterium tumefaciens (OD600= 0,5) contendo as construções
MIPpromotor::GFP e 35S::MYB. Foram realizadas coinflitrações com diferentes
combinações de genes selecionados das famílias MYB e MIP. Para a infiltração
foi utilizado uma seringa (sem agulha), onde foi efetuado uma pressão na face
inferior da folha, uma contra-pressão com o dedo no outro lado. A infiltração de
sucesso foi observada como uma área de "molhagem" espalhada na folha (Li,
2011).
As folhas de tabaco coinfiltradas foram analisadas relativamente
quanto à atividade do gene gfp, que codifica a ‘Green Fluorescent Protein’
(GFP), sob luz UV. O sinal de fluorescência foi observado com o microscópio
confocal de varrimento a laser.
4 RESULTADOS
Para análise de expressão gênica, 25 genes MYBs foram selecionados
com base no trabalho de Fan et al. (2012). A análise da transcrição temporal
em resposta a infecção por ‘Ca. L. asiaticus' mostrou que todos os CsMYBs
apresentaram padrão de expressão diferencial. Os resultados demonstraram
135
que 36% dos genesnão foram expressos em Valencia ou Limão Rugoso e 20%
dos genes não foram expressos em ambas as variedades (Fig. 1).
De acordo com a análise de expressão, os genes CsMYB81,
CsMYB83, CsMYB126 e CsMYB480 foram selecionados para realização dos
ensaios de coinfiltração em folhas de Nicotiana benthamiana juntamente com
os genesCsPIP1;4 e CsTIP2;1. Os dados de expressão gênica de CsMYBs e
CsMIPs foram submetidos a análise de correlação de Pearson, cuja
significância dos coeficientes (r) foi determinada pelo teste t de student. Os
resultados permitiram evidenciar correlações significativas tanto positivas como
negativas entre CsMYBs e CsMIPs ao longo do período de desenvolvimento da
doença (Tabela 1).
Folhas de Nicotiana benthamiana foram coinfiltradas com diferentes
combinações de genes selecionados das famílias CsMYBs e CsMIPs, sendo
realizadas um total de oito coinfiltrações. Foram realizadas coinfiltrações em
duplicatas em dias diferentes, onde a indução da expressãodo gene gfp sob luz
UV foi aparente em quatro combinações de coinfiltrações: CsTIP2;1:CsMYB81,
CsPIP1;4:CsMYB83, CsTIP2;1:CsMYB83 eCsPIP1;4:CsMYB83 (Fig. 2).
136
Fig. 1.Análise da expressão de MYBs de citros em resposta a infecção por
'Ca. L. asiaticus"em limão rugoso e laranja doce. Níveis de mRNA relativos
(log2) entre plantas infectadas e controle a 0, 7, 17, 34 e semanas após a
infecção para todos os CsMYB, via qPCR. GAPC2 foi usado como controle
endógeno.
137
Tabela 1.Coeficientes de correlação de Pearson entre os dados de co-
expressão de CsMYBs e CsMIPs. A: Amostras de folhas de laranja doce
infectadas por Candidatus Liberibacter asiaticus. B: Amostras de folhas de
limão rugoso infectadas por Candidatus Liberibacter asiaticus. Correlações
significativas apresentam* ou ** para p<0.05 ou p<0.01, respectivamente.
138
Fig. 2. Ensaio de coinfiltração em folhas de Nicotiana benthamiana. A:
coinfiltração CsTIP2;1:CsMYB81; B: coinfiltração CsPIP1;4:CsMYB83; C:
coinfiltração CsTIP2;1:CsMYB83; D: coinfiltração CsPIP1;4:CsMYB83. Seta:
fluorescência indicando a indução de expressão de GFP na área de
coinfiltração.
5 CONCLUSÃO
· Foi possível confirmar a co-expressão via qPCR de alguns genes MYBs e
MIPs em amostras de folhas de limão rugoso e laranja doce infectadas por
Candidatus Liberibacter asiaticus.
· Quatro combinações de coinfiltrações (CsTIP2;1:CsMYB81,
CsPIP1;4:CsMYB83, CsTIP2;1:CsMYB83 e CsPIP1;4:CsMYB83) induziram
a expressão do gene gfp sob luz UV em folhas de Nicotiana benthamiana,
sugerindo que a expressão dos genes MIPs analisados é induzida pelas
respectivas MYBs.
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CONCLUSÃO GERAL
Um total de 34 membros da família gênica MIP (CsMIPs) foi identificado
em C. sinensis e dividido em cinco subfamílias (CsPIPs, CsTIPs, CsNIPs,
CsSIPs e CsXIPs). A expressão gênica de CsMIPs foi observada em diferentes
tecidos sob estresses por seca e sal, bem como com infecção por 'Ca.
Liberibacter asiaticus'. Um papel especial na regulação da expressão gênica é
proposto para a maioria CsMIPs durante déficit hídrico, estresse salino e o
desenvolvimento da doença HLB. Plantas transgênicas de tabaco
superexpressando CsTIP2;1 apresentaram tolerância a estresses hídrico e
salino devido (i) ao maior conteúdo de água nas células, resultando na maior
dissolução do sal e auxiliando na expansão celular, (ii) a atenuação dos danos
oxidativos pela detoxificaçãodo H2O2 e (iii) indução da abertura estomática.
Essas modificações contribuiram para maior capacidade fotossintética, taxa de
transpiração e eficiência do uso da água sob condições de deficiência hídrica,
em comparação com as plantas não-transformadas. Oito genes codificando
GolS foram identificados no genoma de laranja doce e caracterizados no
presente estudo. A expressão gênica de CsGolS foi analisada em diferentes
tecidos de citros sob estresses por deficiência hídrica e sal, bem como com
infecção por 'Candidatus Liberibacter asiaticus'. Plantas transgênicas de tabaco
superexpressando CsGolS6 apresentaram maior capacidade fotossintética,
maior taxa de transpiração e condutância estomática comparado com a
linhagem tipo-selvagem. O gene CsGolS6 desempenha papel importante na
resposta e tolerância de plantas para o estresse a seca. Coletivamente, os
resultados obtidos no presente estudo permitiram ampliar a base de
conhecimento potencialmente aplicável ao melhoramento, bem como facilitar o
desenvolvimento de novas variedades porta enxerto potencialmente úteis para
a citricultura brasileira.
