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UNIVERSIDADE ESTADUAL DE SANTA CRUZ

PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E

BIOLOGIA MOLECULAR

Análise funcional do gene EgPHI-1 (Phosphate induced-1)

de eucalipto em tabaco

AURIZANGELA OLIVEIRA DE SOUSA

ILHÉUS-BAHIA-BRASIL

Fevereiro de 2013


Análise funcional do gene EgPHI-1 (Phosphate induced-1)

de eucalipto em tabaco

Tese apresentada à Universidade

Estadual de Santa Cruz como parte das

exigências para a obtenção do título de

Doutora em Genética e Biologia

Molecular.

Área de Concentração: Genômica

Funcional e Estrutural

Orientador: Dr. Marcio Gilberto

Cardoso Costa

ILHÉUS-BAHIA-BRASIL

Fevereiro de 2013


ANÁLISE FUNCIONAL DO GENE EgPHI-1 (PHOSPHATE INDUCED-1)

DE EUCALIPTO EM TABACO

Tese apresentada à Universidade

Estadual de Santa Cruz como parte das

exigências para a obtenção do título de

Doutora em Genética e Biologia

Molecular.

Área de Concentração:

Genômica Funcional e Estrutural

APROVADA: Ilhéus - Bahia, 25 de fevereiro de 2013.

_________________________________ _______________________________

Drª. Ana Cristina Miranda Brasileiro Dr. Abelmon da Silva Gesteira (EMBRAPA-CENARGEN) (EMBRAPA- CNPMF)

_____________________________ ___________________________

Drª. Fabienne Florence L. Micheli Drª Fernanda Amato Gaiotto (UESC) (UESC)

____________________________

Dr. Marcio Gilberto Cardoso Costa (UESC - Orientador)

ii

À minha GRANDE família, por

sempre ter compreendido as minhas

necessidades e apoiado os meus

sonhos.

OFEREÇO

Ao Dr. Julio Cézar de M. Cascardo

(in memoriam)

DEDICO

iii

AGRADECIMENTOS

À DEUS, a Rocha que me fortalece e a Luz que me sensibiliza.

À Universidade Estadual de Santa Cruz (UESC), em especial ao Programa de

Pós-Graduação em Genética e Biologia Molecular, pela oportunidade.

À Fundação de Amparo à Pesquisa da Bahia (FAPESB), pela bolsa.

Ao Dr. Júlio Cezar de M. Cascardo (in memoriam), pelas lições de uma vida.

Ao Dr. Marcio Gilberto Cardoso Costa, pela confiança e orientação.

Aos Dr. Carlos Priminho Pirovani, Dr. Alex-Alan Almeida e à Drª. Fátima

Cerqueira Alvin, pela coorientação, ensinamentos e amizade.

Ao Dr. André Ferraz (Escola de Engenharia Química – USP, Lorena-SP), pela

recepção e orientação com os experimentos de topoquimica da madeira.

Ao José Moreira, Fernando Masarin e Daielle (Escola de Engenharia Química

– USP, Lorena-SP), pela recepção, suporte técnico, discussões e amizade.

À Fabrícia, pela eficiência e carinho no atendimento da secretaria.

À minha mãe Lilian, pelo amor, confiança e exemplo de vitória.

Ao meu noivo-esposo Vinícius Ferreira, pelo amor, compreensão e motivação.

Ao meu irmão, Adriano, e sua esposa, Zoraide, pelo apoio e amor dedicados.

Aos meus sobrinhos, Theo e João Luca, pela força que despertam em mim.

À família Ferreira, por todo o apoio e amor dedicados nesta jornada.

À Dayse, Joseane, Juliane, Dayane, Priscila, Diana e Amanda pela amizade.

Aos IC’s Thaynara, Lucas, Nathy, Genilson, João e Edson, pela colaboração.

Aos amigos Horley e Leila pelo suporte técnico e dedicação.

À Laís, Luciana Cidade, Ana Camila, Luciana, Luana, Cristina, Fabi, Thaynã,

Jamyle, Lívia, Joyce e Manu, pela parceria na bancada e o essencial carinho.

Aos professores, familiares e amigos que contribuíram para a minha formação

profissional e pessoal.

iv

“Teima filho, é só teimar.”

(Fala de Dona Lindu, mãe de Lula, retirada do filme Lula, o filho do Brasil)

v

ÍNDICE

EXTRATO ................................................................................................................. vii

ABSTRACT ................................................................................................................ ix

LISTA DE FIGURAS .................................................................................................. xi

1. INTRODUÇÃO ........................................................................................................ 1

1.1 Objetivos ........................................................................................................................................... 4

1.1.1 Geral ............................................................................................................................................... 4

1.1.2 Específicos ...................................................................................................................................... 4

2. REVISÃO DE LITERATURA .................................................................................. 5

2.1 O cultivo florestal ............................................................................................................................. 5

2.2 Florestas plantadas no Brasil ........................................................................................................... 6

2.2.1 Eucalipto e a produção de celulose e papel ................................................................................... 8

2.3 Propriedades da madeira x qualidade do papel e celulose ............................................................ 9

2.3.1 Densidade básica .......................................................................................................................... 10

2.3.2 Morfologia da fibra ...................................................................................................................... 11

2.3.3 Teor de holoceluloses (celulose e hemiceluloses) e lignina ......................................................... 11

2.3.4 Conteúdo de extrativos ................................................................................................................ 13

2.4 Ferramentas biotecnológicas aplicadas à qualidade da madeira ................................................. 13

2.4.1 Estudos funcionais relacionados às propriedades da madeira .................................................... 16

2.5 Família PHI-1 ................................................................................................................................... 20

CAPÍTULO 1: Overexpression of a novel PHOSPHATE-INDUCED-1 gene from

Eucalyptus (EgPHI-1) promotes shoot growth and xylem differentiation in

transgenic tobacco ................................................................................................. 24

CAPÍTULO 2: Phosphate induced-1 gene from Eucalyptus (EgPHI-1) enhances

the osmotic stress tolerance in transgenic tobacco ............................................ 60

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CAPITULO 3: Overexpression of an Eucalyptus PHOSPHATE-INDUCED-1

(EgPHI-1) gene alters the chemical composition and topochemical distribution

of lignin in stem xylem of tobacco ......................................................................... 73

4. CONCLUSÕES GERAIS ...................................................................................... 90

5. REFÊNCIAS .......................................................................................................... 91

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EXTRATO

SOUSA, Aurizangela Oliveira de, Universidade Estadual de Santa Cruz, Ilhéus,

Fevereiro de 2013. Análise funcional do gene EgPHI-1 (Phosphate induced-1) de

eucalipto em tabaco. Orientador: Marcio Gilberto Cardoso Costa.

A qualidade da madeira usada para a produção de celulose e papel é

determinada por propriedades adquiridas durante o processo de formação do xilema secundário (xilogênese). Este processo está sob o rígido controle da expressão de genes, muitos dos quais ainda desconhecidos. A comparação dos transcriptomas de xilema de Eucalyptus grandis e E. globulus permitiu identificar alguns destes genes, entre eles o EgPHI-1, o qual codifica uma proteína homologa à PHOSPHATE INDUCED PROTEIN-1 (PHI-1). E. grandis e E. globulus são espécies contrastantes para características como densidade básica, conteúdo de lignina, crescimento e resistências a pragas. Transcritos diferencialmente expressos entre estas espécies podem auxiliar no entendimento das variações fenotípicas observadas, bem como representar um alvo para programas de melhoramento genético em eucalipto e outras arbóreas. A partir desta proposta, o objetivo deste trabalho foi avaliar as possíveis modificações anatomorfológicas e metabólicas, bem como as mudanças nos parâmetros do crescimento, composição química e lignificação promovidas pela superexpressão do gene EgPHI-1 de eucalipto em tabaco (Nicotiana tabacum), de modo a estabelecer relação entre a expressão do gene e a diferenciação do xilema secundário. Análises estruturais e filogenéticas demonstraram que a proteína deduzida EgPHI-1 contém sinal de endereçamento para a via secretora e sítios de glicosilação e fosforilação, sugerindo ser uma proteína apoplástica solúvel que sofre modificações pós-traducionais. O domínio PHI-1 está estruturalmente conservado em EgPHI-1, o que a relaciona com outras proteínas da família PHI-1/EXO. Estudo de expressão em eucalipto submetido a diferentes condições de crescimento mostrou que EgPHI-1 é induzido por ferimento, desidratação e hormônios (auxina e citocininas). Três linhagens de tabaco superexpressando EgPHI-1 constitutivamente foram obtidas. Estas linhagens expressam EgPHI-1 em três diferentes níveis. O padrão de crescimento, as trocas gasosas foliares, a anatomia do xilema de caule e pecíolo, a atividade das enzimas phenylalanine ammonia-lyase (PAL) e peroxidases (POD) foram analisados nas linhagens transgênicas e os resultados comparados com plantas controle não transformadas. A superexpressão de EgPHI-1 favoreceu o crescimento e acúmulo de biomassa seca da parte aérea em detrimento das raízes, bem como alterações na anatomia foliar. O xilema do caule das linhagens

viii

transgênicas apresentou alterações anatômicas, com fibras mais compridas e vasos com maior diâmetro do lúmen. O xilema do pecíolo também apresentou alterações que foram confirmadas por variações da autofluorescência da lignina. A atividade das enzimas PAL e POD foi maior para as linhagens transgênicas. A tolerância ao estresse osmótico induzido por NaCl, polietilenoglicol (PEG) e manitol também foi testada nas linhagens transgênicas. A superexpressão de EgPHI-1 aumentou a tolerância ao estresse osmótico, principalmente o salino, fato avaliado pelo desenvolvimento das raízes e acúmulo de biomassa seca. A tolerância foi correlacionada com o aumento da abundância da proteína endógena BiP nos tecidos. Esta proteína parece ser modulada pela expressão de EgPHI-1. A composição química do caule e a microespectrofotometria ultravioleta (UMSP) de fibras e vasos completaram as análises propostas neste estudo. Os resultados mostraram que houve redução do conteúdo de celulose para todas as linhagens transgênicas, enquanto que a redução do conteúdo de lignina e aumento de hemicelulose ocorreu apenas para uma das linhagens. O conteúdo de extrativos aumentou para duas linhagens. O espectro de absorbância ultravioleta da lignina na camada S2 da parede celular secundária de fibras e vasos do caule foi menor para as plantas transgênicas. A varredura por ultravioleta foi realizada para fibras da planta controle e linhagem transgênica de menor conteúdo de lignina. As imagens geradas demonstram a distribuição da lignina na fibra destas plantas com maior intensidade da absorbância de lignina na porção dos cantos de célula e lamela média para ambas as plantas. No entanto, a parede celular da linhagem transgênica apresentou distribuição de lignina menos uniforme e com menor absorbância. Coletivamente, os resultados demonstram que EgPHI-1 altera a alocação de carbono para crescimento de parte aérea e promove modificações na anatomia do xilema com maior comprimento das fibras e maior diâmetro do lúmen dos vasos. Estes tipos celulares também apresentam redução dos conteúdos de celulose e lignina da parede celular secundária. Esta redução pode ser compensada com o aumento a biossíntese de compostos que compõem os extrativos. Além disto, a expressão de EgPHI-1 modula, pelo menos em parte, a expressão de BiP, uma chaperona que previne a morte celular dos tecidos sob estresse osmótico. Diante disto é possível concluir que a expressão de EgPHI-1 está fortemente relacionada com diferenciação celular do xilema e formação da parede celular secundária. Isto coloca este gene como importante alvo para estudos que visam alterações das propriedades da madeira. Palavras-chave: madeira, xilema secundário, parede celular, celulose, lignina.

ix

ABSTRACT

SOUSA, Aurizangela Oliveira, Universidade Estadual de Santa Cruz, Ilhéus,

February 2013. Functional analysis of EgPHI-1 (Phosphate induced-1) gene from

eucalyptus in tobacco. Advisor: Marcio Gilberto Cardoso Costa.

The quality of wood used for the pulp and paper production is determined by properties acquired during the secondary xylem formation (xylogenesis). This process is under strict gene expression control, with several genes still unknown. Comparative transcriptome analyzes of xylem from Eucalyptus grandis and E. globulus allowed the identification of some of these genes, including EgPHI-1, which encodes a protein homologous to PHOSPHATE INDUCED PROTEIN-1. E. grandis and E. globulus species are contrasting for wood traits such as density, lignin content, growth and resistance to pests. Differentially expressed transcripts between these species may represent the key to clarify the phenotypic variation and indicate a target for genetic improvement programs of eucalyptus and other trees. The aim of this study was to evaluate possible anatomical, morphological and metabolic changes, as well as changes in the growth parameters, chemical composition and lignification promoted by the overexpression of EgPHI-1 from eucalyptus in tobacco (Nicotiana tabacum). Thus, structural and phylogenetic analyzes showed that EgPHI-1 deduced protein contains a signal peptide to secretory pathway and phosphorylation and glycosylation sites, suggesting to be an apoplastic soluble protein which is subjected to post-translational modifications. PHI-1 domain is structurally conserved in EgPHI-1, which associates it to other PHI-1/EXO proteins. Expression studies in eucalyptus maintained under different growth conditions showed that EgPHI-1 is induced by wounding, dehydration and hormones (auxin and cytokinin). Furthermore, three tobacco lines overexpressing EgPHI-1 constitutively were obtained. These lines express the transcript and its corresponding protein in three different levels. Growth patterns, leaf gas exchanges, xylem anatomy of stems and petioles, activity of phenylalanine ammonia-lyase (PAL) and peroxidases (POD) in transgenic lines were analyzed and the results compared with the wild-type (WT). EgPHI-1 overexpression favored the growth and accumulation of shoot dry biomass in detriment of roots, and caused leaf modifications, that probably limited the CO2 fixation. Stem xylem of the transgenic lines showed anatomical changes, with longer fibers and vessels with higher lumen diameter. Petiole xylem also showed changes that were confirmed by autofluorescence of lignin. PAL and POD activities were higher in the transgenic lines. The tolerance to osmotic stress induced by NaCl,

x

polyethylene glycol (PEG) and mannitol were also tested in the transgenic lines. EgPHI-1 overexpression increased the tolerance to osmotic stress, especially salt stress; tolerance was evaluated based on root development and accumulation of dry biomass. The tolerance was correlated with increased endogenous BiP protein expression. This protein seems to be modulated by the expression of EgPHI-1. Stem chemical composition and scanning ultraviolet (UV)-microspectrophotometry (UMSP) of fibers and vessels completed the analysis proposed in this study. The results showed a reduction in the cellulose content for all transgenic lines, while a reduction in the lignin content and an increase in hemicellulose content were observed only for one of the three transgenic lines. Extractives content increased in two transgenic lines. UV absorbance spectra in S2-layer of lignin of fiber and vessel secondary cell walls were lower in the transgenic plants. UV scanning profiles were performed in fibers from WT and transgenic plants with lower lignin content. These UV-images showed more intense lignin absorbance in the cell corners and component middle lamella for WT and transgenic plants. However, cell wall of transgenic plants showed less uniform distribution of lignin and lower absorbance. Collectively, the results demonstrated that EgPHI-1 changes the carbon allocation for shoot growth and promotes changes in the xylem anatomy with longer fibers and vessels with higher lumen diameter. These cell types also show a reduction of cellulose and lignin contents in secondary cell wall. This decrease is compensated by increasing the biosynthesis of extractives compounds. Furthermore, EgPHI-1 expression modulates, at least in part, BiP expression, a chaperone that prevents cell death of the tissues under osmotic stress. In view of this is possible to suggest that EgPHI-1 expression is strongly correlated to xylem cell differentiation and secondary cell wall formation, making EgPHI-1 as an important target for studies aiming at modification of the properties of wood. Key words: wood, secondary xylem, cell wall, cellulose, lignin.

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LISTA DE FIGURAS

Figura 1. Distribuição percentual da área de plantios de Eucalyptus e Pinus

por estado. ABRAF, 2012. ........................................................................................... 6

Figura 2. Crescimento da produção de celulose e papel no Brasil (milhões de

toneladas). BRACELPA, 2011. .................................................................................... 7

Figura 3. Cadeia produtiva do papel e celulose: a qualidade do produto final

depende de ações individuais e conjuntas. Modificado de Dinus; Welt, 1995............. 9

Figura 4. Representação dos principais eventos da xilogênese. Modificado de

Turner; Gallois; Brown, 2007. .................................................................................... 10

Figura 5. Estrutura linear da celulose (fragmento). Modificado de Delmer,

1999. ......................................................................................................................... 12

Figura 6. Estrutura dos resíduos de lignina. Modificado de Whetten; Sederoff,

1995. ......................................................................................................................... 13

Figura 7. Estratégias para identificar e utilizar genes candidatos envolvidos

com características de produção e produtividade visando o melhoramento da

madeira. Modificado de Boerjan, 2005. ..................................................................... 15

Figura 8. Via de bissíntese de lignina. Principais enzimas da via identificadas

por círculos coloridos: PAL - fenilalanina amônialiase; C4H – cinamato 4-hidroxilase;

C3H – coumarato 3-hidroxilase; 4CL, 4-coumarato-COA ligase; e CCR, cinnamoil-

COA redutase; CAD - cinamil álcool desidrogenase; Peroxidases; Lacases.

Modificado de Baucher et al., 1996. .......................................................................... 18

Figura 9. Rede de regulação transcricional da biossíntese de compostos da

parede celular secundária. Fatores transcricionais funcionalmente caracterizados em

Arabidopsis, Populus, Pinus e Eucalyptus. Modificado de Zhong; Lee; Ye, 2010. .... 19

1

1. INTRODUÇÃO

A madeira apresenta elevada importância econômica oferecendo matéria-

prima para os mais variados setores: construção civil, siderúrgica, madeireira e

indústrias de celulose e papel (FENNING; GERSHENZON, 2002). Devido a grande

demanda por este recurso, tem sido cada vez mais necessário o plantio de florestas,

compostas por espécies arbóreas de rápido crescimento e genótipos selecionados

para aumentar a produção e a qualidade do produto final. Estas florestas são uma

alternativa sustentável para atender as necessidades de consumo da população e

reduzir a pressão de desmatamento sobre as florestas naturais (DEL LUNGU; BALL;

CARLE, 2006).

O Brasil se destaca pela manutenção de plantios de eucalipto de alto

desempenho obtidos pelo investimento em tecnologia para geração de híbridos e

clones-elite apropriados principalmente para a produção de celulose e papel. Estes

materiais possuem elevada capacidade de produção média de celulose (44

m3/ha/ano) e apresentam o menor tempo de rotação do setor, apenas sete anos.

Este panorama fez que o Brasil ocupasse a posição de maior produtor mundial de

celulose de mercado de eucalipto e o décimo maior produtor mundial de papel

(BRACELPA, 2011). Contudo, para manter-se competitivo no setor, é imperativa a

realização de ações contínuas e integradas ao longo do processo de produção

(DINUS; WELT, 1995).

A qualidade da madeira usada para a produção de celulose e papel é

determinada por propriedades como densidade básica, comprimento, largura,

espessura e diâmetro do lúmen da fibra, teor de lignina, celulose e hemiceluloses,

conteúdo de extrativos (TRUGILHO et al., 2004; DUTT; TYAGI, 2011). Estas

características são definidas durante a formação do xilema secundário (xilogênese)

2

e estão sob controle espacial e temporal da expressão de genes específicos, muitos

deles ainda desconhecidos (PAUX et al., 2004; FOUCART et al., 2006). Assim, é

grande o interesse científico e comercial pela identificação de genes cuja expressão

esteja relacionada com o desempenho em crescimento e produtividade, qualidade e

valor do produto obtido a partir da madeira (PAUX et al., 2004; PAUX et al., 2005;

RENGEL et al., 2009).

A lignina, terceiro componente mais abundante da madeira, impacta

negativamente o processo de extração e obtenção de celulose em eucalipto. Assim,

os genes que codificam enzimas da via de biossíntese de lignina são os mais

estudados (WHETTEN; SEDEROFF, 1995; PIQUEMAL et al., 1998; BOERJAN;

RALPH; BAUCHER, 2003). Contudo, estudos das vias de biossíntese relacionadas

com a formação da parede celular fornecem uma visão mais integrada da formação

da madeira, acessando informações sobre reguladores transcricionais como NAC e

MYB que participam do processo (OH; PARK; HAN, 2003; GOICOECHEA et al.,

2005; LEROUXEL et al., 2006; MELLEROWICZ; SUNDBERG, 2008; JAMET et al.,

2009; LEGAY et al., 2010; ZHONG; LEE; YE, 2010; AMBAVARAM et al., 2011;

ZHAO; DIXON, 2011).

Eucalyptus grandis e E. globulus, espécies contrastantes para propriedades

da madeira e resistência, foram usadas, entre outras espécies, pelo Projeto

Genolyptus, para obtenção do transcriptoma do xilema de Eucalyptus. E. grandis

apresenta rápido crescimento volumétrico e resistência a pragas. No entanto, a sua

madeira possui baixa densidade e elevado conteúdo de lignina (MYBURG et al.,

2003; MOON et al., 2007). Já E. globulus possui madeira de alta densidade e baixo

conteúdo de lignina. Contudo, as árvores desta espécie apresentam reduzido

crescimento volumétrico e são menos resistentes ao ataque de pragas (ROSA et al.,

2002; MYBURG et al., 2003; JONES; VAILLANCOURT; POTTS, 2006).

A comparação dos perfis transcricionais do xilema de E. grandis e E. globulus

forneceu importantes informações sobre a expressão de genes potencialmente

envolvidos com a formação do xilema secundário e prováveis determinantes de

propriedades da madeira nestas duas espécies (GRATTAPAGLIA, 2004; PASQUALI

et al., 2005). Dentre os genes diferencialmente expressos, o transcrito de EgPHI-1

que codifica uma proteína homologa à PHOSPHATE INDUCED PROTEIN-1 (PHI-1)

de tabaco e que apresentou expressão 7,5-x maior em E. globulus em relação à E.

3

grandis (SOUSA et al., 2011) foi selecionado para o estudo funcional apresentado

neste trabalho.

A seleção de EgPHI-1 pode ser justificada com base nos resultados de

expressão diferencial em espécies vegetais e condições variadas, onde é possível

notar que proteínas ou transcritos com domínio PHI-1 são constantemente relatados.

PHI-1 foi identificado como diferencialmente expresso durante o crescimento

secundário (KO, 2004), diferenciação celular (IWASE et al., 2005), biossíntese de

parede celular vegetal (BAYER et al., 2006) e respostas aos diferentes tipos de

estresses (NORTON et al., 2008; DE VOS; JANDER, 2009; DITA et al., 2009;

MUSTAFA et al., 2009; WU et al., 2010). Contudo, estes trabalhos não

apresentaram estudos funcionais para desvendar a participação de PHI-1 nos

processos identificados.

PHI-1 foi primeiramente identificada em um estudo sobre a participação do

fosfato no processo de divisão celular vegetal, onde foi sugerido o envolvimento de

PHI-1 com a fosforilação de um processo celular não especificado. Como não havia

homologia com outras proteínas descritas nos bancos de dados, PHI-1 foi

considerada como membro de uma nova classe de proteínas (SANO et al., 1999).

Posteriormente, foi proposto que PHI-1 atuava para aliviar as variações do pH

intracelular percebidas como sinal de estresse, mas o modo de ação não fora

esclarecido (SANO; NAGATA, 2002).

Novas informações relacionadas à PHI-1 foram sugeridas a partir da análise

de função do gene EXORDIUM (EXO) em Arabidopsis (FARRAR et al., 2003). EXO

é estruturalmente relacionada à PHI-1 e também está relacionada com o ciclo

celular, funcionando como um componente da via de sinalização de genes

modificadores da parede celular que respondem à brassinosteróide (COLL-GARCIA

et al., 2004; SCHRÖDER et al., 2009). Já o gene EXORDIUM-LIKE 1 (EXL1)

promoveu o crescimento vegetal em condições limitantes de suprimento de carbono

(SCHRÖDER; LISSO; MÜSSIG, 2011). Sob condição de baixa irradiância e estresse

por anoxia, EXL1, EXL2 e EXL4, suprimiram o crescimento induzido por

brassinosteróide e controlaram a alocação de carbono na célula (SCHRÖDER;

LISSO; MÜSSIG, 2012).

A caracterização funcional de EgPHI-1 pode auxiliar a esclarecer algumas das

variações fenotípicas observadas entre as espécies, bem como oferecer um novo

alvo para ser usado em programas de melhoramento genético que tenham como

4

objetivo a melhoria das propriedades da madeira e do padrão de crescimento em

eucalipto, que implicam na qualidade e valor da madeira e seus produtos.

1.1 Objetivos

1.1.1 Geral

Avaliar as possíveis modificações anatomorfológicas e metabólicas, bem

como as mudanças nos parâmetros de crescimento, composição química e

lignificação promovidas pela superexpressão do gene EgPHI-1 de eucalipto em

tabaco, de modo a estabelecer relações entre a expressão do gene e a

diferenciação do xilema secundário.

1.1.2 Específicos

Caracterizar a sequência EgPHI-1 de eucalipto;

Analisar a expressão de EgPHI-1 em eucalipto;

Construir vetor de superexpressão de EgPHI-1 em planta;

Obter a proteína EgPHI-1 recombinante para a produção obtenção de

anticorpos;

Transformar tabaco com vetor de superexpressão de EgPHI-1;

Confirmar a superexpressão de EgPHI-1 em linhagens de tabaco em nível de

mRNA e proteína;

Caracterizar as linhagens transgênicas quanto os parâmetros de crescimento

e trocas gasosas foliares, a anatomia do xilema de caule e pecíolo, a

atividade das enzimas PAL e POD; a tolerância ao estresse osmótico

relacionada ao nível de proteína BiP, a composição química do caule e a

absorbância de lignina em paredes celulares de fibras e vasos, bem como a

distribuição de lignina em fibras do caule.

5

2. REVISÃO DE LITERATURA

2.1 O cultivo florestal

A demanda por madeira e seus derivados tem gerado uma crescente pressão

sobre as florestas naturais e contribuído para a degradação das áreas florestais

mundiais (FENNING; GERSHENZON, 2002; BOERJAN, 2005). Esta demanda se

justifica pela vasta aplicabilidade da madeira, que é usada principalmente como

matéria-prima para construção civil, fabricação de móveis, obtenção de energia pela

queima da lenha e do carvão vegetal, extração de polpa de celulose para a

fabricação de papel e derivados, painéis de madeira industrializada e madeira

mecanicamente processada. Contudo, a capacidade de recuperação das florestas

naturais não acompanha a necessidade humana por seus recursos (PLOMION;

LEPROVOST; STOKES, 2001).

As florestas plantadas, compostas por espécies arbóreas de rápido

crescimento ou genótipos-elite selecionados a partir de ferramentas biotecnológicas,

são uma alternativa sustentável para atender a demanda por madeira e seus

derivados no mundo. A manutenção destas florestas reduz a pressão de

desmatamento sobre as florestas naturais e preserva espécies nativas (DEL

LUNGU; BALL; CARLE, 2006). Além disto, a domesticação de espécies florestais

tem permitido o desenvolvimento das indústrias de processamento de madeira e

agregado avanço tecnológico e econômico aos países onde ocorre o cultivo florestal

(FAO, 2007).

Grande parte da área mundial de florestas plantadas é ocupada com o cultivo

de espécies do gênero Pinus (32%), Cunninghamia (11%) e Eucalyptus (8%) (FAO,

2007). A produção de campo obtida com o cultivo destas e outras espécies arbóreas

6

domesticadas pode atingir a máxima capacidade, uma vez que as florestas

plantadas permitem o manejo da área em um ambiente controlado e o uso de

técnicas biotecnológicas, tais como melhoramento assistido por marcadores,

engenharia genética e propagação in vitro (BOERJAN, 2005).

2.2 Florestas plantadas no Brasil

As florestas plantadas no Brasil ocupam uma área maior do que 6,5 milhões

de hectares, sendo 74,8% correspondente à área de plantios de espécies do gênero

Eucalyptus e 25,2% aos plantios de espécies do gênero Pinus. O setor nacional de

florestas plantadas gera 645,2 mil empregos diretos e 1.475 milhões de empregos

indiretos. Já as atividades econômicas associadas a estas plantações representam

19,2% do saldo da balança comercial brasileira, com 3,1% do total das exportações

nacionais (ABRAF, 2012).

As florestas plantadas brasileiras possuem ampla distribuição geográfica,

sendo que os estados de Minas Gerais, São Paulo, Paraná, Bahia, Santa Catarina,

Mato Grosso do Sul e Rio Grande do Sul detêm 87,7% da área total dos plantios

florestais, como apresentado na figura abaixo (ABRAF, 2012).

Figura 1. Distribuição percentual da área de plantios de Eucalyptus e Pinus por

estado. ABRAF, 2012.

No Brasil, a cadeia produtiva do setor de florestas plantadas caracteriza‑se

pela grande diversidade de produtos, compreendendo um conjunto de atividades

que incluem a produção, a colheita e a transformação da madeira até a obtenção

7

dos produtos finais. A maior parte das plantações (36,1%) é destinada à extração de

celulose, ao passo que os serrados, carvão vegetal, painéis de madeira e

compensados consomem, respectivamente, 15,2%, 10%, 7,4% e 3,7% do total de

madeira. O restante (26,3%) é destinado à produção de lenha e outros produtos

florestais (ABRAF, 2012).

No cenário mundial, o Brasil é o quarto maior produtor de celulose, o maior

produtor de celulose de mercado de eucalipto e o décimo maior produtor de papel. O

sucesso obtido com a produção de celulose e papel no País (Figura 2) se deve

principalmente aos altos níveis de produtividade das plantações de eucalipto e ao

investimento em tecnologia destinada à obtenção de híbridos e clones de alto

desempenho (BRACELPA, 2011). Nos últimos 20 anos, a produção média de

celulose obtida a partir das plantações de eucalipto apresentou um crescimento de

83%, passando de 24 m3/ha/ano para os atuais 44 m3/ha/ano. Além do alto

rendimento, as plantações de eucalipto nacionais apresentam o menor tempo de

rotação do setor, sendo necessários apenas sete anos entre os cortes (BRACELPA,

2011).

Figura 2. Crescimento da produção de celulose e papel no Brasil (milhões de

toneladas). BRACELPA, 2011.

8

2.2.1 Eucalipto e a produção de celulose e papel

Do ponto de vista tecnológico, qualquer matéria-prima fibrosa é passível de

ser utilizada na produção de celulose e papel. Porém, quando analisada sob o

aspecto econômico, uma série de fatores deve ser considerada, alguns dos quais se

referem às características anatômicas, morfológicas, físicas e químicas da madeira

(PILATE et al., 2002).

As espécies do gênero Eucalyptus apresentam rápido crescimento, forma reta

e adaptabilidade aos mais variados climas e solos, o que as torna ideais para as

plantações do setor de celulose e papel (ELDRIDGE et al., 1993). A polpa de

celulose obtida a partir destas espécies é considerada de fibras curtas, sendo ideal

para a fabricação de papel de impressão e escrita e do tipo tissue (papéis sanitários,

toalha de papel e guardanapos), os quais são menos resistentes à tração e ao

arrebentamento, quando comparadas as fibras longas provenientes de coníferas

(BRACELPA, 2011).

A variabilidade genética natural das espécies de eucalipto, bem como a

habilidade destas espécies em formar híbridos tem sido explorada por meio de

programas de melhoramento para identificar genótipos que produzam fenótipos mais

favoráveis para características como crescimento, propriedades da madeira,

resistência a pragas e tolerância ao estresse (POKE et al., 2005). Para regiões de

clima tropical e subtropical, as espécies mais favoráveis para a produção de celulose

e papel são E. grandis, E. urophyla e os híbridos deste cruzamento. Já as regiões de

clima temperado favorecem o desenvolvimento de E. globulus (POKE et al., 2005;

JONES; VAILLANCOURT; POTTS, 2006; MYBURG et al., 2007).

O controle da expressão de genes específicos que regulam espacial e

temporalmente os eventos de formação da madeira (PAUX et al., 2004; FOUCART

et al., 2006) é um dos principais responsáveis pelas variações fenotípicas

percebidas entre as espécies de eucalipto (DUTT; TYAGI, 2011). Assim, é grande o

interesse científico e comercial pela identificação e manipulação de genes

considerados importantes para a determinação das principais características da

madeira, as quais influenciam as propriedades físico-químicas, o desempenho em

crescimento e produtividade, a qualidade do produto obtido e o valor industrial do

material obtido (PAUX et al., 2004; PAUX et al., 2005; RENGEL et al., 2009).

9

2.3 Propriedades da madeira x qualidade do papel e celulose

A qualidade do produto final obtido a partir das plantações florestais

destinadas à produção de papel e celulose depende de ações contínuas e

integradas ao longo do processo (Figura 3). Estas ações devem ser consideradas de

maneira individual e/ou conjunta durante toda a cadeia produtiva (DINUS; WELT,

1995). Contudo, algumas características da madeira são determinadas

biologicamente e a seleção adequada das matrizes pode determinar qualidade e

utilização do produto final (TOMAZELLO FILHO, 1985; QUEIROZ et al., 2004).

Figura 3. Cadeia produtiva do papel e celulose: a qualidade do produto final

depende de ações individuais e conjuntas. Modificado de Dinus; Welt, 1995.

Densidade básica, comprimento, largura, espessura e diâmetro do lúmen da

fibra, teor de lignina e holoceluloses (celulose e hemiceluloses), e conteúdo de

extrativos são propriedades decisivas para a seleção da madeira e ainda para a

determinação do método de extração e produção de celulose e papel (TRUGILHO et

al., 2004; DUTT; TYAGI, 2011). As características que definem estas propriedades

são adquiridas durante a formação do xilema secundário (xilogênese), por meio de

quatro principais eventos que alteram a morfologia das células vasculares (Figura 4).

São eles: expansão celular, deposição de compostos da parede celular secundária,

lignificação e morte celular programada (PLOMION; LEPROVOST; STOKES, 2001;

TURNER; GALLOIS; BROWN, 2007).

10

A expansão celular envolve a formação e modificação da parede celular

primária. Na sequência dos eventos, ocorre a biossíntese e deposição de

polissacarídeos (celulose e hemiceluloses) e proteínas estruturais da parede celular

secundária seguida pela lignificação. Neste processo, a peroxidação dos resíduos

de monolignol dá origem à matriz lignocelulósica. A morte celular programada, com

o colapso do vacúolo e digestão do núcleo por proteases, DNAses e RNAses,

completa as etapas de diferenciação celular para a formação da madeira

(PLOMION; LEPROVOST; STOKES, 2001; DEMURA; FUKUDA, 2007; TURNER;

GALLOIS; BROWN, 2007).

Figura 4. Representação dos principais eventos da xilogênese. Modificado de

Turner; Gallois; Brown, 2007.

2.3.1 Densidade básica

A densidade da madeira reflete a quantidade de matéria lenhosa por unidade

de volume, já a densidade básica refere-se à razão obtida entre o peso seco e o

volume saturado da madeira (TOMAZELLO FILHO, 1985). A avaliação adequada da

densidade básica fornece indicações bastante precisas acerca da impregnação e

rendimento do processo e geralmente está associada às características de

qualidade e de resistências físico-mecânicas da polpa de celulose (QUEIROZ et al.,

2004).

Madeira com alta densidade apresenta maior rendimento de produção, mas

requer maior carga de reagentes para a extração de celulose. Além disto, as fibras

deste tipo de matéria-prima apresentam propriedades de ligação inferiores,

11

tornando-as mais adequadas para a fabricação de papéis tipo tissue. Já as madeiras

de baixa densidade apresentam melhor impregnação e por isto consomem menor

carga de reagentes; no entanto aumentam consideravelmente os custos de

produção industrial por requererem maior consumo específico de madeira com

menor peso de madeira no digestor de processamento. A madeira de baixa

densidade apresenta ainda como característica fibras com alta propriedade de

ligação e por isto, é mais adequada para a fabricação de papéis destinados a

impressão e escrita (ALZATE; TOMAZELLO FILHO; PIEDADE, 2005; DUTT; TYAGI,

2011).

2.3.2 Morfologia da fibra

Sob o aspecto tecnológico, o comprimento e as demais dimensões das fibras

estão relacionados com as propriedades da celulose e do papel (DINUS; WELT,

1995). A partir dessas dimensões, são obtidos diversos coeficientes e índices que se

relacionam, da mesma forma, com as propriedades do produto obtido. De modo

geral, a morfologia das fibras apresenta variação espacial e temporal nas plantas.

Fibras próximas à medula apresentam menor comprimento, largura, espessura da

parede e diâmetro do lúmen. Estas dimensões aumentam com a idade, até atingirem

a estabilização em idade mais avançada (TOMAZELLO FILHO, 1985).

Quanto ao comprimento, verifica-se que as fibras mais longas resultam em

papel com maior resistência. O mesmo pode ser dito com relação à espessura da

parede das fibras. Fibras de parede delgada são achatadas (colapso) mais

facilmente, do que as de parede espessa, proporcionando um aumento nas ligações

interfibras. As demais dimensões das fibras têm apresentado, em diferentes

intensidades, relações com a qualidade da celulose e papel (TOMAZELLO FILHO,

1985; DINUS; WELT, 1995).

2.3.3 Teor de holoceluloses (celulose e hemiceluloses) e lignina

A madeira possui três constituintes macromoleculares predominantes que

formam a parede celular: celulose, hemiceluloses e lignina (MELLEROWICZ;

SUNDBERG, 2008). A proporção destes componentes na parede celular secundária

é considerada determinante para a estrutura, composição química e morfologia da

12

madeira, bem como a qualidade e aplicação do produto obtido (WHETTEN et al.,

2001; YANG et al., 2003).

A celulose é o componente mais abundante da madeira. Trata-se de um

homopolímero de β-1,4-D-glicano (Figura 5), que é sintetizado utilizando como

substratos os resíduos de glicose do tipo UDP-glicose através de ligações β-1,4

lineares (DELMER, 1999; RICHMOND, 2000). A unidade repetitiva da celulose

(composta por duas moléculas de glicose) é conhecida como celobiose e contém

seis grupos hidroxila onde se estabelecem interações do tipo ligações de hidrogênio

intra e intermolecular. Devido a essas ligações de hidrogênio há uma forte tendência

de a celulose formar cristais que a tornam completamente insolúvel em água e na

maioria dos solventes orgânicos (SILVA et al., 2009).

Figura 5. Estrutura linear da celulose (fragmento). Modificado de Delmer, 1999.

As hemiceluloses compõem o segundo composto mais abundante na

madeira, e com natureza altamente amorfa, são bastante hidrofílicas. O termo

hemicelulose é usado para os polissacarídeos que ocorrem normalmente

associados à celulose, para formar uma matriz de ligação cruzada conferindo maior

elasticidade à parede celular vegetal. As hemiceluloses consistem de vários

monossacarídeos polimerizados, incluindo pentoses (como xilose e arabinose),

hexoses (como galactose, glicose e manose), ácido 4-O-metil glucurônico e resíduos

de ácido galactorônico. Em madeiras, a unidade mais abundante das hemiceluloses

é a xilose (SILVA et al., 2009; SCHELLER; ULVSKOV, 2010).

O terceiro composto mais abundante da madeira é a lignina. A lignina é

derivada de três alcoóis hidroxinamílico (monolignóis): hidroxicinamílico p-caumárico,

coniferílico e sinapílico, que polimerizam nas subunidades denominadas: Hidroxifenil

(H), Guaiacil (G) e Sinapil (S), respectivamente (Figura 6). Estas subunidades

13

formam um polímero fenólico amorfo, de estrutura complexa e composição variada

(WHETTEN; SEDEROFF, 1995; BOERJAN; RALPH; BAUCHER, 2003).

Figura 6. Estrutura dos resíduos de lignina. Modificado de Whetten; Sederoff, 1995.

Apesar da importância biológica da lignina para as plantas, o teor e a

composição destes compostos na madeira influencia negativamente o desempenho

da polpação em termos de rendimento e consumo de produtos químicos utilizados

neste processo (DOUGLAS, 1996; DONALDSON; HAGUE; SNELL, 2001; SILVA et

al., 2009). Para a produção de papel de alta qualidade, a lignina necessita ser

extraída por um processo de custos elevados, tanto em termos financeiros quanto

ambientais, por requerer grandes quantidades de energia e reagentes químicos

(PIQUEMAL et al., 1998; BOERJAN, 2005).

2.3.4 Conteúdo de extrativos

Outros componentes de baixa massa molecular (orgânicos ou inorgânicos)

que aparecem em menor proporção na composição da parede celular, tais como

pectinas, polifenóis, taninos, terpenos, estilbenos e flavonoides, também podem

apresentar importante papel na determinação da qualidade da madeira. Estes

compostos formam o pitch que se acumula no maquinário durante o processamento

da madeira e acarretam grandes prejuízos às indústrias de polpa de celulose e de

papel (BARBOSA; MALTHA; CRUZ, 2005)

2.4 Ferramentas biotecnológicas aplicadas à qualidade da madeira

A produção de papel e celulose envolve uma cadeia de ações que visam o

aumento da produtividade e a melhoria da qualidade do produto (DINUS; WELT,

1995). Como, de modo geral, o melhoramento genético de arbóreas é um processo

14

demorado, principalmente devido ao tempo necessário para o estabelecimento das

características entre as gerações (BOERJAN, 2005), a biotecnologia e os estudos

moleculares aparecem como importantes aliados na busca para melhorar

características como densidade básica, morfologia das fibras e teor de compostos,

além de resistência a pragas e tolerância ao estresse ambiental (DINUS; WELT,

1995).

Algumas abordagens podem ser consideradas para a identificação de genes

alvos potencialmente envolvidos com a qualidade da madeira (Figura 7). Nestas

estratégias, as informações genéticas para melhoramento de características de

interesse podem ser acessadas utilizando sistemas-modelo como Arabidopsis,

Populus, Nicotiana e Zinnia, ou ainda por meio de análise de perfil transcricional,

metabólico ou proteico que identifica prováveis alvos e sugerem os possíveis

processos com os quais estes alvos estão envolvidos. O mapeamento de QTLs

(quantitative trait locus) e SSR (simple sequence repeat) relaciona o envolvimento

do genótipo com o fenótipo de interesse na espécie. Já a comparação dos genomas

e anotações gênicas permite identificar a estrutura gênica e delinear mecanismos de

regulação por meio de análises de bioinformática. Após a identificação do gene e

caracterização da função para um fenótipo de interesse, duas vias podem ser

seguidas: modificar clones-elite por engenharia genética ou identificar os alelos

deste gene associados com o fenótipo de interesse e utilizá-los em programas de

melhoramento (BOERJAN, 2005).

15

Figura 7. Estratégias para identificar e utilizar genes-candidato envolvidos com

características de produção e produtividade visando o melhoramento da madeira.

Modificado de Boerjan, 2005.

Com o objetivo de estabelecer uma plataforma genômica para o entendimento

das bases de formação da madeira e resistência a doenças em eucalipto, foi

concebido o Projeto Genolyptus (Rede Brasileira de Pesquisa do Eucalyptus). Por

meio desta iniciativa, tecnologias de genômica e pós-genômica poderiam ser

utilizadas em programas de melhoramento agregando avanços biotecnológicos à

produção florestal (GRATTAPAGLIA, 2004). Dentre as informações geradas com o

Projeto Genolyptus, estão os perfis transcricionais do xilema de E. grandis e E.

globulus, espécies contrastantes para determinadas propriedades da madeira,

padrões de crescimento, resistência a pragas e adaptação ao clima tropical

(PASQUALI et al., 2005).

E. grandis Hill ex Maiden. é uma espécie tropical amplamente usada pela

indústria de celulose e papel. As árvores desta espécie, assim como os seus

híbridos, apresentam rápido crescimento volumétrico e relevante resistência a

pragas. No entanto, a sua madeira possui baixa densidade e elevado conteúdo de

lignina, que elevam os custos de produção (MYBURG et al., 2003; MOON et al.,

2007). Já E. globulus Labill. é uma espécie de clima temperado com madeira de alta

Perfil transcricional, metabólico e proteico

16

densidade e menor conteúdo de lignina. Contudo, devido à baixa adaptabilidade e

reduzido crescimento volumétrico, é pouco utilizada em plantações comerciais de

clima tropical. Além disto, esta espécie apresenta alta suscetibilidade ao ataque de

pragas (ROSA et al., 2002; MYBURG et al., 2003; JONES; VAILLANCOURT;

POTTS, 2006).

Com o uso da tecnologia de microarranjos de DNA aplicada para comparação

dos perfis de expressão, foi possível identificar 898 genes diferencialmente

expressos (≥2-x) entre E. globulus e E. grandis durante a xilogênese (SOUSA et al.,

2011). Dentre estes, 471 genes apresentaram mais alta expressão em xilema de E.

globulus, enquanto que 427 genes foram mais expressos em xilema de E. grandis. A

análise dos resultados permitiu identificar importantes alvos para estudos funcionais.

Estes estudos podem esclarecer quais os reguladores genéticos possivelmente

envolvidos com as variações fenotípicas observadas entre as espécies, bem como

podem auxiliar na busca de alelos para programas de melhoramento genético que

tenham como objetivo a melhoria das propriedades da madeira e do padrão de

crescimento em eucalipto que implicam na qualidade e valor do produto (PASQUALI

et al., 2005; BASTOLLA et al., 2006).

2.4.1 Estudos funcionais relacionados às propriedades da madeira

A maioria dos genes isolados e caracterizados em eucalipto está

principalmente envolvida com a formação da madeira, mas especificamente

relacionada à biossíntese de lignina. Este interesse se justifica pela importância

econômica da madeira e pelos impactos negativos promovidos pela lignina ao

processo de extração de celulose (WHETTEN; SEDEROFF, 1995; PIQUEMAL et al.,

1998; BOERJAN; RALPH; BAUCHER, 2003).

A via de biossíntese de lignina (Figura 8) tem sido a mais bem caracterizada

(FERGUS; GORING, 1970; SMART; AMRHEIN, 1985; DONALDSON; HAGUE;

SNELL, 2001; CHIANG, 2006; COLEMAN et al., 2008; AMBAVARAM et al., 2011) e

muitos genes que codificam as enzimas envolvidas na via (BLOUNT et al., 2000;

PINÇON et al., 2001; MÖLLER et al., 2006; VOELKER et al., 2010) e também

fatores de transcrição que regulam a expressão destes genes (BAUCHER et al.,

1996; TAMAGNONE et al., 1998; GOICOECHEA et al., 2005; TOHGE et al., 2005;

17

LEGAY et al., 2010; THAKUR; AGGARWAL; SRIVASTAVA, 2012) têm sido

clonados e o padrão de expressão analisado.

Para a 4-coumarato:CoA ligase (4CL) foram transformados híbridos de álamo

(Populus tremuloides Michx.) com a sequência anti-senso do gene para

silenciamento do mesmo. Houve redução de 45% do conteúdo de lignina e aumento

de 15% no nível de celulose. Como a massa total de lignina-celulose se manteve

inalterada, sugeriu-se um mecanismo compensatório para o metabolismo primário e

secundário durante o crescimento vegetal (HU et al., 1999). Já a redução da

expressão do acido caféico 3-O-metiltransferase (COMT) em álamo (Populus

tremula x Populus alba) reduziu substancialmente os níveis de lignina e aumentou o

conteúdo de celulose. No entanto, devido a alterações do grau de condensação da

lignina, as plantas se mostraram menos propícias à degradação industrial da lignina

(JOUANIN et al., 2000).

A função da cinamil álcool desidrogenase (CAD) na via de biossíntese da

lignina foi testada pela supressão da expressão do gene em híbridos (Populus

tremula x Populus alba). Embora não tenha ocorrido redução do conteúdo de lignina,

esta foi mais facilmente extraída. Os resultados apontaram que a modificação da

expressão do gene CAD proporciona madeira com melhor qualidade para a indústria

de celulose e papel (BAUCHER et al., 1996). O silenciamento do gene 4CL reduziu

em 40% do conteúdo de lignina e aumentou em 14% do conteúdo de celulose (LI,

2003). A superexpressão do gene CAld5H aumentou a razão das subunidades S-G

de lignina, sem no entanto alterar a quantidade desse composto. Já a co-

transformação do Cald5H senso e 4CL anti-senso reduziu 52% o conteúdo de

lignina, aumentou 64% a razão S-G e elevou em 30% o conteúdo de celulose (LI,

2003). Os autores concluíram que há um efeito independente, porém aditivo das

enzimas envolvidas com as vias de biossíntese de lignina e celulose (LI, 2003).

18

Figura 8. Via de bissíntese de lignina. Principais enzimas da via identificadas por

círculos coloridos: PAL - fenilalanina amônialiase; C4H – cinamato 4-hidroxilase;

C3H – coumarato 3-hidroxilase; 4CL, 4-coumarato-COA ligase; e CCR, cinnamoil-

COA redutase; CAD - cinamil álcool desidrogenase; Peroxidases; Lacases.

Modificado de Baucher et al., 1996.

Com um enfoque mais integrado, a formação da madeira tem sido esclarecida

por meio de estudos das vias de biossíntese envolvidas com a formação da parede

celular, principalmente com relação aos fatores transcricionais que regulam a

expressão dos genes de interesse (OH; PARK; HAN, 2003; GOICOECHEA et al.,

2005; LEROUXEL et al., 2006; MELLEROWICZ; SUNDBERG, 2008; JAMET et al.,

2009; LEGAY et al., 2010; ZHONG; LEE; YE, 2010; AMBAVARAM et al., 2011;

19

ZHAO; DIXON, 2011). Estes estudos têm identificado diversos reguladores

transcricionais (NAC e MYB) da síntese e deposição dos principais compostos

(celulose, hemiceluloses e lignina) da parede celular secundária e já permitem

elaborar modelos de redes de regulação (Figura 9).

Figura 9. Rede de regulação transcricional da biossíntese de compostos da parede

celular secundária. Fatores transcricionais funcionalmente caracterizados em

Arabidopsis, Populus, Pinus e Eucalyptus. Modificado de Zhong; Lee; Ye, 2010.

No modelo de rede de regulação da biossíntese de componentes da parede

secundária, o primeiro nível é ocupado pelo grupo de reguladores NACs que

orientam a expressão dos fatores transcricionais downstream e estão fortemente

relacionados à regulação da biossíntese dos componentes da parede celular

secundária (ZHAO; DIXON, 2011). Os fatores de transcrição com domínio NAC são

reguladores transcricionais específicos de plantas e atuam em importantes eventos

biológicos, como crescimento, desenvolvimento e resposta ao estresse (OLSEN et

al., 2005). O genoma de Arabidopsis contém pelo menos 114 NACs e no mínimo 10

deles estão intimamente relacionados à regulação da síntese de componentes da

20

parede celular secundária para a diferenciação de fibras (SND1/WND), vasos

(VND6-7) e espessamento de parede (NST1/2) (WILSON et al., 2009).

No segundo nível de regulação está o MYB46 e seus homólogos funcionais

(MYB83, MYB20, MYB4, MYB2 e MYB3), que atuam amplamente na ativação do

programa de biossíntese da parede celular secundária (GOICOECHEA et al., 2005;

LEGAY et al., 2010). As ações destes reguladores são concentradas e permitem a

ativação coordenada dos genes os quais coordenam a síntese, transporte e

deposição dos principais componentes da parede celular secundária, (celulose, xilan

e lignina). A regulação coordenada da via de biossíntese de lignina é mediada por

fatores transcricionais MYB (DEMURA; FUKUDA, 2007; ZHONG; LEE; YE, 2010).

Os estudos funcionais têm sido de fundamental relevância na identificação de

reguladores transcricionais da biossíntese de componentes da parede celular. No

entanto algumas questões ainda são pertinentes, uma delas seria descobrir quais os

sinais que regulam a ativação dos reguladores transcricionais para a ativação do

programa de síntese dos principais compostos da parede celular (ZHONG; LEE; YE,

2010). Para esta questão já foi sugerido que hormônios (auxina, citocininas,

giberelinas e brassinosteróides) são sinais importantes para a ativação da

diferenciação dos elementos vasculares (CLOUSE; SASSE, 1998; MÜSSIG;

FISCHER; ALTMANN, 2002; DEMURA; FUKUDA, 2007; ZHONG et al., 2008;

DAYAN et al., 2010; KIM et al., 2012).

A regulação da transcrição para a formação da parede secundária pode

envolver ainda um conjunto de ativadores, repressores e reguladores tipo feedback.

Esclarecer estes reguladores e sinais e quais são seus alvos e suas inter-relações

fornecerá uma visão mais ampla da complexa rede de regulação transcricional para

ativação da biossíntese para a formação da parede secundária (ARMSTRONG et

al., 2004; CHINNUSAMY; SCHUMAKER; ZHU, 2004; DEMURA; FUKUDA, 2007;

DEMURA; YE, 2010; AMBAVARAM et al., 2011).

2.5 Família PHI-1

Abordagens como as realizadas pelo Projeto Genolyptus oferecem uma gama

de informações sobre reguladores transcricionais e transcritos que podem ajudar a

21

entender algumas das razões para as diferenças fenotípicas encontradas na

madeira, tais como densidade básica, produção de extrativos e capacidade de

produção de polpa (POKE et al., 2005). Entre os transcritos identificados como

diferencialmente expressos entre E. grandis e E. globulus está a sequência que

codifica para a proteína homóloga à PHOSPHATE INDUCED PROTEIN-1 (PHI-1).

Este gene foi denominado EgPHI-1 e mostrou expressão 7,5-x maior em E. globulus

do que em E. grandis (SOUSA et al., 2011).

O gene PHI-1 foi primeiramente identificado e isolado em um estudo que

investigou a participação do fosfato no processo de divisão celular vegetal (SANO et

al., 1999). Células BY-2 de tabaco (Nicotiana tabacum) foram cultivadas por oito dias

e depois transferidas para meio de cultura desprovido de fosfato, onde foram

mantidas por três dias. Na ausência de fosfato o ciclo celular de BY-2 se manteve

em estado estacionário. Após a readição de fosfato, ao meio de cultura, as células

retomaram o ciclo celular. A análise de expressão diferencial entre as duas

condições de cultivo levou a identificação de um transcrito altamente expresso após

a adição do fosfato. Esta sequência foi caracterizada e o gene nomeado de acordo

com a condição na qual foi induzido, phosphate-induced (PHI)-1 (SANO et al., 1999).

A presença da sequência Lys-Gly-Ala na porção N-terminal da proteína PHI-1

sugeriu haver relação entre esta proteína e proteínas ATPases de membrana

plasmática de alguns fungos (Neurospora crassa, Saccharomyces cerevisiae e

Schizosaccharomyces pombe) e também de Arabidopsis, sendo provável o

envolvimento PHI-1 com a atividade de fosforilação de algum processo de celular

não identificado. Além desta, não foi identificada, naquele momento, homologia entre

a proteína PHI-1 e qualquer outra proteína descrita nos bancos de dados, sugerindo

que PHI-1 pertence a uma nova classe de proteínas (SANO et al., 1999).

A continuidade dos estudos demonstrou que a adição de ácido ascórbico

(ABA) à cultura BY-2 também induzia a expressão do gene PHI-1. A adição de ABA

promove acidificação do citoplasma, da mesma maneira como o fosfato promove

mudanças de pH. Assim foi proposto que PHI-1 responde à mudanças do pH

citoplasmático (SANO; NAGATA, 2002). Estas variações de pH seriam provocadas,

provavelmente, por alterações dos níveis de prótons co-transportados para dentro

das células junto com o fosfato. As mudanças de pH seriam interpretadas pelas

células como um indicador de estresse e como resposta à este estímulo haveria a

expressão de PHI-1. PHI-1 poderia então, atuar para aliviar as variações do pH

22

intracelular, contudo o seu modo de ação não foi esclarecido (SANO; NAGATA,

2002).

A identificação do gene EXORDIUM (EXO) em Arabidopsis acrescentou

novas informações à classe de proteínas PHI-1. EXO é uma proteína

estruturalmente relacionada à proteína PHI-1 de tabaco, com 76% de homologia

entre as duas sequências (FARRAR et al., 2003). Células vegetais embrionárias em

divisão, meristemas apicais e folhas jovens apresentaram elevada expressão do

gene EXO, tendo sugerido que EXO está relacionado à manutenção de células

meristemáticas (FARRAR et al., 2003). EXO, assim como PHI-1, estaria relacionada

ao ciclo celular, contudo, EXO atuaria por outra via, também não identificada

(FARRAR et al., 2003).

Posteriormente, foi sugerido que EXO possui atividade regulatória das

proteínas modificadoras da parede celular (KCS1, Exp5, AGP4 e N-TIP). Estas

proteínas têm a expressão ativada por EXO em resposta à brassinosteróide (COLL-

GARCIA et al., 2004). Assim, a provável função de EXO é mediar a expansão celular

por meio da regulação transcricional de genes envolvidos com crescimento induzido

por brassinosteróide (SCHRÖDER et al., 2009).

A função do gene EXORDIUM-LIKE 1 (EXL1) de Arabidopsis também está

sendo esclarecida. EXL1 apresenta 67% de identidade e 79% similaridade com a

proteína EXO e possui o domínio PHI-1 conservado. A expressão de EXL1

promoveu o crescimento mesmo em condições limitantes de suprimento de carbono

(SCHRÖDER; LISSO; MÜSSIG, 2011). EXL1 foi importante para a adaptação das

plantas às condições de baixa irradiância e estresse por anoxia. Sugeriu-se que

EXL1, assim como os homólogos EXL2 e EXL4, suprime o crescimento induzido por

brassinosteróide e controla a alocação de carbono na célula (SCHRÖDER; LISSO;

MÜSSIG, 2012)

Além das proteínas EXO, proteínas ou transcritos com domínio PHI-1 têm

sido identificados em diferentes trabalhos que visam esclarecer a expressão

diferencial em plantas. De acordo com estes estudos, é possível verificar

modificações dos níveis de expressão de membros da família PHI-1 em diferentes

condições de desenvolvimento, o que sugere o envolvimento dos representantes

PHI-1 com eventos como crescimento secundário (KO, 2004), diferenciação celular

(IWASE et al., 2005), biossíntese de parede celular vegetal (BAYER et al., 2006) e

respostas aos diferentes tipos de estresses (NORTON et al., 2008; DE VOS;

23

JANDER, 2009; DITA et al., 2009; MUSTAFA et al., 2009; WU et al., 2010). Contudo,

estes trabalhos não apresentam estudos funcionais para o provável membro da

família PHI-1 identificado.

24

CAPÍTULO 1:

Overexpression of a novel PHOSPHATE-INDUCED-1 gene

from Eucalyptus (EgPHI-1) promotes shoot growth and xylem

differentiation in transgenic tobacco

(Artigo submetido à Physiologia Plantarum em 21/12/2012)

25

Overexpression of a novel PHOSPHATE-INDUCED-1 gene from Eucalyptus

(EgPHI-1) promotes shoot growth and xylem differentiation in transgenic tobacco

Aurizangela O Sousa1, Elza Thaynara C M Assis

1, Rochele Patrícia Kirch

2, Delmira C

Silva1, Alex-Alan F de Almeida

1, Carlos P Pirovani

1, Fátima C Alvim

1, Giancarlo Pasquali

2,

Marcio G C Costa1δ

1 Center for Biotechnology and Genetics, Biological Sciences Department, State

University of Santa Cruz – UESC, Ilhéus, BA, 45662-900, Brazil

2 Molecular Biology and Biotechnology Department, Institute of Biosciences, Federal

University of Rio Grande do Sul – UFRGS, Porto Alegre, RS, 91501-970, Brazil

δ Corresponding author

e-mail: [email protected]

mailto:[email protected]

26

Abstract

Phosphate-induced protein 1 (PHI-1) comprises an emerging and widely distributed

class of proteins firstly isolated from phosphate-treated phosphate-starved tobacco cell

cultures. Comparative transcriptome of xylem cells from Eucalyptus species of contrasting

phenotypes for wood quality and growth traits led to the identification of a differentially

expressed PHI-1 homologue in E. globulus (EgPHI-1). Here, we have further characterized

EgPHI-1 in order to determine its possible involvement in processes affecting xylem

differentiation and plant growth. In silico analyses indicated that EgPHI-1 is a novel PHI-

1/EXO protein family member that contains phosphorylation sites and whose encoding gene

is under the control of cis-elements associated with cell division and response to light,

hormones and stress. EgPHI-1 expression is induced by wound, auxin, cytokinin and

dehydration treatments. Overexpression of EgPHI-1 in transgenic tobacco altered the

partitioning of biomass, favoring its allocation to shoots in detriment of roots. The stem of

transgenic plants showed anatomical alterations evidenced by increased xylem vessel lumen

diameter and fiber length, while their leaves exhibited strongly lignified vessels. A significant

increase in the activity of phenylalanine ammonia-lyase (PAL) and peroxidase (POD) was

observed in the transgenic plants. Leaf gas exchange parameters were also changed in

transgenic plants, apparently due to a limitation of CO2 fixation in the leaf mesophyll. Taken

together, our results demonstrate that EgPHI-1 is a new PHI-1/EXO protein that mediates

shoot growth and xylem differentiation, presumably by acting in signaling pathways that

control cell division and differentiation in response to both endogenous and environmental

cues.

Introduction

Eucalyptus is the dominant genus of woody plants in the planted forests of many

tropical and subtropical regions, constituting an important source of renewable energy and

raw materials for pulp and paper industry (Bernard 2003). Many species of this genus are

distinguished by their fast growth, straight trunks, and wood quality with attractive properties,

adaptability to different soils and climates, and easy vegetative propagation (Bernard 2003,

Eldridge et al. 1993, Myburg et al. 2007). These include E. grandis and E. globulus, species

with contrasting characteristics of growth and wood quality. E. grandis is a species native to

tropical and subtropical regions that exhibits a rapid growth, but produces a low density

27

wood. On the other hand, E. globulus is a species from temperate climates that produces a

high density wood, but exhibits a slow growth (Bernard 2003, Eldridge et al. 1993, Myburg et

al. 2007).

As part of a multi-institutional effort to identify genes responsible for the growth

patterns and wood properties intrinsic to each Eucalyptus species, a comparative analysis

between the xylem transcriptomes of E. grandis and E. globulus was previously carried out

using high density microarrays (Pasquali et al. 2005). This study led to the identification of

898 differentially expressed genes (fold-change ≥2) between the two Eucalyptus species.

Annotation and functional classification of these genes showed their probable functions and

relationships with the key processes and cellular events that take place during xylem

formation. One of the differentially expressed genes, which appeared to be 7.5 times more

expressed in E. globulus than in E. grandis, encodes a predicted protein with high similarity

to phosphate-induced protein 1 (PHI-1). PHI-1 was first identified in cell cultures of tobacco

as a protein involved in the phosphate-induced cell cycle activity (Sano et al. 1999, Sano and

Nagata 2002).

More recently, a gene identified by T-DNA mutagenesis in Arabidopsis, called

EXORDIUM (EXO), was found to encode a protein structurally related to PHI-1 of tobacco.

EXO was predominantly expressed in embryo cells in division, apical meristems and young

leaves, suggesting its role in the maintenance of meristematic cells (Farrar et al. 2003). EXO

and EXORDIUM-like (EXL) genes of Arabidopsis were also identified as mediators of

growth and cell expansion promoted by brassinosteroids (BR) (Coll-Garcia et al. 2004,

Schröder et al. 2009). Although they do not show similarities to any protein with known

function, PHI-1/EXO are a widely distributed class of proteins, present in coniferous,

monocotyledonous and dicotyledonous plants, as well as in mosses and soil bacteria (Dellagi

et al. 2000, Sano et al. 1999, Schröder et al. 2009). In plants, PHI-1 has been reported to be

present among the differentially expressed genes under conditions of secondary growth (Ko

2004), suspension cultures of dedifferentiated cells (Iwase et al. 2005), protein fractions

isolated from plant cell walls (Bayer et al. 2006), response to fungal infection (Mustafa et al.

2009, Wu et al. 2010) and insect damage (De Vos and Jander 2009), growth in contaminated

soils (Norton et al. 2008) and in association with weeds (Dita et al. 2009).

Since previous experimental evidence indicated its differential expression between

Eucalyptus species of contrasting wood properties and growth characteristics, we have tested

28

the hypothesis that E. globulus PHI-1 (EgPHI-1) affects xylem differentiation and/or plant

growth. To test this, we have further characterized the genomic and deduced amino acid

sequences of EgPHI-1 and generated transgenic tobacco plants expressing it constitutively.

Our results showed that EgPHI-1 is a new member of the PHI-1/EXO protein family involved

in the processes of shoot growth, and xylem differentiation. The results also indicated that

EgPHI-1 has a potential to be used in biotechnological strategies that propose modifications

in plant growth and wood properties of Eucalyptus and other woody species.

Materials and methods

Sequence analysis and phylogenetic tree construction

The EgPHI-1 cDNA sequence was obtained by DNA sequencing of a xylem cDNA

library of E. globulus (Pasquali et al. 2005). The Expasy platform tools

(http://expasy.org/tools/) were used to calculate/deduce the different nucleotide and amino

acid sequence properties of EgPHI-1. Comparative analyses were performed between EgPHI-

1 and other 43 amino acid sequences with conserved domain PHI-1/EXO (Schröder et al.

2009) by multiple sequence alignment using the default parameters of ClustalW (Thompson

et al. 1994). Phylogenetic analysis was performed using the maximum likelihood method

based on the JTT matrix-based model (Jones et al. 1992) of MEGA5 software (Tamura et al.

2011). The tree with the highest log likelihood was chosen and shown. The bootstrap test was

conducted with 1000 replicates and statistical values presented as percentages in the branches.

The 1.5-kb 5'-upstream promoter region of EgPHI-1 (locus Eucgr.F00356) available in the

Eucalyptus reference genome database (http://www.phytozome.net/eucalyptus.php) was used

to analyze the regulatory cis-elements employing the PlantCare software

(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

E. grandis seedling treatments

Hormonal and stress-inducing treatments were applied to 4-month-old E. grandis

plants grown in vitro in ¼ Murashige & Skoog medium (MS, Invitrogen, USA) solidified

with 0.7% (w/v) Phytoagar (Duchefa, The Netherlands) at 26 oC and a photoperiod of 16 h.

Groups of 3-4 plantlets were separately sprayed three times with 0.2 mg L-1

kinetin, 2 mg L-1

1-naphthaleneacetic acid (NAA) or water as control. Other groups of 3-4 plantlets were

http://expasy.org/tools/

http://www.phytozome.net/eucalyptus.php

http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

29

subjected to dehydration stress or mechanical wounding by, respectively, leaving the culture

flasks opened overnight in the growth chamber or clenching leaves and stems with a flat

forceps followed by water sprays and overnight incubation in the growth chamber. All plants

were harvested 24 h post-treatment, immediately frozen and stored in liquid nitrogen. One

group of (control) plants was frozen in liquid nitrogen without any treatment.

Plasmid construction and generation of transgenic tobacco plants

The complete EgPHI-1 cDNA sequence identified in a xylem library of E. globulus

was released from the pSPORT1 cloning vector by SalI/NotI digestion reaction. The purified

~1.2 kb fragment was subcloned in sense orientation in the XhoI/NotI sites of the pUC118

vector, between the promoter and terminator sequences of the cauliflower mosaic virus

(CaMV) 35S. The construction 35S::EgPHI-1 was released from pUC118 by BamHI/HindIII

double digestion reaction and cloned into the binary vector pCAMBIA 2301 (CAMBIA,

Australia). This vector contains the neomycin phosphotransferase (nptII) and β-glucuronidase

(uidA) genes under the control of CaMV 35S promoter. The plasmid construction was

introduced into Agrobacterium tumefaciens EHA-105 by direct DNA uptake. Preparation of

leaf-disk explants of wild-type (WT) tobacco (Nicotiana tabacum cv. Havana),

Agrobacterium transformation and plant regeneration were carried out as previously described

(Horsch et al. 1985). Three transgenic lines derived from distinct transformation events were

selected on appropriate media by their resistance to kanamycin, positive reaction in GUS

histochemical assays (Jefferson 1989) and by PCR amplification of nptII and uidA genes.

Transgenic plants were maintained on MS medium (Murashige and Skoog 1962) containing

kanamycin sulfate (100 mg L-1

) until rooting. The same procedure was performed to WT

plants, but they were maintained on MS medium without kanamycin sulfate. Primary

transformants (T0) were transferred to soil and grown in greenhouse at air temperature,

relative humidity and daily photon irradiance conditions of 26 ± 2 °C, 80 ± 3% and 12 ± 2

mol m-2

d-1

, respectively, in order to obtain the T1 generation. Resistance to kanamycin in T1

progeny produced by self-pollination was used to select transformed plants. Kanamycin-

resistant T1 plants were transferred to soil and grown in greenhouse for further analyses.

Three replicates of each kanamycin-resistant T1 transgenic line and WT plants were used in

all experiments. All plants were maintained under the same conditions of growth in

greenhouse.

30

RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was isolated from E. grandis seedlings and leaves of kanamycin-resistant

T1 and WT tobacco plants using the PureLinkTM

Plant RNA Reagent (Invitrogen, USA),

according to manufacturer's instructions. The purity and concentration of total RNA samples

were determined spectrophotometrically and by agarose gel electrophoresis. Total RNA

samples were treated with RNase-free DNase I (Fermentas, USA) to remove any

contaminating genomic DNA, following manufacturer's recommendations. Total RNA treated

with DNase I was used in cDNA synthesis with the M-MLV Reverse Transcriptase (Promega,

USA), according to manufacturer's instructions.

Steady-state EgPHI-1 mRNA levels were estimated using the ABI 7500 Real Time

PCR System (Applied Biosystems, USA) and the SYBR Green PCR Master Mix (Applied

Biosystems, USA), according to manufacturer's recommendations. Quantitative real-time

PCRs were performed using three biological and three experimental replicates. The genes

encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH; AJ133422.1) and ribosomal

protein L23A (RibL23A) (de Oliveira et al. 2012) were used as internal reference genes for

normalizing EgPHI-1 expression in tobacco and in E. grandis, respectively. A negative

control without cDNA template was included for each primer pair. Primers sequences were:

GAPDH (5’-TGTCAATGAGAAAGAATAC-3’/ 5’-AGACCCTCAACAATTCCAAA-3’),

RibL23A (5’-AAGGACCCTGAAGAAGGACA-3’/ 5’-CCTCAATCTTCTTCATCGCA-3’),

and EgPHI-1 (5’-GCTCTGACCAGTTTCCGAAAA-3’ /

5’CCGGAACAGAGGTTAGGTAAGCT-3’). The relative expression values were calculated

using the 2-∆∆Ct

method (Livak and Schmittgen 2001).

Gas leaf exchange and growth measure

Kanamycin-resistant T1 and WT tobacco plants grown for 90 days under greenhouse

conditions were used in the analysis of leaf gas exchange parameters, which included net

photosynthetic rate (A), leaf transpiration (E), stomatal conductance to water vapor (gs) and

ratio to internal (Ci) and atmospheric (Ca) concentration of CO2 (Ci/Ca) in leaves. The gas

exchange parameters were measured in the early morning (08:00 – 09:00 AM) using a

portable photosynthesis system LI-6400 (Li-Cor) equipped with an artificial light resource

31

(6400-02B RedBlue). All measurements were made on the third, fully expanded mature leaf

from the apex of three replicates of each plant. Instantaneous water-use efficiency was

calculated by the ratio to A/E values, while the intrinsic water-use efficiency was obtained by

the ratio to A/gs values.

The same kanamycin-resistant T1 and WT plants mentioned above were used to

measure plant height, root length and volume, leaf number per plant and leaf area, which was

calculated by the ratio to total leaf area and leaf dry biomass. Root volume was measured

using the intact root system to displace the water column in a graduated cylinder. Total leaf

area was determined using the LI-3100 area meter (Li-Cor). Leaves, stems and roots were

separated and kept in an air-circulation stove at 75 °C until a constant weight was obtained.

These materials were used to determine the dry biomass of the different plant parts.

Xylem anatomy

Stem samples (third internode from the apex) were collected from 90-days-old

kanamycin-resistant T1 and WT tobacco plants maintained under the same growth conditions

described above. Samples were fixed in 70% ethanol and used for xylem anatomical analysis.

Free-hand stem cross sections were cleared and stained with 1% (v/v) astra blue and 1% (v/v)

safranin. Stem samples were also used to obtain isolated fibers. Plant material for this purpose

was maintained in a solution of glacial acetic acid and hydrogen peroxide (1:1) for 48 h at 60

°C. The resulting fibrous complex was washed and stained with 1% (v/v) safranin.

Microscopic slides were prepared with the double staining cross sections, as well as

with isolated staining fibers. Slides were viewed under a light microscope (Olympus - CX41)

and photographed with a digital camera (Olympus - C7070) attached to the equipment. Xylem

anatomy was also observed at cross sections of the base of the petioles (third fully expanded

leaves from the apex). Lignin autofluorescence of the vessels was detected using an

epifluorescence microscope (Leica DMRA2) equipped with a camera (Leica MPS60) using

340-380 nm excitation wavelength and 400 nm barrier filters.

Images were analyzed employing the ImageJ 1.44 software (http://imagej.nih.gov.ij).

It was performed 135 measurements of fiber length. Double staining from stem xylem images

were used to determine the vessel lumen diameter. Ten measurements were performed at

http://imagej.nih.gov.ij/

32

different locations in each image. Three biological replicates were used, each containing three

slides.

Enzyme assays

The extracts for measuring phenylalanine ammonia-lyase (PAL) and peroxidase

(POD) activities were obtained from lyophilized stem samples that were macerated in the

presence of liquid nitrogen and 1% (w/w) PVP. Specific extraction buffers for each enzyme

were used to homogenize 50 mg of the macerated material; for PAL, 5 mL of 100 mM

sodium borate buffer pH 7.0, while for POD, 2 mL of buffer composed of 50 mM potassium

acetate pH 5.0 and 1 mM EDTA. The homogeneous mixtures were sonicated using a probe

(Ultrasonic Processor - GEX130) for 1 min (six cycles of 10 s with 20 s intervals between

cycles) at 70% amplitude. The whole procedure with samples was carried out in ice bath.

Extracts were centrifuged for 35 min at 4 °C (16,000 × g) and supernatants were recovered.

For PAL activity assay, an aliquot of protein extract (0.8 mL) was mixed to 2 mL

reaction buffer composed of 50 mM sodium borate buffer pH 8.8 and 6 mM L-phenylalanine.

The mixture was incubated at 40 °C for 1 h. PAL activity was determined by the conversion

of L-phenylalanine into trans-cinnamic acid with the increase in absorbance at 290 nm. All

activity assays were carried out in three replicates for each sample. For POD activity assay, an

aliquot of the protein extract (150 µL) was mixed to 150 µL reaction buffer composed of 50

mM potassium acetate pH 5.0, 20 mM guaiacol and 0.8 M hydrogen peroxide. POD activity

was measured spectrophotometrically at 25 °C for a period of 2 min, with 10 s intervals. The

absorbance at 470 nm due to the consumption of guaiacol was converted into mmol h–1

g–1

(DM) by the equation y = 0.0189 + 0.1284 x (R2 = 0.99) from a POD-guaicol standard curve

(Rehem et al. 2011).

Recombinant protein production and antibody preparation

Production and purification of the EgPHI-1 protein was achieved by cloning the

encoding gene into pET-32a. Briefly, a 1.2 kb fragment was released from the pSPORT1

cloning vector by NcoI/HindIII digestion. EgPHI-1 protein from amino acids 40 to 312 (EGP)

was expressed as a N-fusion to the TRX-HIS-S tag region of the expression vector pET-32a

in Escherichia coli strain Rosetta DE3. Synthesis of the recombinant protein was induced by

33

adding 0.4 mM IPTG to bacterial culture at 18 ºC. After 16 h, cells were harvested and EGP

recombinant protein was affinity purified using Ni2+

-agarose (Qiagen, USA) according to

manufacturer’s instructions. The protein tag was removed by enzymatic digestion with

thrombin and the EGP purified protein was used as an antigen for anti-EGP antibody

production in rabbits, which were immunized with three subcutaneous injections at 2-weeks

intervals.

Protein extraction and immunoblotting analysis

Total protein was extracted from lyophilized leaf samples that were macerated in

liquid nitrogen and 1% (w/w) PVP by the phenol/SDS method (Pirovani et al. 2008). About

100 mg of the resulting powder was homogenized with 2 mL of acetone. The homogeneous

mixture was centrifuged at 16,000 × g for 3 min at 4 °C. The pellet recovered was washed two

times with icy acetone and then more two times with 10% (w/v) TCA in acetone. The mixture

was homogenized and sonicated under refrigeration for 15 min (pulses of 10 s with 20 s

intervals between each cycles) at 70% amplitude (Ultrasonic Processor - GEX130). The

sonicated material was centrifuged at 16,000 × g for 3 min at 4 °C and the pellet recovered

was washed with 80% icy acetone. After wash, the homogenized material was centrifuged at

16,000 × g for 3 min at 4 °C. The pellet was dried under vacuum, and 100 mg of the acetone

dry powder was homogenized in 0.8 mL of SDS dense buffer [100 mM Tris-HCl pH 8.0, 2%

(w/v) SDS, 30% (w/v) sucrose and 5% (v/v) 2-mercaptoetanol] and 0.8 mL of phenol

saturated with Tris, pH 8.0. The phases were then separated by centrifuging at 16,000 × g for

3 min at 4 °C. The protein fraction was allowed to precipitate for 12 h at -20°C by adding 5

volumes of 0.1 M ammonium acetate in icy methanol. The precipitated material was collected

by centrifugation at 16,000 × g for 5 min at 4 °C and washed two times with 0.1 M

ammonium acetate in icy methanol and once with 80% icy acetone. The protein extract was

dried at room temperature and solubilized in 100 μL rehydration buffer (7 M urea, 2 M

thiourea, 2% (w/v) CHAPS, 0.002% (w/v) bromophenol blue). The insoluble residue was

removed by centrifugation, and the total protein concentration was determined using the 2-D

Quant Kit (GE Healthcare,USA) according to manufacturer’s instructions. Equivalent

amounts of total protein (40 mg) were resolved by 12.5% SDS-PAGE and transferred to

nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen, USA) according to

manufacturer’s instructions. The membrane was blocked with 5% (w/v) nonfat dry milk in

TBS-T [100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% (v/v) Tween 20]. EgPHI-1 was

34

detected using the purified antibody anti-EGP at a 1:2,000 dilution, followed by a goat anti-

rabbit IgG conjugated to alkaline phosphatase (Sigma, USA) at a 1:10,000 dilution. The

activity of alkaline phosphatase was assayed using 5-bromo-4-chloro-3-indolyl phosphate and

p-nitroblue tetrazolium (Promega, USA). Protein was quantified from membrane imaging

using the GelQuant.Net 1.8.0 software (www.biochemlabsolutions.com) and the values of

transgenic lines were calculated in relation to the WT protein levels.

Statistical analysis

Statistical analysis was performed with the software BioEstat 5.3

(http://www.mamiraua.org.br/), which tested the experiments in a completely randomized

design. Statistical differences between WT and transgenic lines and between treated and

untreated E. grandis seedlings were assessed based on the analysis of variance (ANOVA) and

means were separated by Student’s t-test, with a critical value of p≤0.05, p≤0.01 and p≤0.001.

Results

Identification and sequence analysis of EgPHI-1

The EgPHI-1 cDNA sequence characterized in this study was 100% similar to coding

sequence of the locus Eucgr.F00356 in the E. grandis genome sequence database

(http://www.phytozome.net/eucalyptus.php). Therefore, we assumed that EgPHI-1

corresponds to this locus in the only reference genome sequence available for Eucalyptus.

EgPHI-1 was 1,234 bp in length and contained an open reading frame (ORF) of 951 bp

encoding a deduced protein of 316 amino acid residues, with a theoretical isoelectric point of

8.06 and a calculated molecular weight of 33.1 kDa. A signal peptide possibly involved in the

secretory pathway targeting to the endoplasmic reticulum was identified in the N-terminal

portion of the EgPHI-1 deduced amino acid sequence, with a probable cleavage site between

residues G27 and S28 (Fig. 1A). The absence of hydrophobic regions to form transmembrane

helices along the sequence suggests that EgPHI-1 is a soluble protein possibly localized in the

apoplast. Specific sites of glycosylation (O-β-GlcNAc, T257 and T264), serine (S57, S70,

S120, S147 and S310), threonine (T65, T84 and T213) and tyrosine phosphorylation (Y248)

and phosphorylation by protein kinase C (PKC, T104) were also identified in the deduced

amino acid sequence of EgPHI-1 (Fig. 1A).

http://www.biochemlabsolutions.com/

http://www.mamiraua.org.br/

http://www.phytozome.net/eucalyptus.php

35

Promoter analysis and expression of EgPHI-1 in E.grandis

Analysis of the 1.5 kb 5'-upstream promoter region of EgPHI-1 indicated the presence

of several regulatory cis-elements (Fig. 1B). Interestingly, among the elements identified are

those involved in the response to light (Sp1 [CC(G/A)CCC], GAG-motif [AGAGATG], Box

II [ACACGTTGT], I-box [GATATGG/CCTTATCCT] TCT-motif [TCTTAC], GA-motif

[ATAGATAA], GATA-motif [(AAG)GATA(GGA/AGG)] and G-box

[(TAA/C)ACGT(G/T)]). In addition, two as-2-box cis-elements [GATAATGATG] involved

in tissue-specific expression in shoots were also identified. The promoter region of EgPHI-1

also has one element which putatively regulates the circadian rhythm (Circadian

[CAANNNNATC]), two binding sites of MYB (MRE [AACCTAA] and MBS [CGGTCA]),

two response elements to abscisic acid (ABA; ABRE [(GCA/C)ACGTG(TC)]) and two

response elements to methyl jasmonate (MeJA; CGTCA-motif [CGTCA] and TGACG-motif

[TGACG]). Other cis-elements found in our analysis were as follow: two ARE elements

[TGGTTT], which are essential for anaerobic induction, one HSE element

[AGAAAATTCG], which responds to heat stress, and one TCA-element [TCAGAAGAGG],

an important element for the responses induced by salicylic acid (Fig. 1B).

To examine some of the possible conditions that induce EgPHI-1 expression in E.

grandis, seedlings were grown in vitro and subjected to four different treatments. Expression

analysis showed that EgPHI-1 transcription can be induced by mechanical wound, NAA,

kinetin and dehydration (Fig. 1C). E. grandis seedlings treated with NAA showed the highest

EgPHI-1 expression compared with the other treatments. EgPHI-1 expression in the kinetin-,

mechanical wound- and dehydration-treated seedlings was nearly twice as high as that of

untreated seedlings (p0.05; Fig. 1C).

PHI-1 domain and similarity analysis of EgPHI-1

The major structural feature of EgPHI-1 is a region of 260 amino acid residues (Y44

to S314) harboring the PHI-1 domain [Pfam 04674] (Fig. 1A). The EgPHI-1 and other 43

homologous amino acid sequences previously described (Schröder et al. 2009) were aligned

for sequence comparison and phylogenetic analysis (Fig. 2A). Our results indicated that

EgPHI-1 belongs to a subset of proteins different from the previously characterized tobacco

PHI-1 and Arabidopsis EXO (Sano et al. 1999, Farrar et al. 2003). EgPHI-1 grouped into a

36

clade that includes proteins from Oryza sativa and Vitis vinifera (Fig. 2A). EgPHI-1 shares

64% similarity with AAM08535 of O. sativa and 75% similarity with CAO61694 of V.

vinifera. A high degree of conservation among these sequences can be visualized in Fig. 2B.

Growth and biomass partitioning altered in EgPHI-1-overexpressing transgenic

plants

Aiming to clarify the possible role of EgPHI-1 in the processes affecting wood

properties and plant growth, transgenic tobacco plants were generated containing the gene

construct shown in Fig. 3A. Analysis of the EgPHI-1 transcript and protein levels was carried

out in three 35S::EgPHI-1 transgenic lines by reverse transcription-quantitative PCR (RT-

qPCR; Fig. 3B) and western blot (Fig. 3C), respectively. The transgenic line L3 showed the

highest levels of EgPHI-1 transcripts, whereas L2 presented an intermediate EgPHI-1 mRNA

steady-state levels. Transgenic L1 plant exhibited the lowest transcript levels of EgPHI-1,

although still much higher than the WT plants (Fig. 3B). Similar profiles among transgenic

plants were also observed at protein levels, as shown in Fig. 3C.

Analysis and measures of growth of transgenic and WT plants were performed 90

days after their maintenance under normal growth conditions in greenhouse. The transgenic

lines of higher EgPHI-1 expression were taller and had more leaves than WT plants (Figs. 4A,

B). However, all transgenic plants exhibited smaller leaf area than WT plants (Fig. 4C).

Measurements of dry biomass of different plant organs showed that EgPHI-1 caused

significant changes in the allocation of biomass. All transgenic plants showed a significant

increase in leaf biomass (Fig. 4D), while for the stem biomass only L3 exhibited a significant

increase (p0.05; Fig. 4E). On the other hand, root biomass was significantly reduced in all

transgenic plants (Fig. 4F). A reduction in the length of roots was observed only for L1 (Fig.

4G). In contrast, the roots of all transgenic plants showed a higher volume than the WT (Fig.

4H). Although important changes were observed in the proportion of dry biomass among the

different organs of transgenic plants (Fig. 4I), their total biomass did not show significant

variation (p0.05) when compared to WT plants. These results clearly demonstrate that the

overexpression of EgPHI-1 alters the partitioning of biomass among different plant organs,

favoring its allocation to the shoots.

37

EgPHI-1 changes stem anatomy and fibers

In order to evaluate the effect of EgPHI-1 on stem anatomy, cross-sections of the third

internode from the apex were collected and analyzed under light microscopy. Anatomical

alterations were observed in transgenic plant stems as compared to the WT (Fig. 5A).

Transgenic plants L1 exhibited a differential activity of the vascular cambium, with greater

differentiation of primary xylem cells. Primary xylem vessel elements of L2 presented a

peculiar lignification, as it thickened in the periclinal region of the cell walls. Transgenic

plants L3 showed an intense process of secondary xylem formation, consisting mostly of

secondary cell walls in relation to WT plants (Fig. 5A). EgPHI-1 also produced changes in the

morphology of stem vessels and fibers. Transgenic plants exhibited a significant increase in

fiber length (Fig. 5B). A significant increase in the lumen diameter of the vessels was

observed in two (L2 and L3) out of the three transgenic plants analyzed (Fig. 5C).

EgPHI-1 affects the activity of enzymes involved in lignin biosynthesis

The activities of phenylalanine ammonia-lyase (PAL) and peroxidase (POD), two

important enzymes involved in the lignin biosynthetic pathway, were analyzed in stem

samples of transgenic and WT plants. The results demonstrated that the overexpression of

EgPHI-1 promoted significant alterations in the activity of these enzymes (Figs. 6A, B). PAL

activity was significantly (p0.05) higher in all transgenic as compared to WT plants (Fig.

6A), while POD activity was significantly (p0.05) higher in the L2 and L3 transgenic plants

(Fig. 6B).

EgPHI-1 alters lignin deposition in the leaf xylem

Cross-sections of the base of the petioles of transgenic and WT plants were observed

by epifluorescence microscopy to evidence the lignified structures in leaf xylem. The

autofluorescence exhibited by these structures showed that the overexpression of EgPHI-1

also promoted important changes in the lignin deposition in leaf xylems (Fig. 7). In general,

the xylem lignified of transgenic plants L1 and L3 were more organized and closer to each

other, resulting in a more compact structure. The size and shape of the cells of these two

transgenic lines also showed higher linearity and symmetry in comparison to WT plants.

Transgenic plants L2 showed a more pronounced compression of the xylem cells; however,

38

linearity in the shape of the cell walls was less evident (Fig. 7). Observations of the

electronically modified images (black & white color) to highlight the lignified areas (blue

fluorescence) demonstrated that the intensity of fluorescence varied among transgenic plants

according to EgPHI-1 expression levels, suggesting the occurrence of variation of lignin

content or deposition in the cell walls of the xylem in transgenic plants (Fig. 7).

EgPHI-1 affects the leaf CO2 assimilation

The effects of EgPHI-1 overexpression on leaf gas exchange parameters of transgenic

and WT plants were also evaluated. For this evaluation, the mean values of irradiance,

temperature and relative humidity recorded during the experimental period were 1,000 ± 0.7

µmol photons m-2

s-1

, 26 ± 0.2 oC and 70 ± 3 %, respectively. The overexpression of EgPHI-1

significantly reduced (p0.05) the net photosynthetic rate (A) in all transgenic plants (Fig.

8A). Leaf transpiration (E) was reduced in L1 (-6.4%) and L2 (-13.7%) transgenic plants,

while L3 showed an increase of 7.0% in this parameter compared to WT plants (Fig. 8B). The

stomatal conductance to water vapor (gs) did not change significantly (p0.05) in transgenic

plants compared to WT (Fig. 8C), with the exception of L3. The ratio to internal (Ci) and

atmospheric (Ca) concentration of CO2 (Ci/Ca) in leaves increased significantly in all

transgenic plants (Fig. 8D). The reduction of A and the increase of Ci/Ca, without changing

the value of gs, suggests that EgPHI-1 overexpression limits, in some way, the ability of the

mesophyll to assimilate CO2. Transgenic plants also showed lower intrinsic (A/gs) and

instantaneous (A/E) water-use efficiencies as compared to WT plants (Figs. 8E, F).

Discussion

In silico analyses indicated that EgPHI-1 is a soluble protein with a signal peptide

related to the secretory pathway, suggesting that it is an apoplastic protein potentially

associated with the plant cell wall, as reported for EXO/EXL proteins (Schröder et al. 2009).

The different post-translational modification sites present in EgPHI-1, such as glycosylation

and phosphorylation sites, suggest that this protein may play a functional role by the

phosphorylation/dephosphorylation promoted by kinases and specific phosphatases, as

reported for tobacco PHI-1 (Sano et al. 1999).

39

The identification of known cis-elements in the promoter region of EgPHI-1 may

provide important clues about its regulation. For example, the presence of ABRE cis-elements

suggests that EgPHI-1 can be regulated by ABA (Xiong and Zhu 2003), as reported for

tobacco PHI-1 (Schröder et al. 2009). The presence of MBS and MRE cis-elements also

suggests that EgPHI-1 is regulated by transcription factors of the MYB family, which have

been implicated in regulating the secondary growth in Arabidopsis and the lignification

process and flavonoid biosynthesis in Antirrhinum (Tamagnone et al. 1998, Oh et al. 2003).

The remainder cis-elements identified suggest that the expression of EgPHI-1 may be under

light and circadian control (Saibo et al. 2009) and also may be induced under anaerobic

conditions (Shinozaki et al. 2003, Heis et al. 2011), heat (Haralampidis et al. 2002), injuries

caused by insects and fungal pathogens (Wang et al. 2011, Zhang et al. 2011) and salicylic

acid (Ohtake et al. 2000). The presence of cis-elements regulated by light, circadian cycle and

anaerobic conditions was reported for EXL1, which is expressed especially under low

irradiance conditions, prolonged darkness and anoxia (Schröder et al. 2011, Schröder et al.

2012).

Induction of EgPHI-1 expression by NAA and kinetin indicates a positive feedback

loop between EgPHI-1 and auxin/cytokinin signalling. Auxin and cytokinin are considered

essential for early xylem differentiation during both normal development and wounding (Ye

2002, Pesquet et al. 2005, Turner et al. 2007). The wounding treatment further demonstrated

that EgPHI-1 is a wound-inducible gene, suggesting its involvement in jasmonic acid

(JA)‑dependent and/or ‑independent wound signaling pathways (León et al. 2001). The

induction of its expression by dehydration-stress demonstrates that EgPHI-1 can also

participate in the regulation of abiotic stress responses through an ABA-dependent signaling

pathway, as suggested by the presence of ABREs in its promoter region (Yamaguchi-

Shinozaki and Shinozaki 2006, Gomez-Porras et al. 2007, Fujita et al. 2011). ABA-induced

PHI-1 expression was shown in phosphate-starved BY-2 tobacco cells (Sano and Nagata

2002).

The PHI-1domain present in the EgPHI-1 deduced amino acid sequence is a long and

structurally conserved region that identifies the PHI-1/EXO protein family and whose

function and mechanism of action have not been clarified yet. However, members of this

family have been implicated in processes of cell expansion and proliferation (Farrar et al.

2003, Schröder et al. 2009, Schröder et al. 2011). EgPHI-1 displays considerable similarity to

40

tobacco PHI-1 (Sano et al. 1999) and Arabidopsis EXO/EXL (Schröder et al. 2009), but direct

comparisons among these and other PHI-1/EXO sequences showed that EgPHI-1 belongs to a

subgroup that is divergent and distinct from those PHI-1/EXO of tobacco and Arabidopsis

and includes orthologues of grape and rice. Thus, EgPHI-1 represents a new member of PHI-

1/EXO protein family with functions or mode of action that may differ, at least in part, from

the previously characterized members of this family.

EgPHI-1 overexpression promoted significant changes in the growth parameters of

transgenic plants, including height, leaf number, area and biomass, and root volume and

biomass. Measurements of dry weight indicated that EgPHI-1 did not alter the total biomass

of transgenic plants in comparison with WT plants. However, it changed the biomass

partitioning, which was preferentially allocated to shoots as opposed to roots. These results

contrast with those obtained for EXO of Arabidopsis, in which its overexpression promoted

both shoot and root growth (Schröder et al. 2009). With the increase in leaf biomass and leaf

number per plant in the EgPHI-1 transgenic lines, there was a reduction in the leaf area,

suggesting an increase in leaf thickness or density, probably attributable to enhanced vascular

differentiation. Transgenic tobacco plants exhibited a higher root volume than WT plants,

which also suggests the production of thicker roots caused by enhanced vascular

differentiation. However, the decreased root biomass in transgenic plants indicates that the

EgPHI-1-induced anatomical modifications also change the patterns of assimilate export,

which are channeled to shoots probably due to the increased xylem vessel lumen diameter of

stem and leaf. Since hydraulic conductivity is directly related with vessel diameter, the

EgPHI-1-induced changes may favor water and solute transport through the xylem and,

hence, improve shoot growth. Besides the increase in vessel lumen diameter, vessel collapse

was not observed in transgenic plants probably due to its increased lignification. To withstand

with the pressure generated by water transport through the xylem, vessel walls must be

strengthened by the deposition of lignin which enables the maintenance of xylem integrity and

hydraulic architecture (Smart and Amrhein 1985). The vessel wall collapse can limit the

supply of water and affect the growth and morphology of the plants (Piquemal et al. 1998).

Analysis of stem anatomy in transgenic plants indicates that EgPHI-1 mediates the

differentiation of xylem cells, the development and elongation of fibers and the process of

lignification. This is the first report describing the involvement of a PHI-1/EXO protein

family member in such developmental processes. Nevertheless, the expression of genes

41

encoding cell wall modifying proteins such as KCS1, Exp5, AGP4, δ-TIP and WAK-1 has

been previously reported to be EXO-dependent (Coll-Garcia et al. 2004, Schröder et al. 2009).

It is believed that the EXO protein promotes cell expansion via modulation of the gene

expression of this set of genes (Farrar et al. 2003, Coll-Garcia et al. 2004). KCS1 is a protein

involved in wax biosynthesis (Todd et al. 1999). Exp5 is an expansin involved in cell wall

loosening and break of non-covalent bonding between cellulose and hemicellulose (Cosgrove

2005). AGP4 is an extracellular arabinogalactan protein that is implicated in many plant

growth and developmental processes (Steinmacher et al. 2012). The δ-TIP protein is a plant

aquaporin that changes the membrane water permeability (Forrest and Bhave 2008). WAK1 is

a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular

domain which interacts with cell wall pectins, playing a role in the development and cell

expansion (Decreux and Messiaen 2005). The relationship between EgPHI-1 and these cell

wall modifying proteins, if any, remains to be examined in future investigation.

The cell wall is composed mainly of cellulose, hemicellulose and lignin, and the

proportion of these compounds is critical to the secondary cell wall formation (Zhong et al.

2010). Variations in the lignin autofluorescence intensity were observed among transgenic

plants accordingly to the levels of EgPHI-1 expression, suggesting the involvement of

EgPHI-1 in the regulation of enzymes involved in lignin biosynthesis. Analysis of PAL and

POD confirmed our hypothesis of increased activity of these enzymes in transgenic plants.

PAL catalyzes the first step of the general phenylpropanoid pathway, providing the precursors

for the biosynthesis of lignin, flavonoids, stilbenes and many other phenolic compounds

(Whetten and Sederoff 1995). On the other hand, POD catalyzes the last enzymatic step in the

lignin biosynthesis, the dehydrogenation of p-coumaryl alcohols that are deposited in the

secondary cell wall (Harkin and Obst 1973, Piquemal et al. 1998). These data suggest that

EgPHI-1 may act as a component in the signaling pathway that controls cell wall

biosynthesis. The presence of MRE/MBS cis-elements (Dubos et al. 2010) in the EgPHI-1

promoter region also corroborates with this hypothesis. AtMYB52, AtMYB54, AtMYB69

and AtMYB103 are considered positive regulators of lignin, cellulose and xylan biosynthesis,

as well as of fiber cell wall thickening in Arabidopsis (Zhong et al. 2008).

More noteworthy, the overexpression of EgPHI-1 significantly reduced the net

photosynthetic rate without causing significant changes in gs, except for transgenic line L3,

which also showed a significant increase in E leaf transpiration. Transgenic plants showed a

42

significant increase in the Ci/Ca ratio. These results suggest that the reduction of A in

transgenic plants may be due to the limitation of CO2 assimilation in the leaf mesophyll. This

limitation may be biochemical, caused by changes in enzyme activities involved in

carboxylation reactions (Bernacchi et al. 2003), or morphological, caused by leaf anatomy

changes (Terashima et al. 2001, Hanba et al. 2002). Despite the decreased A in transgenic

plants, reduction in the total biomass production or carbon assimilation was not observed in

comparison with WT plants.

In conclusion, the overexpression of EgPHI-1 in transgenic tobacco improves shoot

growth and xylem differentiation. EgPHI-1 presumably acts in signaling pathways which

control cell division and differentiation in response to both endogenous and environmental

signals. The modulation of complex traits such as plant growth and wood formation is of

central interest for biotechnological applications in forestry, where it is desirable to maximize

the above ground carbon.

Acknowledgements

The authors are very grateful to Dr. Marco Antônio Costa and Rodolfo Telles F.

Menezes from Cytogenetics Laboratory (State University of Santa Cruz – UESC, Ilhéus-BA,

Brazil) by the technical support. They also thank Dr. Amanda F. S. Mendes and Nathália dos

Santos Lima (State University of Santa Cruz – UESC, Ilhéus-BA, Brazil) for their

contributions in the enzyme assays and fiber measuring, respectively. This work was

supported by Financiadora de Estudos e Projetos (FINEP, Brazilian Ministry of Science and

Technology - MCT) [grant number 2101063500]; Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq, MCT) [Grant numbers 50.6348/04-0, 578632/08-0,

311361/09-9]; and the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB,

Secretariat of Science, Technology and Innovation – SECTI) [grant number BOL1569/2010].

References

Bayer EM, Bottrill AR, Walshaw J, Vigouroux M, Naldrett MJ, Thomas CL, Maule AJ

(2006) Arabidopsis cell wall proteome defined using multidimensional protein

identification technology. Proteomics 6: 301-311

43

Bernacchi CJ, Pimentel C, Long SP (2003) In vivo temperature response functions of

parameters required to model RuBP-limited photosynthesis. Plant Cell Environ 26: 1419-

1430

Bernard M (2003) Eucalyptus: a strategic forest tree. In: Wei R-P, Xu D (Eds.) Eucalyptus

plantations: research, management and development. Singapure: World Scientific. p. 3-18.

Coll-Garcia D, Mazuch J, Altmann T, Müssig C (2004) EXORDIUM regulates

brassinosteroid-responsive genes. FEBS Lett 563: 82-86

Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev Mol Cell Biol 6: 850-861

de Oliveira LA, Breton MC, Bastolla FM, Camargo SdS, Margis R, Frazzon J, Pasquali G

(2012) Reference genes for the normalization of gene expression in Eucalyptus species.

Plant Cell Physiol 53: 405-422

De Vos M, Jander G (2009) Myzus persicae (green peach aphid) salivary components induce

defence responses in Arabidopsis thaliana. Plant Cell Environ 32: 1548-1560

Decreux A, Messiaen J (2005) Wall-associated kinase WAK1 interacts with cell wall pectins

in a calcium-induced conformation. Plant Cell Physiol 46: 268-278

Dellagi A, Birch PRJ, Heilbronn J, Avrova AO, Montesano M, Palva ET, Lyon GD (2000) A

potato gene, erg-1 is rapidly induced by Erwinia carotovora ssp atroseptica, Phytophthora

infestans, ethylene and salicylic acid. J Plant Physiol 157: 201-205

Dita MA, Die JV, Ronàn B, Krajinski F, KüSter H, Moreno MT, Cubero JI, Rubiales D

(2009) Gene expression profiling of Medicago truncatula roots in response to the parasitic

plant Orobanche crenata. Weed Res 49: 66-80

Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB

transcription factors in Arabidopsis. Trends Plant Sci 15: 573-581

Eldridge KG, Davidson J, Harwood CE, van Wyk G (1993) Eucalypt domestication and

breeding. Oxford: Clarendon Press.

Farrar K, Evans IM, Topping JF, Souter MA, Nielsen JE, Lindsey K (2003) EXORDIUM - a

gene expressed in proliferating cells and with a role in meristem function, identified by

promoter trapping in Arabidopsis. Plant J 33: 61-73

Forrest K, Bhave M (2008) The PIP and TIP aquaporins in wheat form a large and diverse

family with unique gene structures and functionally important features. Funct Integr

Genomics 8: 115-133

Fujita Y, Fujita M, Shinozaki K, Yamaguchi-Shinozaki K (2011) ABA-mediated

transcriptional regulation in response to osmotic stress in plants. J Plant Res 124: 509-525

44

Gomez-Porras J, Riano-Pachon D, Dreyer I, Mayer J, Mueller-Roeber B (2007) Genome-wide

analysis of ABA-responsive elements ABRE and CE3 reveals divergent patterns in

Arabidopsis and rice. BMC Genomics 8: 260

Hanba YT, Kogami H, Terashima I (2002) The effect of growth irradiance on leaf anatomy

and photosynthesis in Acer species differing in light demand. Plant Cell Environ 25: 1021-

1030

Haralampidis K, Milioni D, Rigas S, Hatzopoulos P (2002) Combinatorial interaction of cis

elements specifies the expression of the Arabidopsis AtHsp90-1 gene. Plant Physiol 129:

1138-1149

Harkin JM, Obst JR (1973) Lignification in trees: indication of exclusive peroxidase

participation. Science 180: 296-298

Heis M, Ditmer E, de Oliveira L, Frazzon AP, Margis R, Frazzon J (2011) Differential

expression of cysteine desulfurases in soybean. BMC Plant Biol 11: 166

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and

general method for transferring genes into plants. Science 227: 1229-1231

Iwase A, Ishii H, Aoyagi H, Ohme-Takagi M, Tanaka H (2005) Comparative analyses of the

gene expression profiles of Arabidopsis intact plant and cultured cells. Biotechnol Lett 27:

1097-1103

Jefferson RA (1989) The GUS reporter gene system. Nature 342: 837-838

Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices

from protein sequences. Comput Appl Biosci 8: 275-282

Ko JH (2004) Plant body weight-induced secondary growth in Arabidopsis and its

transcription phenotype revealed by whole-transcriptome profiling. Plant Physiol 135:

1069-1083

León J, Rojo E, Sánchez-Serrano JJ (2001) Wound signalling in plants. J Exp Bot 52: 1-9

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time

quantitative PCR and the 2−ΔΔCt

method. Methods 25: 402-408

Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco

tissue cultures. Physiol Plant 15: 473-497

Mustafa BM, Coram TE, Pang ECK, Taylor PWJ, Ford R (2009) A cDNA microarray

approach to decipher lentil (Lens culinaris) responses to Ascochyta lentis. Australas Plant

Pathol 38: 617-631

45

Myburg AA, Potts BM, Marques CMP, Kirst M, Gion J-M, Grattapaglia D, Grima-Pettenati J

(2007) Eucalyptus. In Kole CR (Ed) Genome mapping and molecular breeding in plants,

vol 7. Berlin: Springer. p. 115-160

Norton GJ, Nigar M, Williams PN, Dasgupta T, Meharg AA, Price AH (2008) Rice–arsenate

interactions in hydroponics: a three-gene model for tolerance. J Exp Bot 59: 2277-2284

Oh S, Park S, Han K-H (2003) Transcriptional regulation of secondary growth in Arabidopsis

thaliana. J Exp Bot 54: 2709-2722

Ohtake Y, Takahashi T, Komeda Y (2000) Salicylic acid induces the expression of a number

of receptor-like kinase genes in Arabidopsis thaliana. Plant Cell Physiol 41: 1038-1044

Pasquali G, Bastolla FM, Pazzini F, Kirch RP, Pizzoli G, Carazzole MF, Brondani RV,

Coelho ASG, Grattapaglia D, Brommonschenkel SH, Pappas Jr GJ, Pereira GAG,

Cascardo JCM (2005) Sequencing and differential expression of xylem specific genes from

two Eucalyptus species with highly contrasting wood properties. In Annals of IUFRO Tree

Biotechnology, Pretoria, South Africa. p. 22

Pesquet E, Ranocha P, Legay S, Digonnet C, Barbier O, Pichon M, Goffner D (2005) Novel

markers of xylogenesis in Zinnia are differentially regulated by auxin and cytokinin. Plant

Physiol 139: 1821-1839

Piquemal J, Lapierre C, Myton K, O’connell A, Schuch W, Grima-Pettenati J, Boudet A-M

(1998) Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin

profiles in transgenic tobacco plants. Plant J 13: 71-83

Pirovani CP, Carvalho HAS, Machado RCR, Gomes DS, Alvim FC, Pomella AWV,

Gramacho KP, Cascardo JCdM, Pereira GAG, Micheli F (2008) Protein extraction for

proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora

perniciosa, the causal agent of the witches' broom disease. Electrophoresis 29: 2391-2401

Rehem BC, Almeida A-AF, Santos IC, Gomes FP, Pirovani CP, Mangabeira PAO, Corrêa

RX, Yamada MM, Valle RR (2011) Photosynthesis, chloroplast ultrastructure, chemical

composition and oxidative stress in Theobroma cacao hybrids with the lethal gene Luteus-

Pa mutant. Photosynthetica 49: 127-139

Saibo NJM, Lourenço T, Oliveira MM (2009) Transcription factors and regulation of

photosynthetic and related metabolism under environmental stresses. Ann Bot 103: 609-

623

Sano T, Kuraya Y, Amino S-I, Nagata T (1999) Phosphate as a limiting factor for the cell

division of tobacco BY-2 cells. Plant Cell Physiol 40: 1-16

46

Sano T, Nagata T (2002) The possible involvement of a phosphate-induced transcription

factor encoded by Phi-2 gene from tobacco in ABA-signaling pathways. Plant Cell Physiol

43: 12-20

Schröder F, Lisso J, Lange P, Müssig C (2009) The extracellular EXO protein mediates cell

expansion in Arabidopsis leaves. BMC Plant Biol 9: 1-12

Schröder F, Lisso J, Müssig C (2011) EXORDIUM-LIKE 1 promotes growth during low

carbon availability in Arabidopsis. Plant Physiol 156: 1620-1630

Schröder F, Lisso J, Müssig C (2012) Expression pattern and putative function of EXL1 and

homologous genes in Arabidopsis. Plant Signal Behav 7: 22-27

Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression

in the drought and cold stress responses. Curr Opin Plant Biol 6: 410-417

Smart CC, Amrhein N (1985) The influence of lignification on the development of vascular

tissue in Vigna radiata L. Protoplasma 124: 87-95

Steinmacher DA, Saare-Surminski K, Lieberei R (2012) Arabinogalactan proteins and the

extracellular matrix surface network during peach palm somatic embryogenesis. Physiol

Plant 146: 336-349

Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C (1998)

The AmMYB308 and AmMYB330 transcription factors from Antirrhinum regulate

phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell 10: 135-154

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular

evolutionary genetics analysis using maximum likelihood, evolutionary distance, and

maximum parsimony methods. Mol Biol Evol 28: 2731-2739

Terashima I, Miyazawa S-I, Hanba YT (2001) Why are sun leaves thicker than shade leaves?

Consideration based on analyses of CO2 diffusion in the leaf. J Plant Res 114: 93-105

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of

progressive multiple sequence alignment through sequence weighting, position-specific

gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680

Todd J, Post-Beittenmiller D, Jaworski-Jan G (1999) KCS1 encodes a fatty acid elongase 3-

ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. Plant J 17:

119-130

Turner S, Gallois P, Brown D (2007) Tracheary element differentiation. Annu Rev Plant Biol

58: 407-433

47

Wang Y, Liu G, Yan X, Wei Z, Xu Z (2011) MeJA-Inducible expression of the heterologous

JAZ2 promoter from Arabidopsis in Populus trichocarpa protoplasts. Journal Plant Dis

Protect 2: 69-74

Whetten R, Sederoff R (1995) Lignin biosynthesis. Plant Cell 7: 1001-1013

Wu J, Zhang Y, Zhang H, Huang H, Folta KM, Lu J (2010) Whole genome wide expression

profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing

technology. BMC Plant Biol 10: 234

Xiong L, Zhu J-K (2003) Regulation of abscisic acid biosynthesis. Plant Physiol 133: 29-36

Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular

responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57: 781-803

Ye ZH (2002) Vascular tissue differentiation and pattern formation in plants. Annu Rev Plant

Biol 53: 183-202

Zhang L, Xi D, Luo L, Meng F, Li Y, Wu C-A, Guo X (2011) Cotton GhMPK2 is involved in

multiple signaling pathways and mediates defense responses to pathogen infection and

oxidative stress. FEBS J 278: 1367-1378

Zhong R, Lee C, Ye Z-H (2010) Evolutionary conservation of the transcriptional network

regulating secondary cell wall biosynthesis. Trends Plant Sci 15: 625-632

Zhong R, Lee C, Zhou J, McCarthy RL, Ye Z-H (2008) A battery of transcription factors

involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell

20: 2763–2782.

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Figure Captions

Figure 1. Sequence analysis, promoter region and expression of EgPHI-1. (A)

Nucleotide sequence of the EgPHI-1 coding sequence (top line numbered on the left) and

deduced amino acid residues (bottom line numbered on the right). Sequence underlined in

blue represents a putative signal peptide. Red diamond indicates the probable cleavage point

of the signal peptide. Sequences underlined in black, orange and green represent the specific

phosphorylation sites for serine, threonine and tyrosine, respectively. A protein kinase C

(PKC) putative phosphorylation site is indicated by a purple square. Amino acid residues in

red represent possible glycosylation sites. The sequence highlighted in gray represents the

PHI-1 functional domain (Pfam04674). (B) Cis-elements and motifs in the EgPHI-1 promoter

region. Potential regulatory sequences identified in the 1.5 kb 5'-upstream promoter region of

EgPHI-1 are represented as follows: closed circles, Sp1; black box, GAG-motif; gray arrow to

right, TCT-motif; solar circle, circadian; open boxes, I-Box; inverted black triangles, G-Box;

open diamond, box II; open triangles, ABRE; gray diamonds, ARE; black triangle, GA-motif;

gray triangle, HSE; black arrow to left, MBS; gray arrow to left, TCA-element; black arrows

to right, MRE; gray boxes, GATA-motif; gray circles, as-2-box; closed diamond, TATA. (C)

Expression of EgPHI-1 in E. grandis seedling subjected to different treatments. Total RNA

was isolated from E. grandis seedlings treated (wound, NAA, kinetin and dehydration) and

untreated. Relative gene expression was assessed by RT-qPCR in three biological replicates

and three technical replicates per plant. Expression was calculated by the 2-∆∆Ct

method and

values were normalized against RibL23A. Bars represent the mean ± SE of three replicates

(n=3). Statistically significant differences at p≤0.05 (*), p≤0.01 (**) and p≤0.001 (***)

between treated and untreated E. grandis seedling are indicated.

Figure 2. Phylogenetic relationships and primary structure conservation of EgPHI-1.

(A) Phylogenetic tree based on EgPHI-1 deduced amino acid sequence and other PHI-1/EXO

homologous sequences described (Schröder et al. 2009). The EgPHI-1 sequence is marked by

a green diamond. Tobacco PHI-1 sequence is marked by a red diamond. EXO/EXL sequences

are marked by blue diamonds. The numbers on the branches indicate the bootstrap value (%).

Black dot indicates the mid-point rooting. (B) Conservation between EgPHI-1 amino acid

residues and the two nearest amino acid sequences (O. sativa AAM08535 and V. vinifera

CAO61694). Minimum and maximum conservation between amino acid residues are

represented by color range from brown to yellow and by numbers, respectively. *Indicates

49

omissions to regions with low conservation. Lateral numbers represent the size of the aligned

fragment. Non-colored amino acid residues represent variations in the sequence.

Figure 3. The EgPHI-1 gene construction and expression in transgenic tobacco. (A)

Schematic diagram of the 35S::EgPHI-1 gene construction used in Agrobacterium-mediated

genetic transformation of tobacco. EgPHI-1 cDNA was placed in sense orientation into

plasmid pCAMBIA 2301 under the control of the CaMV 35S promoter and terminator. (B)

Relative EgPHI-1 expression in transgenic plant lines. Total RNA was isolated from leaves of

transgenic (L1, L2 and L3) and WT plants. Relative gene expression was assessed by RT-

qPCR in three biological replicates and three technical replicates per plant. Expression was

calculated by the 2-∆∆Ct

method and values were normalized against GAPDH. (C) Relative

protein levels in transgenic plant lines. Equal amounts of total proteins extracted from leaves

of transgenic (L1, L2 and L3) and WT plants were separated by SDS-PAGE and

immunoblotted with anti-EGP antibody. Levels of EgPHI-1 were measured in three technical

replicates per plant. Protein levels were quantified from membrane imaging and the values of

transgenic lines were calculated in relation to the WT protein levels. Bars represent the mean

± SE of the three replicates (n=3). Statistically significant differences at p≤0.05 (*) and

p≤0.001 (***) between transgenic and WT plants are indicated.

Figure 4. Growth of transgenic tobacco overexpressing EgPHI-1. Transgenic (L1, L2

and L3) and WT plants grown for 90 days under greenhouse conditions were used to perform

measurements of height (A), leaf number per plant (B), specific leaf area (C), leaf dry

biomass (D), stem dry biomass (E), root dry biomass (F), root length (G), root volume (H)

and total dry biomass (I). Bars represent the mean ± SE of three replicates (n=3). Statistically

significant differences at p≤0.05 (*), p≤0.01 (**) and p≤0.001 (***) between transgenic and

WT plants are indicated.

Figure 5. Stem anatomy of transgenic tobacco overexpressing EgPHI-1. (A) Stem

cross-section of transgenic (L1, L2 and L3) and WT plants grown for 90 days under

greenhouse conditions. The cross-sections were stained with 1% (v/v) astra blue and 1% (v/v)

safranin, specific dyes for cellulose and lignin, respectively. The double staining cross

sections were viewed under a light microscope. VC, vascular cambium; V, vessels; X, xylem;

F, fibers; P, phloem. Magnification bars represent 100 µm. (B) Fiber length. (C) Vessel lumen

diameter. Fibers were obtained for stem samples (third internode from the apex) of plants

50

grown for 90 days in a greenhouse by the chemical maceration method. Macerated material

was washed and stained with 1% (v/v) safranin. Fiber slides were viewed under a light

microscope. Bars represent the mean ± SE of the three technical replicates (n=135). Three

biological replicates were used for the vessel measurement, each containing three slides, with

10 measurements made per slide. Bars represent the mean ± SE of the three replicates (n=30).

Statistically significant difference at p≤0.01 (**) and p≤0.001 (***) between the transgenic

and WT plants are indicated.

Figure 6. Activity of lignin biosynthesis-related enzymes in transgenic tobacco

overexpressing EgPHI-1. (A) PAL activity in stems of the transgenic (L1, L2 and L3) and

WT plants. Changes in the absorbance values at 290 nm min–1

g–1

indicate the conversion of

phenylalanine to trans-cinnamic acid by PAL activity. (B) POD activity in stems of the

transgenic (L1, L2 and L3) and WT plants. Variations in the absorbance values at 470 nm

min–1

g–1

(DM) indicate the consumption of guaiacol by POD activity. Protein extracts were

obtained from freeze-dried stems of plants grown for 90 days in greenhouse under ideal

growing conditions. Bars represent the mean ± SE of three replicates (n=3). Statistically

significant differences at p≤0.05 (*) and p≤0.01 (**) between the transgenic and WT plants

are indicated.

Figure 7. Lignin autofluorescence in vessels of transgenic tobacco overexpressing

EgPHI-1. Lignin autofluorescence in vessels of the base of petioles of transgenic (L1, L2 and

L3) and WT plants. Cross sections were performed at the petioles of the third fully expanded

leaf from the apex of the plants grown for 90 days under greenhouse conditions. The

autofluorescence was detected using 340-380 nm excitation wavelength and 400 nm barrier

filters (I). To highlight lignified areas (blue fluorescence), images were electronically

modified (black & white color) (II). Magnification bars represent 100 µm.

Figure 8. Physiological analyses in transgenic tobacco overexpressing EgPHI-1.

Measurements of net photosynthetic rate (A), transpiration (B), stomatal conductance to water

vapor (C), Ci/Ca ratio (D), intrinsic (E) and instantaneous (F) water-use efficiencies were

performed in transgenic (L1, L2 and L3) and WT plants. Measurements were performed on

the third fully expanded leaf from the apex of plants grown for 90 days under greenhouse

conditions. Bars represent the mean ± SE of the three replicates (n=3). Statistically significant

51

difference at p≤0.05 (*) and p≤0.001 (***) between the transgenic and WT plants are

indicated.

52

Figure 1

Untreated Would NAA Kinetin Dehydration

53

Figure 2

54

Figure 3

55

Figure 4

56

Figure 5

57

Figure 6

58

Figure 7

59

Figure 8

60

CAPÍTULO 2:

Phosphate induced-1 gene from Eucalyptus (EgPHI-1)

enhances the osmotic stress tolerance in transgenic tobacco

(Artigo aceito para publicação na Genetics and Molecular Research em 01/02/2013)

61

Phosphate induced-1 gene from Eucalyptus (EgPHI-1) enhances the osmotic stress

tolerance in transgenic tobacco

A.O. Sousa, E.T.C.M. Assis, C.P. Pirovani, F.C. Alvim, M.G.C. Costa

Centro de Biotecnologia e Genética, Departamento de Ciências Biológicas,

Universidade Estadual de Santa Cruz – UESC, Ilhéus, BA, 45662-900, Brazil

Corresponding author: M.G.C. Costa

E-mail: [email protected]

Fax: +55 73 3680-5226

Running title: Involvement of EgPHI-1 gene in osmotic stress tolerance


62

ABSTRACT

Environmental stresses such as drought, freezing and high salinity induce osmotic

stress on plant cells. The plant response to osmotic stress involves a number of physiological

and developmental changes, which are possible, in part, by the modulation of expression of

specific genes. Phosphate induced-1 (PHI-1) was firstly isolated from phosphate-treated

phosphate-starved tobacco cell cultures as a stress-inducible gene presumably related to

intracellular pH maintenance; however the role of the PHI-1 gene product has not yet been

clarified. A gene encoding a predicted protein with high similarity to tobacco PHI-1, named

EgPHI-1, was previously identified in Eucalyptus by comparative transcriptome analysis of

xylem cells from species of contrasting phenotypes for wood quality and growth traits. Here,

we show that the overexpression of EgPHI-1 in transgenic tobacco enhances the tolerance to

osmotic stress. In comparison with the wild-type (WT) plants, the EgPHI-1 transgenic plants

showed a significant increase in root length and biomass dry weight under NaCl-, PEG- and

mannitol-induced osmotic stresses. The enhanced stress tolerance of transgenic plants was

correlated with the increased endogenous protein levels of the molecular chaperone binding

protein BiP, which in turn was correlated with the EgPHI-1 expression level in the different

transgenic lines. These results provide evidences about the involvement of EgPHI-1 in

osmotic stress tolerance, via modulation of BiP expression, and also pave the way for its

future use as a candidate gene for engineering tolerance to environmental stresses in crop

plants.

Key words: intracellular pH, phosphorylation, abiotic stress, chaperone, endoplasmic

reticulum, cell death.

INTRODUCTION

Environmental stresses such as drought, freezing and high salinity are often

interconnected, since they may induce similar cellular damage manifested primarily as

osmotic stress. As environmental stress conditions seriously affect crop production in many

parts of the world, the molecular and cellular processes underlying the acclimation of plants to

osmotic stress have attracted much interest (Zhu, 2002; Mahajan and Tuteja, 2005; Vásquez-

Robinet et al., 2010). To cope with osmotic stress, plants developed a number of

physiological and developmental mechanisms, including alteration of life cycle, inhibition of

shoot growth and enhancement of root growth, adjustment of ion transport, carbon

metabolism and the synthesis of compatible solutes (Xiong and Zhu, 2002). Many of these

changes are possible by the modulation of gene expression of specific genes, which represents

an important approach for engineering tolerance in plants (Brinker et al., 2010).

A complex apparatus of signaling factors is required for transcription of stress-

inducible genes. These factors perceive the environment variations and transduce the signals

through the cells. Changes in the intracellular pH are one of the first indicators of osmotic

stress perceived by the cells (Netting, 2000). Metabolism, transport and signaling are

homeostatic processes fully dependent on the regulation of intracellular pH in plant cells

(Sakano, 1998). Regarding this point, the phosphate induced-1 (PHI-1) gene, first identified

in cell cultures of tobacco (Sano et al., 1999), has attracted considerable interest. Despite its

sequence similarity to EXORDIUM (EXO) gene of Arabidopsis, PHI-1/EXO do not show

similarities to any gene with known function. Tobacco PHI-1 was initially presumed to have a

role in phosphate-induced cell cycle re-entry (Sano et al., 1999). Then, the PHI-1 gene

product was also inferred to have a role in alleviating changes of intracellular pH caused by

63

stress conditions in the cells (Sano and Nagata, 2002). However, the exact role of the PHI-1

gene product has not yet been clarified.

Comparative analysis between the xylem transcriptomes of Eucalyptus grandis and E.

globulus, using high density microarrays, led to the identification of a differentially expressed

gene encoding a predicted protein with high similarity to tobacco PHI-1 (Pasquali et al.,

2005). This gene, named EgPHI-1, was 7.5-× more expressed in E. globulus than in E.

grandis (Pasquali et al., 2005), Eucalyptus species of contrasting characteristics of growth and

wood properties. E. grandis is a species native to tropical and subtropical regions that exhibits

a rapid growth, but produces a low density wood. On the other hand, E. globulus is a species

from temperate climates that produces a high density wood, but exhibits a slow growth when

cultivated in tropical regions (Eldridge et al., 1993; Bernard, 2003; Myburg et al., 2007).

Considering the economic and ecological relevance of the genus Eucalyptus, understanding

the cellular function of stress-inducible genes is of central interest for biotechnological

applications in forestry.

In this study, we investigated the relation between EgPHI-1 and osmotic stress.

Transgenic tobacco plants overexpressing EgPHI-1 were generated and subjected to osmotic

stress induced by NaCl, mannitol and polyethylene glycol (PEG). The results indicated that

EgPHI-1 enhances the osmotic stress tolerance of transgenic plants, in a mechanism that

involves the modulation of expression of the molecular chaperone binding protein BiP, an

important component of endoplasmic reticulum (ER) that protects the cell against stresses.

MATERIAL AND METHODS

Plant material

The EgPHI-1 coding sequence identified in a xylem cDNA library of E. globulus was

released from the pSPORT1 cloning vector by SalI/NotI digestion. The purified ~1.2 kb

fragment was subcloned in sense orientation between the promoter and terminator sequences

of the cauliflower mosaic virus (CaMV) 35S of the pUC118 vector, using the XhoI/NotI

restriction sites. The resulting 35S::EgPHI-1 construct was released from pUC118 by

BamHI/HindIII double digestion and inserted into the same restriction sites of the binary

vector pCAMBIA 2301 (CAMBIA, Australia), which also contains the neomycin

phosphotransferase (nptII) and β-glucuronidase (uidA) genes under the control of CaMV 35S

promoter. The plasmid construction was introduced into Agrobacterium tumefaciens EHA105

by direct DNA uptake. The construction was transferred to wild-type (WT) tobacco

(Nicotiana tabacum cv. Havana) by Agrobacterium tumefaciens-mediated genetic

transformation, as previously described (Horsch et al., 1985). Three transgenic lines (L1, L2

and L3) representing distinct transformation events were selected on appropriate media by

their resistance to kanamycin, positive reaction in GUS histochemical assays (Jefferson,

1989), PCR amplification of nptII and uidA genes and by reverse transcription-quantitative

real-time PCR (RT-qPCR) (Sousa AO, Assis ETCM, Kirch RP, Silva DC, Almeida A-AF,

Pirovani CP, Alvim FC, Pasquali G and Costa MGC, unpublished results). Seeds from WT

and kanamycin-resistant T1 transgenic plants for the osmotic stress experiments were derived

from plants grown in a greenhouse under identical conditions.

Osmotic stress experiments

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Seeds from WT and transgenic plants were sterilized and plated on MS medium

(Murashige and Skoog, 1962) solidified with 0.7% agar. After two days in a cold room (4°C),

the plates were transferred to a growth chamber with a long day light regime (16 h day, 140

μmol m-2

s-1

, 27±2°C; 8 h night, 27±2°C) and grown in a randomized manner during 15 days.

After this period, nine plants of similar sizes of each WT and transgenic lines were transferred

to MS medium supplemented with the stress inductors NaCl, mannitol or PEG, in

concentrations sufficient to impose a water potential of -1MPa. The WT and transgenic plants

were transferred to MS medium without stress inductors in the control treatment. The plants

were maintained in a growth chamber during the next 25 days under the same conditions

described above. At the end of this period, phenotypic aspects such as leaf wilting, chlorosis,

and necrotic lesions were analyzed as well as the root length and the biomass dry weight of

individual plants. To determine the biomass dry weight, the plants were kept in an air-

circulation stove at 75 °C until a constant weight. All treatments were performed in

experimental triplicate.

Protein extraction and western blot analysis

Leaves of control and stressed WT and transgenic plants were used for BiP

immunoblot analyses. These plants were grown for 60 days in a greenhouse under identical

normal conditions. Lyophilized leaves were macerated in liquid nitrogen and 1% (w/w) PVP

by the phenol/SDS method (Pirovani et al., 2008). The concentration of total protein was

determined using the 2-D Quant Kit (GE Healthcare, USA) according to manufacturer’s

instructions. Equivalent amounts of total protein (20 mg) were separated in SDS-12.5%

PAGE and transferred to nitrocellulose membrane using the iBlot Dry Blotting System

(Invitrogen, USA) according to manufacturer’s instructions. Membranes were probed with the

polyclonal antibody against the carboxyl region of soybean BiP (Figueiredo et al., 1997) at a

1:2,000 dilution. As secondary antibody was the goat anti-rabbit IgG conjugated to alkaline

phosphatase (Sigma, USA). The 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and p-

nitroblue tetrazolium (NBT) (Promega, USA) were used as substrate for colorimetric reaction

from activity of alkaline phosphatase. BiP levels were quantified from membrane images

using the GelQuant.Net 1.8.0 software (www.biochemlabsolutions.com) and the values of

transgenic lines were calculated in relation to the WT protein levels.

Statistical Analysis

All the measurements were repeated at least three times from different individual

plants. Statistical differences between WT and transgenic lines were assessed based on the

analysis of variance (ANOVA) and means were separated by Student’s t-test, with a critical

value of p≤0.05 and p≤0.01.

RESULTS

Overexpression of EgPHI-1 enhances osmotic stress tolerance in transgenic

tobacco

We analyzed the effects of EgPHI-1 overexpression, at the whole plant level, on the

response of plants to osmotic stress induced by NaCl, mannitol and PEG. The transgenic lines

showed increased tolerance to the different stresses as compared with the WT tobacco, even

25 days after the stress treatments (Figure 1). While the WT tobacco exhibited small leaves

with chlorotic aspect and a reduced growth in NaCl treatment, as compared with the control

http://www.biochemlabsolutions.com/

65

treatment, it did not affect the growth of transgenic plants. Although PEG and mannitol

treatments affected in some extension the growth of transgenic plants, as compared with the

control treatment, their effects were more severe in the WT tobacco. PEG promoted a slightly

chlorosis in the transgenic plants, while the WT plants became completely necrotic in this

treatment. Mannitol promoted a more severe chlorosis and reduced t growth in the transgenic

plants as compared with the control treatment. It is interesting to note that under control

conditions (without stress inductor), the transgenic plants exhibited a higher growth than the

WT plants.

A direct comparison between stressed WT and transgenic plants demonstrated that the

transgenic plants showed a significantly higher root length and biomass dry weight than the

WT plants in all stress conditions (Figure 2). The only exception was found in the mannitol

treatment, in which the root length of transgenic plants was smaller than the WT plants.

However, the biomass dry weight of transgenic plants was ~3-fold higher than the WT plants

in this treatment. The transgenic lines L1, L2 and L3 showed root length, respectively, 3.7-,

4.7- and 4.1-fold higher than the WT plants under NaCl treatment, while their biomass dry

weight in this treatment were, respectively, 3.7-, 6.4- and 3.5-fold higher than the WT plants.

For PEG treatment, the transgenic lines L1, L2 and L3 showed an increase of, respectively,

123-, 142- and 81-fold in root length and 7-, 12- and 9-fold in biomass dry weight as

compared with WT plants.

EgPHI-1-mediated osmotic stress tolerance correlates with the BiP expression

The deduced amino acid sequence of EgPHI-1 is predicted to contain a signal peptide

in its N-terminal portion targeting to the endoplasmic reticulum (ER) (data not shown).

Therefore, the constitutive overexpression of EgPHI-1 in the transgenic plants may disrupt the

ER homeostasis. This phenomenon usually results in the induction of ER-molecular

chaperones, such as BiP, which has been demonstrated to mediate drought tolerance in

transgenic plants (Alvim et al., 2001; Valente et al., 2009). To examine whether the observed

osmotic stress tolerance of transgenic tobacco plants overexpressing EgPHI-1 correlates with

BiP accumulation in plant tissues, the abundance of BiP was determined in leaves of WT and

transgenic plants by western blot analysis. All the transgenic plants evaluated showed a

remarkable increase in BiP accumulation as compared with WT tobacco (Figure 3). This

increase was of 21%, 45% and 85% for L1, L2 and L3, respectively. Interestingly, such an

increase in BiP accumulation was proportional to the expression levels of EgPHI-1 in the

transgenic lines. The transgenic line L3 was shown to accumulate the highest levels of

EgPHI-1 transcripts, L2 an intermediate level and L1 the lowest transcript levels (Sousa AO,

Assis ETCM, Kirch RP, Silva DC, Almeida A-AF, Pirovani CP, Alvim FC, Pasquali G and

Costa MGC, unpublished results).

DISCUSSION

The results presented here show that the EgPHI-1 expression modulates plant

development and response to hyperosmotic conditions. The transgenic plants showed higher

root length and biomass dry weight than the WT plants in control (non-stress) conditions. A

similar phenotype was observed in transgenic Arabidopsis plants overexpressing EXO,

another member of PHI-1 family. EXO induced a high growth of transgenic plants at the first

stages of development by promoting the cellular expansion process (Coll-Garcia et al., 2004).

Thus, our observations suggest a connection between EXO and EgPHI-1 functions.

66

NaCl, mannitol and PEG induce hyperosmotic stress by different ways. NaCl

penetrates through the membranes and causes ionic stress and toxicity due to Na+ excess;

mannitol also has the ability to penetrate through the membranes, but it not induces ionic

stress, while PEG is a non-ionic and non-penetrating agent that decreases the water potential

of the nutrient solution without causing toxicity (Gangopadhyay et al., 1997a,b). Therefore,

the use of these three stress inducers allowed to compare plant responses to osmotic stress by

disturbance of water relations, changes in the absorption and utilization of essential nutrients

and accumulation of toxic ions (Yokoi et al., 2002).

Transgenic tobacco plants expressing EgPHI-1 showed a significantly improved

tolerance to osmotic stress induced by NaCl, mannitol and PEG as compared with WT plants.

They displayed an increased root length and biomass dry weight in the different osmotic

stress conditions, whereas the WT tobacco showed progressive chlorosis, severe growth

inhibition and necrosis at the same stress conditions tested. The only exception was observed

in the mannitol treatment, which inhibited the root length of transgenic plants as compared

with the WT plants. This result suggests that EgPHI-1 may facilitate the penetration of

mannitol through the root cell membranes and/or to be involved in cellular processes in the

root that are negatively affected by the presence of mannitol.

The tolerance of plants to salt stress has been usually attributed to their ability to

accumulate Na+ in intracellular compartments (He and Yu, 1995; Lutts et al., 1996). This

mechanism allows a better plant growth under salt stress (Thomas et al., 1992). The enhanced

tolerance of EgPHI-1-expressing transgenic tobacco plants to NaCl treatment may be related

to the increased intracellular compartmentalization of Na+ or to the alleviation of intracellular

pH changes caused by salt stress, as suggested for tobacco PHI-1 (Sano and Nagata, 2002).

Tobacco PHI-1 was postulated to play a role in alleviating changes of intracellular pH that are

caused by stress, such as high concentrations of phosphate (Sano and Nagata, 2002), by its

involvement in one of the two possible mechanisms: (i) intracellular proton exclusion from

cells via an ATP-driven proton pump and (ii) proton consumption through pH-sensitive

decarboxylation reactions in organic acid metabolism (Sakano, 1998).

Most interestingly was the observation that BiP protein levels increased proportionally

to the EgPHI-1 expression levels in the transgenic lines. Thus, our results provide evidence

that EgPHI-1 enhances the osmotic stress tolerance by a mechanism that involves, at least

partially, the modulation of BiP expression. BiP is an ER resident molecular chaperone that

plays a central role in ER stress signaling by sensing alterations that affect protein folding and

assembly in the ER environment (Malhotra and Kaufman, 2007). In addition to its role as an

ER molecular chaperone, BiP overexpression in plants has also been shown to increase their

tolerance to drought stress (Alvim et al., 2001; Valente et al., 2009). The drought tolerance

mediated by BiP has not been associated with the typical short-term and long-term avoidance

responses or with other well-known tolerance mechanisms (Valente et al., 2009), but rather

with the prevention of stress-induced cell death by negatively regulating the stress-induced N-

Rich Protein (NRP)-mediated cell death response (Reis et al., 2011). The increased BiP levels

in transgenic plants overexpressing EgPHI-1 may be a result of ER stress triggered by the

input of EgPHI-1 protein in the ER. High protein input in ER overloads the folding machinery

and generates an elevation of unfolded proteins in the ER lumen, resulting in the induction of

ER-molecular chaperones such as BiP (Malhotra and Kaufman, 2007). The induction of many

chaperone and co-chaperone genes was observed in transgenic rice expressing BiP1 and

associated with the ER stress response (Wakasa et al., 2011). These observations provide a

fundamental basis for EgPHI-1-mediated osmotic stress tolerance that can be exploited for

67

biotechnological applications in forestry and also extended to other economically important

crops.

ACKNOWLEDGMENTS

The authors are very grateful to Dr. Elizabeth P.B. Fontes from Departamento de

Bioquímica e Biologia Molecular, BIOAGRO, Universidade Federal de Viçosa, by providing

the polyclonal antibody against BiP protein. This work was supported by Financiadora de

Estudos e Projetos (FINEP, Brazilian Ministry of Science and Technology - MCT) [grant

number 2101063500], Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq, MCT) [grant numbers 50.6348/04-0, 578632/08-0, 311361/09-9] and the Fundação de

Amparo à Pesquisa do Estado da Bahia (FAPESB, Secretariat of Science, Technology and

Innovation – SECTI) [grant number BOL1569/2010].

REFERENCES

Alvim FC, Carolino SM, Cascardo JC, Nunes CC, et al. (2001). Enhanced accumulation of

BiP in transgenic plants confers tolerance to water stress. Plant Physiol. 126: 1042-

1054.

Bernard M (2003). Eucalyptus: a strategic forest tree. In: Eucalyptus plantations: research,

management and development (Wei R-P and Xu D, eds.) World Scientific, Singapure,

3-18.

Brinker M, Brosché M, Vinocur B, Abo-Ogiala A, et al. (2010). Linking the salt

transcriptome with physiological responses of a salt-resistant populus species as a

strategy to identify genes important for stress acclimation. Plant Physiol. 154: 1697-

1709.

Coll-Garcia D, Mazuch J, Altmann T and Müssig C (2004). EXORDIUM regulates

brassinosteroid-responsive genes. FEBS Lett. 563: 82-86.

Eldridge KG, Davidson J, Harwood CE and van Wyk G (1993). Eucalypt domestication and

breeding. Clarendon Press, Oxford, United Kingdom.

Figueiredo JEF, Cascardo JCM, Carolino SMB, Alvim FC, et al. (1997). Water-stress

regulation and molecular analysis of the soybean BIP gene family. Braz. J. Plant

Physiol. 9: 103-110.

Gangopadhyay G, Basu S and Gupta S (1997a). In vitro selection and physiological

characterization of NaCl- and mannitol-adapted callus lines in Brassica juncea. Plant

Cell Tiss. Org. Cult. 50: 161-169.

Gangopadhyay G, Basu S, Mukherjee B and Gupta S (1997b). Effects of salt and osmotic

shocks on unadapted and adapted callus lines of tobacco. Plant Cell Tiss. Org. Cult.

49: 45-52.

He DY and Yu SW (1995). In vitro selection of a high-proline producing variant from rice

callus and studies on its salt tolerance. Acta Phytophysiol. Sin. 21: 65-72.

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, et al. (1985). A simple and general method

for transferring genes into plants. Science 227:1229-1231.

Jefferson RA (1989). The GUS reporter gene system. Nature 342: 837-838.

Lutts S, Kinet JM and Bouharmont J (1996). Effects of salt stress on growth, mineral nutrition

and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.)

cultivars differing in salinity resistance. Plant Growth Regul. 19: 207-218.

Mahajan S and Tuteja N (2005). Cold, salinity and drought stresses: an overview. Arch.

Biochem. Biophys. 444: 139-158.

68

Malhotra JD and Kaufman RJ (2007). The endoplasmic reticulum and the unfolded protein

response. Semin. Cell. Dev. Biol. 18: 716-731.

Murashige T and Skoog F (1962). A revised medium for rapid growth and bio assays with

tobacco tissue cultures. Physiol. Plant. 15: 473-497.

Myburg AA, Potts BM, Marques CMP, Kirst M, et al. (2007). Eucalyptus. In: Genome

mapping and molecular breeding in plants: forest trees (Kole CR, ed.). Springer,

Berlin, 115-160.

Netting AG (2000). pH, abscisic acid and the integration of metabolism in plants under

stressed and non-stressed conditions: cellular responses to stress and their implication

for plant water relations. J. Exp. Bot. 51: 147-158.

Pasquali G, Bastolla FM, Pazzini F, Kirch RP, et al. (2005). Sequecing and differential

expression of xylem specific genes from two Eucalyptus species with highly

contrasting wood properties. In: Programme and abstracts of the IUFRO Tree

Biotechnology 2005, Pretoria, 22.

Pirovani CP, Carvalho HAS, Machado RCR, Gomes DS, et al. (2008). Protein extraction for

proteome analysis from cacao leaves and meristems, organs infected by

Moniliophthora perniciosa, the causal agent of the witches' broom disease.

Electrophoresis 29: 2391-2401.

Reis PAA, Rosado GL, Silva LAC, Oliveira LC, et al. (2011). The binding protein BiP

attenuates stress-induced cell death in soybean via modulation of the N-rich protein-

mediated signaling pathway. Plant Physiol. 157: 1853-1865.

Sakano K (1998). Revision of biochemical pH-stat: involvement of alternative pathway

metabolisms. Plant Cell Physiol. 39: 467-473.

Sano T, Kuraya Y, Amino S-I and Nagata T (1999). Phosphate as a limiting factor for the cell

division of tobacco BY-2 cells. Plant Cell Physiol. 40: 1-16.

Sano T and Nagata T (2002). The possible involvement of a phosphate-induced transcription

factor encoded by Phi-2 gene from tobacco in ABA-signaling pathways. Plant Cell

Physiol. 43: 12-20.

Thomas JC, Armand RL and Bohnert HJ (1992). Influence of NaCl on growth proline and

phosphoenol pyruvate carboxylase level in Mesembryanthemum crystallinum

suspension cultures. Plant Physiol. 98: 626-631.

Valente MAS, Faria JAQA, Soares-Ramos JRL, Reis PAB, et al. (2009). The ER luminal

binding protein (BiP) mediates an increase in drought tolerance in soybean and delays

drought-induced leaf senescence in soybean and tobacco. J. Exp. Bot. 60: 533-546.

Vásquez-Robinet C, Watkinson JI, Sioson AA, Ramakrishnan N, et al. (2010). Differential

expression of heat shock protein genes in preconditioning for photosynthetic

acclimation in water-stressed loblolly pine. Plant Physiol. Bioch. 48: 256-264.

Wakasa Y, Yasuda H, Oono Y, Kawakatsu T, et al. (2011). Expression of ER quality control-

related genes in response to changes in BiP1 levels in developing rice endosperm.

Plant J. 65: 675-689.

Xiong L and Zhu JK (2002). Molecular and genetic aspects of plant responses to osmotic

stress. Plant Cell Environ. 25: 131-139.

Yokoi S, Bressan RA and Hasegawa PM (2002). Salt stress tolerance of plants. JIRCAS Work.

Rep. 25-33.

Zhu J-K (2002). Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol.

53: 247-273.

69

Figure captions

Figure 1. Phenotypes of tobacco plants exposed to different osmotic stress treatments.

Fifteen-days-old wild-type (WT) and transgenic (L1, L2 and L3) tobacco plants were

subjected to osmotic stress (-1.0 MPa) induced by NaCl, PEG and mannitol. Plants were

maintained in medium without stress inductors in the control treatment. Photographs were

taken 25 days after the treatments.

Figure 2. Root length (left) and biomass dry weight (right) of EgPHI-1 expressing

transgenic tobacco plants under different stress treatments. Measurements were performed in

WT and transgenic (L1, L2 and L3) plants subjected to osmotic stress induced by NaCl, PEG

and mannitol (white bars) or maintained under control (non-stress) conditions (black bars), 25

days after the treatments. Bars represent the mean ± SE of the three replicates (n=27).

Statistically significant differences at p≤0.05 (*) and p≤0.01 (**) between the WT and

transgenic plants grown under control or stress conditions are indicated.

Figure 3. BiP protein levels in WT and transgenic tobacco. A. Total protein extract.

Equal amounts of total protein isolated from leaves of WT and transgenic (L1, L2 and L3)

plants were separated by SDS-PAGE. B. Western blot of BiP protein. Total protein extracts

were immunoblotted with anticarboxyl BiP antibody. C. Relative BiP level. BiP protein levels

were quantified from membrane images using the GelQuant.Net 1.8.0 software and the values

of transgenic lines (L1, L2 and L3) were calculated in relation to WT protein levels. Bars

represent the mean ± SE of the three replicates (n=3). Statistically significant differences at

p≤0.05 (*) and p≤0.01 (**) between the WT and transgenic plants are indicated.

70

Figure 1

71

Figure 2

72

Figure 3

73

CAPITULO 3:

Overexpression of an Eucalyptus PHOSPHATE-INDUCED-1

(EgPHI-1) gene alters the chemical composition and topochemical

distribution of lignin in stem xylem of tobacco

(Brief communication a ser submetido à Transgenic Research)

74

Overexpression of an Eucalyptus PHOSPHATE-

INDUCED-1 (EgPHI-1) gene alters the chemical

composition and topochemical distribution of lignin

in stem xylem of tobacco

Aurizangela O. Sousa, André Ferraz, Marcio G. C. Costa

Aurizangela O. Sousa, Marcio G. C. Costa ()

Center for Biotechnology and Genetics, Biological Sciences Department, State University of

Santa Cruz – UESC, Ilhéus, 45662-900, Bahia, Brazil

e-mail: [email protected]

André Ferraz

Department of Chemical Engineering, School of Engineering of Lorena, University of São

Paulo- USP, Lorena 116, 12602-810, São Paulo, Brazil


75

Abstract

Genetic manipulation of specific biosynthetic pathways can provide insights into the growth and development of

plants. To explore such a possibility, a gene preferentially expressed in xylem of Eucalyptus globulus (EgPHI-1)

was overexpressed in transgenic tobacco and the resulting effects upon the stem xylem were analyzed. Chemical

methods were used to determine the overall amount of cellulose, hemicelluloses and lignin in stems of transgenic

and wild-type (WT) plants. In comparison with the WT, the transgenic plants exhibited alterations in the

contents of these compounds, which ranged from a reduction of cellulose in all transgenic lines to a decrease of

lignin and increase of hemicellulose and extractives depending on the transgenic line. Additionally, scanning

ultraviolet (UV)-microspectrophotometry (UMSP) was used to characterize the in situ distribution of lignin

within individual cell wall layers of different cell types. UV absorbance spectra analyses of lignin in S2-layers of

fiber and vessel secondary walls revealed a decrease in the lignin absorbance values of transgenic plants, as

compared with the WT. UV scanning profile of fibers provided the topochemical distribution of lignin. These

UV-images clearly indicated a more intense lignin absorbance in the cell corners and compound middle lamella

in both WT and transgenic plants. However, the transgenic plants showed a less uniform lignin distribution and

lower lignin absorbance intensity in S2-layers than the WT. Collectively, these data demonstrate that EgPHI-1

regulates the cell wall chemical composition in individual cell types, which makes it an important target for

genetic manipulation of xylem properties in plants.

Keywords EXO/PHI-1, cell wall, secondary xylem, cellulose, lignin

Introduction

Fibers, tracheary elements, and tracheids or vessels are specialized cells types that compose the wood or

secondary xylem (Plomion et al. 2001). The xylem cells form thick secondary cell walls (SCWs) composed

mainly by cellulose, hemicellulose and lignin (Boerjan et al. 2003). The deposition of these compounds on the

SCW determines the dimensions of the xylem cells, while the properties of these cells depend on the chemical

and mechanical properties of the SCWs. The SCWs have multiple layers (S1-S2) that differ in microfibril

organization and in ratios of cellulose to matrix (lignins, hemicellulose, and pectin) components (Mellerowicz

and Sundberg 2008). The rigidity of the SCW is conferred by lignins, which are fundamentals for structural

support and impermeability required for transport of water and nutrients over long distances (Leplé et al. 2007).

Lignins are the second most abundant component of wood and are also essential in determining the quality of

plants for use as raw materials, mainly for pulp and paper industries. The presence of lignins is a limiting factor

for pulp and paper production, since they need to be extracted from the wood by chemical methods that are

expensive and reduce the pure cellulose fibers production (Peter et al. 2007).

The deposition of lignin in the SCWs requires transcriptional regulators to coordinate the expression of hundreds

of genes participating in this process (Andersson-Gunnerås et al. 2006; Zhong et al. 2010). Several efforts have

been dedicated to developing genetically engineered trees, with the emphasis on reducing lignin quantity to

improve wood pulp production efficiency (Thakur et al. 2012; Hu et al. 1999; Mandrou et al. 2012; Quang et al.

2012; Zhong et al. 2003; Pilate et al. 2002). Although the roles of most genes involved in secondary xylem

formation have been elucidated, our knowledge about the SCW biosynthesis and its integration into plant

76

metabolism and connection with wood quality is still fragmentary. To gain a more in-depth knowledge were

performed comparative analysis between xylem transcriptomes of Eucalyptus grandis and E. globulus, two

species with contrasting characteristics of growth and wood quality (Pasquali et al. 2005). E. globulus produces a

wood of high density and relatively low lignin content, which contribute to a better pulp yield.

Among the differentially expressed genes found in the xylem of E. globulus was one encodes a polypeptide with

high similarity to PHOSPHATE-INDUCED PROTEIN 1 (PHI-1) from tobacco and to EXORDIUM (EXO) and

EXORDIUM-like (EXL) proteins from Arabidopsis. PHI-1/EXO comprises an emerging and widely distributed

family of proteins involved in signaling pathways that control cell division and differentiation in response to

hormonal and environmental signals (Sano et al. 1999; Sano and Nagata 2002; Farrar et al. 2003; Coll-Garcia et

al. 2004; Schröder et al. 2009; Schröder et al. 2011, 2012). E. globulus PHI-1 (EgPHI-1) is the first reported

member in PHI-1/EXO family to be associated with the different xylem cell wall properties in woody plants. By

using conventional analytical methods and scanning UV-microspectrophotometry (UMSP), a powerful technique

that provides information on the distribution of lignin within individual cell wall layers of various cell types

(Chabannes et al. 2001; Koch and Kleist 2001), we report here that the overexpression of EgPHI-1 changes the

cell wall composition and the topochemical distribution of lignin in stem xylem of transgenic tobacco.

Materials and methods

Plant material

The wild-type (WT) and three transgenic tobacco lines (L1, L2 and L3), representing distinct events of

Agrobacterium-mediated genetic transformation, were examined in the present study. EgPHI-1 overexpression at

different levels has been confirmed in the different transgenic lines by reverse transcription quantitative real-time

PCR (RT-qPCR) and western blot analyses [electronic supplementary material (ESM) Fig. S1]. The different

analyses were performed in stems of mature plants grown for 60 days in a greenhouse, under identical normal

conditions. All the details concerning the cloning of EgPHI-1 gene, generation of Agrobacterium constructions,

obtention and culture of the transgenic lines have been previously described (Sousa et al. submitted paper)

Determination of chemical composition of stem

Stems (total length) of 60 days-old transgenic and WT plants were harvested and kept in an air-circulation stove

at 75 °C until a constant weight was obtained. Three grams of dry stems were milled to pass through a 0.84-mm

screen. Milled samples were extracted with 95% ethanol for 6 h in a Soxhlet apparatus. The percentage of

extractives was determined on the basis of the dry weights of the extracted and non-extracted milled samples.

The hydrolysis of ethanol-extracted samples was performed with 72% sulfuric acid at 30°C for 1 h as described

previously (Ferraz et al. 2000). The acid solutions resulting were diluted with water and the mixture was heated

at 121°C/1 atm for 1 h. Thereafter, the residual materials were cooled and filtered (glass filter number 3). The

solids were dried to constant weight at 105°C and determined as insoluble lignin. The absorbance of the filtrated

at 205 nm was used to determine soluble lignin concentration. Monomeric sugar concentrations in the soluble

fraction were determined by HPLC (HPX87H column; Bio-Rad, USA) at 45°C and an elution rate of 0.6 ml min-

1with 5 mmol l

-1 sulfuric acid. Temperature-controlled refractive index detector at 35°C was used to detect

sugars. Xylose, mannose and galactose were eluted at the same time, under these conditions, and appeared as a

77

single peak. Glucose, xylose, arabinose and acetic acid were used as external calibration standards. No

corrections were performed because of the sugar-degradation reactions that take place during acid hydrolysis.

The factors used to convert sugar monomers to anhydromonomers were 0.90 for glucose to glucan and 0.88 for

xylose to xylan, and arabinose to arabinan. Acetyl content was calculated as the acetic acid content multiplied by

0.72. This procedure was conducted in triplicate. Glucose was reported as cellulose after correction by the

hydrolysis factor, and the other sugars and acetic acid were used to calculate the hemicellulose content in the

samples.

Sample preparations for topochemical analyses

Samples of the stem base taken from transgenic and WT plants were dehydrated in a graded series of acetone

and embedded in Spurr’s epoxy resin (Spurr 1969). Sections 1 μm thick were cut with an ultramicrotome

equipped with a diamond knife. Thereafter, the sections were transferred to quartz microscope slides, thermally

fixed and covered with a quartz cover slip.

Cellular scanning by UV-microspectrophotometry (UMSP)

The UMSP analyses were carried out using a ZEISS MSP800 equipped with a scanning stage, allowing the

determination of image profiles at constant wavelengths using the scan programs (Koch and Kleist 2001). The

SCW of fibers and vessels in stem xylem of transgenic and WT plants were analyzed by photometric point

measurements (spot size l μm2) in a wavelength range between 240 nm and 400 nm, with a TIDAS MSP 800

microspectrometer (J&M Analytics) equipped with TIDASDAQ software. This program evaluates the UV

absorbance point spectra of the lignified SCW. For the assessment of UV absorbance point spectra, 20 and 10

point measurements for fibers and vessels, respectively, of each plant were carried out. Individual fibers of WT

and L2 were selected and scanned for the UV-image profiles at constant wavelength of 280 nm (absorbance

maximum of softwood lignin). The UV-image profiles showed the lignin distribution in SCW of selected fibers

of stem xylem. The scan program digitizes rectangular fields with a local geometrical resolution of 1 μm² and

photometrical resolution. These are converted in a color scales (green to red) to visualization of the absorbance

intensities. The scans can be depicted as a histogram of statistical evaluation for the semi-quantitative lignin

distribution.

Statistical analysis

Statistical significance between WT and transgenic plants was determined by analysis of variance (ANOVA)

followed by Student’s t-test. A probability (P) of 0.05 or less was taken to indicate statistical significance.

Results are expressed as mean ± SD for (n) experiments.

Results

Lignin and cellulose contents

Three tobacco transgenic lines overexpressing EgPHI-1 gene were evaluated according to their overall lignin

and cellulose contents in stems (Table 1). Interestingly, all the transgenic lines showed a significant reduction in

cellulose content as compared with WT plants. The percentages of decrease in cellulose contents were of ~

78

4.7%, 5% and 2.5%, respectively, for L1, L2 and L3 as compared with the WT. A significant decrease in the

lignin content, of ~6%, was observed for L2, while there were no significant alterations in the content of this

compound in the other transgenic lines. The cellulose/lignin ratio increased in L2, while these ratios were

observed to decrease for L1 and L3.

Hemicellulose and extractives

The levels of the xylan, arabinan and acetic acid were used to calculate the hemicellulose content in stems of

transgenic and WT plants (Table 2). The xylan content varied differently among the transgenic lines when

compared with WT plants. There was an increase of this compound in L1 (9.5%), while a reduction was

observed in L2 (-1.8%). Similarly, a significant increase in the arabinan content was observed in L2 (21.6%),

while no significant differences were observed in L1 and L2. There were also no significant differences in the

acetic acid content between transgenic and WT plants. The sum of xylan, arabinan and acetic acid levels to

estimate the hemicellulose contents was significantly increased (7.9%) in L1. The content of extractives obtained

with the ethanol-95% extraction was observed to vary significantly (p≤0.05) in some transgenic lines (Fig. 1). A

high increase was observed in L2 (19.6%) and L3 (9.1%), while L1 did not show significant differences in

comparison with WT plants (p≤0.05).

UV absorbance spectra of lignin in fiber and vessel SCWs

UV absorbance point spectra within a wavelength range between 240 nm and 400 nm was used to analyze the

lignin content in cell wall layers of individual cell types, such as fibers and vessels (Fig. 2). Typical profiles of

lignified S2-layers of fibers (Fig. 2a) and vessels (Fig. 2b) were generated for transgenic and WT plants. Fibers

and vessels of L2 and L3 (Figs. 2a,b) showed a lower UV absorbance value than the WT around 280 nm, the

absorption maximum of the spectra corresponding to non-conjugated units in lignins. Conversely, fibers of L1

exhibited a similar UV absorbance value than the WT at 280 nm, but its vessels displayed a much higher UV

absorbance value than the WT at this point of spectra. These data indicate a reduction of lignin content in fiber

and vessel SCWs in two out three transgenic plants evaluated.

Topochemical distribution of lignin in fiber SCWs

UV scanning profiles of lignin distribution in fiber SCWs were compared between L2 and WT plants (Fig. 3).

L2 was selected for scanning UMSP because reduction in lignin content was chemically and

microspectrophotometrically demonstrated. The color indicates different intensities of UV-absorbance at a

constant wavelength of 280 nm (absorption maximum of softwood lignin). These UV-image profiles were

produced from selected fiber sections of WT and L2 plants (Figs. 3a,b). The scanning UV-images of these cells

indicated that WT and L2 fibers had different absorbance levels, with the most intense absorbance seen in the

cell corners (CC) and compound middle lamella (CML) for both plants (Figs. 3c,d). The UV-image for L2 fiber

showed a diffuse lignin distribution in S2-layer, with relatively low absorbance intensity (Fig. 3c). On the other

hand, the S2-layer of WT fiber showed a more uniform lignin distribution, with higher absorbance intensity (Fig.

3d). Average lignin absorbance values of the each image were calculated from the frequency histograms.

According to histogram statistical evaluation, the average lignin absorbance value at 280 nm was higher in WT

(0.20) than in L2 (0.16) fibers (Figs. 3e,f).

79

Discussion

Reduction of lignin content has been demonstrated in transgenic plants; however, a compensatory mechanism

use to promote cellulose accumulation in these plants (Hu et al. 1999; Ambavaram et al. 2011). In addition, plant

species with severe lignin or cellulose reductions (>20%) have been observed to show a decreased stem height

and diameter, leaf size and altered leaf shape, causing a retarded growth and development (Legay et al. 2010;

Joshi et al. 2011; Piquemal et al. 1998; Goicoechea et al. 2005; Zhong et al. 2003; Leplé et al. 2007). In the

present study, the reduction of cellulose in the EgPHI-1-expressing transgenic plants was accompanied by a

reduction of lignin content in fibers and vessels of two out three transgenic lines analyzed. Such a reductions

were not severe (< 10%) and did not adversely affect the phenotype of transgenic lines. Therefore, the resulting

phenotype produced by EgPHI-1 overexpression is distinct from those previously reported for other transgenic

plants and should be of particular interest for biotechnological application.

Hemicelluloses are polysaccharides present in plant cell walls that play an important biological role by shaping

and strengthening the cell wall by interaction with cellulose and, in some cell walls, with lignin (Scheller and

Ulvskov 2010). Although the interaction of hemicelluloses and cellulose has been shown to play a central role in

the cell walls, in our analyses a low correlation was observed between the variations in hemicelluloses and

cellulose contents (r2 values for linear correlations were lower than 0.24). For example, the major hemicellulose

component, xylan, varied differently among the transgenic lines, probably due to the different levels of EgPHI-1

expression in each transgenic line. Arabinan, another hemicellulose component important to flexibility of cell

walls (Jones et al. 2003), increase only in L2. Thus, our results demonstrate that the reduction of cellulose was

not compensated by hemicellulose carbohydrates synthesis, indicating that the effects of EgPHI-1 expression are

less predictable on hemicellulose content than on cellulose and/or lignin contents.

EgPHI-1 overexpression of increased the extractive proportion in dry biomass of stems from two transgenic

lines. This increase may be the result of an increase in the content of waxy materials and low molar mass

aromatics (Aharoni et al. 2004), or even small amounts of oligosaccharides dissolved during the extraction

procedure. It has been proposed that lignin/cellulose reduction may cause a change in carbon flow that is

directed into biosynthesis of other constituents of primary or secondary cell walls (Turner et al. 2007).Thus, it is

probable that the lower amount of cellulose and lignin in transgenic lines is mass balanced by other components

present in the fraction of extractives, but that were unidentified under the analytical conditions used in this work.

UV-microspectrophotometry enabled us to determine lignin content and distribution within individual cell wall

layers of specific cell types. Two transgenic lines demonstrated lower UV absorbance on S2-layers of fiber and

vessel SCWs at 280 nm. Softwood lignin, as tobacco, presents absorption maximum at 280 nm (Koch and Kleist

2001) and the reduction of absorbance valuess observed in the transgenic lines indicates that EgPHI-1

overexpression reduce the lignification in both cell types. UV scanning profiles showed differences in the

topochemical distribution of lignin in fibers of transgenic and WT plants. Although the areas of higher and lower

lignin contents were virtually the same in both plants, with CML and CC exhibing the highest absorbances and

SCW the lowest, the lignification patterns were quite distinct. WT plants showed a uniform pattern of lignin

deposition, whereas a diffuse lignification pattern was observed in transgenic plants. The same areas of higher

and lower lignin contents were reported in xylem tissue of Pinus radiate (Möller et al. 2006). CML has been

observed to contain much higher lignin levels than the S2-layer. For example, in Populus lignin comprises 68%

80

of the CML compared to only 6–8% and 25% of the S2-layers of fibers and vessel elements, respectively

(Donaldson et al. 2001).

High lignin depletion in vessel walls in the secondary xylem was observed to reduce the plant capacity to resist

the compressive forces associated with water conduction, showing a tendency towards collapsing (Coleman et al.

2008). In transgenic aspen plants, the cellulose reduction in the conductive xylem resulted in stems weak and

prone to collapse, promoting hydraulic limitations that, in turn, caused the dwarfed (Joshi et al. 2011). These

reported modifications and limitations were not observed in transgenic lines overexpressing EgPHI-1during the

period that the plants were maintained in greenhouse. Taken together, our data clearly indicate that of EgPHI-1

expression regulates the synthesis of cell wall components in specific cell types, without adversely affecting the

phenotype of transgenic plants. Such modifications are of central interest for biotechnological applications in

forestry and biofuel crops.

Acknowledgements

The authors thank José Moreira da Silva (School of Engineering of Lorena, University of São Paulo- USP, São

Paulo - Brazil) for the technical assistance in chemical and microscopic analyses. We are also are thankful to Dr.

Fernando Masarin for helping in the discussions. This work was supported by Financiadora de Estudos e

Projetos (FINEP, Brazilian Ministry of Science and Technology - MCT) [grant number 2101063500], Conselho

Nacional de Desenvolvimento Científico e Tecnológico (CNPq, MCT) [grant numbers 50.6348/04-0,

578632/08-0, 311361/09-9] and the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB, Secretariat

of Science, Technology and Innovation – SECTI) [grant number BOL1569/2010].

Electronic supplementary material

The supplementary data of this article can be found in the online version at DOI:

References

Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE Clade of AP2 domain

transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance

when overexpressed in Arabidopsis. Plant Cell Online 16 (9):2463-2480. doi:10.1105/tpc.104.022897

Ambavaram MMR, Krishnan A, Trijatmiko KR, Pereira A (2011) Coordinated activation of cellulose and

repression of lignin biosynthesis pathways in rice. Plant Physiol 155 (2):916-931.

doi:10.1104/pp.110.168641

Andersson-Gunnerås S, Mellerowicz EJ, Love J, Segerman B, Ohmiya Y, Coutinho PM, Nilsson P, Henrissat B,

Moritz T, Sundberg B (2006) Biosynthesis of cellulose-enriched tension wood inPopulus: global

analysis of transcripts and metabolites identifies biochemical and developmental regulators in

secondary wall biosynthesis. Plant J 45 (2):144-165. doi:10.1111/j.1365-313X.2005.02584.x

Boerjan W, Ralph J, Baucher M (2003) Lignin Biosynthesis. Annu Rev Plant Biol 54 (1):519-546.

doi:10.1146/annurev.arplant.54.031902.134938

Chabannes M, Ruel K, Yoshinaga A, Chabbert B, Jauneau A, Joseleau J-P, Boudet A-M (2001) In situ analysis

of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial

patterns of lignin deposition at the cellular and subcellular levels. Plant J 28 (3):271-282.

doi:10.1046/j.1365-313X.2001.01159.x

Coleman HD, Samuels AL, Guy RD, Mansfield SD (2008) Perturbed lignification impacts tree growth in hybrid

poplar - A function of sink strength, vascular integrity, and photosynthetic assimilation. Plant Physiol

148 (3):1229-1237. doi:10.1104/pp.108.125500

81

Coll-Garcia D, Mazuch J, Altmann T, Müssig C (2004) EXORDIUM regulates brassinosteroid-responsive

genes. FEBS Lett 563 (1):82-86

Donaldson L, Hague J, Snell R (2001) Lignin distribution in coppice poplar, linseed and wheat straw.

Holzforschung, vol 55. doi:10.1515/hf.2001.063

Farrar K, Evans IM, Topping JF, Souter MA, Nielsen JE, Lindsey K (2003) EXORDIUM– a gene expressed in

proliferating cells and with a role in meristem function, identified by promoter trapping in Arabidopsis.

The Plant J 33 (1):61-73. doi:10.1046/j.1365-313X.2003.01608.x

Ferraz A, Baeza J, Rodriguez J, Freer J (2000) Estimating the chemical composition of biodegraded pine and

eucalyptus wood by DRIFT spectroscopy and multivariate analysis. Bioresour Technol 74 (3):201-212.

doi:10.1016/s0960-8524(00)00024-9

Goicoechea M, Lacombe E, Legay S, Mihaljevic S, Rech P, Jauneau A, Lapierre C, Pollet B, Verhaegen D,

Chaubet-Gigot N, Grima-Pettenati J (2005) EgMYB2, a new transcriptional activator from Eucalyptus

xylem, regulates secondary cell wall formation and lignin biosynthesis. Plant J 43 (4):553-567.

doi:10.1111/j.1365-313X.2005.02480.x

Hu W-J, Harding SA, Lung J, Popko JL, Ralph J, Stokke DD, Tsai C-J, Chiang VL (1999) Repression of lignin

biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotechnol 17

(8):808-812

Jones L, Milne JL, Ashford D, McQueen-Mason SJ (2003) Cell wall arabinan is essential for guard cell function.

PNAS 100 (20):11783-11788. doi:10.1073/pnas.1832434100

Joshi CP, Thammannagowda S, Fujino T, Gou J-Q, Avci U, Haigler CH, McDonnell LM, Mansfield SD,

Mengesha B, Carpita NC, Harris D, DeBolt S, Peter GF (2011) Perturbation of wood cellulose synthesis

causes pleiotropic effects in transgenic Aspen. Mol Plant 4 (2):331-345. doi:10.1093/mp/ssq081

Koch G, Kleist G (2001) Application of scanning UV microspectrophotometry to localise lignins and phenolic

extractives in plant cell walls. Holzforschung, vol 55. doi:10.1515/hf.2001.091

Legay S, Sivadon P, Blervacq A-S, Pavy N, Baghdady A, Tremblay L, Levasseur C, Ladouce N, Lapierre C,

Séguin A, Hawkins S, Mackay J, Grima-Pettenati J (2010) EgMYB1, an R2R3 MYB transcription

factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar.

New Phytol 188 (3):774-786. doi:10.1111/j.1469-8137.2010.03432.x

Leplé J-C, Dauwe R, Morreel K, Storme V, Lapierre C, Pollet B, Naumann A, Kang K-Y, Kim H, Ruel K,

Lefèbvre A, Joseleau J-P, Grima-Pettenati J, De Rycke R, Andersson-Gunnerås S, Erban A, Fehrle I,

Petit-Conil M, Kopka J, Polle A, Messens E, Sundberg B, Mansfield SD, Ralph J, Pilate G, Boerjan W

(2007) Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping

reveals effects on cell wall polymer metabolism and structure. Plant Cell Online 19 (11):3669-3691.

doi:10.1105/tpc.107.054148

Mandrou E, Hein P, Villar E, Vigneron P, Plomion C, Gion J-M (2012) A candidate gene for lignin composition

in Eucalyptus: cinnamoyl-CoA reductase (CCR). Tree Genet Genomes 8 (2):353-364.

doi:10.1007/s11295-011-0446-7

Mellerowicz E, Sundberg B (2008) Wood cell walls: biosynthesis, developmental dynamics and their

implications for wood properties. Curr Opin Plant Biol 11 (3):293-300. doi:10.1016/j.pbi.2008.03.003

Möller R, Koch G, Nanayakkara B, Schmitt U (2006) Lignification in cell cultures of Pinus radiata: activities of

enzymes and lignin topochemistry. Tree Physiol 26 (2):201-210. doi:10.1093/treephys/26.2.201

Pasquali G, Bastolla FM, Pazzini F, Kirch RP, Pizzoli G, Carazzole MF, Brondani RV, Coelho ASG,

Grattapaglia D, Brommonschenkel SH, Pappas Jr GJ, Pereira GAG, Cascardo JCDM (2005) Sequecing

and differential expression of xylem specific genes from two Eucalyptus species with highly contrasting

wood properties. In: Institute FAAB (ed) IUFRO Tree Biotechnol 2005, Pretoria, South Africa, 2005.

Programme and abstracts of the IUFRO Tree Biotechnol 2005, p 22

Peter GF, White DE, Torre RDL, Singh R, Newman D (2007) The value of forest biotechnology: a cost

modelling study with loblolly pine and kraft linerboard in the southeastern USA. Int J Biotechnol 9

(5):415-435

Pilate G, Guiney E, Holt K, Petit-Conil M, Lapierre C, Leple J-C, Pollet B, Mila I, Webster EA, Marstorp HG,

Hopkins DW, Jouanin L, Boerjan W, Schuch W, Cornu D, Halpin C (2002) Field and pulping

performances of transgenic trees with altered lignification. Nat Biotechnol 20 (6):607-612

Piquemal J, Lapierre C, Myton K, O’connell A, Schuch W, Grima-pettenati J, Boudet A-M (1998) Down-

regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic

tobacco plants. Plant J 13 (1):71-83. doi:10.1046/j.1365-313X.1998.00014.x

Plomion C, Leprovost G, Stokes A (2001) Wood Formation in Trees. Plant Physiol 127 (4):1513-1523.

doi:10.1104/pp.010816

Quang T, Hallingbäck H, Gyllenstrand N, Arnold S, Clapham D (2012) Expression of genes of cellulose and

lignin synthesis in Eucalyptus urophylla and its relation to some economic traits. Trees 26 (3):893-901.

doi:10.1007/s00468-011-0664-5

82

Sano T, Kuraya Y, Amino S-i, Nagata T (1999) Phosphate as a Limiting Factor for the Cell Division of Tobacco

BY-2 Cells Plant Cell Physiology 40 (1):1-16

Sano T, Nagata T (2002) The possible involvement of a phosphate-induced transcription factor encoded by Phi-2

gene from tobacco in ABA-signaling pathways. . Plant and Cell Physiol 43 (1):12-20.

doi:10.1093/pcp/pcf002

Scheller HV, Ulvskov P (2010) Hemicelluloses. Annu Rev Plant Biol 61 (1):263-289. doi:doi:10.1146/annurev-

arplant-042809-112315

Schröder F, Lisso J, Lange P, Müssig C (2009) The extracellular EXO protein mediates cell expansion in

Arabidopsis leaves. BMC Plant Biology 9 (20):1-12. doi:10.1186/1471-2229-9-20

Schröder F, Lisso J, Müssig C (2011) EXORDIUM-LIKE1 promotes growth during low carbon availability in

Arabidopsis. Plant Physiol 156 (3):1620-1630. doi:10.1104/pp.111.177204

Schröder F, Lisso J, Müssig C (2012) Expression pattern and putative function of EXL1 and homologous genes

in Arabidopsis. Plant Signal Behav 7 (1):22-27. doi: 10.4161/psb.7.1.18369

Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26

(1–2):31-43. doi:http://dx.doi.org/10.1016/S0022-5320(69)90033-1

Thakur AK, Aggarwal G, Srivastava DK (2012) Genetic modification of lignin biosynthetic pathway in Populus

ciliata Wall. via Agrobacterium-mediated antisense CAD gene transfer for quality paper production.

Natl Acad Sci Lett 35 (2):79-84. doi:10.1007/s40009-012-0018-x

Turner S, Gallois P, Brown D (2007) Tracheary element differentiation. Annu Rev Plant Biol 58 (1):407-433.

doi:doi:10.1146/annurev.arplant.57.032905.105236

Zhong R, Lee C, Ye Z-H (2010) Evolutionary conservation of the transcriptional network regulating secondary

cell wall biosynthesis. Trends Plant Sci 15 (11):625-632. doi:10.1016/j.tplants.2010.08.007

Zhong R, Morrison WH, Freshour GD, Hahn MG, Ye Z-H (2003) Expression of a mutant form of cellulose

synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis. Plant Physiol 132 (2):786-

795. doi:10.1104/pp.102.019331

Figure Legends

Fig. 1 Extractives content in stem of control and transgenic lines. Dry stem weight percentage of extractives

presents in transgenic lines (L1, L2 and L3) and control no-transformed (WT) plants with ethanol-95%

extraction. Bars represent the mean ± SD of the three experimental replicates (n=3). Same letters do not differ

among the samples at significance level of 0.05.

Fig. 2 UV absorbance point spectra of the lignified cell walls of fibers (a) and vessels (b) of transgenic lines (L1,

L2 and L3) and control no-transformed (WT) plants. At least 20 spectra were recorded from fibers and 10 from

vessels. The mean spectra are shown in this figure. Standard deviations, calculated from the absorbance values

measured at wavelength of 280 nm, were 5% and 4% for fiber and vessel cells, respectively.

Fig. 3 Fibers of stem xylem of control and transgenic line. Fiber selected from control no-transformed plant

(WT, a) and L2 transgenic line (b). UV scanning profiles of lignin distribution in individual fibers of stem xylem

of WT (c) and L2 transgenic line (d). Histograms to lignin distribution in fibers of stem xylem of WT (e) and L2

transgenic line (f) according to absorbance intensity values at 280 nm. Red box represent the spot size (l μm2)

used to scanner the cells. The differently colored pixels (green to red) mark the absorbance intensity at 280 nm.

Magnification bars represent 10 µm. CC: cell corners. CML: compound middle lamella. S2: secondary wall.

84

Table 1 Lignin and cellulose contents in stem of WT and transgenic lines

Plant Total ligninδ Cellulose

δ Cellulose/Lignin

WT 14.83 ± 0.1 32.45 ± 0.1 2.188

L1 15.03 ± 0.1 30.92 ± 0.1*** 2.058

L2 13.94 ± 0.2*** 30.90 ± 0.1*** 2.216

L3 14.53 ± 0.1 31.62 ± 0.1*** 2.175 Data are the means ± SD of three independent experiments. Statistically significant differences at p≤0.001 (***) between the WT and transgenic plants. δ % of dry stem weight.

85

Table 2 Hemicellulose content in stem of WT and transgenic lines

Plant Xylan δ Arabinan

δ Acetic acid

δ Sum

δ

WT 12.45 ± 0.05 0.71 ± 0.02 3.17 ± 0.06 16.33 ± 0.05

L1 13.64 ± 0.09*** 0.70 ± 0.02 3.28 ± 0.08 17.63 ± 0.16***

L2 12.21 ± 0.08* 0.86 ± 0.07** 3.17 ± 0.05 16.25 ± 0.20

L3 12.46 ± 0.13 0.79 ± 0.06 3.16 ± 0.09 16.43 ± 0.14

Data are the means ± SD of three independent experiments. Statistically significant differences at p≤0.05 (*), p≤0.01 (**) and p≤0.001 (***) between the WT and transgenic plants. δ % of dry stem weight.

86

Figure 1

87

Figure 2

88

Figure 3

89

Fig. S1. The EgPHI-1 gene construction and expression in transgenic tobacco. (A) Schematic diagram

of the 35S::EgPHI-1 gene construction used in Agrobacterium-mediated genetic transformation of tobacco.

EgPHI-1 cDNA was placed in sense orientation into plasmid pCAMBIA 2301 under the control of the CaMV

35S promoter and terminator. (B) Relative EgPHI-1 expression in transgenic plant lines. Total RNA was isolated

from leaves of transgenic (L1, L2 and L3) and WT plants. Relative gene expression was assessed by RT-qPCR

in three biological replicates and three technical replicates per plant. Expression was calculated by the 2-∆∆Ct

method and values were normalized against GAPDH. (C) Relative protein levels in transgenic plant lines. Equal

amounts of total proteins extracted from leaves of transgenic (L1, L2 and L3) and WT plants were separated by

SDS-PAGE and immunoblotted with anti-EGP antibody. Levels of EgPHI-1 were measured in three technical

replicates per plant. Protein levels were quantified from membrane imaging and the values of transgenic lines

were calculated in relation to the WT protein levels. Bars represent the mean ± SE of the three replicates (n=3).

Statistically significant differences at p≤0.05 (*) and p≤0.001 (***) between transgenic and WT plants are

indicated.

90

4. CONCLUSÕES GERAIS

EgPHI-1 é um novo membro da classe de proteínas PHI-1/EXO e possui forte

envolvimento com a diferenciação celular e formação da parede celular secundária;

EgPHI-1 altera a alocação de carbono para o crescimento de parte aérea em

detrimento da raiz;

EgPHI-1 participa da biossíntese de componentes da parede celular

secundária de vasos e fibras modificando os conteúdos de lignina e celulose;

EgPHI-1, provavelmente por meio de um mecanismo compensatório, aumenta

a biossíntese de compostos presentes na fração dos extrativos;

EgPHI-1 pode atuar como uma proteína moduladora, pelo menos em parte,

da expressão da chaperona BiP, a qual previne a morte celular dos tecidos sob

estresse osmótico.

91

5. REFÊNCIAS

ABRAF. Anuário estatístico da ABRAF: 2012 ano base 2011. Brasilia: ABRAF. 2012. 150 p. ALZATE, S. B. A.; TOMAZELLO FILHO, M.; PIEDADE, S. M. D. S. Variação longitudinal da densidade básica da madeira de clones de Eucalyptus grandis Hill ex Maiden, E. saligna Sm. e E. grandis x urophylla. Scientia Forestalis, v.68, p.87-95. 2005. AMBAVARAM, M. M. R. et al. Coordinated activation of cellulose and repression of lignin biosynthesis pathways in rice. Plant Physiology, v.155, n.2, February 1, 2011, p.916-931. 2011. ARMSTRONG, J. I. et al. Identification of inhibitors of auxin transcriptional activation by means of chemical genetics in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, v.101, n.41, October 12, 2004, p.14978-14983. 2004. BARBOSA, L. C. A.; MALTHA, C. R. A.; CRUZ, M. P. Composição química de extrativos lipofílicos e polares de madeira de Eucalyptus grandis. Ciência & Engenharia, v.15, p.13-20. 2005. BASTOLLA, F. M. et al. Differential gene expression in two Eucalyptus species. 52º Congresso Brasileiro de Genética. Sbg. Foz do Iguaçu 2006. BAUCHER, M. et al. Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar. Plant Physiology, v.1, n.12, p.1479-1 490. 1996. BAYER, E. M. et al. Arabidopsis cell wall proteome defined using multidimensional protein identification technology. Proteomics, v.6, n.1, p.301-311. 2006. BLOUNT, J. W. et al. Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway. Plant Physiology, v.122, n.1, January 1, 2000, p.107-116. 2000.

92

BOERJAN, W. Biotechnology and the domestication of forest trees. Current Opinion in Biotechnology, v.16, n.2, p.159-166. 2005. BOERJAN, W.; RALPH, J.; BAUCHER, M. Lignin Biosynthesis. Annual Review of Plant Biology, v.54, n.1, p.519-546. 2003. BRACELPA. Relatório estatístico florestal 2010/2011: Associação Brasileira de Celulose e Papel. 2011. 49 p. CHIANG, V. L. Monolignol biosynthesis and genetic engineering of lignin in trees, a review. Environmental Chemistry Letters, v.4, n.3, p.143-146. 2006. CHINNUSAMY, V.; SCHUMAKER, K.; ZHU, J. K. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. Journal of Experimental Botany, v.55, n.395, January 1, 2004, p.225-236. 2004. CLOUSE, S. D.; SASSE, J. M. BRASSINOSTEROIDS: Essential regulators of plant growth and development. Annual Review of Plant Physiology and Plant Molecular Biology, v.49, n.1, p.427-451. 1998. COLEMAN, H. D. et al. Perturbed lignification impacts tree growth in hybrid poplar - A function of sink strength, vascular integrity, and photosynthetic assimilation. Plant Physiology, v.148, n.3, November 2008, p.1229-1237. 2008. COLL-GARCIA, D. et al. EXORDIUM regulates brassinosteroid-responsive genes. FEBS Letters, v.563, n.1, p.82-86. 2004. DAYAN, J. et al. Enhancing plant growth and fiber production by silencing GA 2-oxidase. Plant Biotechnology Journal, v.8, n.4, p.425-435. 2010. DE VOS, M.; JANDER, G. Myzus persicae (green peach aphid) salivary components induce defence responses in Arabidopsis thaliana. Plant, Cell & Environment, v.32, n.11, p.1548-1560. 2009. DEL LUNGU, A.; BALL, J.; CARLE, J. Global planted forests thematic study: results and analysis. In: (Ed.). FAO. Rome, v.38, 2006. Global planted forests thematic study: results and analysis, p.178 DELMER, D. P. Cellulose biosynthesis: Exciting times for a difficult field of study. Annual Review of Plant Physiology and Plant Molecular Biology, v.50, n.1, 1999/06/01, p.245-276. 1999. DEMURA, T.; FUKUDA, H. Transcriptional regulation in wood formation. Trends in Plant Science, v.12, n.2, p.64-70. 2007. DEMURA, T.; YE, Z.-H. Regulation of plant biomass production. Current Opinion in Plant Biology, v.13, n.3, p.298-303. 2010.

93

DINUS, R. J.; WELT, T. Tailoring fiber properties to paper manufacture: recent developments. TAPPI Pulping Conference. Chicago, Illinois: Georgia Institute of Technology. October 1-5, 1995. 16 p. DITA, M. A. et al. Gene expression profiling of Medicago truncatula roots in response to the parasitic plant Orobanche crenata. Weed Research, v.49, p.66-80. 2009. DONALDSON, L.; HAGUE, J.; SNELL, R. Lignin distribution in coppice poplar, linseed and wheat straw. Holzforschung. 55: 379 p. 2001. DOUGLAS, C. J. Phenylpropanoid metabolism and lignin biosynthesis: from weeds to trees. Trends in Plant Science, v.1, n.6, p.171-178. 1996. DUTT, D.; TYAGI, C. H. Comparison of various eucalyptus species for their morphological, chemical, pulp and paper making characteristics. Indian Journal of Chemical Technology v.18, p.145-151. 2011. ELDRIDGE, K. G. et al. Eucalypt domestication and breeding. United Kingdom: Clarendon Press, Oxford. 1993 FAO. State of the world's forests 2007. Rome: Food and Agriculture Organization of the United Nations. 2007. 144 p. FARRAR, K. et al. EXORDIUM– a gene expressed in proliferating cells and with a role in meristem function, identified by promoter trapping in Arabidopsis. The Plant Journal, v.33, n.1, p.61-73. 2003. FENNING, T. M.; GERSHENZON, J. Where will the wood come from? Plantation forests and the role of biotechnology. Trends in biotechnology, v.20, n.7, p.291-296. 2002. FERGUS, B. J.; GORING, D. A. I. The location of guaiacyl and syringyl lignins in birch xylem tissue. Holzforschung - International Journal of the Biology, Chemistry, Physics and Technology of Wood. 24: 113 p. 1970. FOUCART, C. et al. Transcript profiling of a xylem vs phloem cDNA subtractive library identifies new genes expressed during xylogenesis in Eucalyptus. New Phytologist, v.170, n.4, p.739-752. 2006. GOICOECHEA, M. et al. EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. The Plant Journal, v.43, n.4, p.553-567. 2005. GRATTAPAGLIA, D. Integrating genomics into Eucalyptus breeding. Genetic Molecular Research., v.3, p.369-379. 2004. HU, W.-J. et al. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotech, v.17, n.8, p.808-812. 1999.

94

IWASE, A. et al. Comparative analyses of the gene expression profiles of Arabidopsis intact plant and cultured cells. Biotechnology Letters, v.27, n.15, p.1097-1103. 2005. JAMET, E. et al. Cell wall biogenesis of Arabidopsis thaliana elongating cells: transcriptomics complements proteomics. BMC Genomics, v.10, n.1, p.505. 2009. JONES, T. H.; VAILLANCOURT, R. E.; POTTS, B. M. Detection and visualization of spatial genetic structure in continuous Eucalyptus globulus forest. Molecular Ecology, v.16, n.4, p.697-707. 2006. JOUANIN, L. et al. Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity. Plant Physiology, v.123, p.1363–1373. 2000. KIM, T.-W. et al. Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature, v.482, n.7385, p.419-422. 2012. KO, J. H. Plant body weight-induced secondary growth in Arabidopsis and its transcription phenotype revealed by whole-transcriptome profiling. Plant Physiology, v.135, n.2, p.1069-1083. 2004. LEGAY, S. et al. EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar. New Phytologist, v.188, n.3, p.774-786. 2010. LEROUXEL, O. et al. Biosynthesis of plant cell wall polysaccharides — a complex process. Current Opinion in Plant Biology, v.9, n.6, p.621-630. 2006. LI, L. Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proceedings of the National Academy of Sciences, v.100, n.8, p.4939-4944. 2003. MELLEROWICZ, E.; SUNDBERG, B. Wood cell walls: biosynthesis, developmental dynamics and their implications for wood properties. Current Opinion in Plant Biology, v.11, n.3, p.293-300. 2008. MÖLLER, R. et al. Lignification in cell cultures of Pinus radiata: activities of enzymes and lignin topochemistry. Tree Physiology, v.26, n.2, February 1, 2006, p.201-210. 2006. MOON, D. H. et al. Comparison of the expression profiles of susceptible and resistant Eucalyptus grandis exposed to Puccinia psidii winter using SAGE. Functional Plant Biology, v.34, n.11, p.1010. 2007. MÜSSIG, C.; FISCHER, S.; ALTMANN, T. Brassinosteroid-regulated gene expression. Plant Physiology, v.129, n.3, p.1241-51. 2002. MUSTAFA, B. M. et al. A cDNA microarray approach to decipher lentil (Lens culinaris) responses to Ascochyta lentis. Australasian Plant Pathology, v.38, n.6, p.617-631. 2009.

95

MYBURG, A. A. et al. Comparative genetic linkage maps of Eucalyptus grandis , Eucalyptus globulus and their F 1 hybrid based on a double pseudo-backcross mapping approach. TAG Theoretical and Applied Genetics, v.107, n.6, p.1028-1042. 2003. MYBURG, A. A. et al. Eucalyptus. In: K. C. R. Heidelberg (Ed.). Genome mapping & molecular breeding in plants: Forest trees Springer, v.7, 2007. Eucalyptus NORTON, G. J. et al. Rice–arsenate interactions in hydroponics: a three-gene model for tolerance. Journal of Experimental Botany, v.59, n.8, May 1, 2008, p.2277-2284. 2008. OH, S.; PARK, S.; HAN, K.-H. Transcriptional regulation of secondary growth in Arabidopsis thaliana. Journal of Experimental Botany, v.54, n.393, December 1, 2003, p.2709-2722. 2003. OLSEN, A. N. et al. NAC transcription factors: structurally distinct, functionally diverse. Trends in Plant Science, v.10, n.2, p.79-87. 2005. PASQUALI, G. et al. Sequecing and differential expression of xylem specific genes from two Eucalyptus species with highly contrasting wood properties. IUFRO Tree Biotechnology 2005. Pretoria, South Africa: Programme and abstracts of the IUFRO Tree Biotechnology 2005, 2005. 22 p. PAUX, E. et al. Transcript profiling of Eucalyptus xylem genes during tension wood formation. New Phytologist, v.167, n.1, p.89-100. 2005. PAUX, E. et al. Identification of genes preferentially expressed during wood formation in Eucalyptus. Plant Molecular Biology, v.55, p.263–280. 2004. PILATE, G. et al. Field and pulping performances of transgenic trees with altered lignification. Nature Biotechnology, v.20, n.6, p.607-612. 2002. PINÇON, G. et al. Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth. Phytochemistry, v.57, n.7, p.1167-1176. 2001. PIQUEMAL, J. et al. Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. The Plant Journal, v.13, n.1, p.71-83. 1998. PLOMION, C.; LEPROVOST, G.; STOKES, A. Wood Formation in Trees. Plant Physiology, v.127, n.4, p.1513-1523. 2001. POKE, F. S. et al. Genomic Research in Eucalyptus. Genetica, v.125, n.1, p.79-101. 2005.

96

QUEIROZ, S. C. S. et al. Influência da densidade básica da madeira na qualidade da polpa kraft de clones híbridos de Eucalyptus grandis W. Hill ex Maiden X Eucalyptus urophylla S. T. Blake. Revista Árvore, v.28, p.901-909. 2004. RENGEL, D. et al. A new genomic resource dedicated to wood formation in Eucalyptus. BMC Plant Biology, v.9, n.1, p.36. 2009. RICHMOND, T. Higher plant cellulose synthases. Genome Biology, v.1, n.4, p.3001.1–3001.6. 2000. ROSA, C. A. B. et al. Comportamento da madeira de Eucalyptus globulus com diferentes teores de lignina para produção de celulose kraft. 35º Congresso e Exposição Anual de Celulose e Papel. São Paulo: Associação Brasileira Técnica de Celulose e Papel 2002. SANO, T. et al. Phosphate as a Limiting Factor for the Cell Division of Tobacco BY-2 Cells Plant Cell Physiology, v.40, n.1, p.1-16. 1999. SANO, T.; NAGATA, T. The possible involvement of a phosphate-induced transcription factor encoded by Phi-2 gene from tobacco in ABA-signaling pathways. . Plant and Cell Physiology, v.43, n.1, January 15, 2002, p.12-20. 2002. SCHELLER, H. V.; ULVSKOV, P. Hemicelluloses. Annual Review of Plant Biology, v.61, n.1, p.263-289. 2010. SCHRÖDER, F. et al. The extracellular EXO protein mediates cell expansion in Arabidopsis leaves. BMC Plant Biology, v.9, n.20, p.1-12. 2009. SCHRÖDER, F.; LISSO, J.; MÜSSIG, C. EXORDIUM-LIKE1 promotes growth during low carbon availability in Arabidopsis. Plant Physiology, v.156, n.3, July 1, 2011, p.1620-1630. 2011. SCHRÖDER, F.; LISSO, J.; MÜSSIG, C. Expression pattern and putative function of EXL1 and homologous genes in Arabidopsis. Plant Signaling and Behavior, v.7, n.1, January, p.22-27. 2012. SILVA, R. et al. Aplicações de fibras lignocelulósicas na química de polímeros e em compósitos. Quimica Nova, v.32, n.3, p.661-671. 2009. SMART, C. C.; AMRHEIN, N. The influence of lignification on the development of vascular tissue in Vigna radiata L. Protoplasma, v.124, n.1, p.87-95. 1985. SOUSA, A. O. et al. Comparative transcriptome analysis reveals key gene expression differences between the xylems of Eucalyptus globulus and Eucalyptus grandis. Ilhéus: Universidade Estadual de Santa Cruz 2011. TAMAGNONE, L. et al. The AmMYB308 and AmMYB330 transcription factors from Antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. The Plant Cell Online, v.10, n.2, February 1, 1998, p.135-154. 1998.

97

THAKUR, A. K.; AGGARWAL, G.; SRIVASTAVA, D. K. Genetic modification of lignin biosynthetic pathway in Populus ciliata Wall. via Agrobacterium-mediated antisense CAD gene transfer for quality paper production. National Academy Science Letters, v.35, n.2, 2012/04/01, p.79-84. 2012. TOHGE, T. et al. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. The Plant Journal, v.42, n.2, p.218-235. 2005. TOMAZELLO FILHO, M. Variação radial da densidade básica e da estrutura anatômica da madeira do Eucalyptus saligna e E. grandis. IPEF, v. 29, p.37-45. 1985. TRUGILHO, P. F. et al. Classificação de clones de Eucalyptus sp visando à produção de polpa celulósica. Revista Árvore, v.28, n.6, p.895-899. 2004. TURNER, S.; GALLOIS, P.; BROWN, D. Tracheary element differentiation. Annual Review of Plant Biology, v.58, n.1, p.407-433. 2007. VOELKER, S. L. et al. Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar. Plant Physiology, v.154, n.2, p.874-886. 2010. WHETTEN, R.; SEDEROFF, R. Lignin biosynthesis. The Plant Cell Online, v.7, n.7, July 1, 1995, p.1001-1013. 1995. WHETTEN, R. et al. Functional genomics and cell wall biosynthesis in loblolly pine. Plant Molecular Biology, v.47, n.1, p.275-291. 2001. WILSON, D. et al. SUPERFAMILY—sophisticated comparative genomics, data mining, visualization and phylogeny. Nucleic Acids Research, v.37, n.suppl 1, January 1, 2009, p.D380-D386. 2009. WU, J. et al. Whole genome wide expression profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing technology. BMC Plant Biology, v.10, p.234. 2010. YANG, J. et al. Novel gene expression profiles define the metabolic and physiological processes characteristic of wood and its extractive formation in a hardwood tree species, Robinia pseudoacacia. Plant Molecular Biology, v.52, n.5, 2003/07/01, p.935-956. 2003. ZHAO, Q.; DIXON, R. A. Transcriptional networks for lignin biosynthesis: more complex than we thought? Trends in Plant Science, v.16, n.4, p.227-233. 2011. ZHONG, R.; LEE, C.; YE, Z.-H. Evolutionary conservation of the transcriptional network regulating secondary cell wall biosynthesis. Trends in Plant Science, v.15, n.11, p.625-632. 2010.

98

ZHONG, R. et al. A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell, v.20, n.10, p.2763–2782. 2008.

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