Análise computacional de genes associados ao metabolismo de

UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
DEPARTAMENTO DE GENÉTICA
PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E
BIOLOGIA MOLECULAR
DISSERTAÇÃO DE MESTRADO
ANÁLISE COMPUTACIONAL DE GENES ASSOCIADOS AO
METABOLISMO DE FIXAÇÃO DE NITROGÊNIO NO
FEIJÃO-CAUPI (Vigna unguiculata) E CANA-DE-AÇÚCAR
(Saccharum spp.)
GABRIELA SOUTO VIEIRA DE MELLO
RECIFE
2009
1
UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
DEPARTAMENTO DE GENÉTICA
PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E BIOLOGIA MOLECULAR
DISSERTAÇÃO DE MESTRADO
ANÁLISE COMPUTACIONAL DE GENES ASSOCIADOS AO
METABOLISMO DE FIXAÇÃO DE NITROGÊNIO NO FEIJÃOCAUPI (Vigna unguiculata) E CANA-DE-AÇÚCAR (Saccharum spp.)
GABRIELA SOUTO VIEIRA DE MELLO
Dissertação
apresentada
ao
Programa de Pós-graduação em
Genética e Biologia Molecular
da Universidade Federal de
Pernambuco como requisito para
obtenção do grau de Mestre em
Genética pela UFPE
Orientadora: Profª. Drª. Ana Maria Benko-Iseppon
Co-orientador: Prof. Dr. Tercílio Calsa Júnior
RECIFE
2009
2
Mello, Gabriela Souto Vieira de
Análise computacional de genes associados ao metabolismo de
fixação de nitrogênio no feijão-caupi (Vigna unguiculata) e cana-de-açúcar
(Saccharum spp.) / Gabriela Souto Vieira de Mello. – Recife: O Autor, 2009.
160 folhas : il., fig., tab.
Dissertação
(mestrado)
–
Universidade
Pernambuco.CCB. Genética, 2009.
Federal
de
Inclui bibliografia e anexo.
1. Genética Molecular 2. Bioinformática 3. Feijão-Caupi 4.
Cana-de-açúcar I Título.
577.21
CDU (2.ed.)
UFPE
572.8
CDD (22.ed.)
CCB – 2009-142
3
4
“Mude suas opiniões, sustente seus princípios; troque suas folhas, mas
mantenha intacta suas raízes”
Victor Hugo
5
Agradecimentos
A Deus por tudo e por ter colocado tantas pessoas maravilhosas na minha vida.
À Minha mãe, Uitamira pelo suporte moral e financeiro, por estar presente em todos
os momentos da minha vida, pelo eterno incentivo e por manter nossa família unida que, junto
com meu irmão João, me deram forças para correr atrás de tudo que eu sempre quis.
Ao Meu pai, Ricardo José (in memorian), pelo exemplo de vida e força espiritual.
À minha avó Irene pelo maravilhoso acolhimento em sua casa.
Aos meus padrinhos Roberto Vieira de Mello e Maristela Ferraz por sempre
acreditarem em mim.
A todos os meus familiares, todos vocês, cada um de uma forma peculiar,
iluminaram meu caminho.
À minha irmã de coração Petra, que me ensinou tudo com uma enorme paciência, por
sempre estar ao meu lado nas piores horas, pelas risadas, pelos maravilhosos momentos de
descontração. Por TUDO, sem ela esse trabalho nunca teria sido feito.
Aos meus amigos Marcela Randau, Mirella Soares e Moacyr Barreto por terem
enchido minha pós-graduação de momentos felizes e descontraídos, os levarei para sempre na
minha memória.
Ao Túlio pelo apoio incondicional, por sempre me ajudar com uma palavra de
conforto e uma idéia, por tanto me fazer rir e principalmente pela eterna paciência.
À minha orientadora Profa. Ana Maria Benko-Iseppon pela oportunidade e por me
ensinar a acreditar mais em mim.
Ao meu co-orientador Tercílio Calsa Júnior pelos ensinamentos.
A todos os meus amigos e membros do Laboratório de Genética e Biotecnologia
Vegetal que tornaram o andamento desse mais agradável.
A Carol e Luís pelos ensinamentos e principalmente a Nina pelas incontáveis ajudas e
por sempre me apoiar.
Aos professores do Programa de Pós-Graduação em Genética.
À CAPES pela bolsa concedida durante o desenvolvimento do projeto.
6
SUMÁRIO
Item
Página
LISTA DE ABREVIATURAS
.........
VIII
LISTA DE FIGURAS
.........
XI
LISTA DE TABELAS
.........
XII
RESUMO
.........
XVI
ABSTRACT
.........
XIII
INTRODUÇÃO
.........
15
CAPÍTULO 1. Revisão da literatura
.........
17
1.1. Interações Planta-microoganismo
.........
18
1.2. Fixação Biológica de Nitrogênio (FBN)
.........
19
1.2.1. Importância Econômica e Ambiental
.........
19
1.2.2. Mecanismos da FBN em Angiospermas
.........
21
1.2.3. FBN em Vigna unguiculata
.........
23
1.2.4. FBN em Saccharum sp.
.........
23
1.2.5. Aspectos Genéticos da FBN
.........
24
1.2.6. Principais Nodulinas Primárias
.........
25
1.2.7. Principais Nodulinas Secundárias
.........
32
.........
38
1.3.1. Importância Econômica
.........
38
1.3.2. Origem e Distribuição Geográfica
.........
39
1.3.3. Melhoramento do Feijão-Caupi
.........
40
1.3.4. Aspectos Botânicos e Genéticos
.........
41
1.3.5. Projetos HarvEST, NordEST e CGKB
.........
42
.........
44
1.4.1. Importância Econômica
.........
44
1.4.2. Origem e Distribuição Geográfica
.........
45
1.4.3. Melhoramento da Cana-de-Açúcar
.........
45
1.4.4. Aspectos Botânicos e Genéticos
.........
46
1.4.5. Projeto SUCEST
.........
47
1.5. Análise Bioinformática
.........
49
1.3. O Feijão-Caupi
1.4. A Cana-de-Açúcar
6
1.5.1. Retrospectiva e Aplicações Atuais
.........
49
1.5.2. Bancos de Dados, Ferramentas e Programas
.........
50
.........
52
CAPÍTULO 2 - Computational Analysis of Genes Associated with .........
Symbiotic Nitrogen Fixation in the Cowpea (Vigna unguiculata)
Transcriptome - Artigo a ser enviado para a revista Genetics and
Molecular Research.
CAPÍTULO 3 - Expression of Nodulins in Sugarcane Transcriptome .........
Revealed by Computational Analysis - Artigo a ser enviado para a revista
Genetics and Molecular Research.
70
2. Referências Bibliográficas
CONCLUSÕES GERAIS
ANEXO
.........
115
156
157
7
LISTA DE ABREVIATURAS
APC
Anaphase Promoter Complex (Complexo Promotor da Anáfase)
ATP
Adenosina Trifosfato
BLAST
Basic Local Alignment Search Tool (Ferramenta Básica de Alinhamento Local
de Seqüências)
CCaMK
Calcium/Calmodulin Dependent Protein Kinase-like (Quinase CálcioCalmodulina-Dependente
CCS52
Cell Cycle Switch Protein (Proteína Interruptora do Ciclo Celular)
CD
Conserved Domain (Domínio Conservado)
CDK
Cyclin-Dependent Kinase (Quinase Ciclina-dependente)
cDNA
Complementary
Complementar)
DDBJ
DNA Database of Japan (Banco de dados de DNA do Japão)
DEFH125
Deficient Homolog 125 (Homólogo Deficiente 125)
DMI
Does Not Make Infection (Não Realiza a Infecção)
DMT
Divalent Metal Transporter (Transportador de Metais Divalentes)
EMBL
European Molecular Biology Laboratory (Laboratório Europeu de Biologiao
Molecular)
EMBRAPA
Empresa Brasileira de Pesquisa Agropecuária
ENOD
Early Nodulin (Nodulina Primária)
EST
Expressed Sequence Tag (Etiqueta de Seqüência Expressa)
FAPESP
Fundação de Amparo à Pesquisa do Estado de São Paulo
FBN
Fixação Biológica do Nitrogênio
GenBank
Banco de Genes do NCBI
GOGAT
Glutamato Sintase
Desoxyribonucleic
Acid
(Ácido
Desoxirribonucléico
VIII
8
GS
Glutamina Sintase
IITA
International Institute of Tropical Agriculture (Instituto Internacional de
Agricultura Tropical)
KEGG
Kyoto Encyclopedia of Genes and Genomes (Enciclopédia de Genes e
Genomas de Kyoto)
LRR
Leucine Rich Repeats (Repetições Ricas em Leucina)
LysM
Lysin Motif (Motivo de Lisina)
MADS-box
Box of the Proteins MCM1 from Saccharomyces cerevisiae, AGAMOUS from
Arabidopsis thaliana, DEFICIENS from Antirrhinum majus and SRF from
Homo sapiens (Conjunto das Proteínas MCM1 de Saccharomyces cerevisiae,
AGAMOUS de Arabidopsis thaliana, DEFICIENS de Antirrhinum majus e
SRF de Homo sapiens).
MEGA
Molecular Evolutionary Genetics Analysis (Análises Genéticas e Evolução
Molecular)
MFS
Major Facilitator Superfamily (Superfamília de Facilitadores Principais)
MIP
Major Intrinsic Protein (Proteína Intrínseca Principal)
MS
Membrana do Simbiossomo
MtAnn
Annexin from Medicago truncatula (Anexina de Medicago truncatula)
N
Nitrogênio
NASA
National Aeronautics and Space Administration (Agência Espacial Norte
Americana)
NCBI
National Center for Biotechnology Information (Centro Norte Americano dee
Biotecnologia e Informação)
NFP
Nod Factor Perception (Percepção do Fator Nod)
NFR
Nod Factor Receptor (Receptor de Fator Nod)
NH4+
Íon Amônia
NIN
Nodule Inception Protein (Proteína do Início da Nodulação)
NJ
Neighbor Joining (Agrupamento por Vizinhança)
9
IX
NO3-
Íon Nitrato
NOD
Nodulina
NORDEST
Rede Nordeste de Biotecnologia
NORK
Nodulation Receptor Kinase (Receptor Quinase da Nodulação)
Nramp
Natural resistance-associated macrophage protein (Proteína do Macrófago
Associada à Resistência Natural)
NSP
Nodulation Signaling Pathway (Via de sinalização da Nodulação)
ONSA
Organization for Nucleotide Sequencing and Analysis (Organização para
Sequenciamento e Análise de Nucleotídeos)
ORF
Open Reading Frame (Quadro Aberto de Leitura)
PDB
Protein Data Base (Banco de Dados de Proteínas)
PIR
Protein Information Resources (Recursos de Informações Protéicas)
RNA
Ribonucleic acid (Ácido Ribonucléico)
SAGE
Serial Analysis of Gene Expression (Análise Serial da Expressão Gênica)
SUCEST
Sugarcane EST Project (Projeto EST da Cana-de-açúcar)
SYMRK
Symbiosis Receptor-Like Kinase (Receptor Quinase da Simbiose)
UPGMA
Unweighted Pair Group Method with Arithmetic Mean (Método não
Polarizado de Agrupamentos aos Pares com Médias Aritméticas)
X
10
LISTA DE FIGURAS
CAPÍTULO 1
Figura 1. Visão geral do ciclo do nitrogênio
20
Figura 2. Representação esquemática da via de transdução de sinal ativado pelos
fatores Nod, bem como os tipos e as principais proteínas encontradas nos
29
CAPÍTULO 2
Figura 1: Dendrograms generated after Maximum Parsimony analysis
showing relationships among conserved domains in early nodulins (A) ENOD8
and (B) Annexin sequences including Vigna unguiculata orthologs.
83
Figura 2. Dendrograms generated after Maximum Parsimony analysis
showing relationships considering conserved domains of late nodulins (A)
Sucrose synthase and (B) Glutamine synthase sequences with Vigna
unguiculata orthologs.
84
Figura 3. General distribution of transcripts found in the NordEST libraries.
(A) Prevalence of early nodulin genes. (B) Prevalence of late nodulin genes.
86
Figura 4. Comparative prevalence of early and late nodulins genes in the
cowpea NordEST libraries.
86
Figura 5. Expression pattern of cowpea transcripts to the here studied nodulins
genes. (A) Graphic representation of the early nodulins CCS52a, Annexin,
NSP1, DMI3, ENOD8 and NORK clusters. (B) Graphic representation of the
late nodulins NOD70, SS, NOD26, NOD35, GS and Lgb.
88
CAPÍTULO 3.
Figura 1. (A) Comparative prevalence of early and late nodulins genes in the
SUCEST libraries. (B) Prevalence of reads per nodulin category.
127
Figura 2. Prevalence of sugarcane nodulins in the SUCEST libraries. (A)
Occurrence of the early nodulins reads (B) Occurrence of the late nodulins
reads.
128
Figura 3. Differential display of standard sugarcane transcripts representing
selected nodulin genes. Graphic A represents the expression of early nodulins
and graphic B represents the late nodulins.
130
11
XI
LISTA DE TABELAS
CAPÍTULO 1
Tabela 1. Descrição sucinta das bibliotecas geradas no projeto NORDEST
43
Tabela 2. Descrição sucinta das bibliotecas geradas no projeto SUCEST
48
CAPÍTULO 2
Tabela 1. Type and features of nodulin genes used as query against the cowpea
databases.
112
Tabela 2. Main cowpea clusters significantly similar to known nodulins.
tBLASTn results including the best match of each nodulin type.
113
Tabela 3. Conserved domains description of the best hits in cowpea database for
each nodulin type.
114
CAPÍTULO 3.
Tabela 1. Description of the SUCEST libraries.
121
Tabela 2. Type and features of nodulins genes used as query against the
Sugarcane database.
154
Tabela 3. Main sugarcane clusters similar to nodulins genes. tBLASTn results
and sequence evaluation of sugarcane nodulins genes including the best match of
each gene.
155
12
XII
RESUMO
A fixação biológica de nitrogênio tem sido um dos principais focos de interesse no que se
refere à nutrição mineral vegetal, sendo explorada na agricultura como uma fonte
ecologicamente benigna de nitrogênio, além de reduzir o uso de fertilizantes químicos, que
aumentam o custo da produção e causam danos ao meio ambiente. Nesse contexto, destacase a relação simbiótica entre bactérias da família Rhizobiaceae e raízes de leguminosas que
permitem à planta, através dos nódulos radiculares, a absorção do nitrogênio fornecida pela
bactéria, enquanto esta faz uso dos fotossintatos e de um ambiente microaeróbico fornecido
pela planta. Esse trabalho teve como objetivo identificar, através de ferramentas
computacionais, seqüências dos genes que participam da fixação de nitrogênio (NORK,
DMI3, NIN, NSP1, Anexina, CCS52a, ENOD40, ENOD8, NOD26, DMT1, NOD70,
Glutamina sintase, Leghemoglobina, NOD35 e Sucrose sintase) nos transcriptomas de
Vigna unguiculata e de Saccharum officinarum. Foi possível a identificação de 263
ortólogos às nodulinas estudadas no transcriptoma do feijão-caupi, com destaque para as
Leghemoglobinas que corresponderam a 95% dos clusters identificados. Com relação aos
genes estudados na cana-de-açúcar, foram observados 195 clusters ortólogos, apresentando
em sua maioria uma alta similaridade com nodulinas de outras monocotiledôneas. De uma
forma geral, o estudo pôde constatar a presença das nodulinas em todos os tecidos
analisados, com diferentes níveis de expressão. A maioria dos transcritos se encontrava nas
bibliotecas de folhas infectadas e de raiz sob estresse salino no caso do caupi e em flores e
raízes no caso da cana. Quando analisadas através de alinhamentos múltiplos, as nodulinas
oriundas de diferentes organismos e aquelas encontradas no caupi apresentaram maior
semelhança entre espécies pertencentes à mesma classe. Com relação às Angiospermas em
geral, a família Fabaceae foi separada do restante, confirmando a função divergente e
especifica destes genes no grupo. Os resultados do presente estudo sugerem o
envolvimento das nodulinas em vias amplamente conservadas do desenvolvimento vegetal,
confirmando o papel multifuncional desses genes além da interação benéfica com
microorganismos. De uma forma geral, esse trabalho tem potencial para colaborar com o
desenvolvimento de marcadores moleculares para o melhoramento das espécies estudadas,
assim como para o entendimento da abundância, diversidade e evolução destes genes. O
estudo pode ainda fornecer meios de elucidar os mecanismos envolvendo esses genes em
outras vias, que não a de fixação, promovendo não só o controle adicional desses processos,
como também uma possível expansão dessa vantajosa relação para plantas nãoleguminosas de importância econômica.
Palavras-chave: Fixação
bioinformática; EST.
biológica
de
nitrogênio;
feijão-caupi;
cana-de-açúcar;
XIII
13
ABSTRACT
The biological nitrogen fixation has been one of the main targets in what refers to plants’
mineral nutrition, being explored in agriculture as an environmentally benign nitrogen
source besides the fact that it can reduces the use of chemical fertilizers, which increases
the cost of the production and causes damages to the environment. In this context, the
symbiotic relationship between bacteria of the Rhizobiaceae family and leguminous roots is
distinguished, enabling the absorption of nitrogen by the plant supplied by the bacteria in
root nodules, while this microorganism makes use of the fotoshyntates and the
microaerobic environment provided by the plant. This work aimed to identify, through
computational tools, gene sequences involved in the nitrogen fixation (including NORK,
DMI3, NIN, NSP1, Annexin, CCS52a, ENOD40, ENOD8, NOD26, DMT1, NOD70,
Glutamine synthase, Leghemoglobin, NOD35 and Sucrose synthase) in the transcriptomes
of Vigna unguiculata and Saccharum officinarum. It was possible to identify 263 orthologs
to the nodulins studied in the cowpea transcriptome, highlighting the Leghemoglobins that
corresponded to 95% of the clusters found. Regarding the genes studied in sugarcane, 195
ortholog clusters could be observed, often presenting high similarity with monocot
nodulins. In a general view, it was possible to find the presence of the nodulins in all
analyzed tissues, with different expression levels. Most of the transcripts were in the
libraries of infected leaves and roots under salt stress in cowpea and of flowers and roots in
the case of sugarcane. When analyzed through multiple alignments, the nodulins from
different organisms and those found in cowpea showed greater similarity among species
that belonged to the same class. Regarding the Angiosperms in general, the Fabaceae
family was separated from the others, confirming the divergence and specific function of
these genes within this group. The results of the present study suggest the involvement of
these nodulins in highly conserved pathways of the plant development, confirming the
multifunctional role of these genes besides the beneficial interaction with microorganisms.
In a general view, this work has the potential to collaborate with the development of
molecular markers for the improvement of the species studied, as well as for the
understanding of the abundance, diversity and evolution of these genes. The study may also
provide ways to elucidate the mechanisms involving these genes in other pathways, besides
nitrogen fixation, promoting not only the additional control of this process, but also a
possible expansion of this beneficial relationship to economically important nonleguminous plants.
Palavras-chave: Nitrogen biological fixation; cowpea; sugarcane; bioinformatic; EST.
14
XIV
INTRODUÇÃO
A fixação biológica de nitrogênio (FBN) é um dos principais processos no que
se refere à nutrição mineral das plantas, sendo por isso extensivamente explorada na
agricultura. Entretanto, essa importante fonte primária de nitrogênio tem perdido espaço
nas recentes décadas com o aumento do uso de fertilizantes químicos, tornando a
agricultura moderna extremamente dependente e dispendiosa a custos acima de US$ 300
milhões por ano, considerando todo o planeta. Além disso, a produção e aplicação desses
fertilizantes têm se tornado um grande problema devido aos métodos ineficientes
empregados na sua utilização, culminando muitas vezes em níveis inaceitáveis de poluição
em reservatórios de água e na eutrofização de lagos e rios, bem como no seu alto custo,
impossibilitando sua utilização por agricultores de baixa renda.
O atual e expansivo interesse no desenvolvimento sustentável e nas fontes de
energia renováveis tem atentado para o fato de que a FBN é ecologicamente correta,
podendo sua maior exploração reduzir o uso de combustíveis fósseis, auxiliando assim no
reflorestamento e na reutilização de terras degradadas, através do enriquecimento de
nitrogênio disponível no solo. Portanto, torna-se imprescindível conhecer os mecanismos
que envolvem esse processo em cultivares que apresentem não apenas uma alta capacidade
de FBN, como também uma melhor adaptação às condições ambientais adversas.
Dentre as plantas com FBN eficiente, o feijão-caupi (Vigna unguiculata (L.)
Walp.) destaca-se pelo seu alto valor protéico, tratando-se de uma cultura muito utilizada
tanto na alimentação humana, como na alimentação de animais de criação, devido à sua
rusticidade e destacável capacidade de adaptação em ambientes com estresse hídrico,
térmico e salino. Portanto em diversas condições ambientais, o feijão-caupi pode ser
utilizado como adubo verde, uma vez que apresenta uma eficiente fixação biológica de
nitrogênio em associação com bactérias dos gêneros Rhizobium e Bradyrhizobium.
Por outro lado, a cana-de-açúcar (Saccharum officinarum), por ser uma
monocotiledônea, não apresenta processos de FBN tão eficientes como o feijão-caupi, e
associa-se principalmente com bactérias endofíticas como Glucanobacter diazotrophicus e
Herbaspirillum seropedicae que fornecem para a cana hormônios vegetais e nitrogênio.
Trata-se certamente de uma das culturas economicamente mais importantes para o homem,
sendo cultivada em regiões tropicais e subtropicais em mais de 80 países. O Brasil é
15
responsável por aproximadamente 25% de toda a produção mundial, tendo o estado de
Pernambuco como um dos maiores produtores do país, onde ocupa 40% da economia local.
A importância da cana pode ser atribuída a sua múltipla utilização, podendo ser empregada
in natura, sob a forma de forragem, para alimentação animal ou como matéria-prima para
fabricação de vários produtos, destacando-se o açúcar e álcool.
Nesse contexto, torna-se evidente a necessidade de maiores informações e novos
conhecimentos sobre os mecanismos genéticos utilizados por essas plantas na fixação de
nitrogênio, uma vez que não há uma estimativa, em termos econômicos, da contribuição
destas na FBN.
O presente trabalho teve como objetivos principais identificar e caracterizar as
sequências dos principais genes responsáveis pela fixação de nitrogênio no feijão-caupi e
na cana-de-açúcar, analisando sua estrutura, seu perfil de expressão diferencial e
comparando-as com as demais sequências de bancos de acesso restrito, bem como àquelas
descritas na literatura e depositadas em bancos de dados públicos.
16
Capítulo 1
Revisão de Literatura
______________________________________________________________________
17
1. REVISÃO DA LITERATURA
1.1. Interações Planta-Microorganismo
Uma grande variedade de interações ocorre entre plantas e microorganismos,
como vírus, bactérias, fungos e nematóides, sendo algumas prejudiciais para as plantas,
resultando em bilhões de dólares perdidos por ano com os danos e os tratamentos com
fungicidas e pesticidas. Outras relações são simbiônticas e benéficas, por aumentarem a
absorção e utilização de nutrientes e/ou o crescimento vegetal. Essas relações ocorrem
devido às trocas dinâmicas de múltiplos sinais entre as plantas e os microorganismos, que
podem resultar na resistência ou susceptibilidade à infecção ou simbiose (Birch e Kamoun,
2000).
Uma das mais importantes interações vantajosas entre planta e microorganismo
é a realizada entre as raízes das leguminosas e bactérias rizobiais, com a formação do
nódulo radicular responsável pela fixação biológica de nitrogênio. Entretanto, a associação
com fungos micorrízicos também merece destaque, uma vez que estes são simbiontes
vegetais ancestrais (Simon et al., 1993) e colonizam cerca de 90% das plantas terrestres
(Zhu et al., 2005). Ademais, outras associações simbióticas são realizadas com os
microrganismos presentes na rizosfera, sendo estas de suma importância em processos
como a decomposição, mineralização, desnitrificação, armazenamento e mobilização de
nutrientes e solubilização de fosfato (Khan et al., 2007).
Os organismos que habitam o interior das plantas, em tecidos como folhas,
ramos e raízes, os quais não produzem estruturas externas visíveis, são denominados
endofíticos (Azevedo e Araújo, 2007); compreendendo principalmente fungos e bactérias,
que comparados aos microrganismos patogênicos, não causam prejuízos à planta
hospedeira (Neto et al., 2003). A presença destes microorganismos endofíticos já foi
constatada em inúmeras espécies vegetais de interesse econômico, como algodão (Misaghi
e Donndelinger, 1990), milho (Araújo et al., 2000), cana-de-açúcar (Rosenblueth et al.,
2004), soja (Kuklinsky-Sobral et al., 2004), arroz (Sandhiya et al., 2005) e cacau (Rubini et
al., 2005).
Vários efeitos positivos foram atribuídos à presença dos organismos
endofíticos em plantas hospedeiras, como a promoção do crescimento vegetal (Tsavkelova
18
et al., 2007), a fixação de nitrogênio, a supressão do desenvolvimento de nematóides (Sturz
e Kimpinski, 2004), a indução de resistência sistêmica (Madhaiyan et al., 2004) e a
proteção das plantas contra herbívoros (Schardl et al. 2004).
1.2. Fixação Biológica de Nitrogênio (FBN)
1.2.1. Importância Econômica e Ambiental
O processo pelo qual o nitrogênio circula através das plantas e do solo pela
ação de organismos vivos é conhecido como ciclo do nitrogênio (Figura 1), que é
considerado um dos ciclos mais importantes nos ecossistemas terrestres, uma vê que o
nitrogênio participa da composição de muitas moléculas, como ácidos nucléicos e proteínas
sendo considerado, com exceção da água, o nutriente mais limitante para o crescimento
vegetal. Apesar de ser requerido em quantidades significativas pelos seres vivos, este
elemento é encontrado na natureza sob uma forma quimicamente estável devido à presença
de uma tripla ligação N-N, o que limita sua utilização imediata, requerendo sua
transformação para uma forma combinada que facilite sua assimilação (Sprent e Sprent,
1990).
As plantas utilizam o nitrogênio sob a forma de íon nitrato (NO3-) ou íon
amônio (NH4+) para a formação dos aminoácidos; entretanto a absorção e disponibilidade
natural dessas formas se dão principalmente pela decomposição de plantas e animais, o que
impossibilita utilização das mesmas na agricultura intensiva. Com essa carência de
fertilização natural é necessária a adição de fertilizantes químicos nas plantações, gerando
dependência de fontes externas, aumento do custo de produção e podendo inclusive causar
danos ao meio ambiente. Nesse contexto, destaca-se a importância da fixação biológica de
nitrogênio, caracterizada pela relação simbiótica entre bactérias e micorrizas com as raízes
vegetais (Raven et al., 2001).
A FBN não é apenas reconhecida como uma estratégia vantajosa para os
legumes, ela também é uma importante alternativa para o enriquecimento do solo,
considerando-se a inclusão desses vegetais nas culturas de rotação como uma eficiente
metodologia para aumentar os estoques de nitrogênio total do solo, com consequente
19
melhoria do mesmo e da produtividade das culturas (Vezzani, 2001). Sua utilização se dá,
geralmente, no pré-cultivo, onde a plantação de leguminosas precede a cultura principal,
que se beneficia posteriormente com a mineralização do nitrogênio. Atualmente essa
prática tem sido muito utilizada por pequenos agricultores, que não podem custear a
fertilização artificial, ou ainda por sistemas de produção orgânicos, onde não é permitida a
adição de adubos químicos sintéticos (Calegari, 2000).
A FBN é tolerada pela necessidade do organismo em contraste aos fertilizantes
químicos que são geralmente aplicados em grandes doses, sofrendo 50% de lixiviação, o
que não apenas eleva os gastos, mas também culmina em sérios problemas de poluição,
particularmente nos reservatórios de água (Zahran, 1999). Assim, as consequências
econômicas aliadas às ambientais, tornam evidente a necessidade de investimentos em
pesquisas que objetivem compreender os mecanismos fisiológicos, bioquímicos e
moleculares da FBN, de modo que os conhecimentos obtidos possam beneficiar tanto o
setor agrícola quanto as estratégias de reflorestamento.
Fig 1. Visão geral do ciclo do nitrogênio
20
1.2.2. Mecanismos da FBN em Angiospermas
Na natureza, a FBN pode ser realizada por diferentes grupos de microorganismos
procarióticos, dentre os quais se destacam as bactérias do solo da família Rhizobiaceae,
pertencente aos gêneros Bradyrhizobium, Azorhizobium e Rhizobium, denominadas
genericamente de rizóbios. Os rizóbios caracterizam-se pela capacidade de interação
simbiótica com o sistema radicular de leguminosas, por meio da formação de estruturas
denominadas nódulos radiculares (Jordan, 1984).
Nos nódulos radiculares, esses microorganismos fornecem aos vegetais nitrogênio
através da ação da nitrogenase, sob a forma de NH4+. Esses íons são então convertidos nos
aminoácidos glutamina e glutamato pela ação das enzimas vegetais glutamina sintetase e a
glutamato sintase, respectivamente. A planta, por sua vez, fornece à bactéria produtos da
fotossíntese e um ambiente microaeróbico ideal para a nitrogenase, que é sensível ao
oxigênio (Spaink, 2000).
O processo de fixação que ocorre nos nódulos das raízes compõe a última etapa de
um processo de desenvolvimento, iniciando-se com o reconhecimento molecular nos pêlos
radiculares das plantas, permitindo apenas a entrada dos mesmos e impedindo a
colonização por microorganismos oportunistas (Parniske e Downie, 2003).
O primeiro passo na interação molecular entre a planta e a bactéria é a detecção pelo
rizóbio dos compostos fenólicos quimiotáticos, denominados flavonóides, secretados pelas
raízes das plantas (Oldroyd e Downie, 2004). Os sinais dos flavanóides são reconhecidos
pelos reguladores transcricionais Nod rizobiais (fatores Nod), proteínas que se ligam às
moléculas sinalizadoras da planta, ativando a expressão dos genes responsáveis pela
fixação de nitrogênio (Long, 1996).
Os fatores Nod induzem muitas respostas nas células radiculares durante o processo
de reconhecimento do rizóbio, ou seja, no estágio que antecede a simbiose, como mudanças
nos filamentos de actina próximos à extremidade do pêlo radicular, a despolarização da
membrana celular, o aumento do pH citoplasmático, a indução dos picos de cálcio, a
ativação da divisão das células corticais radiculares e a indução da expressão de genes
específicos nos tecidos epidermais e corticais (Long, 2001). Entretanto, como a variedade
química externa dos fatores Nod é característica de cada rizóbio, esse reconhecimento
21
ocorre de modo específico, uma vez que os diferentes fatores determinam a especificidade
entre a planta hospedeira e a bactéria simbiótica (Perret et al., 2000).
A partir do reconhecimento, o rizóbio penetra nas raízes através da formação de
uma estrutura tubular chamada canal ou via de infecção, a qual atravessa a epiderme e o
córtex da raiz formando o nódulo primário. A bactéria é então liberada através do canal de
infecção no citoplasma das células e, em paralelo, inicia-se uma divisão celular na região
cortical da raiz, levando à formação do nódulo maduro (Franssen et al., 1992). Neste, a
bactéria aumenta e diferencia-se na forma nitrogênio-fixante, conhecida como bacteróide.
Esses bacterióides são cercados por uma membrana vegetal especializada, que permite a
troca metabólica, formando o simbiossomo (Oldroyd e Downie, 2004).
Uma vez que o nódulo é induzido, a planta utiliza um sistema de controle
homeostático para regular o número de nódulos em formação. Em legumes, este controle é
alcançado por mecanismos regulatórios conhecidos como auto-regulação da nodulação
(Gresshoff, 1993), onde um sinal inicial dos nódulos é desenvolvido durante o tempo
necessário para estabelecer o ciclo de realimentação após o qual cada nódulo primordial é
iniciado, mas falha no desenvolvimento (Gresshoff, 2003).
A FBN ocorre em apenas 10 das cerca de 380 famílias de angiospermas, sendo
encontrada em mais de 90% das leguminosas pertencentes às subfamílias Mimosoideae e
Papilionoideae, bem como em 30% das Caesalpinioideae (Sprent e Sprent, 1990).
Entretanto, a simbiose radicular nitrogênio-fixante também pode ser observada em alguns
membros das famílias Betulaceae, Casuarinaceae, Coriariaceae, Datiscaceae, Elaeagnaceae,
Myricaceae, Rhamnaceae, Rosaceae e Ulmaceae (Mullin et al., 1990).
Além da simbiose rizobial, cerca de 90% das plantas terrestres realizam uma
associação endossimbiótica com fungos micorrízicos arbusculares, pertencentes à ordem
Glomales (Brundrett, 2002). Essa interação com micorrizas compartilha muitas
características com a simbiose rizobial durante a sinalização (Oldroyd et al., 2005). Essa
interação envolve a invasão das hifas fúngicas nas células corticais radiculares, criando os
arbúsculos, onde parecem acontecer trocas de nutrientes. Essa relação planta-fungo permite
uma melhor absorção do fosfato, nitrogênio e outros macro e micronutrientes presentes no
solo (Hodge et al., 2001; Rausch et al., 2001). Além disso, também cianobactérias
22
pertencentes ao gênero Nostoc realizam FBN com plantas do gênero Gunnera, entretanto
essa relação ocorre de forma extracelular (Parniske, 2000).
1.2.3. FBN em Vigna unguiculata
Na fixação de nitrogênio em V. unguiculata, como nas leguminosas, há o
reconhecimento dos rizóbios pelas células radiculares, com posterior formação dos nódulos
primários e maduros. O feijão-caupi realiza a FBN com bactérias dos gêneros Rhizobium e
Bradyrhizobium (Fernandes et al., 2003), apresentando associação com pelo menos seis
espécies: B. japonicum (Jordan, 1984), B. elkanii (Kuykendall et al., 1992), Sinorhizobium
fredii (Lajudie et al., 1994), S. xinjiangensis (Chen et al., 1988) e R. hainanense (Chen et al.,
1997) e R. tropici IIA (Zilli et al., 2006).
Com relação à eficiência de fixação de nitrogênio no feijão-caupi, estudos têm
mostrado principalmente as espécies B. japonicum e B. elkanii como as espécies que
apresentam maior competência em prover um suprimento adequado de nitrogênio para a
nutrição da maioria das leguminosas herbáceas, em regiões de clima tropical (Moreira e
Siqueira, 2002).
1.2.4. FBN em Saccharum sp.
A cana-de-açúcar, em comparação com as leguminosas, interage com bactérias
nitrogênio-fixantes de uma maneira muito singular. Algumas bactérias endofíticas já foram
isoladas
em
cana,
incluindo
Gluconacetobacter
diazotrophicus,
Herbaspirillum
seropedicae e H. rubrisubalbicans; foram observadas colônias nos espaços intercelulares e
nos tecidos vasculares da maioria dos órgãos da cana infectada, sem causar mudanças
anatômicas visíveis ou sintomas de doenças (Reinhold-Hurek e Hurek, 1998).
Apesar de escassas as informações sobre quais mecanismos estão envolvidos no
estabelecimento desse tipo particular de interação e quais moléculas mediam a sinalização
entre planta e bactéria, sabe-se que a planta pode controlar a colonização bacteriana pelo
envio de sinais moleculares apropriados e/ou fornecendo um microambiente favorável para
o estabelecimento das bactérias. Em contrapartida, tais bactérias propiciam um melhor
23
desenvolvimento dos vegetais possivelmente pelo suplemento de nitrogênio (Sevilla et al.,
2001).
Segundo Vargas et al. (2003), muitos dos genes, envolvidos na sinalização plantabactéria durante a associação e no metabolismo do nitrogênio, são provavelmente ativadas
pela bactéria endofítica na etapa inicial da colonização vegetal, o que torna possível à cana
assimilar e metabolizar o nitrogênio fixado pelas bactérias. Esses genes também parecem
atuar como ativadores dos nódulos, uma vez que apresentam homologia com algumas
nodulinas de leguminosas (Nogueira et al., 2001).
1.2.5. Aspectos Genéticos da FBN
Os primeiros estudos genéticos dos mecanismos que envolvem a FBN foram
iniciados na década de 1980, onde os primeiros genes de Rhizobium para fixação de
nitrogênio e para nodulação foram clonados (Spaink et al., 1998). Com a identificação da
grande maioria dos genes bacterianos necessários para a fixação simbiôntica de nitrogênio,
importantes progressos foram conseguidos no que tange à elucidação da contribuição da
planta para essa interação (Long, 2001).
A utilização de Medicago truncatula e Lotus japonicus como plantas-modelo para a
pesquisa com legumes tem acelerado muito o mapeamento genético e o isolamento dos
genes requeridos para a fixação de nitrogênio, especialmente os que estão envolvidos na
sinalização planta-rizóbio e no desenvolvimento do nódulo (Oldroyd e Downie, 2004;
Oldroyd et al., 2005).
A análise diferencial de bibliotecas de cDNA oriundas de raízes nodulares
encontrou genes nódulo-acentuadores, chamados nodulinas, que foram divididos em duas
classes principais: as nodulinas primárias denominadas ENOD (Early Nodulins), como
ENOD2, ENOD12, ENOD40 e nodulinas com funções tardias (late nodulins) associadas
com a fixação de nitrogênio, como leghemoglobulina, glutamina sintetase e NOD35
(Mylona et al., 1995).
Os genes das nodulinas primárias são geralmente expressos durantes os primeiros
estágios da nodulação e parecem estar envolvidos no processo de infecção e/ou na
organogênese nodular, enquanto as nodulinas tardias são principalmente expressas nas
estruturas dos nódulos maduros ao redor do local da fixação de nitrogênio, participando
24
nesse processo (Niebel et al., 1998). Atualmente, muitas nodulinas ainda necessitam de
maiores informações sobre sua estrutura gênica, expressão espacial e até mesmo da
estrutura de seu promotor. Além disso, nenhum estudo com mutantes conseguiu identificar
danos à simbiose, quando comparados com o tipo selvagem/controle (Gresshoff, 2003).
O processo evolutivo que permitiu que algumas plantas, leguminosas ou não,
realizassem a simbiose nodular ainda permanece desconhecido. Entretanto, o fato de
existirem homólogos aos genes ENOD em vegetais não-leguminosos sugere que o
estabelecimento da simbiose nodular envolveu tanto o aparato genético existente num
organismo ancestral, não relacionado à via simbiôntica, quanto à especialização de alguns
genes e/ou o surgimento de novos genes (Szczyglowski e Amyot, 2003). Corroborando
essa hipótese, foram identificados genes de nodulação expressos em tecidos vegetais não
simbiônticos, como por exemplo, o gene ENOD12 que é um componente da parede celular,
também encontrado em caules e flores (Scheres et al., 1990).
Genes MADS-box, envolvidos no mecanismo de crescimento da extremidade do
tubo polínico, estão agora emergindo como importantes reguladores de outros processos
vegetais, incluindo a simbiose nodular (Zucchero et al., 2001). Em adição, o gene nóduloespecífico da alfafa nmhC5, assim como o gene de expressão tardia do pólen DEFH125 e
ZmMADS2 de Antirrhinum majus e Zea mays, respectivamente, pertencem à mesma
categoria dos MADS-box, sendo portanto considerados funcionalmente similares (Theissen
et al., 2000). Dessa forma, o aparato molecular utilizado na formação nodular também
parece estar envolvido em outras funções, considerando-se que outros fatores ainda não
totalmente esclarecidos devem atuar no processo da simbiose (Hirsch et al., 2001).
1.2.6. Principais Nodulinas Primárias
Os genes vegetais que participam do processo de sinalização do rizóbio e de
formação do nódulo radicular durante a simbiose são coletivamente chamados nodulinas
primárias. Apesar do processo inicial da nodulação ainda não ser bem elucidado, muitos
genes já foram descritos como nodulinas primárias, sendo claramente divididos de acordo
com o processo em que participam, como no reconhecimento e transdução dos sinais dos
25
fatores Nod rizobiais, culminando na expressão gênica de outros genes requeridos na
nodulação e na formação do nódulo radicular primordial.
Inicialmente, ocorre a percepção dos sinais rizóbio-derivados pelos receptores de
membrana, principalmente pelas proteínas do tipo quinase LysM (Lysin Motif; Motivos de
Lisina), como NFR1 (Nod Factor Receptor; Receptor do Fator Nod), NFR5 e NFP (Nod
Factor Perception; Percepção do Fator Nod) (Limpens et al., 2003; Madsen et al., 2003;
Radutoiu et al., 2003), havendo o posterior reconhecimento por outros receptores quinase,
como NORK (Nodulation Receptor Kinase; Receptor quinase da Nodulação), SYMRK
(Symbiosis Receptor-like Kinase; Receptor quinase da Simbiose), DMI2 (Does not Make
Infection; Não Realiza a Infecção) e/ou SYM19 (Endre et al., 2002; Stracke et al., 2002;
Mitra et al., 2004; Capoen et al., 2005).
Em seguida, a mensagem é processada via canais iônicos, constituídos por
proteínas como DMI1 de M. truncatula, CASTOR e POLLUX de L. japonicus, ancoradas
nas membranas (Ané et al., 2004; Imaizumi-Anraku et al., 2005; Kanamori et al., 2006),
que ativam proteínas quinases cálcio-calmodulina-dependentes (DMI3 e SYM9) (Lévy et
al., 2004), as quais por sua vez ativam fatores de transcrição dos tipos GRAS (NSP1 e
NSP2; Nodulation-signaling pathway; Via de sinalização da nodulação) e NIN (Nodule
Inception; Início da nodulação), permitindo, desta forma, a expressão de outras nodulinas
(Schauser et al., 1999; Borisov et al., 2003; Kalo et al., 2005; Smit et al., 2005) (Figura 1).
O NORK e seus parálogos que possuem funções e sequências altamente similares,
são compostos por um domínio extracelular com uma sequência única de 400 aminoácidos
e três domínios com repetições ricas de leucina (LRR) que mediam as interações protéicas,
seguidos por um domínio transmembrana e um típico domínio de proteína kinase
serina/treonina intracelular (Shiu e Bleecker, 2001). Essa estrutura geral permite que o
NORK participe da percepção do sinal originado pelas proteínas LysM extracelulares e na
transdução deste através do domínio intracelular (Kistner e Parniske, 2002).
Algumas proteínas que possuem similaridade com o ectodomínio do NORK já
foram encontradas em Arabidopsis, monocotiledôneas e gimnospermas, sugerindo que essa
região deve ter um papel biológico além da nodulação. Entretanto, a função desse segmento
do domínio extracelular ainda permanece desconhecida (Endre et al., 2002). Além disso,
receptores quinases com domínios ricos em repetições de leucina têm sido identificados em
26
numerosas vias de sinalização em plantas, incluindo a percepção de sinais dos patógenos
(Dangl e Jones, 2001).
De uma forma geral, o sinal externo reconhecido pelo NORK ativa a via de ação
dos canais iônicos das membranas do núcleo e de organelas que vão permitir a entrada do
cálcio (Oldroyd e Downie, 2006). O aumento de íons cálcio vai ser reconhecido no núcleo
pelo gene DMI3 que codifica uma proteína quinase cálcio-calmodulina-dependente
(CCaMK), responsável pela transdução do sinal originado pelo aumento de Ca2+,
culminando em mudanças na expressão dos genes implicados na simbiose (Lévy et al.,
2004; Mitra et al., 2004).
A CCaMK já foi identificada em várias espécies vegetais, sendo considerada
multifuncional. Esta proteína é formada por cinco domínios: um serina treonina quinase,
CaM-ligante e três cálcio-ligante EF-hand (Patil et al., 1995) mais um domínio que
promove ligação ao cálcio, que pode ocorrer de duas formas: através da ligação direta com
os domínios EF-hand ou pela ligação indireta com a calmodulina formando um complexo
(Takezawa et al., 1996).
Essas duas vias de ligação com o cálcio devem permitir que esta proteína
decodifique a informação gerada pela variação de cálcio, como as proteínas quinases
calmodulina-dependentes dos sistemas animais, que apresentam uma indução da atividade
da quinase por etapas em resposta à oscilação de cálcio (De Koninck e Schulman, 1998;
Dal Santo et al., 1999).
Essas quinases cálcio-ativadas regulam a expressão dos genes requeridos para o
desenvolvimento do nódulo através da ativação dos fatores de transcrição NSP pertencentes
à família GRAS (Kalo et al., 2005). Entretanto, o modo como essa ativação ocorre não foi
esclarecido, uma das hipóteses sugeridas seria a través da fosforilação da CCaMK,
localizada no núcleo (Smit et al., 2005).
O gene NSP é essencial para as mudanças na expressão gênica induzidas pelos
fatores Nod, como a formação do canal de infecção e a divisão das células corticais
(Catoira et al., 2000; Oldroyd e Long, 2003; Mitra et al., 2004). Além disso, o gene NSP1
participa de outros processos além sinalização primária, sendo requerido possivelmente na
manutenção do desenvolvimento nodular e/ou da infecção (Smit et al., 2005).
27
As proteínas codificadas pelo NSP possuem um domínio GRAS, o qual contém
uma região N-terminal variável e uma C-terminal conservada, que possui cinco domínios
(Kalo et al., 2005; Smit et al., 2005). Esses domínios ocorrem somente em plantas,
apresentando homólogos em muitos vegetais superiores como arroz (Oryza sativa),
arabidopsis (Arabidopsis thaliana), tomate (Lycopersicon esculentum), petúnia (Petunia
hybrida) e lírio (Lilium longiflorum), participando de processos como transdução de sinal,
manutenção do meristema e desenvolvimento vegetal (Bolle, 2004).
Outro fator de transcrição ativado pelo DMI3 é codificado pelo gene NIN
(Schauser et al., 1999), que é responsável pela entrada da bactéria e pelas respostas aos
fatores Nod das células corticais (morfogênese do nódulo) e epidérmicas (infecção e
expressão gênica). Também tem sido proposto que esse gene participa dos sinais
nutricionais, hormonais ou outros endógenos e exógenos durante o processo de nodulação
(Marsh et al., 2007). Entretanto, a recente identificação de proteínas semelhantes à NIN em
arabidopsis e arroz sugere que essas proteínas atuem tanto durante a nodulação, quanto na
sinalização de outros processos (Schauser et al., 2005).
28
PsSYM19
MtDMI2
MsSYMRK
LjNORK
Fatores
Nod
LjPOLLUX
LjCASTOR
MtDMI1
Oscilações do cálcio
Cálcio
Canais iônicos
Oscilações do cálcio
Receptores
Quinase
CCaMK
Receptores LysM
NFR1
NFR5
NFP
NIN
GRAS
Fatores de
transcrição
DMI3
PsSYM9
RNA Pol
Nodulinas
Figura 2. Representação esquemática da via de transdução de sinal ativado pelos fatores Nod,
bem como os tipos e as principais proteínas encontradas nos legumes em cada etapa. Os círculos
azul e amarelo representam a região de citoplasma e o núcleo da célula vegetal respectivamente.
Abraviações: Lj, Lotus japonicus; Ms, Medicago sativa; Mt, Medicago truncatula; Ps, Pisum
sativum. (Desenvolvida pela autora, com base em Oldroyd et al., 2005 e Oldroyd e Downie,
2006).
Além das nodulinas primárias descritas acima, outras proteínas também participam
dos estágios inicias da simbiose rizobial, dentre elas destacam-se a anexina, CCS52A (Cell
cycle switch protein; Proteína interruptora do ciclo celular) e as nodulinas primárias
ENOD40 e ENOD8. A anexina participa da organização da membrana do simbiossomo
cálcio-dependente durante a colonização dos tecidos vegetais. Estudos da localização in situ
da atividade promotora do gene que codifica esta proteína mostraram uma indução no
nódulo primordial, confirmando sua função durante a iniciação ou no estabelecimento das
estruturas endossimbióticas da membrana do siombiossomo (Manthey et al., 2004).
Niebel et al. (1998) mostraram que a expressão do gene Anexina de M. truncatula
(MtAnn1) requer a ativação dos fatores Nod, sendo mais expresso na zona de pré-infecção
do que na zona contendo os canais de infecção, sugerindo que o mesmo está mais
29
implicado na preparação da infecção ou na organogênese do nódulo do que no processo de
infecção em si. Ademais, este estudo mostrou que o MtAnn1 está relacionado com as
mudanças que ocorrem no citoesqueleto celular durante a simbiose, permitindo a ativação
das células corticais e a deformação do pêlo radicular.
A família das anexinas, inclui proteínas identificadas em muitos organismos
eucarióticos, sendo constituída por proteínas cálcio-dependentes fosfolipídios-ligantes
(Raynal e Pollard, 1994; Kaetzel e Dedman, 1995; Moss, 1997). No entanto, em plantas
essas proteínas possuem diferentes características (Clark e Roux, 1995). No aipo, por
exemplo, a anexina foi identificada como uma proteína cálcio-dependente vacúoloassociada (Seals et al., 1994); no algodão ela está associada com a modulação da atividade
calose sintase localizada na membrana plasmática, enquanto no tomate e no milho elas
possuem atividade de ATP-ase (Adenosine triphosphate; Adenosina trifosfato); entretanto
apenas a anexina de tomate é capaz de interagir com a actina do citoesqueleto (McClung et
al., 1994; Calvert et al., 1996).
Essas proteínas geralmente possuem uma região variável N-terminal curta e uma
região central conservada, composta de quatro repetições com cerca de 70 aminoácidos; a
única exceção é a classe VI de animais que contêm oito repetições (Morgan e Fernandez,
1997).
A diferenciação do nódulo primordial começa pela interrupção da divisão celular e
subsequente início de vários endociclos, onde ocorre duplicação do material cromatínico
sem haver divisão celular, culminando no aumento gradual do volume celular, que é
essencial para a multiplicação da bactéria e estabelecimento dos bacterióides (Favery et al.,
2002). Além disso, a amplificação do tamanho do genoma pelos endociclos assegura uma
maior quantidade de genes envolvidos nos processos simbióticos (Foucher e Kondorosi,
2000).
A endoreduplicação é uma estratégia comum no desenvolvimento de órgãos e
tecidos vegetais (Kondorosi et al. 2000; Larkins et al., 2001) e caracteriza-se pela repetição
da fase S do ciclo celular. Uma das maneiras de induzir o fenômeno de poliploidia é
inativado os complexos ciclina/CDK (Cyclin-dependent kinase; Quinase ciclina dependente)
antes do ponto de transição para a fase M (Fang et al., 1998). Com relação à mitose, sua
inibição pode ser conseguida pela ativação precoce do complexo promotor da anáfase (APC;
30
Anaphase promoter complex), responsável pela proteólise ubiquitina-dependente das
ciclinas mitóticas (Tarayre et al., 2004).
Duas isoformas do gene que codificam ativadores APC, CCS52a e CCS52b, foram
identificadas em M. truncatula e A. thaliana (Cebolla et al., 1999; Tarayre et al., 2004).
Enquanto a CCS52a parece ser ortóloga às proteínas Cdhl de animais e de fungos, a
CCS52b é encontrada apenas em tecidos vegetais (Tarayre et al., 2004). Em M. truncatula,
a CCS52a é responsável pela degradação do ciclo mitótico e pela regulação da
endoreduplicação durante a diferenciação celular simbiótica nos estágios finais da
maturação do nódulo (Cebolla et al., 1999; Vinardell et al., 2003).
Assim como o gene CCS52a, o ENOD40 também participa da formação do nódulo
primordial, sendo induzido pelos fatores Nod. Este gene codifica um peptídeo de nove a
treze aminoácidos, que é caracterizada pela ausência de um longo quadro aberto de leitura
(ORF; Open read frame) (Kouchi et al., 1999, Vleghels et al., 2003); seus transcritos foram
detectados não apenas nos nódulos radiculares mas também em tecidos meristemáticos nãosimbióticos, como nas raízes laterais (Papadopoulou et al., 1996; Fang e Hirsch, 1998),
folhas jovens (Asad et al., 1994) e tecidos embrionários (Flemetakis et al., 2000).
A nodulina codificada por este gene associa-se, além da nodulação, a diferentes
processos, uma vez que são expressos em outros tecidos e possuem homólogos em plantas
não leguminosas (Kouchi et al., 1999; Cebolla et al., 1999; Foucher e Kondorosi 2000).
Entretanto o ENOD40, em contraste ao CCS52a, atua no transporte de componentes, como
carboidratos, para as células corticais permitindo a organização apropriada do nódulo
primordial (Charon et al., 1999; Kouchi et al., 1999).
Pesquisas realizadas com RNA de interferência demonstraram que o ENOD40,
apesar de ser requerido na ativação da divisão das células corticais que conduzem à
formação do nódulo primordial (Sousa et al., 2001), não participa do processo de infecção
do rizóbio (Kumagai et al., 2006). Além disso, o fato de sua expressão persistir no nódulo
maduro sugere um possível papel adicional na função nodular (Kouchi e Hata, 1993; Yang
et al., 1993).
Em adição, homólogos desse gene são bem conservados em plantas nãoleguminosas, tendo sido descritos em plantas como tabaco (Nicotiana tabacum; Matvienko
31
et al., 1996) e arroz (Kouchi et al., 1999), indicando uma atuação mais geral no reino
vegetal.
Outra nodulina primária que participa da organogênese do nódulo é a codificada
pelo ENOD8, que pertence a uma família gênica duplicada em tandem, na qual três genes já
foram identificados em M. truncatula (Dickstein et al. 2002). Esse gene codifica uma
proteína com atividade de acetiltranferase associada à membrana do simbiossomo nos
nódulos radiculares (Pringle e Dickstein, 2004; Catalano et al., 2004), que integra a família
GSDL de proteínas lipolíticas encontradas nas membranas de plantas e bactérias.
Muitos estudos têm sido realizados com o objetivo de compreender melhor os
processos envolvidos na sinalização dos fatores Nod e na formação dos nódulos radiculares
das plantas. Esses dados poderão contribuir para o aperfeiçoamento e aumento da eficiência
do processo de fixação biológica de nitrogênio das culturas de interesse agronômico. Além
disso, a identificação da expressão das nodulinas em tecidos e órgãos vegetais que não
realizam simbiose nitrogênio-fixante e a descoberta de diversas nodulinas primárias em
plantas não leguminosas, podem tornar viável a transferência de genes que participam do
processo de nodulação em outras plantas, aumentando, assim, a eficiência destas últimas na
absorção de nitrogênio.
1.2.7. Principais Nodulinas Tardias
A expressão das nodulinas tardias, que participam da troca metabólica entre a
planta e o microssimbionte coincide com o início da fixação de nitrogênio, que é acionada
pela nitrogenase expressa no rizóbio (Schröder et al., 1997). Essa classe de nodulina inclui
proteínas transportadoras de membrana (Kaiser et al., 2003; Jeong et al., 2004) e proteínas
associadas especificamente com a membrana do simbiossomo (MS) (Wienkoop e Saalbach,
2003; Catalano et al., 2004), permitindo o estabelecimento e a manutenção do processo
simbiótico através do fluxo de várias moléculas e íons requeridos pelo bacterióide ou
vegetal (Roberts e Tyerman, 2002).
A principal proteína da MS, integrante da família MIP (Major intrinsic protein;
Proteína intrínseca principal) de proteínas de canais, é codificada pelo gene NOD26 (Dean
et al., 1999). Os membros desta família são caracterizados pela presença de seis domínios
32
α-hélice transmembrana, que formam uma estrutura hélice-loop-hélice (Jung et al., 1994;
Agre et al., 1995).
A família MIP é particularmente diversa em plantas superiores e mais de 30 genes
podem ser encontrados em arabidopsis. Esses genes são divididos em quatro subfamílias:
proteínas intrínsecas tonoplásticas, proteínas intrínsecas da membrana plasmática, proteínas
intrínsecas semelhantes à nodulinas e pequenas proteínas básicas intrínsecas (Johanson et
al., 2001).
A aquaporina NOD26 é encontrada exclusivamente na membrana do simbiossomo,
representando aproximadamente 10% do total de proteínas presentes na mesma (Weaver e
Roberts, 1992). Essa aquaporina, além de ser altamente permeável à água, necessária à
manutenção do equilíbrio osmótico nos nódulos, permitindo também o fluxo de glicerol,
formamida, malato e outros eletrólitos que ajudam na osmoregulação (Rivers et al., 1997).
A NOD26 é fosforilada pela proteína quinase cálcio-dependente da família CDPK apenas
na serina localizada na posição 262 do domínio carboxi-terminal. Essa fosforilação regula a
taxa de transporte do malato através da membrana do simbiossomo (Weaver e Roberts,
1992).
Essa proteína é homóloga a várias proteínas intrínsecas do tipo-canal encontradas
em Escherichia coli (Sweet et al., 1990), fungos (Van Aelst et al.,1991), Drosophila (Rao
et al., 1990) e mamíferos (Kent e Shiels, 1990), sugerindo-se que a alta conservação entre
os aminoácidos nos diferentes organismos para esta característica surgiu a partir de um
ancestral comum (Baker e Saler, 1990).
Outro transportador localizado na MS é codificado pela nodulina tardia DMT1
(Divalent metal transporter; Transportador de metais divalentes), que funciona no
transporte de íons, como zinco, cobre, manganês e principalmente ferro, para o bacterióide.
Nos bacterióides, o ferro participa da formação de inúmeras proteínas envolvidas na
fixação de nitrogênio, incluindo a nitrogenase e os citocromos utilizados na cadeia
transportadora de elétron (Kaiser et al., 2003).
A proteína DMT1 pertencente à família de proteínas de membrana Nramp (Natural
resistance-associated macrophage protein; Proteína do macrófago associada à resistência
natural), sendo induzida nos nódulos no início da fixação de nitrogênio e tem sua expressão
aumentada no nódulo maduro, sugerindo que o ferro seja requerido por enzimas que atuam
33
durante o desenvolvimento e o funcionamento nodular (Kaiser et al., 2003). Transcritos do
DMT1 têm sido encontrados em diversos tipos celulares, sendo sua estrutura altamente
conservada, apresentando homólogos em outras plantas, insetos, microorganismos e
vertebrados (Mims e Prchal, 2005).
Na planta, as leghemoglobinas, uma abundante nodulina que funciona como
transportador de oxigênio para o simbiossomo, são compostas pelo grupo heme (que é rico
em ferro) e pela globina, que é sintetizada pela planta em resposta à infecção bacteriana
(Verma e Long, 1983; Appleby, 1984). A molécula de leghemoglobina é formada antes do
começo da fixação de nitrogênio e atua na eficiência deste processo, uma vez que fornece
um fluxo adequado de oxigênio para a respiração do rizóbio e correto funcionamento do
complexo da nitrogenase bacteriana (Appleby, 1984).
As leghemoglobinas são codificadas nas plantas por uma pequena família gênica
(Laursen et al.,1994), que já foram clonados em Parasponia andersonii (Ulmaceae) e em
plantas que não realizam a FBN, incluindo monocotiledôneas. Por este motivo, Hardison
(1996) sugere que o grupo prostético heme, carreador de oxigênio, exclusivo dos nódulos,
seja um produto especializado oriundo da divergência de uma hemoglobina ancestral
presente antes da separação dos principais reinos.
Embora sejam requeridas para o correto funcionamento dos nódulos radiculares, as
leghemoglobinas não são necessárias para o crescimento e desenvolvimento vegetal na
presença de uma fonte externa de nitrogênio, sugerindo-se que sua expressão ocorra
exclusivamente durante a FBN (Ott et al., 2005). Além disso, a leghemoglobina também
participa nos nódulos radiculares da regulação de outra nodulina tardia, a sucrose sintase.
A forma ativa da sucrose sintase é um tetrâmero composto por monômeros
idênticos, sendo encontrada em abundância nos nódulos onde catalisa a reação reversível da
clivagem da sucrose. Além disso, a sucrose sintase é considerada como o principal
transportador de carboidrato das folhas para os nódulos (Reibach e Streeter, 1983).
A atividade da sucrose sintase no nódulo é modulada por grupos heme livres que se
ligam aos seus monômeros, considerando-se que a concentração desses grupos heme
dependa da ação das leghemoglobinas. Durante a senescência do nódulo, o grupo heme
deve ser liberado das leghemoglobinas, permitindo a inativação da sucrose sintase,
enquanto nos nódulos maduros, a alta concentração desses grupos heme não inibe esta
34
enzima, uma vez que estão ligados à leghemoglobina que possui maior afinidade com estes
(Colebatch et al., 2004).
A sucrose é um importante metabólito para o crescimento e desenvolvimento
vegetal, desempenhando importante função em vários processos fisiológicos, como o
transporte do carbono, a regulação do crescimento e desenvolvimento e transdução de sinal
(Smeekens, 2000). Na FBN é a fonte primária carbono para os tecidos radiculares e para o
bacteróide, fornecendo um esqueleto para o desenvolvimento das estruturas celulósicas
como os canais de infecção e provendo intermediários de carbono para a assimilação do
nitrogênio fixado (Colebatch et al., 2004).
A sucrose sintase é codificada por uma pequena família multigênica em várias
espécies, incluindo ervilha (Barratt et al., 2001), arabidopsis (Baud et al., 2004), batata
(Zrenner et al., 1995) e milho (Duncan et al., 2006). Estudos com plantas mutantes e
transgênicas que apresentaram atividade reduzida da sucrose sintase, demonstraram que
isoformas específicas são essenciais para o metabolismo normal dos diferentes órgãos
(Subbaiah e Sachs, 2001; Ruan et al., 2003).
Em L. japonicus (Horst et al., 2007), arabidopsis (Baud et al., 2004) e arroz
(Harada et al., 2005) a sucrose sintase é codificada por uma família de seis genes, havendo
muitas isoformas em leguminosas como M. truncatula (Hohnjec et al., 1999) e ervilha
(Barratt et al., 2001). A similaridade entre as isoformas da sucrose sintase pertencentes aos
diferentes grupos entre as espécies sugere que os genes que codificam essas isoformas
divergiram há um período relativamente longo, ao menos antes da separação entre mono e
dicotiledôneas (Horst et al., 2007).
O gene NOD70 possui 12 possíveis regiões transmembrana organizadas em dois
grupos que são separados por um grande loop hidrofílico. A proteína codificada por este
gene integra a MS, sendo responsável pelo transporte de nitrato, nitrito e cloreto (Pao et al.,
1998), apresentando-se similar às da superfamília MFS (Major Facilitator Superfamily;
Superfamília de facilitadores principais) de transportadores de membrana, que atuam no
fluxo de vários substratos como açúcar, drogas, íons orgânicos e inorgânicos,
intermediários do ciclo de Krebs, aminoácidos e peptídeos, podendo ser encontrados em
quase todos os organismos (Pao et al., 1998; Szczyglowski et al., 1998).
35
Em adição, a subfamília de proteínas com alto grau de similaridade ao GmNOD70
e LjNOD70 está presente numa grande variedade de espécies vegetais, sugerindo-se que os
vegetais possuam uma subfamília de transportadores ânion/nitrato relacionados com a
NOD70, os quais devem ter um papel mais amplo além da simbiose. Essa idéia é suportada
pela análise de bibliotecas de EST de soja, que revelou a presença de muitas sequências
similares ao GmN70 em outros órgãos além do nódulo (Vincill et al., 2005).
O NOD35 é um homotetrâmero que codifica uma oxidoradutase, a uricase nóduloespecífica (uricase II, EC.1.7.3.3) localizada nos peroxissomos das células não infectadas.
Essa enzima tem um papel essencial na biossíntese do ureído, principal produto que atua no
transporte do nitrogênio fixado dos nódulos para os galhos nas leguminosas tropicais
(Tajima e Kouchi, 1996).
Apesar de tratar-se de uma nodulina tardia, o NOD35 é expresso nas células não
infectadas e por essa razão apresenta mecanismos de regulação completamente diferentes
dos encontrados em outras nodulinas (Mauro e Verma, 1988). Tajima et al. (1991)
identificaram uma quantidade significativa de transcritos destes genes antes do início da
fixação de nitrogênio, que durante o processo aumentou gradativamente, sugerindo que a
indução deste gene seja controlada em duas etapas.
De uma forma geral, o N2 atmosférico é reduzido à amônia pela ação do complexo
enzimático da nitrogenase no rizóbio, que requer um ambiente com baixa concentração de
oxigênio, obtido pela presença de leghemoglobina e pela disponibilidade de um forte
redutor bioquímico, a ferredoxina, fornecido pelo hospedeiro; tais condições são
encontradas nos nódulos funcionais do sistema radicular de leguminosas. Posteriormente, a
amônia, produto final desse processo, é liberada no citoplasma e assimilada pelo ciclo
glutamina sintase/glutamato sintase (GS/GOGAT).
No entanto, quando existe disponibilidade de nitrato no meio ambiente a
leguminosa não estabelece a relação simbiótica. O nitrato absorvido será reduzido à amônia,
pelas enzimas nitrato redutase e nitrito redutase, que posteriormente será assimilada pelo
sistema GS/GOGAT. A partir destes primeiros compostos nitrogenados, glutamina e
glutamato, todos os demais compostos orgânicos nitrogenados são produzidos pela ação de
transaminases. Desta maneira, o sistema radicular é a principal fonte de nitrogênio para os
drenos, sítios de alta demanda de N2 (Camargos, 2002).
36
A amônia é assimilada pelas enzimas vegetais nodulares GS (E.C.6.3.1.2) e
GOGAT (EC 1.4.1.14). A eficiência da assimilação do nitrogênio fixado por essas enzimas
têm um importante papel na produtividade da planta, uma vez que essa via mantém a
amônia em baixas concentrações no citoplasma vegetal. A GS é localizada nos cloroplastos
e citoplasma de folhas e no citoplasma de células de raízes (Oaks e Hirel, 1985), sendo a
GOGAT localizada nos cloroplastos de folhas (Miflin e Lea, 1980) e em plastídeos nas
raízes (Emes e Fowler, 1979).
De acordo com Gonnet e Diaz (2000), a GS catalisa a aminação ATP-dependente
do glutamato, formando glutamina e a GOGAT, por sua vez, catalisa a transferência
redutiva do nitrogênio amida da glutamina para a posição a-ceto do 2-oxoglutarato,
resultando na formação de duas moléculas de glutamato que servem como substrato para a
biossíntese de vários metabólitos nitrogenados como os que são precursores de proteínas e
ácidos nucléicos (Schuller et al., 1986).
Uma simbiose eficaz requer a expressão coordenada dos genes vegetais e
bacterianos. A expressão dos genes GS e GOGAT nos nódulos é influenciada pelo estágio
de desenvolvimento do nódulo e pela presença da amônia produzida pela ação da
nitrogenase (Vance et al., 1988). Entretanto o modo como a amônia produzida pela
nitrogenase regula a GS ainda permanece desconhecido (Suganuma et al., 1999).
Tendo em vista que a síntese da glutamina ocorre via reação da GS e sendo ela o
substrato para a GOGAT, supõe-se que a GS desempenhe um papel central no metabolismo
do nitrogênio. As evidências sugerem que a GS poderia estar sujeita a diversos tipos de
controle, dentre os quais se incluem a repressão e a ativação em resposta a diferentes
aminoácidos e fitormônios (Chanda et al., 1998).
A variabilidade genotípica mensurada pela atividade da GOGAT em alfafa (Jessen
et al., 1988) e da GS em feijão-comum (Hungría et al., 1991), pode servir como possíveis
marcadores que forneçam subsídios aos programas de melhoramento. Além disso, a ação de
outras nodulinas tardias que aumentam a eficácia da FBN também tem sido estudada com o
intuito de melhorar a produtividade das leguminosas.
37
1.3. O Feijão-Caupi
1.3.1. Importância Econômica
As leguminosas compreendem a segunda maior família em impacto econômico
dentre as plantas cultivadas, constituindo aproximadamente 27% da produção mundial
agrícola (Graham e Vance, 2003). Em escala mundial, os legumes contribuem com cerca de
30% das proteínas consumidas por humanos e animais, servindo como fonte primária de
proteínas e vitaminas, sendo capazes de acumular metabólitos secundários, como
isoflavonóides, que são benéficos para a saúde humana. Além de sua importância como
fonte nutricional, estas plantas têm a capacidade única de realizar a fixação biológica de
nitrogênio em associação com rizóbios e micorrizas, excelentes fertilizantes naturais
(Dixon e Sumner, 2003).
Dentre as leguminosas, o feijão-caupi destaca-se por suas características de
rusticidade, versatilidade e adaptabilidade a condições de seca, solos ácidos e alcalinos.
Além disso, este grão é muito tolerante a baixa fertilização devido à sua alta taxa de fixação
de nitrogênio, pela simbiose com micorrizas e rizóbios. Assim, não só pela sua capacidade
de fertilizar naturalmente o solo e suportar condições ambientais desfavoráveis, mas
também por impedir a infecção e a reprodução de organismos oportunistas, o feijão-caupi
pode ser considerado uma cultura de excelência para a agricultura (Fery, 1990; Ehlers e
Hall, 1997).
Além dos benefícios que proporciona ao solo, o feijão-caupi é rico em proteínas
(23-25%), apresentando todos os aminoácidos essenciais, carboidratos (62%), minerais e
vitaminas, além de ter uma grande quantidade de fibras dietéticas e baixos teores de
gordura (com uma média de 2% de teor de óleo) (EMBRAPA, 2008). Além disso, este
feijão é a principal fonte de nutrientes da dieta da população de baixa renda, considerandose que seu cultivo constitua importante meio de sustento da maioria dos pequenos
produtores rurais do norte e nordeste brasileiros.
Praticamente todas as partes da planta são aproveitadas: as sementes, vagens e
folhas são consumidas frescas como vegetais verdes, os grãos podem ser consumidos após
cozimento e o restante da planta pode ser usado como alimento para animais domésticos.
Todas as partes da planta usadas na alimentação são nutritivas e com alto teor protéico,
38
tornando-a extremamente importante para populações de baixa renda, onde muitas pessoas
não têm acesso a outras fontes de proteínas (Magloire, 2005).
Atualmente seu cultivo concentra-se em regiões de clima quente, sendo três
quartos da produção encontrados na África (Shoshima et al., 2005). No Brasil trata-se do
único feijão capaz de sobreviver com sucesso na região norte (alta umidade, muita chuva e
solo argiloso) e no Nordeste (seca, solo arenoso, por vezes salino e muito sol) (Barreto,
1999), onde contribui com cerca de 41% do feijão consumido pela população. Em 2004 os
maiores produtores nacionais dessa cultura foram o Ceará e o Piauí, que produziram
aproximadamente 212.000 toneladas por ano (FNP, 2004).
O feijão-caupi, por sua importância econômica e social, é um organismo de
grande interesse científico. Devido ao seu atributo nutricional superior, versatilidade,
adaptabilidade e produtividade foi escolhido pela agência espacial norte-americana (NASA;
North-American Space Agency) como um dos poucos vegetais a serem pesquisados nas
estações espaciais (Ehlers e Hall, 1997).
1.3.2. Origem e Distribuição Geográfica
As espécies do gênero Vigna estão distribuídas nas regiões tropicais e
subtropicais de todo o mundo, entretanto há controversas sobre seu centro de origem e
diversidade. Pant et al. (1982) sugerem que o provável local de introdução desse legume se
deu na Índia, durante o período neolítico, tendo a Nigéria como centro primário de
diversidade das espécies selvagens (Steele e Mehra, 1980; Ng e Marechal, 1985).
No entanto, Freire-Filho (1988), levando em consideração a elevada taxa de
endemismo e a maior concentração de espécies do gênero Vigna na África, sugere que a
evolução e dispersão deste gênero tenham ocorrido a partir deste continente; entre as
espécies nativas da África, V. unguiculata predomina em algumas regiões e suas formas
selvagens não foram encontradas fora do continente africano. Ainda, Padulosi e Ng (1997)
apontam a região de Transvaal, na República da África do Sul, como a provável região de
especiação de V. unguiculata (L.) Walp.
Na América latina, o feijão-caupi foi provavelmente trazido da Europa e do Oeste
da África pelos colonizadores europeus e pelos escravos africanos durante os séculos 16 e
39
17 (Simon et al., 2007). O processo ocorreu primeiramente nas colônias espanholas e logo
após no Brasil, possivelmente no estado da Bahia e posteriormente para outras regiões do
nordeste brasileiro (Freire Filho et al., 1999).
Mesmo tendo sofrido diversos eventos de introgressão e possuindo uma ampla
variedade de fenótipos entre suas cultivares, o pool gênico do feijão-caupi parece ser bem
limitado, principalmente nas espécies cultivadas; esta leguminosa parece ter passado por
um efeito de gargalo durante sua domesticação. Por essa razão as conclusões sobre sua
origem e distribuição ainda não foram totalmente esclarecidas (Ehlers e Hall, 1997).
1.3.3. Melhoramento do Feijão-Caupi
O feijão-caupi possui características extremamente vantajosas para o seu
melhoramento como: autofecundação, genoma estável (evitando o “escape” de genes) e
ciclo de vida curto (cerca de dois meses) (Saccardo et al., 1992). Entretanto, durante muito
tempo seu melhoramento foi baseado apenas em métodos tradicionais de cruzamento, com
seleção de genótipos adaptados às condições específicas de cada região (Xavier et al.,
2005).
Durante o período de 1970 a 1988 as pesquisas que visavam seu melhoramento
concentraram-se no desenvolvimento de cultivares apenas para o campo. Em 1989,
diversificou-se, incluindo o melhoramento sistemático das cultivares locais e o
desenvolvimento de uma gama de cultivares com o intuito de obter maiores grãos e maior
eficiência na forragem para os sistemas de rotação de culturas (Fatokun et al., 2002).
Atualmente um dos principais objetivos dos programas de melhoramento é o
desenvolvimento de características agronômicas desejáveis, como tolerância aos estresses
abióticos (drogas, salinidade e calor), maior produtividade e resistência à patógenos (Timko
et al., 2007).
O Instituto Internacional da Agricultura Tropical (IITA), localizado na África, é
considerado o mais importante centro de pesquisa com feijão-caupi, entretanto avanços
significantes têm sido alcançados em diferentes regiões do mundo, como na Índia, Mali,
Nigéria, Senegal e, em menor escala, outros países. Recentemente, a Universidade da
40
Califórnia (USA) e a EMBRAPA (BR) também reforçaram e expandiram suas pesquisas
nessa área (Singh et al., 2002).
No Nordeste brasileiro, vários trabalhos têm visado à produtividade, resistência a
vírus, adaptabilidade e estabilidade de genótipos do feijão-caupi baseados em metodologias
que utilizam regressão linear (Finlay e Wilkinson, 1963; Eberhart e Russell, 1966). Esses
estudos têm subsidiado o lançamento de cultivares de feijão-caupi em vários estados
(Freire-Filho et al., 2001; 2002). Embora de suma importância, esses estudos não
priorizaram o alto potencial desse legume em fixar nitrogênio, uma característica capaz de
melhorar a produtividade e manter a fertilidade do solo sem a necessidade de fertilizantes
químicos.
Apesar da grande diversidade de fenótipos com um alto potencial genético e da
importância econômica e social nos países em desenvolvimento, de uma forma geral, o
feijão-caupi ainda permanece como uma cultura pouco explorada, sendo necessários mais
estudos no sentido de tornar seu cultivo mais rentável. Assim, quando comparado a outras
leguminosas como alfafa e soja, poucos esforços e investimentos são dispensados aos
estudos com o feijão-caupi (Singh, 2005).
Atualmente, as ferramentas biotecnológicas modernas podem propiciar ao
feijão-caupi não só condições de competitividade e características que atendam às
necessidades comerciais internacionais (Timko, 2002), como também maiores informações
sobre a estrutura e a composição do seu genoma e proteoma, o que auxiliaria na
interpretação da evolução do clado Phaseoloid/Millettoid e Papilionoideae em geral,
contribuindo substancialmente para o melhoramento dessa cultura (Simon et al., 2007;
Timko et al., 2008).
1.3.4. Aspectos Botânicos e Genéticos
O feijão-caupi é uma angiosperma de cultura autógama (Teófilo et al., 2001),
pertencente à classe Dicotyledoneae, ordem Fabales, família Fabaceae (Leguminosae),
subfamília Faboideae (Papilionoidea), tribo Phaseoleae, subtribo Phaseolinae, gênero Vigna
e espécie V. unguiculata (L.) Walp. (NCBI, 2008). Com relação à sua família, o feijão-
41
caupi apresenta um dos menores genomas, contendo aproximadamente 620 mega pares de
base (Paterson, 2006).
Essa leguminosa apresenta 2n=22 cromossomos, entretanto esse número pode
sofrer variações; em algumas cultivares cerca de 20% das células contêm 23 cromossomos
mitóticos (Benko-Iseppon, 2001; Adetula, 2006). Além disso, sua cariotipagem é
controversa, enquanto Barone e Saccardo (1990) observaram um grande cromossomo, um
muito pequeno e os outros nove distribuídos em três grupos de tamanhos intermediários,
Pignone et al. (1990) descreveu o cariótipo como composto por cinco cromossomos
grandes, cinco médios e um pequeno.
Pouca atenção tem sido dada à caracterização gênica no feijão-caupi (Timko et
al., 2007), observando-se que os maiores progressos na genômica de leguminosas têm sido
realizados com as espécies modelo M. truncatula, L. japonicus e Glycine max (Cronk, et al.,
2006; Sato et al., 2007). Esses organismos contêm não só suas sequências genômicas e
bibliotecas de EST (Expressed Sequence Tag; Etiqueta de sequência expressa)
disponibilizadas em bancos públicos, como também seus mapas físicos e genéticos (Sato et
al., 2007).
1.3.5 Projetos HarvEST, NordEST e CGKB
Atualmente o sequenciamento do genoma e do transcriptoma do feijão-caupi está
sendo desenvolvido por alguns grupos e disponibilizados em bancos de dados públicos,
como Cowpea Genespace/Genomics Knowledge Base (CGKB), responsável pela geração
de sequências através da filtração do DNA genômico metilado (Chen et al., 2007) e o
HarvEST, que gerou mais de 180.000 ESTs (HarvEST, 2008).
Numa iniciativa inovadora, grupos de pesquisa do nordeste brasileiro deram
inicio em 2004 ao projeto NordEST, integrante da rede Renorbio, que visa sequenciar o
primeiro genoma expresso de uma leguminosa (V. unguiculata) no Brasil. Além de ser uma
iniciativa inovadora, o diferencial deste projeto está na construção de bibliotecas
contrastantes para características de estresses bióticos e abióticos.
O sequenciamento do genoma do feijão-caupi é parte do projeto “Genômica
funcional, estrutural e comparativa de feijão-caupi (V. unguiculata)”, que visa obter cerca
42
de 100.000 sequências geradas a partir de diferentes bibliotecas contrastantes (Tabela 1),
com o intuito de identificar genes candidatos e novas sequências potencialmente úteis para
fins de melhoramento da cultura
Tabela 1. Descrição sucinta das bibliotecas geradas no projeto NordEST, incluindo o código da
biblioteca, o número total de ESTs sequenciadas e a descrição da situação na extração do cDNA
Código da Nº Total
biblioteca de ESTs
CT00
288
Controle
Raiz
BM90
1624
Genótipo BR14-Mulato
Folha
IM90
464
Genótipo IT85F coletado 90 minutos após infecção com vírus
Folha
Descrição
Tecido
do mosaico
SS00
1433
Genótipo sensível à salinidade sem estresse salino
Raiz
SS02
2204
Genótipo sensível à salinidade após 2 horas de estresse*
Raiz
SS08
3647
Genótipo sensível à salinidade após 8 horas de estresse*
Raiz
ST00
2500
Genótipo tolerante à salinidade sem estresse salino
Raiz
ST02
3646
Genótipo tolerante à salinidade após 2 horas de estresse*
Raiz
ST08
3142
Genótipo tolerante à salinidade após 2 horas de estresse*
Raiz
* Genótipos submetidos a estresse salino foram cultivados com 200mM NaCl
43
1.4. A Cana-de-Açúcar
1.4.1. Importância Econômica
A cana-de-açúcar é certamente uma das culturas economicamente mais
importantes, sendo cultivada em regiões tropicais e subtropicais em mais de 80 países
(Vettore et al., 2003). A importância da cana pode ser atribuída à sua múltipla utilização,
podendo ser empregada in natura, sob a forma de forragem, para alimentação animal ou
como matéria-prima para fabricação de rapadura, melaço, aguardente, açúcar e álcool
(Novaretti, 1981).
O bagaço é utilizado na fabricação de diversos tipos de papel, de fármacos e na
síntese de compostos orgânicos, com grande número de aplicações na indústria química e
farmacêutica (Pinazza e Alimandro, 2003). Ademais, da sua queima é possível gerar
energia térmica e elétrica (Portal Única, 2008). Do melaço, além do álcool, extraem-se
leveduras, mel, ácido cítrico, ácido lático e glutamato monossódico. Além disso, a partir do
etanol são fabricados polietileno, estireno, cetona, acetaldeído, poliestireno, ácido acético,
éter, acetona e uma gama de produtos químicos extraídos normalmente do petróleo
(Pinazza e Alimandro, 2003).
No Brasil, a cana-de-açúcar foi a primeira cultura introduzida no país, sendo
cultivada inicialmente no litoral nordestino apenas para a produção de açúcar (Szmrecsányi
e Moreira, 1991). A partir da década de 1970, a cana adquiriu maior status com o incentivo
do governo federal para que o setor sucroalcooleiro contribuísse para a solução da crise
energética emergente, frente à sua potencialidade como fonte geradora de energia renovável
(Barela, 2005).
Atualmente a cana é cultivada em quase todos os estados brasileiros. O
agronegócio sucroalcooleiro é responsável por 2,4% do PIB nacional, gerando 3,6 milhões
de empregos diretos e indiretos (Albino et al., 2006). Além disso, dados da EMBRAPA
(2008) estimaram que na safra 2007/2008 foram produzidos 290 milhões tonelada/ano, o
que reafirma o Brasil como o maior produtor mundial de açúcar e álcool (EMBRAPA,
2008).
A área mundial ocupada pelo cultivo da cana-de-açúcar corresponde a seis
milhões de hectares e especula-se que aumente para 9,1 milhões de hectares nos próximos
44
oito anos (Albino et al., 2006). O interesse pela cana tem aumentado bastante no âmbito
internacional, principalmente devido às recentes negociações do protocolo de Kyoto que
visam à redução do efeito estufa, o que dá à atividade canavieira do Brasil um destaque
ambiental altamente positivo, uma vez que o uso energético da cana-de-açúcar evita um
acréscimo anual de mais de 20% do total de emissões de CO2 pela queima de combustíveis
fósseis no país (Macedo, 2001).
1.4.2. Origem, Distribuição Geográfica
O provável centro de origem da cana-de-açúcar é o norte da Índia e estima-se que
sua domesticação pelo homem tenha ocorrido por volta de 2500 A.C., iniciando-se em
Papua Nova Guiné (Brandes, 1956). Seu cultivo se concentra em áreas tropicais e
subtropicais em mais de 80 países ao redor do mundo, estendendo-se em uma ampla faixa
de latitudes desde 35º N até 30º S, bem como em altitudes que variam desde o nível do mar
até mil metros (Magalhães, 1987; SUCEST, 2008)
A cultura da cana foi introduzida nas Américas em 1494 em São Domingos,
enquanto no Brasil, seu plantio teve inicio na Província de São Vicente em 1522 com
híbridos oriundos do cruzamento de S. officinarum e S. basberi, trazidos da Ilha da Madeira.
Dessa mesma ilha, em 1533, Duarte Coelho Pereira introduziu a cana-de-açúcar em
Pernambuco (Artschwager e Brandes, 1958; Bastos, 1987).
Posteriormente, as canas-nobres, termo criado por melhoristas holandeses para se
referir aos genótipos de S. officinarum com alto teor de açúcar, dominaram a economia do
país, sendo prioritariamente utilizadas pelas indústrias de açúcar não só no Brasil como
também no mundo (Dantas, 1960).
1.4.3. Melhoramento da Cana-de-Açúcar
O sucesso do cultivo da cana está atrelado principalmente aos programas de
melhoramento genético, os quais objetivam desenvolver variedades melhor adaptadas às
condições de solo e clima, minimizar os danos causados pelos ataques de pragas, aumentar
45
a resistência às doenças e melhorar as características industriais das variedades (Rosse et al.,
2002).
Em 1887, Soltweld realizou o primeiro cruzamento em cana-de-açúcar obtendo
sementes férteis, demonstrando a viabilidade do melhoramento da cana através de
cruzamentos controlados. Dois anos mais tarde, Harrison e Bowell obtiveram plântulas de
sementes originárias de cruzamentos. Surgiam assim os primeiros programas de
melhoramento genético (Cesnik, 2008).
As variedades atuais, classificadas como Saccharum spp., são híbridos oriundos
de cruzamentos e retrocruzamentos interespecíficos que apresentam um elevado nível de
ploidia e um complexo comportamento meiótico; estima-se que seu genoma apresente de
cinco a dez por cento do genoma das espécies parentais ancestrais (Lu et al., 1994).
No Brasil, os programas de melhoramento foram iniciados a partir do surgimento
de uma epidemia de gomose, doença causada pelo patógeno Xanthomonas axonopodis pv.
vasculorum, na principal variedade do país, resultando em enormes prejuízos (Matsuoka et
al., 1999). Em 1910 foram instaladas as duas primeiras estações experimentais de cana-deaçúcar do Brasil, uma no Rio de Janeiro e outra em Pernambuco. Esta última iniciou em
1913 pesquisas para a obtenção de variedades resistentes à broca Diatraea e ao piolho
Trionymus (Cesnik, 2008).
Segundo Barbosa et al. (2000) nas últimas três décadas houve marcante
contribuição do melhoramento genético no desenvolvimento do setor canavieiro do Brasil,
com ganhos acentuados de produtividade e qualidade. Nesse período, houve mais de 30%
de aumento na média de produtividade da cana-de-açúcar e da recuperação de quilogramas
de açúcar por tonelada de cana moída.
1.4.4. Aspectos Botânicos e Genéticos
A cana-de-açúcar é uma monocotiledônea pertencente à família Poaceae
(gramíneas), tribo Andropogoneae e ao gênero Saccharum que engloba cerca de 30
espécies (EMBRAPA, 2006; NCBI, 2008). É uma cultura semi perene e alógama, com
ciclo de cinco a sete anos, que requer um sistema radicular profundo para aumentar sua
produtividade em solos pouco férteis e com baixa retenção de umidade (Demattê, 2005).
46
Devido à sua origem multiespecífica, a cana-de-açúcar apresenta um dos genomas
mais complexos entre as plantas cultivadas (Ingelbrecht et al., 1999). Ainda, na sua maioria,
as variedades atuais são férteis e possuem número cromossômico variando entre 2n=70 e
2n=130. Essa variação não ocorre somente entre órgãos de uma mesma planta, mas também
entre células de um mesmo tecido (Roach e Daniels, 1987; Portieles et al., 2002).
Atualmente vários projetos de genômica da cana-de-açúcar estão sendo
desenvolvidos por diferentes grupos de pesquisa em todo o mundo. A Austrália e os EUA
além de desenvolverem projetos para o mapeamento e a aplicação de marcadores de DNA
têm sequenciado, juntamente com outros países como a África do Sul, a França e o Brasil,
mais de 300.000 ESTs (Carson e Botha, 2000; Casu et al., 2001; Grivet e Arruda, 2001;
Perrin e Wigge, 2002). As informações geradas por esses programas têm sido úteis no
mapeamento comparativo da família Poaceae, fazendo uso de marcadores comuns que
hibridizam em cana, arroz, milho, trigo e aveia, entre outras (SUCEST, 2008).
1.4.5. Projeto SUCEST
No Brasil, em 1999, a rede ONSA (Organization for Nucleotide Sequencing and
Analysis; Organização para Seqüenciamento e Análise de Nucleotídeos) deu inicio ao
projeto SUCEST (Sugarcane Expressed Sequence Tag Project; Projeto EST da Cana-deaçúcar) que tinha como principal objetivo identificar 50.000 genes através do
sequenciamento do genoma expresso da cana a partir de clones randômicos oriundos de 26
bibliotecas de cDNA extraídas de diversos órgãos e tecidos em diferentes estágios de
desenvolvimento (Tabela 2).
Atualmente este banco de dados disponibiliza um total de 291,689 ESTs,
agrupadas em 43,141 clusters, que podem ser utilizadas para a identificação da composição
gênica da cana e determinação da expressão diferencial em cada biblioteca (SUCEST,
2008).
47
Tabela 2. Descrição sucinta das bibliotecas geradas no projeto SUCEST, incluindo o código das bibliotecas, o
número total de ESTs, descrição dos tecidos e condições de extração dos cDNAs
(Fonte: Banco de Dados do SUCEST, http://sucest.lad.ic.unicamp.br/en/).
Código da
biblioteca
AD1
Nº Total de
ESTs
18137
AM1, AM2
28128
Infecção de tecidos de plantas cultivadas in vitro por Glauconacetobacter
diazotroficans
Meristema apical
CL6
11872
Calos tratados por 12h à 4ºC e 37ºC no escuro e no claro
FL1, FL2, FL3,
83899
Flor em diferentes estágios de desenvolvimento
HR1
12000
LB1, LB2
18047
Infecção de tecidos de plantas cultivadas in vitro por Herbaspirilum
diazotroficans
Ramo lateral de plantas adultas
LR1, LR2
18141
Primórdio foliar
LV1
6432
Crescimento foliar in vitro
NR1, NR2
768
Todas as bibliotecas
RT1, RT2, RT3
31487
Ápice radicular e 0,3 cm a partir do ápice radicular em plantas maduras
RZ1, RZ2, RZ3
24096
Tansição raiz-caule de plantas jovens
SB1
16318
Colmo
SD1, SD2
21406
Desenvolvimento de sementes
ST1, ST3
20762
Primeiro ou quarto internó do caule de plantas jovens
Descrição
FL4, FL5, FL8
48
1.5. Análise Bioinformática
1.5.1. Retrospectiva e Aplicações Atuais
A bioinformática, surgida no início dos anos 80, combina os conhecimentos da
matemática, estatística, ciência da computação, biologia e química, com o objetivo de
administrar e analisar grande quantidade de dados biológicos (Borém e Santos, 2001;
Carraro e Kitajima, 2002). Este ramo da ciência adquiriu maior visibilidade a partir da
produção massiva de sequências gênicas e protéicas oriundas do Projeto Genoma Humano.
A grande quantidade de dados gerados exigia recursos computacionais cada vez mais
eficientes para o armazenamento e análise destes dados. Assim, a bioinformática passou a
desempenhar papel essencial em outros projetos genoma (Prosdocini et al., 2002).
Atualmente através da bioinformática é possível manipular uma grande diversidade
de dados biológicos; os programas e algoritmos desenvolvidos são capazes de armazenar,
processar, analisar, decifrar estruturas, traçar relações entre moléculas e vias e interpretar
grande quantidade de informações (Borém e Santos, 2001).
No Brasil, o Laboratório de Bioinformática da Unicamp foi pioneiro no
desenvolvimento e aplicação de várias ferramentas computacionais à pesquisa genômica.
Em 2000, foi responsável pela montagem in silico do genoma da bactéria Xyllela fastidiosa,
o primeiro sequenciado no país (Simpson et al., 2000). Posteriormente, vários outros
centros de bioinformática surgiram no Brasil e diversas redes nacionais e regionais de
sequenciamento de genomas foram criadas, como o Laboratório Nacional de Computação
Científica em Petrópolis, onde funciona o Centro de Bioinformática do Projeto Genoma
Brasileiro.
Projetos envolvendo genomas expressos também se encontram em andamento no
país, como o Projeto Genoma Humano do Câncer da FAPESP (Fundação de Amparo à
Pesquisa do Estado de São Paulo) e o Projeto do Schistosoma mansoni, realizado pela Rede
Genoma de Minas Gerais (Santos e Ortega, 2003).
49
1.5.2. Bancos de Dados, Ferramentas e Programas
Com a geração massiva de sequências nucleotídicas e protéicas tornou-se necessária
a criação de bancos de dados capazes de armazenar essa grande quantidade de dados e que
permitissem o acesso dessas informações pelos diferentes grupos de pesquisas. Atualmente
diversos bancos de dados públicos e privados foram criados e, além do acesso aos dados
depositados, vários deles disponibilizam informações importantes sobre as sequências
armazenadas e ferramentas úteis para sua manipulação (Morais, 2003).
O GenBank (Banco de Genes), hospedado no NCBI (National Center for
Biotechnology Information; Centro Nacional para Informação Biotecnológica), é um banco
de dados americano que permite acesso irrestrito à sequências nucleotídicas e protéicas de
grande variedade de organismos. Atualmente, este banco encerra 96.400.790 sequências,
contabilizando 97.381.682.336 bases (NCBI, 2008). Além disso, o GenBank faz parte da
rede de Colaboração de Base de Dados de Sequências Nucleotídicas Internacional a qual
compreende os bancos de dados japonês (DDBJ, DNA Database of Japan; Banco de Dados
de DNA do Japão), europeu (EMBL, European Molecular Biology Laboratory;
Laboratório Europeu de Biologia Molecular) e americano (GenBank). A criação desta rede
permite a estes bancos a troca contínua de dados, de forma que os mesmos sejam
atualizados periodicamente (NCBI, 2008).
Além desses, destacam-se o PDB (Protein Data Bank; Banco de Dados de
Proteínas), o PIR (Protein Information Resource; Recursos de Informações Protéicas) e o
KEGG (Kyoto Encyclopedia os Genes and Genomes; Enciclopédia de Genes e Genomas de
Kyoto) que também mantêm um constante intercâmbio de dados (Tateno et al., 2002;
Prosdocini, et al., 2002).
Ademais, o NCBI também disponibiliza outras bases de dados, como o UniGene,
que agrupa todas as sequências oriundas de transcriptomas e o RefSeq, que reúne somente
as sequências mais representativas de um transcrito. Além dos bancos de dados, o NCBI
disponibiliza informações sobre taxonomia, genomas completos, mapas gênicos, estruturas
protéicas e o PubMed, uma ferramenta de busca bibliográfica (Benson et al., 2000).
Concomitantemente à criação dos bancos de dados, várias ferramentas e programas
foram desenvolvidos com o intuito de analisar as sequências continuamente geradas pelos
50
projetos de sequenciamento. Entre as principais ferramentas destacam-se o BLAST (Basic
Local Alignment Search Tool; Ferramenta de Busca por Alinhamento Local), utilizado na
busca de sequências através da similaridade de bases ou aminoácidos (Altschul et al.,
1990), bem como o ORF-finder, que pode traduzir sequências nucleotídicas em todos os
seis quadros abertos de leitura (NCBI, 2008).
Enquanto o BLAST está envolvido em análises locais de similaridade, o programa
CLUSTAL executa alinhamentos múltiplos, tanto a partir de sequências de nucleotídeos
quanto de aminoácidos, que levam em consideração análises globais de similaridade. Além
disso, o programa permite a construção de cladogramas e fenogramas, para inferência
filogenética e fenética, que podem ser visualizados, por exemplo, no programa TreeView
(Page, 1996; Thompson et al., 1997).
O MEGA (Molecular Evolutionary Genetics Analysis - Programa para Análise
Genética Moleculares Evolutivas) permite a análise de caracteres evolutivamente
informativos. Além disso, ele permite calcular matrizes de distância genética e analisar a
composição de sequências nucleotídicas e protéicas. O programa também disponibiliza
algoritmos como UPGMA (Unweighted Pair Group Method with Arithmetic Means;
Método não polarizado de Agrupamentos aos Pares com Médias Aritméticas) (Sneath e
Sokal, 1973), NJ (Neighbor-Joinning; Agrupamento por Vizinhança) (Saitou e Nei, 1987) e
máxima parcimônia (Eck e Dayhoff, 1966; Fitch, 1971), permitindo a realização de
inferências filogenéticas e fenéticas através da construção de dendrogramas (Sudhir et al.,
2004).
Outro programa bastante utilizado pelos bioinformatas é o CLUSTER, que permite
a análise de sequências genômicas e de dados gerados por experimentos de microarray,
SAGE (Serial analysis of Gene Expression; Análise Serial da Expressão Gênica), EST,
entre outros, incluindo ferramentas de auto-organização de mapas, agrupamento de médias
K (K-Means Clustering) e clusterização hierárquica, que permite o estudo do perfil de
expressão in silico dos genes (Eisen et al., 1998).
51
2. Referências Bibliográficas
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Capítulo 2
Artigo Científico
______________________________________________________________________
Analysis of Genes Associated with Symbiotic Nitrogen Fixation
in the Cowpea (Vigna unguiculata) Transcriptome
Artigo a ser submetido à revista Genetic and Molecular Research
70
Analysis of Genes Associated with Symbiotic Nitrogen Fixation in the Cowpea (Vigna
unguiculata) Transcriptome
Gabriela Souto Vieira-Mello; Petra Barros dos Santos; Nina da Mota Soares-Cavalcanti;
Ana Carolina Wanderley-Nogueira; Tercílio Calsa-Júnior; Ederson Akio Kido and Ana
Maria Benko-Iseppon
Universidade Federal de Pernambuco, Center for Biological Sciences (CCB),
Departamento de Genética, Laboratório de Genética e Biotecnologia Vegetal e
Laboratório de Genética Molecular, Recife, PE, Brazil.
Short running title: Nitrogen Fixation Genes in Cowpea Transcriptome.
Key words: data mining, early nodulins, late nodulins, expressed sequence tags, salinity
stress.
Corresponding Author:
Ana Maria Benko-Iseppon, UFPE, CCB, Departamento de Genética, Laboratório de
Genética e Biotecnologia Vegetal, Av. Prof. Moraes Rego, s/nº; 50732-970, Recife, PE,
Brazil. E-mail: [email protected]
71
ABSTRACT:
Legumes have a special ability to establish endosymbiosis with soil rhizobia, forming new
organs, called nodules, where nitrogen fixation occurs. These processes, including the
nodule development and establishment, are associated with the spatially and temporally
regulated expression of nodule-enhanced transcripts, the nodulins, classified in early and
late, according with their temporal expression and the role they play in nitrogen fixation.
This work aimed to identify candidate sequences to early (Annexin, DMI3, NSP1, NORK,
CCS52A, NIN, ENOD40 and ENOD8) and late (NOD26, NOD70, Glutamine synthase,
Leghemoglobin, NOD35, Sucrose synthase and DMT1) nodulins in the collection of
cowpea ESTs under diverse conditions available in NordEST and HarvEST databeses,
using bioinformatic tools. The 263 candidates sequences found (139 from early nodulins
and 124 for late nodulins) have shown similarity with the respective genes in other legumes.
The hierarchical clustering analysis revealed higher expression of early nodulins transcripts
in libraries extracted from leaves of IT85F genotype collected with 90 minutes after mosaic
viruses infection (IM90) and from root of genotype tolerant to salinity after 8 hours of
stress (ST08). In the case of the late nodulins, the libraries of salinity sensitive plants
submitted to salt stress (after eight and two hours, respectively SS08 and SS02) presented
the most representative expression. Multiple alignments showed relative conservation
regarding the nodulins in different organisms. The dendrogram revealed a consistent branch
including most dicot taxa separated from monocots. In the Annexin dendogram the legumes
were placed as outgroup, while in the ENOD8, Glutamine synthase and Sucrose synthase
dendrograms the Fabaceae family was separated from other dicots, suggesting that these
proteins presented divergent evolution during Magnoliophyta group evolutionary process.
The present work aimed to bring valuable information for future in vitro and in vivo assays,
as well as for development of molecular markers for genetic breeding and mapping
purposes of cowpea, allowing a better understanding of diversity and evolution of the genes
involved in nitrogen fixation.
72
INTRODUCTION
Biological nitrogen fixation reduces N2 to ammonium, being the largest source of
available nitrogen for life on earth (Newton, 2000). Much of this ammonium comes from
symbiotic nitrogen fixation (SNF) by rhizobia within legume root nodules. The
Leguminosae is one of the most successful families of land plants, mainly because of SNF,
which enables legumes to colonize soils that contain little or no available nitrogen. This
feature, together with the nutritious and protein-rich seeds that they produce, placed
legumes as an essential part of traditional and modern agriculture (Colebatch et al., 2004).
Leguminous plants are able to grow under nitrogen-limiting conditions because of
their ability to establish endosymbiosis with soil bacteria, collectively called rhizobia.
During this interaction new organs, called nodules, are formed in the root plant, allowing
the fixation of atmospheric nitrogen to supply the plant with ammonium. In return, the
microsymbionts obtain photosynthates and an environment with low concentration of
oxygen required by nitrogenase action (Spaink, 2000). These recognition events allow the
invasion of the host as well as the formation of a primary nodule; these two processes occur
in parallel and eventually merge when infection threads release bacteria into the cytoplasm
of the newly formed primordial cells (Parniske and Downie, 2003). In this process, the
bacteria become enclosed by a plant-derived membrane, the symbiosome membrane (SM),
and bacteroid differentiation precedes the metabolic phase of symbiosis (Van de Velde et
al., 2006).
Among cultivated legumes, cowpea (Vigna unguiculata (L.) Walp) arises as an
important crop for dryland areas, despite of its abilities to grow under adverse soil and
climatic conditions (Martins et al., 2003), playing an important role in cropping systems in
73
sub-Saharan Africa, Asia, Central and South America (Singh et al., 1997) especially
because cowpea nodules are very resistant to high temperatures (Simões-Araújo et al.,
2002). Despite of such qualities, little information is available about the genetic background
of this crop regarding nitrogen fixation under normal or stress conditions.
With the identification of the majority of the bacterial genes required in the SNF,
important progress has been made in the elucidation of the genetic mechanisms used by the
plants in this interaction (Long, 2001). In the past few years, different expression profiling
strategies were pursued to identify symbiotically induced genes co-activated during early
and late stages of nodulation (Küster et al., 2007).
Nodule development is associated with the spatially and temporally regulated
expression of a number of nodule-enhanced transcripts (referred to as nodulins) that aid in
the establishment of the symbiosis (Stougaard, 2000), including a number that encode
membrane transport proteins (Kaiser et al., 2003; Jeong et al., 2004) and proteins associated
with the symbiosome membrane (Catalano et al., 2004).
Plant nodulins were classified into two mainly classes according to its expression
moment and the role that they play in the SNF. The early nodulins are generally expressed
during the early stages of nodulation and seem to be involved in the infection processes
and/or nodule organogenesis, while the late nodulins are expressed in mature nodules,
acting in the nitrogen fixation itself (Niebel et al., 1998).
In general, strategies like EST sequencing, construction and analysis of cDNA
libraries, in silico profiling of symbiosis-related gene expression through mining
comprehensive EST collections, and experimental approaches have been carried out mainly
in two model legumes: M. truncatula (Barker et al., 1990) and Lotus japonicus (Handberg
and Stougaard, 1992). In these models and additionally in soybean (Glycine max; Lee et al.,
74
2004), such approaches have enabled comprehensive analysis of gene expression profiles
during the nodulation process (Colebatch et al., 2004). However, cowpea still lacks
comprehensive studies like those, with few nodulins evaluated up to date.
The present work aimed to evaluate nodulin genes transcriptionally activated in
the cowpea transcriptome under diverse experimental conditions, including in silico
expression profiling, gene structure and evolution, as compared with available information
from other plants deposited in public databases.
MATERIAL AND METHODS
Protein sequences derived from full length cDNA sequences from legumes were
used as seed sequences (Table 1), being obtained in FASTA format at NCBI database
(National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov), including
most representative genes from the nodulin family (Annexin, DMI3, NIN, NSP1, NORK,
CCS52A, ENOD8, ENOD40, NOD26, DMT1, NOD70, GS, Leghemoglobin, NOD35 and
Sucrose synthase).
For each seed sequence a tBLASTn (Altschul et al., 1997) was performed against the
cowpea
databases
(NordEST;
http://www.gentrop.ufpe.br/vigna
and
HarvEST;
http://www.harvest-web.org/, together including 202,066 ESTs), considering a cut off of 1e10
. The obtained clusters from both databases were assembled to avoid redundancies in
BLAST results. The contigs were built using the EGAssembler program available at
http://egassembler.hgc.jp/.
75
Reverse alignments of selected clusters were made at the NCBI database using the
BLASTx tool (http://www.ncbi.nlm.nih.gov/BLAST/) in order to confirm the similarity
between the cowpea sequences and the available sequences at GenBank. After that, the
clusters were translated using the ORFfinder program at NCBI homepage. The presence and
integrity of the conserved domains (CDs) was analyzed using RPS_BLAST tool (Altschul et
al., 1997).
For each gene, the cowpea sequences that presented CDs were selected together with
the sequences found using the BLINK tool (NCBI), aiming to generate multiple alignments
at CLUSTALx program. These analyses allowed a structural comparison of conserved and
divergent sites among cowpea sequences and other organisms. The BLINK tool permitted
the inclusion of sequences from different organisms additional to those obtained by
BLASTx, but only those with significant alignments and CDs with adequate structural
composition.
The phylogenetic analysis was performed using the MEGA (Molecular Evolutionary
Genetic Analysis) program, Version 4 for Windows (Kumar et al., 2004) using Maximum
parsimony method, with bootstrap of 2,000 replications and Pairwise deletion for the
treatment of GAPs during the alignments, generating a consensus tree with a cut-off of 50
(50% more parsimonious trees).
Only sequences from the NordEST database were used to perform an expression
profile of cowpea nodulins, since most libraries from HarvEST project (60%) were
constructed with a mixture of tissues from V. unguiculata, bringing less information about
spatial and temporal expression of nodulin transcripts. The Hierarchical Clustering Analysis
and Reordered Data Matrices, performed by CLUSTER and TREEVIEW (Eisen et al., 1998)
76
was carried out with normalized data and allowed the study of clusters expression patterns.
Dendrograms including both axes (using the weighted pair-group for each gene class and
library) were generated by the TreeView program (Eisen et al., 1998). On the graphics
(Figure 5), yellow represented no expression and red all degrees of expression. A
preliminary analysis of nodulin distribution patterns in cowpea libraries was verified by
direct correlation of the reads frequency of each cluster in various NordEST cDNA libraries
using normalized data.
RESULTS
1. Cowpea Orthologs
1.1 Early Nodulins
After trimming redundant clusters, the search for early nodulins at cowpea database
revealed 139 candidates, with e-values ranging from 1e-159 to 5e-10 (Tables 2 and 3). The
annexin results showed high similarity (5e-150 to 3e-31) with four clusters, of which one
presented the four annexin CDs complete, while in two these domains were incomplete,
whilst in one sequence no CD was found. All clusters found in tBLASTn presented best
matches with their respective protein after BLASTx analysis at the GenBank, showing
similarity with M. truncatula, except the third match, which presented similarity with
annexin protein from Fragaria x ananassa.
Regarding DMI3, the cowpea transcriptome presented eight candidates, of which
four presented the S_TKc CD complete, three presented two EFh CD complete and two one
EFh CD complete, however none of them showed the three domains together (S_TKc +
77
EFh + EFh). Furthermore, the reverse alignments revealed that most candidates had best
matches with Fabaceae family.
The tBLASTn results of nodulin NIN showed eight candidates, including three with
full PB1_NLP domain and one with partial domain; while in four clusters no domain could
be found. Two presented similarity with Oryza sativa, while the other six showed higher
similarity with legume species.
In the searches for NSP1 candidates it was possible to note the presence of seven
clusters; however, these candidates presented low degree of similarity (≤ 6e-22). The
clusters found had the full GRAS conserved domain in three, while in the last four this
domain was incomplete. On the other hand, these sequences matched with homologous
proteins from Phaseolus vulgaris, Castanea sativa and Solanum lycopersicum with high
similarity (e-values from 0.0 to 2e-63).
With respect to NORK gene, the cowpea transcriptome revealed the presence of 61
candidates, with e-values ranging from 1e-159 to 5e-24, of which 20 and 40 presented the
PKc_Tyr domain complete and incomplete, respectively, while in one no domain was
found. In general, the NORK orthologs had higher similarity with Dicotyledonous plants,
mainly of Fabaceae family; however, four sequences matched with the Monocotyledonous
O. sativa.
The CCS52A nodulin presented nine cowpea sequences with best e-value of 3e-114;
the full WD40 domain was presented in two clusters and partial domain in four clusters,
while in three no domain was obtained. The BLASTx results revealed higher identity of all
sequences with legume proteins, except one that aligned to an A. thaliana sequence.
The searches for ENOD8 candidates revealed 38 sequences (e-values 3e-113 to 1e-10),
in which the SGNH_plant_lipase_like domain seemed complete in 21 and incomplete in 16
78
clusters. Interestingly, BLASTx results showed similarity mainly with A. thaliana and O.
sativa, but no match was found with organisms from Fabaceae family.
In relation to ENOD40 candidates, three clusters with e-values between 2e-98 and 4e24
were observed. All candidates had the desired domain RRM complete and matched with
M. truncatula ENOD40 protein.
1.2 Late Nodulins
In a general view, the evaluation of late nodulins revealed the presence of 124
candidates (with e-values ranging from 0.0 to 8e-11), from which 55.6% showed the
searched domains Tables 2 and 3). All the eight candidates of DMT1 presented a partial
Nramp CD with e-values from 3e-131 to
5e-25
. In addition, 50% of the candidate sequences
showed similarity with soybean (Glycine max), while the others matched with A. thaliana
and S. lycopersicum.
The search for GS orthologous revealed seven candidates, from which three and
one presented both domains, Gln-synt_N and Gln-synt_C, complete and incomplete,
respectively, another one with only the Gln-synt_C incomplete CD and one with only Glnsynt_C incomplete CD and a single candidate without domain. After reverse alignments all
clusters showed similarity to their respective protein from legume, mainly P. vulgaris and
Vigna aconitifolia.
In relation to the Leghemoglobin gene, 49 clusters were found with e-values
ranging from 6e-47 to 4e-17. The full globin CD was found in 40 candidates being
incomplete in eight, while one presented no domain. After BLASTx, all clusters matched
with legumes, with V. unguiculata represented as the most similar organism.
79
Regarding the NOD26 analysis, tBLASTn pointed 25 candidates with e-values
between 2e-145 to 2e-10. From these candidates 15 presented the complete MIP domain, in
seven this CD was incomplete and in three no conserved domain was found. After reverse
alignments all sequences were similar to NOD26 protein from Fabaceae family.
The data mining results for NOD70 revealed 15 sequences with significant
homology (e-values 3e-91 to 9e-12), being four and six with the Nodulin-like domain
complete and incomplete, respectively, and five without domain. After BLASTx analysis it
was possible to observe the sequences presenting similarity with their respective protein,
being of the Brassicaceae and Rutaceae families the most common; with A. thaliana and
Poncirus trifoliata representing 73.3% of the obtained sequences, while just two were
similar to G. max.
The cowpea NOD35 candidates totalized only two sequences with high degree of
similarity. One presented the full Uricase domain, which was similar to the NOD35 protein
of G. max, while the other showed this domain incomplete and presented similarity to the
respective protein of P. vulgaris.
Finally, 14 candidates to SucSin gene could be identified, with e-values between 0.0
and 8e-11, from which 71.42% showed similarity with sequences from Fabaceae members.
The two sequences with complete Sucrose_synth domain were similar to the legumes Vigna
radiata and Pisum sativum, while 12 sequences showed similarity with other legumes, as
Vicia faba, G. max, and non-legumes plants, as Beta vulgaris, A. thaliana and Citrus unshiu,
being seven with the desired domain incomplete and five with no domain.
80
2. Dendrograms
The multiple alignments generated during this work used proteins of the early
nodulins: Annexin and ENOD8; and late nodulins: Glutamine synthase and Sucrose
synthase. In the results a high degree of conservation was perceived among the nodulin
sequences from diverse organisms.
In the resulted dendrograms it was possible to observe the grouping of different
organisms (fungi, protists, plants and animals) in separated clades according their kingdom
classification. All analyzed dendrograms placed sequences from monocots and dicots in
different clades, with a clear segregation between the Fabaceae family and plants from
other groups.
The generated dendrograms early nodulins are show in the figure 1. In the ENOD8
dendogram (Figure 1A) it was possible to distinguish two groups; the first one comprising
protozoans (I), as outgroup, and the other plants species (II). The group II showed two
subclades, grouping monocots (IIa) and dicots (IIb) in different branches. In the dicot group
it was possible to see a clear separation (dashed line) between legumes and non-legumes of
the Plantaginaceae and Apiaceae families (Figure 1A).
The annexins dendrogram placed Fungi as an outgroup (Branch I), and grouped
animals and plants in two clades (I and II respectively) according to their higher taxonomic
classification (plant, animal and fungi kingdoms). In group III the Fabaceae family (IIIb)
was separated from dicots (dashed line), which was placed together with the monocots Zea
mays and O. sativa, but in a separated subclades (dotted line) (Figure 1B).
Regarding dendrograms for late nodulins (Figure 2), both analyzed sequence groups
(sucrose synthase and glutamine synthase), showed organisms grouped in accordance to
their taxonomic classification. Thus, it was possible to note a clear separation at the
81
Magnoliopsida class with monocots and dicots placed in distinct branches, whereas within
this last class the Fabaceae family appeared separated from other dicots (dashed line).
The sucrose synthase dendrogram (Figure 2A) presented the dicot clade subdivided
into two subclades, one comprising the Asterid subclass (Cichorium intybus and members
of Solanacea family) and the other comprising the Rosid subclass (A. thaliana, Alnus
glutinosa, Citrus unshiu and members of the Fabaceae family).
82
A
B
III
Arabidopsis thaliana
II
I
Figure 1: Dendrograms generated after Maximum Parsimony analysis showing relationships among
conserved domains in early nodulins (A) ENOD8 and (B) Annexin sequences including Vigna unguiculata
orthologs. Numbers in the base of branches indicate bootstrap values.
83
A
B
Figure 2: Dendrograms generated after Maximum Parsimony analysis showing relationships considering
conserved domains of late nodulins (A) Sucrose synthase and (B) Glutamine synthase sequences with
3. unguiculata
Distributionorthologs.
of ESTs in
the NordEST
Libraries
Numbers
in the base
of clades indicate bootstrap values.
Vigna
84
The distribution of the 581 reads in the nine libraries was analyzed, allowing the
identification of 73 early and 508 late nodulins. Moreover, all libraries of the NordEST
database presented at least one transcript of each nodulin class, with exception of the IM90
library (Leaves of IT85F genotype collected with 90 minutes after mosaic viruses
inoculation), where no transcripts from the late nodulins could be detected.
After direct counting of the early nodulin transcripts, a higher prevalence of
transcripts could be observed in ST08 library (roots of tolerant plants to salinity after 8
hours salt stress), followed by SS08 library (roots of salinity sensitive plants after 8 hours
of salt stress), that together represented 54% of the early nodulins, while the control library
(CT00, no stress) had the lower representation (1%) (Figure 3A).
Regarding the distribution of late nodulins, it was possible to note that ST02
library (roots of salinity tolerant plants after 2 hours of salt stress) showed the higher
abundance (38%) of transcripts, whilst the five libraries (ST00, roots of salinity tolerant
plants without salt stress; ST08; SS00, roots of salinity sensitive plants without salt stress;
BM90, leaves of BR14-Mulato genotype and CT00 negative control) presented few reads
totalizing together just 13% (Figure 3B).
In the comparison of the total number of reads found in the NordEST libraries
several differences regarding the two nodulin categories could be observed, especially in
respect to the SS02 (roots of sensitive plants to salinity after 2 hours of stress), SS08 and
ST02 libraries, which showed a difference of more than 85% of read content between both
nodulin types (Figure 4).
85
A
B
Figure 3: General distribution of transcripts found in the NordEST libraries. (A) Prevalence of early nodulin
genes. (B) Prevalence of late nodulin genes. Abbreviations for libraries: CT00 (Negative Control); BM90
(Leaves of BR14-Mulato genotype); IM90 (Leaves of IT85F genotype collected with 90 minutes after mosaic
viruses infection); SS00 (Root of salinity sensitive plant without salt stress); SS02 (Root of salinity sensitive
plant after 2 hours of stress); SS08 (Root of salinity sensitive plant after 8 hours of stress); ST00 (Root of
salinity tolerant plant without salt stress); ST02 (Root of salinity tolerant plant after 2 hours of stress); ST08
(Root of salinity tolerant plant after 8 hours of stress).
Figure 4: Comparative prevalence of early
and late nodulins genes in the cowpea
NordEST libraries. Numbers outside
columns refer to the total of reads found in
each library. Abbreviations for libraries:
CT00 (Negative Control); BM90 (Leaves of
BR14-Mulato genotype); IM90 (Leaves of
IT85F genotype collected with 90 minutes
after mosaic viruses infection); SS00 (Root
of salinity sensitive plant without salt
stress); SS02 (Root of salinity sensitive
plant after 2 hours of stress); SS08 (Root of
salinity sensitive plant after 8 hours of
stress); ST00 (Root of salinity tolerant plant
without salt stress); ST02 (Root of salinity
tolerant plant after 2 hours of stress); ST08
(Root of salinity tolerant plant after 8
hours of stress).
86
4. Expression Pattern
The hierarchical clustering analysis, made after data normalization, allowed an
evaluation of expression intensity and co-expression or co-regulation of different NordEST
libraries and protein families. In this analysis two early nodulins (NIN and ENOD40) and
one late nodulin (DMT1) were not evaluated since they were absent in the database.
Interestingly, considering the expression each nodulin gene separately none of them
presented transcripts in all libraries of Nordest project; as examples, in relation to all early
nodulins, just the annexin candidates presented transcripts in the control (CT00) library.
Regarding the late nodulins studied, they were completely absent of the IM90 library
(leaves of IT85F genotype collected with 90 minutes after mosaic viruses infection).
In relation to the early nodulins, the higher expression was detected in IM90 and
ST08 libraries, followed by BM90 and SS08 libraries, while the ST00 library showed the
lower expression, presenting transcripts only for the NORK and ENOD8 genes, with 12
and four reads, respectively. Furthermore, in the grey dendogram showing a spatial coexpression among libraries, it was possible to observe a stronger relation among
ST08/BM90, SS08/SS02 and ST00/IM90 libraries. Regarding the co-expression of early
nodulins (pink dendogram), the analysis revealed the clustering of ENOD8/NORK + DMI3
genes (Figure 5A).
For late nodulins an almost complete absence of expression was observed in BM90
and SS00 libraries, with exception of NOD26, which presented 19 and 21 transcripts from
BM90 and SS00, respectively, and NOD70, with seven reads from SS00, while the
prevalence of expression was clear in SS08, SS02, ST02 and CT00 libraries. It is
interesting to note that the Leghemoglobin gene showed a higher expression in these
libraries and a co-expression with the group NOD35/GS.
87
The NOD26 gene presented reads distributed in all NordEST libraries, except in the
IM90 library; the higher transcription was observed at SS08 and SS02 libraries. Also, this
gene presented co-expression with the sucrose synthase gene (Figure 5B).
A
B
Figure 5: Expression pattern of cowpea transcripts to the here studied nodulins genes. (A) Graphic
representation of the early nodulins CCS52a, Annexin, NSP1, DMI3, ENOD8 and NORK clusters.
(B) Graphic representation of the late nodulins NOD70, SS, NOD26, NOD35, GS and Lgb. Darker
red quadrants indicate higher expression in the corresponding tissue/library, lighter red/orange lower
expression, and yellow represents no expression. Black dendrograms reflect the relationships among
libraries and pink dendrograms the relationship among nodulins. Abbreviations: GS, Glutamine
Synthase; SS, Sucrose synthase; Lgb, Leghemoglobin; CT00 (Negative Control); BM90 (Leaves of
BR14-Mulato genotype); IM90 (Leaves of IT85F genotype collected with 90 minutes after mosaic
viruses infection); SS00 (Root of salinity sensitive plant without salt stress); SS02 (Root of salinity
sensitive plant after 2 hours of stress); SS08 (Root of salinity sensitive plant after 8 hours of stress);
ST00 (Root of salinity tolerant plant without salt stress); ST02 (Root of salinity tolerant plant after 2
hours of stress); ST08 (Root of salinity tolerant plant after 8 hours of stress).
88
DISCUSSION
1. Cowpea orthologs
Several nodulins orthologs have been described in non-legumes, mainly arabidopsis
and rice, both presenting whole genome sequencing available (Miyao et al., 2007; Zhu et al.,
2006). In addition, a number of legume genes that are required for nodulation are also
essential for the symbiotic associations with arbuscular mycorrhizal (AM) fungi, which are
established in more than 80% of flowering plants. These two associations share several
common features, such as genetically controlled microbial infection by the host plant,
transcriptional activation of a common set of host genes and formation of an intracellular
plant-microbe interface where the nutrient exchange occurs (Oldroyd and Downie, 2004).
Furthermore, it has been hypothesized that the nitrogen-fixing root nodule
symbiosis evolved from part of the existing mechanisms for the AM symbiosis (the ancient
association), considering that the legumes have recruited preexisting genes to make a
functional nodule organogenesis (Heckmann et al., 2006; Smit et al., 2005) with the nonlegume orthologs of these common components maintaining equivalent biological
functions to their legume counterparts (Chen et al., 2007).
Thus, as expected, some here analyzed early nodulins presented similarity to nonlegumes sequences. In the results for the DMI3, NIN and NORK early nodulins it can be
observed that cowpea sequences matched with rice orthologs sequences that are required in
signaling during the AM symbiosis (Chen et al., 2007; Dangl and Jones, 2001; Godfroy et
al., 2006; Schauser et al., 2005). Notably, arabidopsis lacks the orthologs for some early
nodulins that play a significant role in the symbiosis signaling, like NORK and DMI3 (Zhu
et al., 2006). Such gene deletions in arabidopsis (and likely the lineage leading to the
89
Brassicaceae family) explain the inability of some Brassicaceae species to form symbiotic
associations with mycorrhizal fungi and with rhizobia (Stacey et al., 2006).
However, orthologs regarding other early nodulins have been described in the
arabidopsis genome, such as CCS52A and ENOD8, acting in this Brassicaceae respectively
as cell cycle controller and lipase (Brick et al., 1995; Cebolla et al., 1999; Tarayre et al.,
2004). The lack of cowpea clusters similar to ENOD8 in the literature, is probably due the
low amount of Fabaceae sequences of this nodulin deposited in NCBI. In addition, the
analysis of NOD70, DMT1 and Sucrose Synthase late nodulins also revealed similarities
between selected cowpea clusters and arabidopsis sequences. Transcripts of the DMT1
transmembrane protein have been found in different cell types, bearing highly conserved
structure and homology to other plants, including non-legumes (Mims and Prchal, 2005).
Regarding the NOD70 gene, the here generated cowpea sequences showed low
degree of similarity with legumes, probably due to the low amount of sequences deposited
in the GenBank. This is in accordance to Vincill et al. (2005) that evaluated a subfamily of
membrane transport proteins with a higher degree of similarity to GmN70 in a variety of
plant species; similarly, in our results, the NOD70 nodulin matched with non-legumes,
mainly with arabidopsis nodulins.
The sucrose is an important metabolite for the plant growth and development, acting
in several physiological processes beyond the supply of carbon to the bacteroids, like
growth regulation, signal transduction and genetic expression. Considering these functions,
sucrose synthase was found in a variety of plant species (Smeekens, 2000; Sturm et al.
1999). This enzyme is encoded by a small multigenic family present in several plants, such
as potato (Zrenner et al., 1995), corn (Duncan et al., 2006), arabidopsis (Baud et al., 2004)
90
and rice (Harada et al., 2005), with several isoforms found in legumes like M. truncatula
(Hohnjec et al., 1999) and pea (Barratt et al., 2001).
Regarding the annexin early nodulin, the low number of clusters found in the
cowpea database, as expected, was consistent with the role of this gene, that is implicated in
the preparation for infection or nodule organogenesis, rather than in the infection process
itself in legumes (Niebel et al., 1998) The low amount of cowpea cluster candidates can be
explained by the fact that the cowpea sequencing projects used not only young but also
mature plants, which presents the nodules formed and, consequently, lower annexin activity.
Besides the role in the symbioses, annexins from non-legumes are associated with
different cellular processes. In arabidopsis it has been proposed that annexins are part of the
oxidative stress response, while in strawberry studies using annexin cDNA sequences
revealed that the expression during fruit maturation was enhanced (Wilkinson et al., 1995).
Niebel et al. (1998) showed that the M. truncatula sequence (used in this work as seed
sequence) is similar to strawberry annexin. In our work, the cowpea cluster aligned with the
strawberry and alfalfa sequences, probably sharing the same functions in the symbiosis
process.
The NSP1, consisting of the highly conserved GRAS domain, constitute a family of
plant-specific proteins that play roles in various developmental processes such as signal
transduction, meristem maintenance and development. The fact that putative orthologs exist
in a variety of plants, such as A. thaliana, Populus trichocarpa (Smit et al., 2005), potato
and lettuce, indicates a more ancient function for this gene than the symbiosis (Bolle, 2004).
Consistent with these roles, Heckmann et al. (2006) reported that the Nicotiana
benthamiana (Solanaceae family) NSP1-like gene can function in the Nod factor-signaling
pathway, however its ability to activate downstream gene expression is unlikely to be direct
91
in terms of transcriptional activation of nodulation-specific promoters. Instead, it seems
more probable that it acts to activate some aspect of the gene induction pathway that is
common to several genes.
This data indicated that the conserved domains necessary for perception and
activation in the NSP1 protein have been conserved among legumes and non-legumes
(Heckmann et al., 2006). In addition, the Castanea sativa SCARECROW-LIKE protein,
which is comprised by the GRAS domain, play a role during the earliest stages of
adventitious root formation (Sanchez et al., 2007). Together, these facts can explain the
similarity found between the cowpea clusters and non-leguminous plants.
The reverse alignments results for early nodulin ENOD40 and late nodulins
Glutamine synthase, NOD26, NOD35 and leghemoglobin revealed high similarity with
sequences from the Fabaceae family, as expected; the evolutionary proximity from these
organisms is a strong evidence that the cowpea genome presents an abundant and diverse
set of genes involved in nitrogen fixation.
In a general view, the analyses revealed that the cowpea nodulins share high
similarities with nodulins from other legume plants and showed that significant proportion
of nodule-specific functions are performed by recruiting genes common to non-legumes
plants (Fedorova et al., 2002).
2. Dendrograms
Significant proportion of nodule-specific functions are performed by recruiting
preexisting genes common to non-legumes plants (Heckmann et al., 2006), and it is now
known that many of the genes for nodulation have been acquired following duplication of
those with related functions. Therefore, it is not known whether all legumes present these
92
extra genes and, if so, why they are not expressed in non-nodulating forms (Sprent, 2007).
In addition, non-legume orthologs of these common components likely maintain equivalent
biological functions to their legume counterparts (Chen et al., 2007). Thus it is suggested
that in legumes some factor enabling to form nitrogen-fixing symbioses have arose,
generating this group with different properties from the others angiosperms.
The multiple alignments with cowpea and nodulin sequences from other species
showed high degree of conservation among sequences. As expected, the generated
dendrograms reflected the evolutionary history of plants. Most legumes stayed as separated
subclades within the dicots, confirming the hypothesis of Doyle and Luckow (2003) that
nodulation specific genes arose within the legume family already among the earliest
lineages.
The ENOD8, belonging to the GDSL family, is a hydrolytic protein with esterase
and lipase activities (Upton and Buckley, 1995) found in plant and bacteria; however this
protein is not completely conserved in all organisms (Pringle and Dickstein, 2004). In our
dendrogram (Figure 1A) a clear segregation of the bacteria (clade I) and plant (clade II)
groups in monophyletic groups was evident. Regarding the plant kingdom, exclusive
synapomorphies characterized the monocots (IIa) and the dicots (IIb) classes. Within dicots
the legumes (dashed line) appeared as a monophyletic group, as expected, since these
proteins are strongly involved in nitrogen fixation process, being associated with the
symbiosome membrane in root nodules (Catalano et al., 2004).
Numerous orthologous to ENOD8 have been found in plants that are not able to fix
nitrogen, being all probably induced by exogenous signals or are regulated in a tissue
specific manner. In Daucus carota cell cultures, a GDSL gene with 55% identity to ENOD8
encoded a secreted glycoprotein (van Engelen et al., 1995) that was induced by the plant
93
pathogen Sclerotinia sclerotiurum (Bertinetti and Ugalde, 1996). In addition, transcripts of
the ENOD-like genes were identified in roots, stems and flowers of flowering plants,
suggesting that these genes might have roles in the development of different organs
involved principally in the regulation of plant development, morphogenesis, secondary
metabolites synthesis and defense responses (Ling et al., 2006).
Annexins show different properties and diverse intracellular localizations including
association with plasma or organelle membranes, cytoplasm and nuclei, for example. They
play different roles in several organisms (Clark and Roux, 1995; Raynal and Pollard, 1994;
Moss, 1997) including plants (Clark and Roux, 1995). Members of the annexin family are
composed by a variable N-terminal region and a highly conserved C-terminal core, with
exception of the animal VI class, which contains eight repetitions of the annexin domain
(Morgan and Fernandez, 1997). However, plant annexins share common biological
activities and functions with their animal counterparts, such as the ability to bind to F-actin
(Hu et al., 2000) and to stimulate Ca2+-dependent exocytosis (Carroll et al., 1998) or to
function as GTPase (Shin and Brown, 1999). The expression of both animal and plant
annexins can be regulated during the cell cycle, suggesting that they have a potential role
during cell division (Hawkins et al., 2000). Moreover, in animals annexins have been
implicated in the transduction pathways of mitogenic signals, in membrane trafficking
processes such as exocytosis, in interactions with cytoskeletal elements, or in the formation
of voltage-dependent, ion-selective calcium channels (Raynal and Pollard, 1994). The here
obtained annexin dendrogram is in accordance to this divergent evolution, showing animal
annexins (branch II) as a monophyletic group and also as a sister-group of plants, probably
because these two kingdons share synarqueomorphic characters.
94
Previous studies of annexin evolution indicated a single clade (ANXC) composed
by fungal and mycetozoan annexins (Morgan and Fernandez., 1997). Braun et al. (1998)
viewed that the N. crassa annexin homologue is most closely related to the annexin
homologue of Dictyostelium discoideum, suggesting a phylogenetic link between cellular
slime molds and true fungi, what is also in accordance with our findings.
According to Moss (1997) and Morgan and Fernandez (1997), plant annexins make
up a monophyletic cluster whose members generally lack amino-terminal domains and
functional calcium-binding sites in their second and third repeats. As seen in the present
results, the non-legume families (Brassicaceae and Malvaceae) formed a paraphyletic
merophyletic group. In addition, we can see the presence of specific features in annexins
from monocots and dicots, which resulted in the separation of these classes in smaller
clades, as expected. In non legume plants annexins have also been reported to be associated
with different cellular processes. Annexins purified from plant species such as tomato,
maize, cotton and celery presented different characteristics (Clark and Roux, 1995); for
example, a cotton annexin was associated with the modulation of callose synthase activity
located in plasma membrane (Andrawis et al., 1993), while maize annexins showed
ATPase activity (McClung et al., 1994). Moreover, their role in the oxidative stress
response has been proposed since an A. thaliana annexin-encoding cDNA was able to
complement an Escherichia coli mutant unable to grow in the presence of high
concentrations of H2O2 (Gidrol et al., 1996). This function divergence could justify the
diverging positions among legume and non legume annexins, also here observed.
With respect to the legumes, in the phylogeny the cowpea sequence behaved like a
sister-group (IIIb), what was expected since the Fabaceae family (M. truncatula and V.
unguiculata) present annexins with distinct characteristics (almost all associated to nodule
95
formation) from other plants (Manthey et al., 2004). The function of alfalfa annexin, for
example, may be related to the changes occurring in the cellular cytoskeleton during the
nodulation process (Niebel et al., 1998).
The Sucrose Synthase dendrogram (Figure 2A) presented plants (II) and
cyanobacteria (I) as a monophyletic group, due to the difference in the functions of sucrose
in these two organisms. While in plants sucrose works as important metabolite for vegetal
grown and development and as a primary carbon source for the bacteroids (in the case of
legumes) (Smeekens, 2000), in cyanobacteria the sucrose is often synthesized in response
to salt or osmotic stress and is thought to help to maintain osmotic balance and to stabilize
protein and membrane structure and function (Hagemann and Marin, 1999). However,
Lunn (2002) suggested that sucrose is synthesized by the same route, via sucrose synthase,
in both organisms, and hypothesized that plants inherited the sucrose metabolism from a
unicellular organism, cyanobacterial endosymbiont, predicting a horizontal genetic transfer
and parallel evolution, which reflects in the suitability of sucrose for a transport function.
The sucrose synthase is encoded by a small multigene family represented by
different isoforms between the plant species; for example, in L. japonicus this gene is
encoded at least by six genes (Horst et al., 2007), the same number of genes reported for
arabidopsis (Baud et al., 2004) and rice (Harada et al., 2005; Huang et al., 1996). Three
sucrose synthase isoforms of M. truncatula are closely related to the three pea sucrose
synthase isoforms and in both organisms a similar expression pattern is observed (Barratt et
al., 2001; Hohnjec et al., 1999). In addition, a phylogenetic analysis of plant sucrose
synthase genes has been reported previously, and their data clearly show that plant sucrose
synthase genes can be classified into at least three major branches: one monocot group and
two dicots groups (Sturm et al., 1999).
96
In our dendrogram a clear segregation of monocot and dicot can be identified, all
sharing synarqueomorphic characteristics. Besides this, there is a clear separation between
legumes and non legumes into two subclades. The results reflected the evolution process in
which this protein has been through, suggesting that these isoforms diverged during a
relatively long period, at least before the divergence between mono and dicotyledonous
plant groups (Horst et al., 2007). In monocots some isoforms of sucrose synthase
constitutes a critical link in biosynthesis of developing endosperm (Komatsu et al., 2001),
besides the fact that the sucrose synthase gene is expressed in many tissues, including
seedling roots and shoots, endosperm and embryo (Chourey et al., 1998).
Regarding the legumes in the Magnoliopsid clade, Horst et al. (2007) described
different results about the relative contributions of sucrose synthase in carbon metabolism
in the nodule, suggesting that there may be species-specific differences in sucrose
metabolism in different legume nodules. This may explain the fact that legumes, as a
monophyletic group, showed distinct characteristics between temperate (V. faba, P. sativum
and M. truncatula) and tropical species (G. max, V. unguiculata and P. vulgaris); the
transported products in the nitrogen fixation process differs between these two organisms
groups. In temperate legumes, the principal transported product is Asn, with activity of Asn
synthetase is enhanced in nodules of these plants (Atkins et al., 1984). In legumes of
tropical origin, the major transported solutes are the ureides, allantoin and allantoic acid,
since in the nodules of these species the activity of the ‘de novo’ purine pathway and
enzymes of purine oxidation is exceptionally high. Among other, these characteristics are
described as differences between tropical and temperate legumes, which can justify the
separation of these species, also confirmed by the present results.
97
The multiple alignments with glutamine synthase proteins from different organisms
(Figure 2B) showed a high degree of conservation. In the generated dendrogram distinct
monophyletic groups in branches I, II, III and IV were evident, including Archeae, Metazoa,
Fungi and Plant, respectively. It is interesting to note that organisms from Archeae were
placed as outgroup, sharing a synarqueomorphic features with the eukaryotes. Regarding
Eukaryotes, similar patterns were found by Saccone et al. (1995).
Moreover, the obtained results were expected, since the GSI form of glutamine
synthase has been found only in prokaryotes, whereas the GSII form is found in all
eukaryotes and in bacteria belonging to Rhizobiaceae (Shatters et al., 1989), Frankiaceae
(Rochefort and Benson, 1990), and Streptomycetaceae (Kumada et al., 1990), suggesting
that these two gene forms share a very old common ancestor (Turner and Young et al.,
2000). In relation to the Eukaryotes, metazoa, fungi and plant were grouped together in the
same subclade, a result similar to the found by Saccone et al. (1995), which constructed a
molecular phylogeny based in GS enzymes.
In higher plants GS is an octameric enzyme that occurs in diverse isoenzymatic
forms with their subunits encoded by members of a small multigene family (Temple et al.,
1998). These GS isoforms are located in the cytosol and chloroplast, assimilating ammonia
produced by different physiological processes in distinct organs (Ortega et al., 1999). In
leaves, chloroplastic GS function to assimilate primary ammonia reduced from nitrate and
also to reassimilate ammonia released during photorespiration (Lam et al., 1996). In roots,
GS assimilates ammonia (or NO3-) derived directly from the soil or, in the case of legumes,
are fixed by bacteroids (Lea and Ireland, 1999) whilst in the cotyledons it reassimilates
ammonia released by the breakdown of nitrogenous reserves during germination (Swarup et
al., 1990). These multiple isoenzymes have been shown to be differentially expressed in
98
both developmental- and organ-specific manner (McGrath and Coruzzi, 1991; Peterman
and Goodman, 1991), explaining the plant branch (IV) in the generated dendogram, which
followed the traditional phylogeny, placing monocot and dicots in different subclades (IVa
and IVb).
The GS gene family has been particularly well characterized in leguminous plants
in which a crucial role is played by the cytosolic GS in the assimilation of ammonium
released by nitrogen-fixing bacteria within the infected cells of the nodule (Stanford et al.,
1993). Indeed, in several legume species the expression of one or more cytosolic GS genes
has been shown to be induced during nodule development (Cullimore and Bennett, 1992).
Moreover, the separation of tropical and temperate legume species in the dendogram was
expected since nodule GS regulation includes additional tissue-specific and developmental
(Morey et al., 2002).
3. Expression pattern
The rapidly expanding field of genomics provides vast opportunities for evaluating
the coordinated functioning and expression of thousands of genes (Lockhart and Winzeler,
2000). In addition, differentially expressed sequence tags (ESTs) have been isolated and
characterized from effective root nodules of M. truncatula, with a number of identified
genes showing enhanced expression in plant-rhizobium symbiosis (Györgyey et al., 2000).
As originally defined, nodulin genes are those expressed exclusively in nodules
(Legocki and Verma, 1980). However, over the last several years, this presumption has
been reviewed and modified since a number of nodulin genes have been detected in other
plant organs, although with limited expression (Kapranov et al., 1997; Mathesius et al.,
99
2001). Usually, they are members of protein families that play a role in nodule functioning,
but are also active in other physiological processes (Nogueira et al., 2001).
Moreover, several environmental conditions are limiting factors to the growth and
activity of the N2-fixing plants. In the Rhizobium-legume symbiosis, the process of N2
fixation is strongly related to the physiological state of the host plant (Zahran, 1999).
Therefore, an effective nitrogen fixation is limiting by factors, such as salinity, unfavorable
soil pH, nutrient deficiency, mineral toxicity, temperature extremes, inadequate
photosynthesis and plant diseases that impose limitations on the vigor of the host legume
(Brockwell et al., 1995; Peoples et al., 1998). Together, these factors cause changes in the
activation/deactivation of some genes, modifying the expression pattern in certain tissues
and organs.
The effects of salt on nitrogen fixation of legumes have been examined in several
studies, revealing a reduction of N2-fixing activity by salt stress usually attributed to a
reduction in respiration of the nodules, in cytosolic protein production, specifically
leghemoglobin and reduction in the activity of the ammonium assimilation enzymes
glutamine synthetase and glutamate synthase (Cordovilla et al., 1995). In addition,
increasing salt concentrations may have a detrimental effect on soil microbial populations
affecting the fixation rates and effectiveness (Thies et al., 1991).
Additional modifications in the plant host during salt stress were studied by Zahran
and Sprent (1986), which described that soybean root hairs showed little curling or
deformation when inoculated with Bradyrhizobium japonicum in the presence of 170 mM
NaCl, with complete suppression of the nodulation at 210 mM NaCl. They also observed a
reduction in the bacterial colonization and root hair curling of V. faba, where the proportion
of root hairs containing infection threads was reduced by 30%.
100
According to Tejera et al. (2004) to cope the adverse effects of salinity stress
common beans (P. vulgaris) increase the root to shoot ratio, decreasing the content of dry
plant biomass and the nodule number. However, as observed in most cultivated crops, the
salinity response of legumes varied greatly and depended on factors as climatic conditions,
soil properties, stage of growth and species. For example, V. faba, P. vulgaris and G. max
are more tolerant to salinity than other legumes, like P. sativum (Cordovilla et al., 1995).
Our results revealed that some nodulins presented low expression in libraries
submitted to salt stress, as expected, since nitrogen fixation is affected by salinity. The
annexin, Glutamine Synthase and NOD35 genes showed a higher expression in the control
library (CT00; roots in hydropony in the absence of salt stress), probably due to the basal
expression of nodulins under non stressed conditions, while transcripts from the DMI3 and
NOD70 nodulins were found in abundance in the salinity sensitive genotype without salt
stress (SS00), both extracted from root, tissue directly involved in nodulation. Surprisingly,
the early nodulins NSP1 and CCS52A presented a high expression in the SS08 and ST02
libraries, respectively.
The observed abundance of ENOD8 and NORK early nodulins in libraries extracted
from leaves of both BR14-Mulato (BM90) and IT85F (IM90) genotypes collected 90
minutes after mosaic virus infection was expected, since many nodulin genes participate in
developmental processes and are also expressed in diverse tissues from other plants (Bauer
et al., 1996; Papadopoulou et al., 1996). In A. thaliana, for example, the ENOD8
homologous presented higher expression in anthers tissues (Peng and Dickstein, 1994). By
other hand, the high expression of NORK cowpea candidates in libraries constructed from
infected leaves can be explained by the fact that this gene encodes a transmembrane protein
with structural analogy to receptor kinases involved in molecular signaling or disease
101
resistance (De Mita et al., 2007; Jones and Jones, 1994). However, currently available
genetic maps of legumes are limited due to the lack of markers tightly linked to nitrogenfixation, since almost all described markers are focused mainly in resistance to parasites
(Bukar et al., 2004; Ouédraogo et al., 2002).
The creation of a large-scale EST database of M. truncatula offered a the possibility
to prospect genes in silico, whose expression are specific for or greatly enhanced by
symbiosis, allowing the identification of genes that were proposed to be up- or down
regulated in the root nodule. Using this approach, it was possible to identify some
important nodulins in cowpea transcriptome and also to observe their expression pattern
under stress conditions.
The identified sequences represent valuable resources for the development of
markers for molecular breeding and gene-specific markers for nodulation in cowpea and
other related legumes, enriching the genetic, physiological and metabolical data related to
the nitrogen fixation.
102
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111
Table 1. Type and features of nodulin genes used as query against the cowpea databases. The genes are grouped in two nodulin types, Early
nodulin (orange background) and late nodulin (green background) with respective gene name, accession number at NCBI, protein size in amino
acids (aa), organism, with respective domains.
Gene
name
Accession
number
Size
(aa)
Organism
Database
Annexin
CAA75308
313
DMI3
Q6RET7
523
NIN
CAB61243
878
NSP1
ABK35066
542
NORK
CAD10811
925
CCS52A
AAY58271
487
ENOD8
AAL68832
381
Medicago
truncatula
Medicago
truncatula
Lotus
japonicus
Lotus
japonicus
Medicago
truncatula
Lotus
japonicus
Medicago
truncatula
ENOD40
CAD48198
261
DMT1
AAO39834
516
GS
Q43785
356
Lgb
CAA38024
162
NOD26
AAT35231
310
NOD70
AAW51884
598
SucSin
P13708
805
NOD35
BAA19672
309
Medicago
truncatula
Glycine
max
Medicago
sativa
Medicago
sativa
Medicago
truncatula
Glycine
max
Glycine
max
Glycine
max
Conserved Domain 1
Name
Size
Begin
(aa)
Annexin
66
14
End
79
Conserved Domain 2
Name
Size
Begin
(aa)
Annexin
66
86
End
150
Conserved Domain 3
Name
Size
Begin
(aa)
Annexin
66
172
End
232
Conserved Domain 4
Name
Size
Begin
(aa)
Annexin
66
243
End
308
S_TKc
256
11
306
EFh
63
370
460
EFh
63
441
508
-
-
-
-
RWP-RK
52
571
621
PB1_NLP
82
781
862
-
-
-
-
-
-
-
-
GRAS
371
154
532
-
-
-
-
-
-
-
-
-
-
-
-
PKc_Tyr
258
602
868
-
-
-
-
-
-
-
-
-
-
-
-
WD40
289
186
462
-
-
-
-
-
-
-
-
-
-
-
-
SGNH_
plant_
lipase_
like
RRM
315
35
365
-
-
-
-
-
-
-
-
-
-
-
-
74
152
220
-
-
-
-
-
-
-
-
-
-
-
-
Nramp
360
77
439
-
-
-
-
-
-
-
-
-
-
-
-
Glnsynt_N
Globin
82
18
97
259
103
354
-
-
-
-
-
-
-
-
140
20
157
Glnsynt_C
-
-
-
-
-
-
-
-
-
-
-
-
MIP
228
80
268
-
-
-
-
-
-
-
-
-
-
-
-
Nodulinlike
Sucrose_
synth
Uricase
248
27
253
-
-
-
-
-
-
-
-
-
-
-
-
550
7
554
-
-
-
-
-
-
-
-
-
-
-
-
286
15
304
-
-
-
-
-
-
-
-
-
-
-
-
112
Table 2. Main cowpea clusters significantly similar to known nodulins. tBLASTn results including the best match of each nodulin type:
(I) Features and evaluation results with gene name, e-value, cluster size in nucleotides (n), ORF (Open Reading Frame) size in aminoacids (aa), frame ( Fr) and number (#) of hits. (II) Data about BLASTx best alignment: Gi number of NCBI, plant species, e-value and
frame.
(I) Cluster Features and Evaluation
Gene
name
Cluster Nr.
Size
(n)
ORF
(aa)
E-value
Annexin
Contig2698
1153
313
5e-150
DMI3
UP12_145693
1464
403
NIN
UP12_25530
653
NSP1
UP12_1465
NORK
NCBI gi|Nr.
(II) BLASTx Information
aa
Positives Frame
(%)
E-value
Score
3176098
4e-147
525
92
+2
Medicago truncatula
3e-60
91992434
0.0
701
92
+3
Medicago truncatula
189
6e-57
33468530
3e-86
321
83
+2
Lotus japonicus
1586
349
4e-22
89474462
1e-165
587
79
+1
Solanum lycopersicum
Contig1184
1154
284
1e-159
56412259
1e-171
546
92
+3
Sesbania rostrata
CCS52A
UP12_8627
1046
71
3e-114
66932877
2e-106
389
94
+3
Lotus japonicus
ENOD8
UP12_14302
1279
378
3e-113
33147016
4e-124
519
81
+3
Oryza sativa
ENOD40
UP12_6121
1276
253
2e-98
23304837
2e-80
303
80
+2
Medicago truncatula
31322147
4e-129
521
92
+2
Glycine max
Plant Species
DMT1
UP12_13157
1492
312
3e-131
GS
UP12_2868
1344
356
0.0
121345
0.0
654
99
+2
Phaseolus vulgaris
Lgb
VUPISS02004C04
886
145
6e-47
20138590
2e-64
249
100
+1
Vigna unguiculata
NOD26
UP12_17225
1157
301
2e-145
47531135
4e-98
362
90
+1
Medicago truncatula
NOD70
UP12_10450
2342
591
3e-91
57545995
4e-39
167
60
+2
Glycine max
SucSin
UP12_10000
1602
805
0.0
267057
2e-160
1590
99
+1
Vigna radiata
NOD35
UP12_2046
1257
308
5e-169
6175091
5e-164
581
97
+3
Phaseolus vulgaris
113
Table 3. Conserved domains description of the best hits in cowpea database for each nodulin type, including cluster numbers (Nr), gene
name, size in amino-acids (aa), ORF (Open Reading Frame) size in amino-acids (aa), alignment of protein (Ptn), Conserved Domain (CD)
present, integrity (Int) and number (#) of hits with Complete Domain (Com Dom).
Conserved Domain 1
Gene
name
Annexin
DMI3
NIN
NSP1
NORK
CCS52A
ENOD8
Cluster
Nr.
Name
Size
(aa)
ORF
(aa)
Contig2698
Annexin
66
313
S_TKc
256
403
PB1_NLP
82
223
GRAS
302
366
258
452
289
389
315
378
73
253
360
312
82
356
140
145
228
301
248
224
398
550
286
177
UP12_
145693
UP12_
10901
UP12_
9726
UP12_
9214
UP12_
6633
UP12_
14302
ENOD40
DMT1
GS
Lgb
NOD26
NOD70
SucSin
NOD35
UP12_
6121
UP12_
13157
Contig1
VUPISS02
004C04
UP12_
17225
UP12_
12841
Contig2
Contig1
PKc_Tyr
WD40
SGNH_
plant_
lipase_
like
RRM
Nramp
Gln-synt_N
Globin
MIP
Nodulin-like
Sucrose_
synth
Uricase
Align.
Ptn/CD
14/1
79/66
1/46
214/256
130/2
212/83
1/32
250/288
115/3
380/262
96/4
380/285
23/2
358/315
155/1
224/66
1/127
236/360
21/5
97/82
5/1
141/140
74/1
262/205
17/1
223/201
7/1
554/550
14/1
174/162
Conserved Domain 2
Int
#
Com
Dom
Name
Size
(aa)
C
3
Annexin
66
C
4
EFh
63
C
3
-
-
C
3
-
C
20
C
Conserved Domain 3
Int
#
Com
Dom
Name
Size
(aa)
C
2
Annexin
66
C
5
EFh
63
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
C
21
-
-
-
C
3
-
-
I
0
-
C
3
C
Conserved Domain 4
Int
#
Com
Dom
Name
Size
(aa)
Align
Ptn/CD
Int
#
Com
Dom
C
2
Annexin
66
86/1
150/66
C
1
C
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Glnsynt_C
259
103/1
354/258
C
3
-
-
-
-
-
-
-
-
-
-
40
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
C
15
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
C
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
C
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
I
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Alig.
Ptn/CD
243/1
308/66
262/2
321/61
Align
Ptn/CD
170/3
232/65
337/5
394/63
114
Capítulo 3
Artigo Científico
_______________________________________________________
Expression of Nodulins Genes in Sugarcane Transcriptome Revealed
by Computational Analysis
___________________________________________________________
Artigo a ser submetido à revista Genetics and Molecular Research.
115
Identification and Expression of Nodulins in Sugarcane Transcriptome Revealed by In
Silico Analysis
Gabriela Souto Vieira-Mello; Petra Barros dos Santos; Nina da Mota Soares Cavalcanti and Ana
Maria Benko-Iseppon
Universidade Federal de Pernambuco, Centro de Ciências Biológicas, Departamento de
Genética, Laboratório de Genética e Biotecnologia Vegetal, Recife, PE, Brazil.
Short running title: Symbiotic Nitrogen Fixation Genes in Sugarcane Transcriptome.
Key words: data mining, sugarcane, early nodulins, late nodulins, expression pattern
Corresponding Author:
Ana Maria Benko-Iseppon, UFPE, CCB, Departamento de Genética, Laboratório de Genética e
Biotecnologia Vegetal, Av. Prof. Moraes Rego, s/nº; 50732-970, Recife, PE, Brazil. E-mail:
[email protected]
116
ABSTRACT
Nodulin genes have been defined as plant genes that are exclusively induced during nodule
formation in legume plants. Many studies, however, revealed a number of nodulins in nonlegumes, including some monocots, suggesting that these genes play additional roles in plants
besides the nodulation. Sugarcane (Saccharum spp.) establishes a beneficial association with
endophytic nitrogen-fixing bacteria, with some genes involved in these plant-bacteria association
presenting homology with known legume nodulins. In order to gain insight into the role played by
nodulins in sugarcane, we investigated the presence and expression profile of nodulin genes in the
sugarcane transcriptome. In the present work 13 gene coding legume nodulins were selected and
used to search for orthologs in the sugarcane database (SUCEST) using in silico procedures.
Ortolog sequences were identified, translated and their conserved domains (CDs) analyzed (using
BLAST, ORFfinder and RPS_BLAST tools, respectively). To evaluate the expression profile we
used the CLUSTER program, considering tissue of origin and treatment of each library regarding
the available transcripts. We identified 195 candidate contigs in SUCEST database, presenting
significant alignments with known legume nodulins. In silico evaluation revealed higher
expression in FL (flowers), RT (roots) and NR (normalized mix of tissues), confirming the multifunction character of sugarcane nodulins besides the interaction with the endophytic bacteria. The
multiple alignments showed a high homology regarding of the sugarcane candidates with
respective proteins from other plants, mainly monocots, revealing that the genic structure was
relatively conserved among species, probably regarding very ancient genetic processes.
117
INTRODUCTION
Sugarcane is one of the most important sources of sugar and alcohol in the world and is
cultivated in tropical and subtropical areas in more than 80 countries around the globe, especially
in Brazil, where is used mainly for sugar and ethanol production. This crop occupies an area
around one million ha, contributing to 25% of the world’s production (UDOP, 2008). Several
Brazilian sugarcane varieties have the ability to grow with low nitrogen fertilizer inputs.
Historically, this crop has been selected in Brazil for high yields with low inputs of inorganic
nitrogen fertilizer and, unwittingly, for higher contributions of Biological Nitrogen Fixation (BNF)
(Nogueira et al., 2001).
This important Brazilian crop establishes association with endophytic diazotrophic
bacteria, including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and
Herbaspirillum rubrisubalbicans, showing unique features when compared with other nitrogenfixing associations. The bacteria colonize the intercellular spaces and vascular tissues of most
organs of the infected symbiont promoting plant growth, without causing visible plant anatomical
changes or disease symptoms (Baldani et al., 1997; Reinhold-Hurek and Hurek, 1998), possibly
due to effective nitrogen supply (Sevilla et al., 2001). Moreover, it was suggested that the plant
supplies sucrose, among others photosyntates, favoring the endophytic growth (Fuentes-Ramírez
et al., 1999).
It is still unclear which mechanisms are involved in the establishment of this particular
type of interaction and what kind of molecules mediate signaling between plant and bacteria. In
addition, little is known about the role of the plant in this association (Nogueira et al., 2001).
However the fact that distinct sugarcane genotypes have different rates of BNF suggests that plant
118
genetic factors might be controlling the process of bacteria recognition, colonization and/or
nitrogen fixation (Urquiaga et al., 1992).
Nodulins have been defined as plant genes that are exclusively induced during nodule
formation in legume plants (van Kammen, 1984). Many studies, however, revealed a number of
nodulin-related sequences in non-legumes, e.g. those of leghemoglobin (Trevaskis et al., 1997),
uricase II (Takane et al., 1997), ENOD93 (Reddy et al., 1998) and ENOD40 (Kouchi et al., 1999).
Moreover, some non leguminous plants, including rice, were found to have the ability to perceive
lipochitooligosaccharide nodulation signal molecules (Nod factors) produced by the rhizobia
(Reddy et al., 1998). These findings suggest that nodule formation processes are conserved, at
least partially, in non legumes. Inherent nodulation potential of non legumes can probably be
attributed to the existence of nodulin genes in these plants (Reddy et al., 1999).
In addition, some sugarcane genes involved in plant-bacteria signalization during the
association and nitrogen metabolism are probably activated by the endophytic bacteria in the early
steps of plant colonization, allowing sugarcane to assimilate and process the nitrogen fixed by the
bacteria (Vargas et al., 2003). These genes also seem to act as nodule activators, once they present
homology with some legume nodulins (Nogueira et al., 2001).
The investigation of plant gene expression during plant-bacteria associations in sugarcane
transcriptome could be a strategy to unravel the plant molecular mechanisms which are involved
in this particular type of association between plants and diazotrophic bacteria. Large-scale
sequencing of cDNA libraries by the expressed sequence tag (EST) approach has proven to be a
powerful tool to discover new genes and to generate gene expression profiles from different cells
and tissues growing under distinct developmental and physiological conditions (Ohlrogge and
Benning, 2000). In this context, the present work aimed to perform an in silico identification and
characterization of early (Annexin, DMI3, NORK, CCS52A, NIN, ENOD40 and ENOD8) and late
119
(NOD26, NOD70, Glutamine synthase, Leghemoglobin, Sucrose synthase and DMT1) nodulins in
the sugarcane transcriptome (SUCEST project), by using known sequences of legume nodulin as
templates, including an evaluation of the expression profiles of nodulin-related sequences in this
organism.
MATERIALS AND METHODS
The sugarcane ESTs used in the present work are available in the SUCEST database
(www.biotec.icb.ufmg.br/sucest). Information regarding the 31 libraries of the SUCEST project are
described in Table 1 (for further details see Grivet and Arruda, 2001; Vettore et al., 2001).
The identification of sugarcane nodulins was performed by a search using 13 sequences of
known legume nodulins selected from the literature and available at the NCBI databank (Early
nodulins: Annexin, DMI3, NORK, CCS52A, NIN, ENOD40 and ENOD8 and Late nodulins: NOD26,
NOD70, Glutamine synthase, Leghemoglobin, Sucrose synthase and DMT1) (Table 2), against the
SUCEST database using the local tBLASTn tool. After this search, the sugarcane sequences that
matched with nodulin genes with a cut-off of e-10 were used for a homology screening in Genbank
(NCBI) using the BLASTx tool (Altschul et al., 1997). The cluster frame of the tBLASTn alignment
was used to predict the Open Reading Frames (ORFs) for each selected cluster.
Sugarcane
clusters
were
translated
using
the
Orfinder
tool
at
NCBI
(http://www.ncbi.nlm.nih.gov/projects/gorf) and screened for conserved motifs with aid of the RPSBLAST CD-search tool (Altschul et al., 1990). An analysis of nodulin distribution patterns in
sugarcane libraries was verified by direct correlation of the read´s frequency of each cluster in the
SUCEST cDNA libraries, while the prevalence of sugarcane clusters were verified by direct counting
of the reads that composed each cluster, followed by data normalization (considering the total
120
number of reads sequenced in each library) and calculation of the relative frequency (reads per
library). To generate an overall picture of the nodulin expression pattern in sugarcane, a hierarchical
clustering approach (Eisen et al., 1998) was applied using normalized data and a graphic
representation constructed with aid of the CLUSTER program. Dendrograms including both axes
(using the weighted pair-group for each gene class and library) were generated by the TreeView
program (Eisen et al., 1998). On the generated graphics yellow means no expression and brown all
degrees of expression.
Table 1. Description of the SUCEST libraries, including library code, number of ESTs per library and
description of tissues and situations of ESTs extraction. Abbreviations: EST, Expressed Sequence Tag; #,
number.
Library Code
# ESTs
Brief Description
AD1
18137
AM1, AM2
28128
Tissues of plants cultivated in vitro and infected with
Gluconacetobacter diazotroficans
Apical meristem of young plants
CL3, CL4, CL6
11872
Calli treated for 12h at 4ºC and 37ºC in the dark or ligth
FL1, FL2, FL3,
FL4, FL5, FL8
HR1
83899
Flowers at different developmental stages
12000
LB1, LB2
18047
Tissues of plants cultivated in vitro and infected with
Herbaspirilum rubrisublbicans
Lateral buds from mature plants
LR1, LR2
18141
Young leaf
LV1
6432
Leaves from plants grown in vitro
NR1, NR2
768
All normalized tissues
RT1, RT2, RT3
31487
0.3 cm-length roots from mature plants and root apex
RZ1, RZ2, RZ3
24096
Root to shoot zone of young plants
SB1
16318
Stalk bark from mature sugarcane plants
SD1, SD2
21406
Developing seeds
ST1, ST3
20762
First and fourth internodes of young plants
121
RESULTS
1. Sugarcane Orthologs
Using 13 well known nodulin genes as template (Table 2) we could identify 195 candidate
sequences in SUCEST database (that includes 311,493 ESTs), being 129 clusters (1,524 reads) for
early nodulins and 66 clusters (1,646 reads) for late nodulins, with e-values ranging from 0.0 to e-10.
In a general view most analyzed sugarcane clusters showed similarity with monocots, mainly
organisms from Poaceae family, as Oryza sativa (Table 3).
1.1.
Early Nodulins
Regarding annexin a high degree of similarity was found after tBLASTn, with best e-value
4e-152. All nine clusters presented best matches with their respective protein after BLASTx analysis at
the GenBank, two of them with the complete annexin domain. All candidate sequences matched with
monocot plants (Zea mays and Oryza sativa), with exception of two clusters, which presented
similarity with annexins from Arabidopsis thaliana (Brassicaceae) and Cicer arietinum (Fabaceae).
After tBLASTn with the DMI3 seed sequence 25 clusters were identified in the sugarcane
database with e-values varying from 8e-65 to e-10, seven with complete S_TKc domain and 18 with
incomplete domain. After reverse alignments (BLASTx) 19 sugarcane sequences exhibited best
similarity with monocots, including Zea mays (four clusters), Triticum aestivum (one cluster) and
Oryza sativa (14 clusters). The other six sequences were similar to members of dicot families
(Cucurbitaceae, Rosaceae, Fabaceae and Brassicaceae).
Concerning the CCS52A candidates, 12 selected clusters were obtained in the SUCEST
database, with e-values ranging from 2e-130 to 3e-11. After reverse alignment 83.3% presented
similarity with respective protein from O. sativa while the rest was similar to Lotus japonicus and A.
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thaliana. The WD40 conserved domain was complete in three and incomplete in four sequences,
while the rest of the clusters lacked the procured domain.
The search for orthologs to NIN genes revealed the presence of five clusters with high degree
of similarity. In two of these the searched domain RWP-RK was complete, in one this domain was
incomplete and in two no domain was identified. In the BLASTx analysis the clusters showed
similarity to the respective protein from O. sativa and L. japonicus, with e-values ranging from 7e-147
to 8e-23.
The NORK analysis revealed the higher number of similar clusters, presenting 46, with the
best e-value equal to 2e-101. In the BLASTx results 93.5% of all selected clusters showed similarity
with monocots, mainly O. sativa, while 6.5% were similar to A. thaliana. Regarding the integrity of
the PKc_Tyr conserved domains, in 28 and 18 clusters they were complete and incomplete,
respectively.
The 28 putative ENOD8 obtained using BLASTn at SUCEST database showed high
similarity with O. sativa proteins after BLASTx, with more than 75% of the selected clusters similar
to this monocot. 12 clusters presented the procured SGNH_plant_lipase_like domain complete. A
similar result was observed in the reverse alignment to the ENOD40. In this last one, four clusters
were selected in SUCEST database, three showing high similarity with a respective protein from O.
sativa, with the RRM domain found complete and incomplete in three and one clusters, respectively.
1.2.
Late Nodulins
After trimming redundant clusters, the search for late nodulins in sugarcane database also
revealed a high degree of similarity with monocot ortholog proteins, highlighting DMT1 and NOD70,
in which all found clusters were similar to the respective protein as observed after BLASTx. With
respect to DMT1, all eleven selected clusters showed similarity with proteins from O. sativa,
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however just one presented the complete domain, while seven beard incomplete domains and three
clusters lacked the procured domains. Regarding the NOD70 results, only two clusters were similar
to proteins from the dicots Glycine max (Fabaceae family) and Poncirus trifoliata (Rutaceae family),
while the other 13 were similar to O. sativa proteins, being three with complete domain, five with
incomplete domain and seven with no domain.
Considering the Glutamine synthase (GS) results, all selected clusters exhibited best matches
with sugarcane proteins after BLASTx analysis at the GenBank. Two GS candidates displayed the
Gln-synt_N conserved domain (CD) complete, five incomplete and one had no domain. In the other
hand, only two clusters were found in the Leghemoglobin (Lgb) tBLASTn results, from which one
presented the Globin conserved domain complete. In BLASTx results this two candidates presented
high similarity with hemoglobin protein from Z. mays.
Sucrose synthase (SS) and NOD26 candidates revealed similarity mainly with other monocots.
In the case of SS five from 13 selected sequences were similar to respective protein from legumes
plants G. max (4) and Pisum sativum (1) and the remainder nine showed best matches with S.
officinarum (2), O. sativa (2), Sorghum bicolor (1), Z. mays (2) and Bambusa oldhamii (1).
Considering the searched CD, 70% of selected cluster presented the Sucrose_synth domain
incomplete, while 15% presented this domain domain complete; the same percentage was also found
for the cluster with no CD. In relation to NOD26 candidates, 16 clusters were obtained after
tBLASTn, with the reverse alignments confirming that all sequences were similar to
monocotyledonous plants (Z. mays, S. bicolor, O. sativa and S. officinarum) with e-values ranging
from 1e-131 to 3e-85. Regarding the integrity of the procured conserved domain (MIP), in eight it was
found complete, in two incomplete and in six the domain was absent.
After trimming redundant clusters, the search for late nodulins in sugarcane database also
revealed a high degree of similarity with monocot ortholog proteins, highlighting DMT1 and NOD70,
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in which all found clusters were similar to the respective protein as observed after BLASTx. With
respect to DMT1, all eleven selected clusters showed similarity with proteins from O. sativa,
however just one presented the complete domain, while seven beard incomplete domains and three
clusters lacked the procured domains. Regarding the NOD70 results, only two clusters were similar
to proteins from the dicots Glycine max (Fabaceae family) and Poncirus trifoliata (Rutaceae family),
while the other 13 were similar to O. sativa proteins, being three with complete domain, five with
incomplete domain and seven with no domain.
Considering the Glutamine synthase (GS) results, all selected clusters exhibited best matches
with sugarcane proteins after BLASTx analysis at the GenBank. Two GS candidates displayed the
Gln-synt_N conserved domain (CD) complete, five incomplete and one had no domain. In the other
hand, only two clusters were found in the Leghemoglobin (Lgb) tBLASTn results, from which one
presented the Globin conserved domain complete. In BLASTx results this two candidates presented
high similarity with hemoglobin protein from Z. mays.
Sucrose synthase (SS) and NOD26 candidates revealed similarity mainly with other monocots.
In the case of SS five from 13 selected sequences were similar to respective protein from legumes
plants G. max (4) and Pisum sativum (1) and the remainder nine showed best matches with S.
officinarum (2), O. sativa (2), Sorghum bicolor (1), Z. mays (2) and Bambusa oldhamii (1).
Considering the searched CD, 70% of selected cluster presented the Sucrose_synth domain
incomplete, while 15% presented this domain domain complete; the same percentage was also found
for the cluster with no CD. In relation to NOD26 candidates, 16 clusters were obtained after
tBLASTn, with the reverse alignments confirming that all sequences were similar to
monocotyledonous plants (Z. mays, S. bicolor, O. sativa and S. officinarum) with e-values ranging
from 1e-131 to 3e-85. Regarding the integrity of the procured conserved domain (MIP), in eight it was
found complete, in two incomplete and in six the domain was absent.
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2. Distribution of ESTs in SUCEST Libraries
Considering the distribution of the 3,170 nodulin transcripts in the 13 analyzed tissues, in
general a higher prevalence could be observed in flower (FL= 22%), root (RT=13.5%) and stem-root
transition (RZ=10.5%; Figure 1A) tissues. Regarding the correlation of the transcripts distribution
among nodulin classes it is interesting to note that all 29 analyzed libraries from SUCEST database
comprised at least one read while the AD library displayed no difference regarding the number of
reads considering the two classes (early and late); both had 122 reads in total. In counterpart, the RT
library exhibited the highest difference, with the late nodulin class presenting 292 reads and the early
nodulin class 139 reads (Figure 1A).
Considering the correlation of the distribution among reads and nodulins, it was clear that
SS reads were most abundant in the SUCEST libraries, with 879 reads (representing 27.7% of
nodulin transcripts), followed by NORK with 612 reads (19.3% of the total number). The lowest
number of reads was observed for Lgb, with eight reads, representing only 0.25% of all transcripts
found (Figure 1B).
Regarding the individual analysis of the early nodulins (Figure 2A) a higher prevalence in
the FL library was detected, with 381 reads (25%), followed by RZ library, with 196 reads (13%);
however, the expression was also abundant in other libraries, as RT and AM, both representing
9% of the total of reads (139 and 142 reads, respectively). On the other hand, the late nodulin
graphic (Figure 2B) revealed that the FL and RT libraries had the most abundant number of reads,
representing together 37% of all late nodulin transcripts, while the NR and LV had the lowest
representation (2%), with 38 and 30 reads each.
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A
400
350
300
250
Early Nodulins
200
Late Nodulins
150
100
50
0
FL
R T R Z AM S T AD S B S D L B L R HR L V NR C L
B
Figure 1. (A) Comparative prevalence of early and late nodulin genes in the SUCEST libraries. Numbers
in vertical refer to the total of reads. (B) Prevalence of reads per nodulin category. Numbers outside the
columns refer to the absolute number of reads found and below the percentage of reads that compose each
gene category. Abbreviations for libraries: AD: tissues infected by Gluconacetobacter diazotroficans,
AM: Apical meristem; CL: Callus; FL: Flower; HR: tissues infected with Herbaspirillum
rubrisubalbicans; LB: Lateral Bud; LR: Leaf Roll; LV: Leaves; NR: All tissues normalized; RT: Root;
RZ: Stem-Root transition; SB: Stalk Bark; SD: Seeds; ST: Stem.
127
A
B
Figure 2. Prevalence of sugarcane nodulins in the SUCEST libraries. (A) Occurrence of the early
nodulins reads (B) Occurrence of the late nodulins reads. Numbers refer to the percentage of reads in each
library for each nodulin class. Library codes: AD: tissues infected by Gluconacetobacter diazotroficans,
AM: Apical meristem; CL: Callus; FL: Flower; HR: tissues infected with Herbaspirillum
rubrisubalbicans; LB: Lateral Bud; LR: Leaf Roll; LV: Leaves; NR: All tissues normalized; RT: Root;
RZ: Stem-Root transition; SB: Stalk Bark; SD: Seeds; ST: Stem
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3. Expression pattern analysis
All transcripts from the two nodulin classes were used to perform a hierarchical clustering
analysis permitting an evaluation of expression intensity considering the different colors and coexpression among different libraries (black upper dendrogram) or candidates (pink lateral
dendrogram). Considering both graphics (Figure 3) it was evident that early nodulins were more
represented in SUCEST libraries than late nodulins. The in silico expression approach also
revealed a higher expression in flower tissues at different developmental stages, more specifically
FL2 library for early nodulins, and in all tissues normalized for the late nodulins, mainly NR2
library.
Regarding the co-expression among SUCEST libraries in the early nodulin graphic (black
upper dendrogram), some libraries showed a stronger relation, like FL2/RZ2, RT1+LR2/RT2 and
ST3/LB1 (Figure 3A), while in the case of the late nodulins the libraries that showed coexpression were SD2/SD1/LR2 +FL5 and FL2/CL3 (Figure 3B). It is interesting to highlight that
the early nodulins were best represented by reads from tissues of Stem-Root transition (RZ1 and
RZ3) and flower (FL2), totalizing 19.5%, while the late nodulins were prevalent in NR2 library
(35.3%).
Considering the spatial co-expression with transcripts of the nodulin classes (pink lateral
dendrogram), the most representative presence in all tissues was found for NORK, regarding the
early nodulins, and the Suc Sin, considering the late nodulins. In addition the transcripts of the
early nodulins ENOD8, DMI3, Annexin and CCS52A were related, showing a co-expression
among the SUCEST libraries. Concerning the co-expression of late nodulins the analysis revealed
two main groups Suc Sin/GS and DMT1/NOD26.
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Figure 3: Differential display of standard sugarcane transcripts representing selected nodulin genes.
Graphic A represents the expression of early nodulins (NIN, ENOD40, NORK, CCS52A, ENOD8, DMI3
and Annexin) and graphic B represents the late nodulins (Suc Sin, GS, Lgb, NOD35, NOD70, DMT1 and
NOD26). Yellow means no expression and brown means all levels of expression. Library codes: AD1:
tissues infected by Gluconacetobacter diazotroficans; AM1: Apical meristem from mature plants; AM2:
Apical meristem from immature plants; CL3, CL4 and CL6: Pool of calli treated for 12h at 4o e 37oC in
the dark or light; FL1, FL3, FL4, FL5 and FL8: Flowers harvested at different developmental stages;
HR1: tissues infected with Herbaspirillum rubrisubalbicans; LB1 and LB2: Lateral bud from mature
plants; LR1: Leaf roll from immature plants, large insert; LR2: Leaf roll from immature plants, small
insert; LV1: Etiolated leaves from plantlets grown in vitro; NR1 and NR2: all tissues normalized; RT1,
RT2 and RT3: 0.3 cm length roots from mature plants and root apex; RZ1, RZ2 and RZ3: Root to shoot
zone transition of young plants zone 1, 2 and 3; SB1: Stalk bark from mature plants; SD1 and SD2: Seeds
in different stages of development; ST1: Stem, first internodes; ST3: Stem, fourth internodes.
130
DISCUSSION
1. Sugarcane Orthologs
1.1 Early Nodulins
Annexins form a multigene and multifunctional family of amphipathic proteins presenting
a broad taxonomic distribution covering prokaryotes, fungi, protists, plants and higher vertebrates
(Gerke and Moss, 2002; Morgan et al., 2004). Regarding Magnoliophyta this protein is conserved
in both dicotyledonous and monocotyledonous (Smallwood et al., 1992). Concerning their
functions, in legume annexins are upregulated by Nod factors and play a role in nodulation
signaling (Niebel et al., 1998). Besides the role in the symbioses, annexins from non-legumes are
associated with different cellular processes. For example in maize, annexins are considered to be
multifunctional proteins capable of peroxidase activity, elevation of cytosolic calcium and direct
formation of a passive Ca2+- and K+-permeable conductance (Laohavisit et al., 2009). Annexins
have also been documented in plant nuclei where they may participate in DNA replication (Clark
et al., 1998). Another research described a wheat annexin that accumulates in the plasma
membrane in response to cold treatment and may act as a Ca2+ channel (Breton et al., 2000). In
the case of sugarcane Annexin orthologs, the best alignments followed the taxonomic proximity,
since these sequences showed high similarity with the same gene from the Poaceae family (O.
sativa and Z. mays). In addition two sequences presented the conserved domain complete,
indicating the existence and conservation of Annexin genes in sugarcane.
DMI3 is a plant-specific protein that belongs to the CCaMK group of serine-threonine
protein kinases in well-characterized plant genomes, present from the moss Physcomitrella patens
to higher plants including dicots and monocots (Messinese et al., 2007). We found many DMI3
orthologs in sugarcane transcriptome bearing high similarity with previously sequenced genes.
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Most sugarcane CCaMKs presented high similarity with rice sequences. Regarding this
resemblance, it is interesting to note that some authors suggested that legume DMI3 also beard
high similarity to rice and lily (Sathyanarayanan and Poovaiah, 2002; Yang and Poovaiah 2003).
Little is known about the biological role of CCaMKs in plants. The preferential
expression of the lily and rice CCaMKs in developing anthers and root tips (Poovaiah et al., 1999;
Wang and Poovaiah, 1999) has led to the suggestion that they could play a role in mitosis and
meiosis (Yang and Poovaiah 2003). Other authors suggested that a CCaMK is required by
mycorrhized plants to interpret a complex calcium signature elicited in response to fungus signals
(Hrabak et al., 2003; Yang and Poovaiah, 2003). This could be also the case of sugarcane that
besides the interaction with endophytic bacteria, is able to establish mycorrhizal associations
(Reis et al., 1999).
The CCS52A protein is an APC activator involved in mitotic cyclin degradation and in
regulation of endoreduplication, playing a role in cell enlargement during root nodule
organogenesis in legumes (Vinardell et al., 2003). However, this gene is not exclusively
associated with the nodulation process and appears to be a ubiquitous regulator of cell cycle
transition to differentiation in plants cells (Foucher and Kondorosi, 2000). The phylogeny of the
CCS52A follow the classic taxonomic relationship and orthologs of this protein have been found
in various other plant species like L. japonicus, M. truncatula, arabidopsis, tobacco, tomato,
potato, soybean, wheat and rice, indicating a strong conservation of the CCS52A proteins in the
plant kingdom (Cebolla et al., 1999; González-Sama et al., 2006; Vinardell et al., 2003). Despite
of the scarce information regarding the presence of CCS52A in sugarcane, our findings confirm
that this gene is present in this organism since we found clusters with complete domains and best
hits with high degree of similarity with rice.
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The initiation of nodule development has been shown to be dependent on nodule inception
protein (NIN) (Borisov et al., 2003). In addition, NIN also represses spatial expression pattern of
nodulation factors, which may control nodule number (Marsh et al., 2007). Interestingly, the NIN
gene family is found widely among higher plants and algae, including many species that are not
able to promote gaseous nitrogen fixation (Castaings et al., 2009). The most prominent feature of
the NIN protein is a 60-aminoacid-long sequence that is strongly conserved across a variety of
proteins in different plants species. In non-legumes this high conserved region (named RWPQP)
has been predicted to correspond to the DNA binding compound, acting in the dimerization in
non-legumes (Schauser et al., 2005).
Riechman et al. (2000) found that there are no close relatives to the legume NIN proteins
in rice or arabidopsis, instead, these non-legumes presented NIN-like proteins (NLPs) regarding
the closest relatives of legume NINs. In addition, the NLPs are multidomain proteins with a high
degree of conservation; the phylogenetic tree inferred from the NLP alignment suggested that
there are at least three copies of this gene in the common ancestor of mono- and eudicotyledons
(Schauser et al., 2005). Our results confirmed these findings, since the five identified sugarcane
clusters showed a high similarity with rice NLPs, confirming the presence of NIN proteins in
sugarcane.
Sugarcane’s most abundant nodulin regarded the NORK gene class, with 46 clusters. The
extracellular domain of NORK protein includes a unique 400-amino-acid sequence and three
LRR (Leucine Rich Repeat) domains, followed by a transmembrane domain and a typical
serine/threonine protein kinase domain. LRR domains mediate protein interactions and are
thought to be involved in ligand recognition by LRR-RLKs (Leucine Rich Repeat-Receptor-like
kinases), that are required for perception of a liposaccharide nodulation signal in legumes (Endre
et al., 2002; Shiu and Bleecker 2001). Proteins that possess similarity to the unique NORK
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extracellular domain are found in monocots and dicots, suggesting that this region may have a
biological role that is not limited to nodulation (Endre et al., 2002). The RLKs comprise the
largest gene family of receptors in plants, with more than 600 homologs in arabidopsis and 1100
in rice (Shiu et al., 2004). In both organisms these RLKs might have roles in plant development
and in signal transduction during interactions with endophytic organisms and pathogens (Morillo
and Tax, 2006). In addition Vinagre et al. (2006) identified in sugarcane a LRR-RLK whose
expression is regulated in response to interactions with beneficial bacteria. Together, these facts
confirm our findings in sugarcane transcriptome and explain the high number of clusters found.
ENOD8 is a member of the GDSL family of lipolytic enzymes present in plant and
bacteria that have the putative active site serine sequence context, which is not perfectly
conserved in all members of the GDSL gene family (Györgyey et al., 2000). In plants, GDSL
lipase candidates of species like arabidopsis, Rauvolfia serpentina, Medicago sativa, Hevea
brasiliensis and Alopecurus myosuroides have been isolated, cloned and characterized, revealing
that they are conserved between these species (Arif et al., 2004; Cummins and Edwards, 2004;
Oh et al., 2005; Pringle and Dickstein, 2004; Ruppert et al., 2005).
Nogueira et al. (2001) found in infected libraries of SUCEST that sugarcane ENOD8
shows similarity to myrosinase-associated protein (MyAP) related with the plant defense
responses. In our results sequences of ENOD8 protein were also found in non-infected tissues,
suggesting that this protein plays a role in other functions besides the interaction with endophytic
organisms; however, in monocots these functions remain unknown. In dicots, like arabidopsis and
Brassica napus, ENOD8 sequences are specifically expressed in anthers and encodes hydrolytic
proteins; some of which with esterase and lipase activity (Cook and Dénarié, 2000; Peng and
Dickstein, 1994). In addition, the similarity with rice and arabidopsis sequences found in our
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alignments can be explained by the fact that few sequences from other non-legumes are available
in NCBI database
In legumes the ENOD40 is a critical gene responsible for cortical cell divisions leading to
the initiation of nodule development in rhizobial association (Charon et al., 1999), while in the
interaction with arbuscular mycorrhiza plays a role in the fungal growth in the root cortex and
increases the frequency of arbuscule formation (Sinvany et al., 2002). However, ENOD40 is not
exclusively associated with plant-host interactions and possible functions in non-legumes fall into
three possible groups: transport, organogenesis and regulation of phytohormone status. Thus, it
has been suggested that ENOD40 may also have a regulatory role during different stages of plant
development but its precise function is still poorly understood (Ruttink, 2003).
ENOD40 genes can be identified by the presence of regions that are highly conserved
among distantly related plant species (Compaan et al., 2003). In accordance to this fact ENOD40
from O. sativa encodes peptides that are homologous to proteins encoded by the corresponding
genes in legumes, even thought their expression is not associated with symbiotic interactions
(Reddy et al., 1999).
The occurrence of ENOD40 sequences in monocots and different clades within the core
eudicots shows that ENOD40 is an ancient gene that has been maintained in these plants during
divergent evolution (Ruttink, 2003). This gene was also functionally characterized in Z. mays
(Yang et al., 1993); in addition, previous studies have identified isoforms in the sugarcane
genome, using southern analysis (Reddy et al., 1999), confirming the present evidences from
sugarcane transcriptome. Additionally, the low number of clusters found can be explained by
Compaan et al. (2003) that suggested that this gene category is low expressed in most non-legume
plant species.
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1.2 Late Nodulins
DMT1 belongs to the NRAMP (Natural Resistance-Associated Macrophage Protein)
family of metal transporters. For instance, several members of the NRAMP family have been
characterized and have shown to be involved in metal uptake and transport in several organisms
like fungi, animals, plants and bacteria (Cellier and Gros, 2004).
In monocots, like rice, expression studies have revealed that several NRAMP genes are
upregulated upon Fe deficiency, suggesting a role in Fe homeostasis (Thomine et al., 2000;
Bereczky et al., 2003). In our study the majority of sugarcane orthologs exhibited best alignments
with NRAMP proteins of O. sativa what may suggest that these genes can be also related with Fe
homeostasis in sugarcane.
Nodule development is associated with the spatially and temporally regulated expression
of a number of genes that encode membrane transport proteins (Jeong et al., 2004). Muñoz et al.
(1996) showed that NOD70 from monocots has homology with a sulfate transporter and a
possible role in nutrient supply during plant-microbe symbiogenesis. However, Szczyglowski et
al. (1998) showed that NOD70 genes from legumes encode a polytopic membrane protein with
sequence and topology similar to members of the major facilitator superfamily (MFS) of
membrane transporters. Moreover Vincill et al. (2005) showed that the NOD70 from legumes
encode a symbiosome membrane protein that possesses an anion transport activity with selectivity
for nitrate, nitrite, and chloride. Nitrate is an important nitrogen source for plants, being also a
signal molecule that controls various aspects of plant development. In addition to its role as
nutrient, nitrate was shown to act as a signal molecule, which independently of its assimilation,
controls numerous aspects of plant development and metabolism (Wang et al., 2003). In our
results almost all orthologous sequences showed similarity with a nitrate and chloride transporter
from others monocot plants, especially Z. mays, that display a homologue of NOD70 described as
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a nitrate and chloride transporter (Vincill et al., 2005), confirming the importance of this gene
also in sugarcane, since many alignments with similar features to mays could be identified.
With respect to sugarcane GS orthologs, the best alignments showed the procured domain
complete, with most candidates presenting similarity with sugarcane sequences after reverse
alignments, in agreement with the fact that these proteins were already characterized in this
organism (Nogueira et al., 2005). Ours findings can be supported by evidences from other GS
isoforms that have also been found in some non-legumes from temperate climates as Z. mays and
H. vulgare, besides the dicot Lycopersicum esculentum (Becker et al., 1992, Miflin, 1974;
Sakakibara et al., 1992). Some functional studies regarding GS role in monocots revealed that
some GS isoforms are important for normal growth (Hirel et al., 2005) and grain filling in the
case of rice and maize (Shrawat and Good, 2008).
The great similarity of Leghemoglobin candidate clusters was in accordance to the classic
taxonomic relationships, since the significant alignments occurred with Z. mays. One of the
candidates has shown the Globin domain complete, and the existence of this gene in sugarcane is
clear evident, since nonsymbiotic hemoglobin genes have also recently been found in
monocotyledonous plants such as barley, wheat and rice (Andersson et al., 1996; Taylor et al.,
1994) and symbiotic hemoglobin is present in the nitrogen-fixing nodules of both legumes and
nonlegumes plants (Andersson et al., 1997). The function of these plant hemoglobins in
nonsymbiotic tissues is not clear; they may be associated with the transport of oxygen or, as
suggested by Appleby et al. (1990), they may act as oxygen sensors in the signal transduction
pathway for activation of anaerobic genes.
Sucrose synthase catalyzes the reversible conversion of sucrose UDP to UDP-glucose and
fructose and is the central enzyme of carbohydrate metabolism in all plant species. This enzyme is
implicated in a wide variety of processes, including nitrogen fixation (Gordon et al., 1999), starch
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synthesis (Chourey et al., 1998), cellulose biosynthesis (Amor et al., 1995), phloem transport
(Nolte and Koch, 1993) and the ability of storage organs to act as carbon sinks (Zrenner et al.,
1995). In monocots, sucrose synthases play different functions. For example, in rice this enzyme
is induced transcriptionally and translationally in seedlings under oxygen deficiency and its
activity under submerged conditions is significantly higher than under aerobic conditions. In
Potamogeton distinctus the transcription of sucrose synthase is increased in elongating turions
under oxygen deficiency (Harada et al., 2005) while in maize has been implicated in various roles
in the synthesis and degradation of sucrose as well as in the flow of carbon from one organ to
another (Shaw et al., 1994). In addition, parenchyma cells of sugarcane stems accumulate sucrose
up to 20% of their fresh mass or 60% of dry mass in mature internodes (Moore and Maretzki,
1996). The involvement of sucrose synthase in sugarcane metabolism is already proved (Schäfer
et al., 2004) and the great number of sequences found with high homology with this gene
confirms the existence of SUS in sugarcane genome, as expected.
The NOD26-like major intrinsic protein (MIP) is a subfamily of aquaporins, a category of
sequences involved in a number of processes concerning water and solute relations in plants. In
some legumes like soybean, NOD26 has its activity specifically in the peribacteroid membrane of
N2-fixing symbiotic root nodules (Fortin et al., 1987). Homologues of NOD26 have been
identified in plant species that do not develop any N2-fixing symbiosis, but their subcellular
localization is still unclear (Weig et al., 1997). The separation into different functional groups
probably occurred before the monocot-dicot divergence, being suggested that the ancestral gene
of these groups encoded a protein with a specific biological role. The persistence of these groups
in dicots and monocots is also an indication of the crucial importance of MIPs in Angiospermae
(Chaumont et al., 2000).
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Regarding monocots, maize cells contain several NOD26 homologues in their plasma
membrane that have different functions or are differentially regulated (Lopez et al., 2003). Liu et
al. (1994), characterized two rice cDNAs which are homologous to the genes encoding the MIP
family and saw that the expression was enhanced by water stress, salt stress and exogenous
abscisic acid. Together, all these facts explain the great amount of NOD26 homologous sequences
found in sugarcane transcriptome.
2. Expression pattern
Nodulins were initially defined as plant genes that are exclusively induced during nodule
formation and specifically expressed in nodules (Munõz et al., 1996). However, functional
evaluations showed that many of these genes are in fact expressed in nonsymbiotic tissues and/or
during nonsymbiotic conditions (Charon et al., 1999) also presenting a number of homologues in
non-legume plants, as arabidopsis and rice that are unable to form nodules (Chen et al., 2007;
Miyao et al., 2007). Thus, it is hypothesized that nodulin genes have arisen as a result of the
recruitment of pre-existing, non-symbiotic genes which might have roles in other physiological
processes, like controlling growth and development, common to all plants (Andersson et al., 1996;
Mylona et al., 1995). In fact, the presence of nodulin transcripts in non-infected tissues in
SUCEST libraries confirms this hypothesis.
Evaluations recognized that legume genes that are required for nodulation are also
essential for the symbiotic associations with arbuscular mycorrhizal (AM) fungi, which are
established in more than 80% of flowering plants, including monocots as rice (Kistner et al., 2005;
Oldroyd and Downie, 2004). In sugarcane several genes possibly involved in nitrogen metabolism
and plant-bacteria signaling during endophytic diazotrophic association seem to act as noduleenhanced genes (Nogueira et al., 2001). Therefore, the higher expression of the early nodulins
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found in our study, in comparison to the late nodulins, was expected, since this type of nodulins
act mainly in the plant-host signaling pathways.
Regarding the expression pattern of early nodulins, the observed majority of nodulin
transcripts in flower libraries were also expected, since this is the largest SUCEST library with a
higher number of developing stages and sequences, as compared with other tissues. Regarding
NORK results, a significant expression could be detected in most SUCEST libraries; what is in
agreement with the fact that genes encoding RLKs isoforms, besides their roles in organism
interactions, are very closely related to plant developmental processes, being present in tissues
under growth and differentiation, like seeds, plantlets in different stages of development in
flowers, leaves and root-to-shoot transition regions, confirming the crucial importance of this
proteins for plants (Morillo and Tax, 2006). In addition, in rice this isoforms are involved in
hormone reception, growth-factor recognition, the recognition of fungal elicitors and development
of shoot and floral apical meristems (Takayama and Sakagami, 2002), what explain the presence
of sugarcane reads of NORK in many meristematic tissues.
In legumes the expression of ENOD40 is induced within hours of Rhizobium inoculation
and it appears to be critical for proper nodule development; however, transcripts are also localized
in the stem, lateral roots and other tissues in these plants (Charon et al., 1999). Our results have
shown that the ENOD40 sugarcane presented a similar expression pattern as previously found in
rice, in which expression was detected in the developing vascular bundles in the stem (Kawahara
and Chonan, 1968). Additionally, in maize transcripts were detected in roots, leaf and leaf veins,
with the highest expression in the stem (Varkonyi-Gasic and White, 2002).
The occurrence of annexin transcripts in almost all SUCEST libraries occurred in
accordance to Proust et al. (1996) that using Northern-blot analysis revealed that annexins from
plants have a fairly widespread expression. Concerning monocot annexins, Smallwood et al.
140
(1992) showed that the transcripts were found in root tissues, stem and young expanding leaves of
barley, while Carroll et al. (1998) reported that the maize annexin was expressed in root cap cells
and differentiating vascular tissues in roots (Carroll et al., 1998), both similar to annexin
expression in sugarcane found in our analysis.
Besides the nodulins described above, many early nodulins presented an expression
related to organ differentiation in monocots, like DMI3, ENOD8, and CCS52A (Foucher and
Kondorosi, 2000; Messinese et al., 2007; Peng and Dickstein, 1994). Based on the distribution
and prevalence of these early nodulins in sugarcane transcriptome, we can suggest that these
genes also play a role in the organ development in this monocot.
In general the late nodulins are preferentially expressed in mature nodules, acting directly in the
nitrogen fixation (Niebel et al., 1998) what can explain the low number of transcripts found in the
present approach in sugarcane transcriptome that is unable to form nodules. Regarding the
expression pattern of these genes, the highest number recognized regarded the sucrose synthase,
glutamine synthase and NOD26, nodulins required for the development of several tissues.
Studying rice Hirose et al. (2008) detected sucrose synthase transcripts in a wide range of tissues
and at different developmental stages, indicating that they are involved in diverse growth
processes. We found a similar expression pattern in sugarcane transcriptome, with great amounts
of transcripts in the NR2 library (normalized mix of tissues). This gene is also crucial for
enhanced growth in tissue development, explaining the high number of transcripts found in the
early stages of flower development (FL2) and tissues of root to shoot of young plants (RZ). In
addition, this enzyme showed a high activity in others tissues like callus cells and mature
sugarcane internodes (Botha and Black, 2000), in accordance with our findings in CL and ST
libraries.
141
The expression pattern of glutamine synthase and NOD26 was quite similar, with both
presenting reads in almost all SUCEST libraries, what was expected since these genes play an
important role in all plant groups. The high expression of glutamine synthase in sugarcane was in
root tissues. Similar results, were observed in other monocots, like Z. mays and H. vulgare with
significant expression of glutamine synthase isoforms in roots (Becker et al., 1992; Sakakibara et
al., 1992). The NOD26 is an aquaporin that displays a crucial role in water and solute relations in
plants. In agreement with this function, our analysis in sugarcane transcriptome revealed the
prevalence of NOD26 transcripts in tissues that presented a high water flux and requirement like,
seeds (SD), roots (RT), flowers (FL) and internodes (ST). In other monocots, like rice, some
isoforms of this gene have also been localized in organs and tissues with these characteristics, e.g.
vascular tissues, flowers and roots (Fraysse et al., 2005; Senadheera et al., 2009).
In a general view, the fact that different nodulins are expressed in most SUCEST libraries
support the assumption that these genes are expressed not only in nodulation conditions, revealing
the importance of these genes for all angiosperms.
CONCLUDING REMARKS
With aid of bioinformatic tools it was possible to identify all 13 nodulin gene categories out of 195
sugarcane contigs, allowing also inferences regarding their expression pattern. For all nodulin classes
candidates bearing the respective conserved domains could be found in sugarcane, most of them
putatively involved in tissue development and growth, besides plant host interactions. Considering
the low amount of described nodulins in monocots, the identified sequences represent valuable
resources for functional evaluations including expression assays and may lead to significant benefits
for sugarcane production.
142
ACKNOWLEDGMENTS
We thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and
FACEPE (Fundação de Amparo à Pesquisa do Estado de Pernambuco) for the concession of
fellowships. To FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and SUCEST
for the access to the Sugarcane EST data bank.
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Table 2. Type and features of nodulins genes used as query against the Sugarcane databases. The genes are grouped in two nodulins types, Early nodulin (written in
orange) and Late nodulin (written in green) with respective gene name, accession number at NCBI, size of the protein in aminoacids (aa), organism, with respective
domains.
Gene
name
Accession
number
Size
(aa)
Organism
Database
Annexin
CAA75308
313
DMI3
Q6RET7
523
NIN
CAB61243
878
NORK
CAD10811
925
CCS52A
AAY58271
487
ENOD8
AAL68832
381
Medicago
truncatula
Medicago
truncatula
Lotus
japonicus
Medicago
truncatula
Lotus
japonicus
Medicago
truncatula
ENOD40
CAD48198
261
DMT1
AAO39834
516
GS
Q43785
356
Lgb
CAA38024
162
NOD26
AAT35231
310
NOD70
AAW51884
598
SucSin
P13708
805
Medicago
truncatula
Glycine
max
Medicago
sativa
Medicago
sativa
Medicago
truncatula
Glycine
max
Glycine
max
Conserved Domain 1
Name
Size
Begin
(aa)
Annexin
66
14
End
79
Conserved Domain 2
Name
Size
Begin End
(aa)
Annexin
66
86
150
Conserved Domain 3
Name
Size
Begin End
(aa)
Annexi
66
172
232
n
EFh
63
441
508
Conserved Domain 4
Name
Size
Begin End
(aa)
Annexi
66
243
308
n
-
S_TKc
256
11
306
EFh
63
370
460
RWP-RK
52
571
621
82
781
862
-
-
-
-
-
-
-
-
PKc_Tyr
258
602
868
PB1_NL
P
-
-
-
-
-
-
-
-
-
-
-
-
WD40
289
186
462
-
-
-
-
-
-
-
-
-
-
-
-
SGNH_
plant_
lipase_like
RRM
315
35
365
-
-
-
-
-
-
-
-
-
-
-
-
74
152
220
-
-
-
-
-
-
-
-
-
-
-
-
Nramp
360
77
439
-
-
-
-
-
-
-
-
-
-
-
-
Gln-synt_N
82
18
97
259
103
354
-
-
-
-
-
-
-
-
Globin
140
20
157
Glnsynt_C
-
-
-
-
-
-
-
-
-
-
-
-
MIP
228
80
268
-
-
-
-
-
-
-
-
-
-
-
-
Nodulin-like
248
27
253
-
-
-
-
-
-
-
-
-
-
-
-
Sucrose_sy
nth
550
7
554
-
-
-
-
-
-
-
-
-
-
-
-
155
Table 3. Main sugarcane clusters similar to nodulins genes. tBLASTn results and sequence evaluation of sugarcane nodulins genes including the best match of each
gene: (I) Features and evaluation results with sugarcane cluster number, cluster size in nucleotides (n), ORF (Open Reading Frame) size in amino-acids (aa), e-value;
score, frame and numbers (#) of matched clusters. (II) Data about BLASTx best alignment: NCBI GI number and plant species.
Gene name
and expected domain
Annexin
Annexin
DMI3
S_TKc
NIN
PB1_NLP
NORK
PKc_Tyr
CCS52A
WD40
ENOD8
SGNH_plant_lipase_like
ENOD40
RRM
DMT1
Nramp
Glutamine Synthase
Gln-synt_N
Leghemoglobin
Globin
NOD26
MIP
NOD70
Nodulin-like
Sucrose Synthase
Sucrose_synth
(I) Cluster Features and Evaluation
Sugarcane
ORF
Size (n)
e-value
Cluster Nr.
(aa)
(II) BLASTx Information
Score
Frame
# Clusters
NCBI GI Nr.
Plant Species
e-value
SCSBST3098G08.g
1166
314
4,00e-83
541
-1
9
162459661
Zea mays
4,00e-152
SCJLLR1011H04.g
2424
515
8,00e-65
244
1
25
1899175
Cucurbita pepo
0.0
SCQGRT1041A07.g
1254
315
4,00e-38
524
-3
5
56783862
Oryza sativa
7,00e-147
SCVPLB1015A04.g
2973
976
1,00e-101
365
1
46
77548313
Oryza sativa
0.0
SCCCLR1080G07.g
1338
231
2,00e-130
468
-1
12
25446692
Oryza sativa
6,00e-130
SCVPLB1020A04.g
1229
317
100e-60
229
-3
28
51969146
Arabidopsis thaliana
3,00e-76
SCBGLR1047F09.g
1017
203
2,00e-59
178
3
4
42408101
Oryza sativa
2,00e-59
SCBFRZ2017F03.g
2116
263
0.0
385
1
11
108706772
Oryza sativa
0.0
SCJFLR1013F02.g
1743
356
0.0
746
3
9
56681313
Saccharum officinarum
0.0
SCMCRZ3064B09.g
955
171
6,00e-32
132
3
2
125503242
Oryza sativa
3,00e-75
SCEPRZ1008D05.g
2134
317
1,00e-76
282
1
16
162458955
Zea mays
1,00e-131
SCRFLB1055B10.g
1637
486
1,00e-129
457
2
15
28209525
Oryza sativa
5,00e-169
SCCCRZ1002G07.g
3056
816
0.0
1277
-1
13
3915873
Glycine max
0.0
156
CONCLUSÕES GERAIS
• Os transcriptomas do feijão-caupi (NordEST/HarvEST) e de cana-de-açúcar (SUCEST)
apresentaram representantes de todas as nodulinas estudadas.
• A presença e a estrutura das nodulinas no transcriptoma do feijão-caupi sugere que esta
leguminosa apresenta, durante o estabelecimento da fixação biológica de nitrogênio,
mecanismos semelhantes aos encontrados em outras leguminosas modelo, como Lotus
japonicus e Medicago truncatula.
• Em cana-de-açúcar o grande número de sequências ortólogas às nodulinas indica a atuação
destes genes em outras vias metabólicas, além da nodulação, tal como em outras plantas
não leguminosas, sugerindo que o estabelecimento da simbiose nodular envolveu genes
ancestrais, que provavelmente não estavam relacionados à via simbiôntica.
• As nodulinas candidatas de caupi apresentaram maior similaridade com sequências de
plantas leguminosas, enquanto em cana uma maior similaridade foi observada com genes
de outras Monocotiledôneas, em conformidade com a proximidade evolutiva dos
organismos.
• Sequências de organismos que pertencem à mesma família tendem a se agrupar nos
dendrogramas, sugerindo que as nodulinas estejam sujeitas as pressões seletivas,
associadas à evolução divergente.
• Em caupi a maioria dos transcritos foi observada em bibliotecas de folhas infectadas e de
raiz sob estresse salino, enquanto em cana a maioria das reads pertencia às bibliotecas de
flores e raízes, fornecendo indícios de que em Angiospermas esses genes estão
envolvidos em outros processos, além da fixação biológica de nitrogênio.
• As seqüências identificadas neste trabalho representam uma ferramenta valiosa para o
desenvolvimento de marcadores moleculares para o melhoramento das espécies
estudadas, fornecendo meios de elucidar os mecanismos utilizados por esses genes em
outras vias, que não a de fixação, o que permitirá expandir os conhecimentos sobre a
simbiose em plantas não-leguminosas de importância econômica.
157
ANEXO
Instrução para autores
158
159
160
161