Proteômica diferencial em clones de Coffea canephora sob condições de déficit hídrico
Data
2007-02-18
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Universidade Federal de Viçosa
Resumo
A tolerância à seca é o resultado de numerosas características anatômicas, morfológicas e fisiológicas, de natureza constitutiva ou indutiva. Vários mecanismos são responsáveis pela tolerância, e salienta-se a necessidade de que sejam identificados genes e seus produtos que estejam envolvidos nas diferentes respostas que conferem tolerância à seca em café. Este trabalho teve como objetivos verificar a resposta adaptativa de clones de Coffea canephora ao déficit hídrico além de identificar proteínas responsivas a esse estresse . Mudas dos clones 120 (genótipo tolerante à seca) e 109A (genótipo sensível à seca) foram cultivadas em vasos de 12 L e o déficit hídrico foi alcançado suspendendo a irrigação até que as plantas atingissem um potencial hídrico de antemanhã (ψ am ) de -3,0 MPa. A redução do potencial hídrico (ψ am ) nas plantas que foram submetidas ao déficit hídrico foi acompanhada de reduções nas taxas de assimilação líquida do carbono (A) e da condutância estomática (g s ). Foi observado um aumento mais pronunciado no extravasamento de eletrólitos assim como na atividade de enzimas antioxidantes no clone sensível ao déficit hídrico (109A). Para as análises de proteoma, foram adaptados protocolos de extração de proteínas de folha e raiz de café, compatíveis com a eletroforese em gel bidimensional e a identificação de proteínas por espectrometria de massa. Os protocolos adaptados mostraram-se eficientes na retirada de contaminantes não protéicos, em especial polifenóis na extração de proteína de folhas e polissacarídeos na extração de proteínas de raiz. Os géis obtidos mostraram-se reprodutíveis e ausentes de arrastes, apresentando os spots individualizados. Os dados de proteoma indicam que um dos mecanismos de aclimatação ao estresse hídrico usado por Coffea canephora seria a fotorrespiração. Os resultados obtidos indicam a existência de mecanismos de adaptação a um possível dano oxidativo, como o aumento na expressão de alguns chaperones moleculares e de algumas proteínas dos fotossistemas, e a maior expressão de algumas isoformas da subunidade maior da Rubisco. A atividade destas proteínas poderia contribuir para a manutenção do papel antioxidativo da fotorrespiração. Por outro lado, somente no clone tolerante, sob déficit hídrico, foi observada a maior acumulação de uma NADPH-quinona redutase e a manutenção da abundância da glutamina sintase. Estes resultados sugerem que a primeira enzima pode ter importante papel no mecanismo antioxidativo associado à tolerância à seca neste clone e a segunda enzima um importe papel da manutenção da assimilação da amônia e da fotorrespiração nestas condições.
Drought tolerance can result from numerous anatomical, morphological and physiological characteristics that can be from constitutive or inductive nature. Several mechanisms are responsible for the tolerance, and it is pointed out the need that genes and their products involved in the different responses that confer coffee drought tolerance. The aim of this work was the verification of the adaptative responses of Coffea canephora clones to drought stress and identify proteins responsive to that stress. Plants of the clones 120 (drought tolerant genotype) and 109A (drought sensitive genotype) were cultivated in vases of 12 L and water deficit was reached by suspending the irrigation until the plants reached –3.0 MPa predawn hydric potential (ψ am ). The reduction in the ψ am in the plants upon drought stress was accompanied by reductions in liquid carbon assimilation (A) and in estomatical aperture (g s ). A more pronounced increase was observed in electrolytes leakage as well as in the activity of antioxidant enzymes in the sensitive genotype (109A) upon water stress. For proteome analysis, we adapted a protocol for coffee leaf and root protein extraction, which was compatible with two-dimensional gel electrophoresis and protein identification by mass spectrometry. The adapted protocols were efficient in the retreat of non proteic polyfenol contaminants from leaf protein extracts and polysacaridies from root proteins. The processed 2D gels were reproducible, presenting individualized spots, without drags. The proteome data indicated that one of the mechanisms of drought stress acclimatization presented by Coffea canephora would be photorespiration. The obtained results indicated the existence of adaptation mechanisms to a possible damage oxidativo, as the increase in the expression of molecular chaperones and proteins from the photosystems, as well as the increased expression of isoforms of the largest Rubisco subunity. The activiy of these proteins can contribute to the maintenance of the antioxidative role of photorespiration. In contrast, only in the tolerant clone under drough stress, the accumulation of a NADPH-quinona redutase was increase and glutamine sinthase were unaltered. These results suggest that the first enzyme could have important role in the antioxidative mechanism associated to the drought tolerance presented by this clone, and the second enzyme would be associated to the maintenance of the assimilation of the ammonium and of the photorespiration in these conditions.
Drought tolerance can result from numerous anatomical, morphological and physiological characteristics that can be from constitutive or inductive nature. Several mechanisms are responsible for the tolerance, and it is pointed out the need that genes and their products involved in the different responses that confer coffee drought tolerance. The aim of this work was the verification of the adaptative responses of Coffea canephora clones to drought stress and identify proteins responsive to that stress. Plants of the clones 120 (drought tolerant genotype) and 109A (drought sensitive genotype) were cultivated in vases of 12 L and water deficit was reached by suspending the irrigation until the plants reached –3.0 MPa predawn hydric potential (ψ am ). The reduction in the ψ am in the plants upon drought stress was accompanied by reductions in liquid carbon assimilation (A) and in estomatical aperture (g s ). A more pronounced increase was observed in electrolytes leakage as well as in the activity of antioxidant enzymes in the sensitive genotype (109A) upon water stress. For proteome analysis, we adapted a protocol for coffee leaf and root protein extraction, which was compatible with two-dimensional gel electrophoresis and protein identification by mass spectrometry. The adapted protocols were efficient in the retreat of non proteic polyfenol contaminants from leaf protein extracts and polysacaridies from root proteins. The processed 2D gels were reproducible, presenting individualized spots, without drags. The proteome data indicated that one of the mechanisms of drought stress acclimatization presented by Coffea canephora would be photorespiration. The obtained results indicated the existence of adaptation mechanisms to a possible damage oxidativo, as the increase in the expression of molecular chaperones and proteins from the photosystems, as well as the increased expression of isoforms of the largest Rubisco subunity. The activiy of these proteins can contribute to the maintenance of the antioxidative role of photorespiration. In contrast, only in the tolerant clone under drough stress, the accumulation of a NADPH-quinona redutase was increase and glutamine sinthase were unaltered. These results suggest that the first enzyme could have important role in the antioxidative mechanism associated to the drought tolerance presented by this clone, and the second enzyme would be associated to the maintenance of the assimilation of the ammonium and of the photorespiration in these conditions.
Descrição
Dissertação de mestrado defendida na Universidade Federal de Viçosa.
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Citação
GUIMARÃES, B. L. S. Proteômica diferencial em clones de Coffea canephora sob condições de déficit hídrico. 2007. 59 f. Dissertação (Mestrado em Fisiologia Vegetal) - Universidade Federal de Viçosa, Viçosa-MG. 2007.