Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction - under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF - using infiltration-centrifugation procedure - that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.

Bibliographic Details

Main Authors:	Priminho Pirovani, Carlos, Santos Carvalho, Heliana Argôlo, Reboucas Machado, Regina Cele, Santos Gomes, Dayane, Cerqueira Alvim, Fatima, Vilela Pomella, Alan William, Peres Gramacho, Karina, De Mattos Cascardo, Julio Cézar, Guimarães Pereira, Gonçalo Amarante, Micheli, Fabienne
Format:	article biblioteca
Language:	eng
Subjects:	F30 - Génétique et amélioration des plantes, H20 - Maladies des plantes, Moniliophthora, Theobroma cacao, activité enzymatique, extraction, protéine, électrophorèse, organe végétatif des plantes, technique analytique, http://aims.fao.org/aos/agrovoc/c_31727, http://aims.fao.org/aos/agrovoc/c_7713, http://aims.fao.org/aos/agrovoc/c_2604, http://aims.fao.org/aos/agrovoc/c_36910, http://aims.fao.org/aos/agrovoc/c_6259, http://aims.fao.org/aos/agrovoc/c_2523, http://aims.fao.org/aos/agrovoc/c_27991, http://aims.fao.org/aos/agrovoc/c_1513,
Online Access:	http://agritrop.cirad.fr/544968/ http://agritrop.cirad.fr/544968/1/document_544968.pdf
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