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.
Pirovani
Carlos Priminho
CP
UESC, DCB, Laboratório de Genômica e Expressão Gênica, Rodovia Ilhéus-Itabuna, Ilhéus-BA, Brasil.
Carvalho
Heliana Argôlo Santos
HA
Machado
Regina Cele Reboucas
RC
Gomes
Dayane Santos
DS
Alvim
Fátima Cerqueira
FC
Pomella
Alan William Vilela
AW
Gramacho
Karina Peres
KP
Cascardo
Júlio Cézar de Mattos
JC
Pereira
Gonçalo Amarante Guimarães
GA
Micheli
Fabienne
F
D000363:Agaricales / Q000472:pathogenicity; D002099:Cacao / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D018519:Meristem / Q000737:chemistry; D010935:Plant Diseases; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D011489:Protein Denaturation; D020543:Proteome / Q000032:analysis*
D010940:Plant Proteins; D020543:Proteome