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

Abstract

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.

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses

“…The preparation of the acetone dry powder (ADP) from the plant total extract was based on the protocol described by Pirovani et al (2008) (Method A) with modifications (Method B) that are described below (Figure 1). Initially, 1.0 g plant tissue (lyophilized roots) was finely pounded into a powder in liquid nitrogen using a pestle and mortar and intermixed with 7% polyvinylpolypyrrolidone (w/w).…”

“…Protein extraction from ADP was based on the protocol described by Pirovani et al (2008) (Method A) with modifications (Method B). The ADP was resuspended in 10 mL dense sodium dodecyl sulfate (SDS) extracting buffer (30% sucrose, 2% SDS, 0.1 M Tris-HCl, pH 8.0, 5% 2-mercaptoethanol).…”

“…However, cacao has received little attention with respect to proteomic research. Until now, few studies have been carried out to analyze the protein pattern of cacao during its response to different stresses (Pirovani et al, 2008(Pirovani et al, , 2010Rehem et al, 2011). In particular, proteomics offers the possibility of identifying post-translational modifications of proteins, which are largely not predictable from genome sequences.…”

“…To overcome these problems, we developed a new protocol for T. cacao root protein extraction that is based on a protocol for protein extraction from T. cacao leaves and meristems (Pirovani et al, 2008). This protocol attempts to minimize the presence of interfering compounds and has a higher yield of obtained protein.…”

Proteomic response of Moniliophthora perniciosa exposed to pathogenesis-related protein-10 from Theobroma cacao

“…Protein was extracted from M. perniciosa according to Pirovani et al (2008). Protein extracts were subjected to a second cleaning method for total protein purification as described by Meyer et al (1988).…”