Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Plant cell walls possess specific difficulties. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50%) of predicted intracellular proteins. The rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%), belonging to the same functional classes as proteins identified using previously described protocols. The new cell wall preparation described in this paper gives the lowest proportion of proteins predicted to be intracellular when compared to available protocols. The application of its principles should lead to a more realistic view of the cell wall proteome, at least for the weakly bound CWP extractable by salts. In addition, it offers a clean cell wall preparation for subsequent extraction of strongly bound CWP

Cell wall proteins: a new insight through proteomics

Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translational modifications have been characterized in cell wall proteins to date? The purpose of this review is to discuss the experimental results obtained to date using proteomics, as well as some of the new questions challenging future research

Expression of a gene encoding a ribosomal p40 protein and identification of an active promoter site

The promoter of a gene encoding a ribosome-associated protein of 40 kDa from Arabidopsis thaliana (A-p40) was sequenced and the expression of the gene studied. A-p40 was expressed in the same organs and with the same variations as the eukaryotic elongation factor 1α (eEF1A), another gene coding for a protein involved in translation Arabidopsis plants transformed with a β-glucuronidase (GUS) gene driven by the A-p40 promoter confirm that A-p40 is expressed in actively dividing and growing cells. eEF1A promoter-GUS fusions have the same pattern of expression. Comparison of cis-acting elements from A-p40 and eEF1A revealed some common elements. A-p40 promoter deletions and transient gene expression in transfected Arabidopsis protopasts allowed the identification of trap40, a cis-acting element regulating gene expression. Gel retardation experiments indicate that eEF1A and A-p40 are regulated by different cis-acting elements. The role of such elements is discussed.This work was partially supported by EEC project HCM ERBCHRXCT940678, the Centre National de la Recherche Scientifique and Universite Paul Sabatier (Toulouse, France). I.S. was supported by a doctoral scholarship from the Ministere de la Recherche et l’Enseignement Superieur, France.Peer reviewe