Proteomic analysis of chloroplast biogenesis (clb) mutants uncovers novel proteins potentially involved in the development of Arabidopsis thaliana chloroplasts

Abstract

Plant cells outstand for their ability to generate biomass from inorganic sources, this phenomenon takes place within the chloroplasts. The enzymatic machinery and developmental processes of chloroplasts have been subject of research for several decades, and this has resulted in the identification of a plethora of proteins that are essential for their development and function. Mutant lines for the genes that code for those proteins, often display pigment-accumulation defects (e.g., albino phenotypes). Here, we present a comparative proteomic analysis of four chloroplast-biogenesis affected mutants (cla1-1, clb2, clb5, clb19) aiming to identify novel proteins involved in the regulation of chloroplast development in Arabidopsis thaliana. We performed 2D-PAGE separation of the protein samples. These samples were then analyzed by computational processing of gel images in order to select protein spots with abundance shifts of at least twofold, statistically significant according to Student's t-test (P<0.01). These spots were subjected to MALDI-TOF mass-spectrometry for protein identification. This process resulted in the discovery of three novel proteins potentially involved in the development of A. thaliana chloroplasts, as their associated mutant lines segregate pigment-deficient plants with abnormal chloroplasts, and altered mRNA accumulation of chloroplast-development marker genes.

Biological significance: This report highlights the potential of using a comparative proteomics strategy for the study of biological processes. Particularly, we compared the proteomes of wild-type seedlings and four mutant lines of A. thaliana affected in chloroplast biogenesis. From this proteomic analysis it was possible to detect common mechanisms in the mutants to respond to stress and cope with heterotrophy. Notably, it was possible to identify three novel proteins potentially involved in the development or functioning of chloroplasts, also it was demonstrated that plants annotated to carry T-DNA insertions in the cognate genes display pigment-deficient phenotypes, aberrant and underdeveloped chloroplasts, as well as altered mRNA accumulation of chloroplast biogenesis marker genes.

Keywords: Arabidopsis thaliana; Chloroplast development; Comparative proteomics; clb mutants.