Proteome Analysis Of Chlorophyll-deficient Mutant Rice At The Seedling Stage

Leaf color mutant in the higher plant attracts much attention because it results to easily visible trait and has higher mutation frequency. These play important roles in both theoretical research and practical uses. The leaf color mutant phenotype which is clearly distinguished by its characteristics, can serve as a phenotypic marker in plant genetics and breeding studies. It can also be used as an ideal research material on chloroplast development and chlorophyll biosynthesize related gene in functional genomics.In this paper, the morphological characters, chloroplast ultrastructure, proteomics and microarray of W02S and Peiai 64S were studied. W02S is a new male sterile line with the albino green-revertible trait developed from a cross between WS and PTGMS Peiai 64S. WS was developed through anther culture by our research team in 1996 and there after showed stable expression in several generations.The results for the morphologic investigation showed that, up to three-leaf stage, the seedlings of W02S appeared apparently albino but turned into green after the 4th leaves were fully expanded. Peiai64S which was used as a control showed stable chlorophyll content and green color throughout the whole seedling stages. TEM analysis was performed for the leaves collected from the pre-and post green-revertible periods of W02S. During pre green-revertible period, W02S mutant compared to Pei'ai 64S had clear developmental defects such as lack of complete double-layer membrane and the chloroplast thylakoid lamellae. However, during the greening period, complete chloroplast structure, double membrane structure, and internal interconnected grana thylakoids observed in the W02S were the same as that of the control Pei'ai 64S. These results indicated that the cause of albino trait was the changes of the genes related to chloroplast development and chlorophyll biosynthesis that led to the initial thylakoid retardation, rather than changes in other pigments.The protein for analysis of differential protein expression profile between W02S and Peiai64S was extracted at seedling stage. In total 62 proteins were analyzed and identified in the W02SW through MALDI-TOF MS and database searching. Among them 44 were up-regulated proteins and 18 down-regulated proteins. The identified proteins were sorted according to their function. Most of them were involved in signal transduction, plant light responses, osmotic stress regulation, EMP-TCA-ETS metabolic pathways, photosynthesis, photorespiration photophosphorylation etc. This indicated that the albino green-revertible process was regulated by multi-signals and metabolic pathways.To study the differentially expressed genes between the pre-and post green-revertible period of W02S, we examined the mRNA expression changes between the two stages, W02Sw and W02Sg. A total of 5167 rice genes were found to be differentially expressed at their mRNA levels between the two stages. Differential genes expression profile was simultaneously constructed though comparative analysis of gene expression profiles. According to effective classification of the Gene Ontology, these genes could be classified into 17 categories, suggesting that the etiolating process is a regulatory consequence of multiple signal and metabolism pathways.To compare the correlations between the changes in corresponding mRNA and protein expressions, we comprehensively analyzed proteins and mRNA that showed significant differential expressions between W02Sw and W02Sg. We found 5 overlap genes that encode two unknown proteins, one 29 kDa ribonucleoprotein A(cp29A), proteasome alpha subunit and enolase. The expression of four proteins and their corresponding mRNAs showed an upward trend. The only protein and its corresponding mRNA that showed the opposite expression trend was No.37.To obtain complete expression data for the 62 proteins, the mRNA expressions of the 57 proteins, which were not detectable by microarray, were further analyzed by real-time PCR. The results from proteomic analysis, microarray assay and real-time PCR demonstrated a good correlation of the protein differences between W02Sw and W02Sg which in turn corresponded to mRNA differences. Overall, of the 62 proteins, less 20% with upward/downward trends had an opposite mRNA trend; these discrepancies might be due to the modifications of the post-transcription and post-translation processes. The reliability of the experimental results is very high.These new findings provide the basic data that may lead towards the understanding of mechanisms of chlorophyll-deficient phenotype in rice and other plants.