| Plant leaf color mutants not only play an important role in studying photosynthesis of higher plants, chlorophyll biosynthesis, structure, differentiation and development of chloroplast, genetic control, identification of gene function as well as understanding the interaction between nuclear and chloroplast, but also serve as markers in the utilization of heterosis. Brassica juncea L. has many advantages, including a higher drought and heat tolerance, and enhanced resistance to pod-shattering, which is mainly distributed in the highlands of China. L638-y is a natural chlorophyll-deficient mutant discovered and developed from an accession L638-g in B. juncea, which is typical characterized by yellow leaves in whole growing period. In addition, it is unlethality, and belongs to lack of total chlorophyll. In the present study we developed a 2-DE technique for analyzing proteome of leaf color mutants in B. juncea, and then analyzed and compared the leaf proteome of the mutant L638-y and its wild type L638-g at three growth stages, i.e. 3-5 leaves, over wintering and bloting stage. The purpose of this study is to reveal globe proteins variation in leaf of the chlorophyll deficient mutant, and to provide some basic information for identification of the mutant genes. The major results obtained are as follows:1) A 2-DE technique for analyzing leaf proteome of chlorophyll deficient mutant L638-y was developed.In order to efficiently analyze the differentially expressed proteins between the mutant L638-y and its wild type L638-g, the 2-DE system was optimized and developed. That is using 17 cm linear IPG strips with pH ranged from 4 to 7, 11%SDS-PAGE, loading 180μg/350μl, proteins were effectively separated in 2-DE maps. In additions, three protein extraction methods, including TCA/acetone precipitation, Ca2+-phytate methods and an improved polyethylene glycol (PEG) fractionation method were compared. The proteins extracted from L638-y leaves by TCA/acetone precipitation method contained high abundance of RuBisCO. While protein samples prepared with Ca2+-phytate method or the improved PEG fractionation were quite efficient for depletion of RuBisCO, but some proteins were lost in the procedure of Ca2+-phytate fractionation. Finally, 1235±6 protein spots were identified in 2-DE map of the protein sample isolated from the mutant L638-y by the PEG fractionation, which were 330 spots more than those by TCA/acetone precipitation method. By using the PEG fractionation, 190 differentially expressed protein spots were identified between the 2-DE maps of the mutant L638-y and its wild type L638-g, which were 100 spots more than those by TCA/acetone precipitation. Thus, the improved PEG fractionation is more efficient for proteomic analysis of chlorophyll-deficient mutants in B. Juncea.2) Comparative proteomics analysis of differentially expressed proteins between the mutant L638-y and its wild type L638-gTotal proteins in the leaves of L638-y and L638-g at three stages, i.e. 3-5 leaves stage, over wintering period and bloting stage were analyzed using comparative proteomics approach. Total of 124 differentially protein spots were detected identified between L638-y and L638-g. One hundred and one of these 124 differentially expressed proteins were successfully identified by mass spectrometry. These identified proteins were involved in different cell responses and metabolic process, with evident functional tendencies toward carbohydrate metabolism, amino acid and protein synthesis, energy metabolism, signal transduction, DNA repairing, protein folding and degradation, antioxidant mechanism and so on. Based on the quantity variation of these proteins at three developmental stages between L638-y and L638-g, the 101 proteins were divided into five categories by hierarchical cluster analysis. The first category including 8 proteins that related to photosynthesis were down-regulate in L638-y. And the second category (containing 32 proteins) and third category (containing 21 proteins) were related to carbohydrate metabolism, including chlorophyll metabolism, Calvin cycle, photosynthesis and carbonic anhydrase. The proteins included in the second category were down-regulated, and the proteins classified in the third category were up-regulated. The fourth category had 12 proteins, being up-regulated proteins, which mainly involved in energy metabolism and signal transduction. The fifth group contained 16 proteins mainly related to amino acid metabolism and protein synthesis. Interestingly, the quantity of these proteins was similar (very low level) beween L638-y and L638-g at 3-5 leaves and bloting stages, but obviously high in L638-y at over wintering.3) The changes of complex metabolic networks in the chlorophyll deficient mutant L638-yBased on the quantity variation of 101 differentially expressed proteins between L638-y and L638-g, it was elucidated that the metabolic networks were obviously changed in L638-y, including photosynthesis, carbohydrate metabolism and energy metabolism, indicating the existence of complex metabolic regulatory networks in L638-y. |
