| Leaf chlorosis mutation commonly occurred in higher plants. Mutant genes directly or indirectly affect the synthesis or degradation of chlorophyll, and finally lead to decrease in the chlorophyll content of leaves, so these mutants are known as chlorophyll-defecient mutants. Chlorophyll-defecient mutants have great particular value in studying the mechanism of photosynthesis, chlorophyll biosynthesis, chloroplast structure, genetic control of development and differentiation, gene functional identification and understanding the nucleo-cytoplasmic interreaction, it can also serve as a marker traits for crop breeding. Therefore, the development and utilization of new leaf color mutants are of theoretical and practical value.Chlorophyll-defecient L638-y was founded and developed from the normal L638-g line in Brassica juncea L. The mutant characterized with chlorosis from the cotyledon to leaf, no fatal effects, and belong to the total chlorophyll-deficiency mutants. But the biochemical and molecular mechanisms of leaf chlorosis of the mutant remain unclear. In this study, the mutant L638-y and wild-type L638-g were used as materials, to analyze the metabolic intermediates of chlorophyll synthesis and key enzymes of the chlorophyll metabolic pathway, chloroplast soluble proteins and the thylakoid membrane protein components. The purpose of this study is to reveal biochemical mechanisms of chlorophyll deficiency in the mutant L638-y. The major findings are as follows:1. The phenotypes and the content of leaf pigment in chlorosis mutant L638-y at different growth stagesChlorosis phenomenon has always existed in mutant L638-y from the appearance of true leaves to flowering stage. However, the mutant possesses a trend of regreening from middle leaves to older leaves at the bottom. From the 3-5 leaf stage to flowering period, the total chlorophyll and carotenoid contents of leaves in L638-y are significantly lower than those of its wild-type L638-g.2. Determination of chlorophyllase (Chlase) activity in different growth stages of L638-y and wild-type L638-g showed that the leaves Chlase activity of the mutant L638-y at the 3-5 leaf stage was significantly lower than that of the wild type (P <0.01), while there was no difference in Chlase activity between L638-y and wild-type L638-g at over wintering and regreen period. It is shown that the reduction of Chl content in the mutant L638-y is not due to the degradation of chlorophyll.3. By measuring content of the metabolic intermediates in chlorophyll synthesis pathway from the leaves of the mutant L638-y and wild-type L638-g , and comparison the content of various intermediates of the mutant with it wild type, from ALA-PBG-Urogen III - Coprogen III-Proto IX-Mg-proto IX- Pchlide-Chlide-Chla-Chbelb, it was showed that the contents of the intermediates ALA, PBG, Urogen III, Coprogen III's in mutant leaves were significantly higher than those in leaves of the wild-type, while the contents of the intermediates Proto IX, Mg -proto IX, Pchlide, Chlide, Chla, Chlb in mutant leaves were significantly lower than those in leaves of the wild-type. Further, activities of 4 enzymes involved in synthesis metabolism of chlorophyll in L638-y and L638-g was determination, and the results showed that the ALAD activity in leaves between L638-y and L638-g was no difference. While the activity of BPGD in leaves of L638-y was significantly higher than that of L638-g. The most notable was that the activity of COPX and PPOX in leaves of L638-y was significantly lower than those of L638-g. These results were agreement with the measurement of intermediates in chlorophyll synthesis pathway. All of these indicated that chlorophyll synthesis in the mutant L638-y was blocked, and the blocking sites located on the steps from Coprogen III to Proto IX. The mutant may be a new type of chlorsis mutant different from the others previously reported.4. Analysis of soluble proteins and chloroplast thylakoid membrane proteins in chloropast of mutant L638-y and wild-type L638-g showed that there were difference in composition and content of soluble proteins molecular weight lower than 55kD. In additions, there were significantly different in soluble protein components of chloroplast in leaves from different position of mutant L638-y. There were significantly different in compositions and contents of thylakoid membrane proteins between L638-y and L638-g, molecular weight ranging from 14 to 55kD. Compared with L638-g, in L638-y, different components of thylakoid membrane proteins were lost and their contents decreasing in leaves from different parts. Two-dimensional electrophoresis (2-DE) of chloroplast soluble proteins from central leaves of L638-y and L638-g indicated that there were seventeen differentially expressed protein spots identified between the 2-DE maps of L638-y and L638-g. Of the 17 differentially proteins, fourteen proteins were up-regulated two timesor more in the mutant, and three proteins were down-regulated two times in the mutant.In summary, the above results indicated that chlorophyll synthesis in the chlorophyll-defecient mutant L638-y was blocked, and the blocking sites located on the steps from Coprogen III to Proto IX. This mutant may be a new type of chlorsis mutant different from the others previously reported. The mutant L638-y will have a particular value in studying the mechanism of photosynthesis, chlorophyll biosynthesis, chloroplast structure, genetic control of development and differentiation, gene functional identification and understanding the nucleo-cytoplasmic interreaction,etc.
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