Effects Of EGY1-defect On PSⅡ Stability And Leaf Senescence In Arabidopsis Thaliana

Since EGY1 is a membrane-associated and ATP-independent metalloprotease that is localized in the chloroplast,it maybe plays roles in the function of photosynthesis.In the study,the homozygous egy1 mutants of Arabidopsis thaliana were obtained through reverse genetics analysis.The effects of EGY1-defect on function of photosynthesis and leaf senescence were studied by using colorimetry,antibody preparation,two-dimensional gel electrophoresis,pull-down assays,RT-PCR,Western Blot,Northern Blot,and spectrum-technique.The detailed results were summarized as follows:1.Compared to wild type,egy1 mutants were yellow-green and had a slight decrease in growth rate.In egy1 mutants,Fv/Fm,the chlorophyll and photosystemâ…¡(PSâ…¡) contents significantly decreased with increasing leaf age,suggesting that the function of photosynthesis was damaged.Immunoblot analyses further showed that the accelerated reductions in the abundance of D1,D2 and LHCâ…¡occurred with leaf age in egy1 mutants.These results suggested that the EGY1 defection resulted in PSâ…¡damage.2.Thylakoid membrane proteins were separated by BN-PAGE and SDS-PAGE,and then were used to immunoblot analyses.The results showed that the migration of EGY1 was consistent with that of PSâ…¡complex.Pull-down assays suggested that EGY1 was associated with PSâ…¡and colocalized with D2 protein.3.The synthesis of PSâ…¡was almost unchanged in egy1 mutants(about 90%of wild type).It is impossible that the markedly impair function of photosynthesis was due to the slight decrease in PSâ…¡synthesis.Thus,PSâ…¡stability was measured.It was reported that PSâ…¡stability was significantly reduced when egy1 mutants were treated by hot,cold and/or different salt stress.At the same time,PSâ…¡was less stable under high light in egy1 mutants than in wild-type plants.These results suggested that the defection of EGY1 resulted in the loss of photosynthesis function through decreasing the PSâ…¡stability. 4.Leaf survival and soluble protein contents decreased whereas ion leakage of leaf increased significantly with increasing leaf age in egy1 mutants.Furthermore,the yellow-green phenotype of egy1 mutants was accelerated after darkness treatment for 3 days.These results suggested that the defection of EGY1 could induce the accelerated leaf senescence.Northern blotting analysis showed the transcript levels of senescence-associated genes(SAG12,SAG24 and SEN4)increased more and photosynthesis genes(CAB and RBCS)decreased more in egy1 mutants compared to wild-type plants,documenting the accelerated leaf senescence in egy1 mutants. According to the changes of photosynthesis function in young leaves of egy1 mutants, we speculated that the accelerated leaf senescence was caused by the damage of photosynthesis function.5.The egy1 seedlings didn't show hyper-responsive phenotype to exogenously applied sugar,indicating that a hexokinase-dependent sugar-signalling pathway might not be affected by the loss of EGY1.That meaned the accelerated leaf senescence in egy1 mutants was hexokinase-independent sugar-signalling pathway or hormone -dependent signal pathways.In a word,the roles and detailed mechanisms of EGY1 on photosynthesis function and leaf senescence in Arabidopsis thaliana were studied for the first time.These results are helpful to people's understand in chloroplast proteases,and provide us important information to comprehend the molecular mechanism of photosynthesis function and leaf senescence in plants and other systems.