Involvement The Of SG1 In Chloroplast Development And The Suppressor Identification And Related Research Of Topp4-1 In Arabidopsis Related Function N Arabidopsis

Part?:Chloroplast is a crucial organelle in higher plants.It is essential for fixation of CO₂ and also for biosynthesis of carbon skeletons,fatty acids,pigments,and amino acids from inorganic nitrogen.In this study,a new gene,SG1,was identified in a slow-greening mutant?sg1?isolated from an ethylmethanesulphonate-mutagenized population of Arabidopsis.The newly formed leaves of sg1 were initially albino,but gradually turned pale green.After 3 weeks,the leaves of the mutant were as green as those of the wild-type plants.Transmission electron microscopic observations showed that the mutant displayed delayed proplastid to chloroplast transition which caused the slow greening phenotype in the mutant.The results of map-based cloning showed that SG1 encodes a chloroplast-localized tetratricopeptide repeat-containing protein.GUS staining and qRT-PCR data demonstrated that SG1 gene expresses in all tissues,particularly in young green tissues;and also the expression level of SG1 is regulated by light.qRT-PCR revealed that the SG1 mutation disrupted the expression levels of chloroplast development associated genes.Western blotting also showed that the protein levels of ribulose bisphosphate carboxylase large subunit?RbcL?and small subunit?RbcS?were different in different stage leaves of sg1,and they were significantly inconsistent with their mRNA levels.We further constructed sg1 gun and sg1 gun4 double mutants,and found that gun1 and gun4 ameliorated the slow-greening phenotype of sg1 and partially restored the disrupted expression patterns of chloroplast-associated gene.Taken together,the results suggest that SG1 is required for chloroplast development in Arabidopsis.Part?:PP1 phosphatase plays very important function in plant growth and development;however,its specific function remains to be further clarified.Based on topp4-1 mutant,our laboratory has revealed some important functions of PP1 family member TOPP4.To further reveal the function of TOPP4 in plant growth and development,the present study took advantage of EMS mutagenesis and screened the suppressor 2562 topp4-1 double mutant which can restore the dwarf and leaf curl phenotypes of topp4-1.The current study is focused on the suppressor and related functions of TOPP4.By detecting TOPP4 gene and protein expression pattern in 2562mutant,we found that TOPP4 gene expression pattern,protein localization and expression level were not changed in 2562 mutant,suggesting that 2562 restore topp4-1 phenotypes does not depend on the regulation of dominant negative effect of topp4-1 but depends on other signaling pathway.Exogenous auxin response of 2562and 2562 topp4-1 were normal as wild type while,2562 could only partially restored GA response defective phenotype of topp4-1.By mixed pool genomic sequence and map based cloning we identified that 2562 encodes a new unknown function R protein.Through trypan blue staining and qRT-PCR,we found that 2562 topp4-1double mutant could recover the cell death and PR genes up regulation phenotypes in topp4-1.Meanwhile,in order to investigate the influences of subcellular localization on the function of topp4-1,we fused topp4-1 protein with different localization signals.Then,we over-expressed these different localization constructs into wild-type plants.We found that only cytoplasmic localization topp4-1 transgenic plants showed cell death and PR genes up regulation phenotypes.In addition,mos3 topp4-1 double mutant exacerbated the dwarf phenotype of topp4-1,and the double mutant showed infertility;it speculated that TOPP4 and MOS3 may in the parallel signaling pathways as over expression of MOS3 in topp4-1 and TOPP4 in mos3 did not affect the mutant phenotypes.