The Mechanism Of Arabidopsis EMB1270 Regulating Chloroplast Development And A New Method Of PPR Protein Function Analysis

Pentatricopeptide-repeat proteins(PPRs)are characterized by tandem arrays of a degenerate 35-amino-acid(PPR motifs).By specifically binding a single strand RNA,most PPRs participate in regulating post-transcriptional gene expression in organelles,include RNA cleavage,splicing,editing and stability etc.PPRs are conserved in all eukaryotes,but extremely expanded in higher plants.Studies have shown that chloroplast development is regulated by a lot of PPR proteins via changing chloroplast gene expression.In this study,we obtained a weak allele of EMB1270,namely emb1270-2,by screening mutants with chloroplast development defects.The mutant displays yellow cotyledons and virescent leaves at the seedling stage,and a lower maximum photochemical quantum yield of PSII(F_v/F_m).EMB1270encodes a chloroplast-localized PPR protein with 24 PPR motifs,belonging to the P subfamily.The knockout mutant,emb1270-1,was embryonic lethal.We found that in the emb1270-2 mutant,the splicing efficiency of the chloroplast clpP1 intron 2 and ycf3 intron 1 was significantly decreased.Further analysis showed that EMB1270 can interact with two chloroplast-localized splicing factors,CAF1 and CFM2.We thus speculate that EMB1270 cooperates with CAF1 and CFM2 to specifically regulate the splicing of clpP1 intron 2 and ycf3 intron 1.Considering that many knockout mutants of PPR proteins are embryonic lethal,it is difficult to investigate biological functions of these PPR proteins.Instead,the knockdown transgenic plants are usually employed.In this study,we found that overexpressing a fragment of a PPR gene in wild type plants led to co-suppression of its endogenous gene expression,which is dependent on post-transcriptional gene silencing(PTGS).The phenotype of co-suppression lines was similar to the knockdown plants of the corresponding PPR gene.We thus provided a new method for studying the function of embryonic lethal PPR genes by constructing their co-suppression lines in Arabidopsis.Using this method,we created the co-suppression lines for an embryonic lethal PPR protein with unknown functions,EMB976.And by analyzing these co-suppression lines,possible target genes of EMB976 were disclosed.