Functional Study Of MTERF9 In Arabidopsis Chloroplast Localization

Chloroplast gene expression is crucial for plant photosynthesis and development,but the mechanisms that regulate it are still unclear.In mammals,the mitochondrial transcription termination factor(mTERF)family plays important roles in the process of mitochondria gene expression,but information relating to their roles in photosynthetic organisms remains scarce.In this study,we foucus the mTERF9,a member of mTERF family in Arabidopsis thaliana.We obtained the T-DNA insertion homozygous mutant mterf9.Loss of mTERF9 results in stunted growth and obvious yellow phenotype.We generated the transgenic plant expressing mTERF9 fused with4×MYC tag.We performed immunolocalization analysis of mTERF9 within chloroplasts.The chloroplast,stromal and thylakoid fractions were subjected to immunoblot analysis with specific MYC antibody.Most of the mTERF9 protein was detected in the stromal fractions.The steady-state levels of thylakoid and stromal proteins were examined by immunoblot analyses.The results showed that protein contents of Psad,Psba,Rbcl and Rbcs were substantially reduced.The levels of plastid-encoded polymerase(PEP)-dependent chloroplast genes were reduced,whereas those of nucleus-encoded polymerase(NEP)-dependent chloroplast genes increased in the mutant by qRT-PCR analysis.However,Northern blot assays indicated the expression of psbA,rbcL and accD were not changed obviously,but the transcript pattern of the polycistron petD was affected,the levels of petD transcript containing intron were increased in the mterf9 mutant.The result showed that mTERF9 involved in RNA splicing.We further tested the chloroplast RNA editing events in mterf9.The sequencing of chloroplast RNA editing sites showed that the editing of six sites(atpF,clpP,ndhB-2,ndhB-3,ndhB-6 and ndhB-7)was affected in mterf9.RNA immunoprecipitation(RIP)analysis showed that mTERF9 is associated with the RNA transcript nearby ndhB-6 and ndhB-7.These results suggested that mTERF9 protein is required for RNA editing and splicing.Therefore,mTERF9 protein is an important factor for post-transcriptional regulation in chloroplast in Arabidopsis thaliana.