A Novel Nucleus-plastids Dual Localization Transcription Factor,BAI,Regulates Chloroplast Development In Arabidopsis Thaliana

Photosynthesis uses light energy to convert carbon dioxide and water into organic matter while releasing oxygen.Chloroplast is the site of photosynthesis.Most of the photosystem components are encoded by nuclear genes,and about 10% are encoded by chloroplast genes.Chloroplast contains two types of RNA polymerases for gene transcription: the bacterial type plastid-encoded RNA polymerase(PEP)and a phage type nuclear-encoded RNA polymerase(NEP).Current researches suggest that HEMERA(HMR),also known as PLASTID TRANSCRIPTIONALLY ACTIVE 12(PTAC12),is a crucial component of PEP supercomplex.As a single-subunit phage-type RNA polymerase,NEP is encoded by SCABRA3(SCA3).The deletion mutants of HMR and SCA3 display varying degrees of leaf albino and yellowish phenotypes.Chloroplast RNA polymerases play vital roles in chloroplast biogenesis,but how chloroplast RNA polymerases transcribe plastidial genes remains enigmatic.This project uses an Arabidopsis mutant bai(in Chinese "albino")as the research object to conduct the following research by using the technical methods of biochemistry and molecular biology:1)The gene account for the mutant albino,BAI,was mapped by using a combination of mapbased cloning and genome resequencing.Bioinformatics analysis showed that BAI is a plastidsnucleus double-localized C2H2 zinc finger transcription factor encoded by nuclear gene.2)Chloroplast morphology was severely damaged in the mutant bai by comparing the ultrastructure of chloroplasts of the wild type and bai by transmission electron microscopy analysis.BAI is mainly expressed in ungerminated seeds by analyzing the transcript expression of BAI in various tissue parts of the wild type,as well as GUS histochemical staining.Moreover,it was determined that BAI was localized in both the nuclei and the chloroplast through confocol and immunoblotting of BAI-e GFP transgenic plants.3)The interacting proteins of BAI were found by co-immunoprecipitation combined with mass spectrometry.HMR was selected from the mass spectrometry results.Afterwards,the interaction between BAI and HMR was verified by bimolecular fluorescence complementation.4)The downstream target genes regulated by BAI were searched by using chromatin immunoprecipitation.Sequencing results showed that BAI could bind to a CTT motif.And a gene encoding NEP,SCA3,was identified as the downstream target gene of BAI.After analyzing the promoter regions of SCA3 and HMR,Ch IP-seq results demonstrated that BAI is able to directly bind to the CTT-rich regions of the promoters of SCA3 and HMR.SCA3 and HMR are downstream target genes of BAI.5)The expression levels of key plastidial genes that depend on PEP and NEP in the wild type and bai were analyzed.The results showed that the expressions of PEP-dependent genes were significantly reduced in bai,while the expressions of NEP-dependent genes were enhanced.These indicated that PEP function is impaired in mutant bai.In summary,this study found an unreported C2H2 zinc finger transcription factor and explored the molecular mechanism of its involvement in the regulation of chloroplast gene transcription.Our results not only provide new insights into the regulation of photosystem development,but also fill in the uncharted territory of this specific area.