Screening And Functional Identification Of BPG2/YL1 Interacting Proteins In Arabidopsis

Chloroplast is a kind of unique organelle in plant cells.Many important metabolic pathways in plant cells are carried out in chloroplasts.Solar energy could be convert into chemical energy for cell growth and plant development through photosynthesis.Most chloroplast proteins are encoded by nuclear genomes and transferred from cytoplasmic ribosomes to chloroplasts.A few chloroplast proteins are encoded by genes from chloroplast genome and synthesized by chloroplast ribosome in chloroplasts.The assembly and functional defects of chloroplast ribosome not only affects the synthesis of chloroplast genome-encoding proteins,but also affects the development and activity of chloroplast.Therefore,the assembly and translation regulation of chloroplast ribosome is crucial for cell activity,plant growth and development.BPG2 is a nuclear genome-encoding Yqe H GTPase located in chloroplast.It contains a zinc finger domain and four GTP combined domains.bpg2 mutant has a yellow leaves phenotype,indicating that BPG2 is necessary for the normal development of chloroplasts.Because BPG2 can specifically regulate the accumulation of 16 S r RNA and 23 S r RNA in chloroplasts,it may affect chloroplast development by regulating the assembly and translation activities of chloroplast ribosomes.In the process of plants adapting to the external environment,plant cells need to integrate numerous external and internal signals to adjust the life activities inside the cells,including the regulation of chloroplast gene expression.Previous study indicated that the expression of BPG2 could be regulated by light and Brz,and might as an important regulatory factor in BR signaling pathway for the regulation of chloroplast development.However,the mechanism of BPG2 playing roles in chloroplast development remains to be studied.In this study,two potential BPG2 interacting proteins,RABe1 b and PRPS10 a,were identified using Co-IP and Yeast two-hybrid techniques,providing a basis for further understanding the roles of BPG2 in regulating chloroplast ribosome assembly and chloroplast development.The main results of this study were summarized as follows:1.BPG2-GFP gene was transformed into Arabidopsis: Plasmids of p CAMBIA2300-BPG2-GFP was constructed and transgenic plants of Arabidopsis were obtained by agrobacterium mediated transformation.After antibiotic screen,11 homozygous transgenic lines were obtained to provide materials for the immunoprecipition experiment.2.Co-IP was used to identify BPG2 interacting proteins: GFP antibody was used to precipitate BPG2-GFP interacting proteins from protein extracts of transgenic plants expressing p CAMBIA2300-BPG2-GFP.BPG2 specific polyclonal antibodie was used to obtain BPG2 potential interaction proteins from protein extracts of wild-type plant,and the above immunco-precipitated proteins were further identified by mass spectrometry.Through the analysis of the mass spectrometry results,several chloroplast localization proteins including RABe1b(AT4G20360)were preliminarily identified.This study focuses on the interaction between BPG2 and RABe1 b and the functional analysis of RABe1 b.3.BPG2 interacting proteins were identified by yeast two-hybrid: p GBKT7-BPG2 bait plasmid was constructed and was used for yeast two-hybrid library screening.Through sequencing,sequence alignment,protein localization and functional analysis of the positive clones,six BPG2 interacting proteins located in chloroplasts were finally identified,include DEP(AT5G53850),DKLA(AT4G00895),PRPS10a(AT3G13120),PIP(AT2G14260),RAB18(AT1G43890)and PGPP1(AT3G58830).The yeast two-hybrid results further showed that PRPS10 a could interact directly with BPG2 in vitro,and showed that PRPS10 a could interacts with BPG2's GTP binding domain.4.RABe1 b function analysis: Plasmids of p CAMBIA2300-RABe1b-GFP was constructed and transgenic plants of Arabidopsis were obtained by agrobacterium mediated transformation.The results of confocal microscopy showed that RABe1 b located in chloroplasts.Through the phenotypic comparison and analysis with the wild type,we found that the RABe1 b over-expression plants have a albinism phenotype with different degrees.RABe1b knock-out causes embryonic death.Through phenotypic analysis on RABe1b knock-down mutant rabe1b-2,we found that its chlorophyll content is significantly lower than that of the wild type.In addition,rabe1b-2 also showed abnormal leaf and primary root growth,indicating that RABe1 b is crucial for plant growth and development.5.Functional analysis of PRPS10a: Plasmids of p CAMBIA2300-PRPS10a-GFP was constructed and transgenic plants of Arabidopsis were obtained by agrobacterium mediated transformation.Confocal microscopy showed that PRPS10a was located in the chloroplast nucleoid.PRPS10 a gene was knocked out in wild type and bpg2-2 mutant plants respectively using CRISPR/Cas9 system.Through phenotypic analysis,we found that prps10 a had variegated cotyledon phenotype,while prps10 a bpg2-2 showed severe yellow cotyledon phenotype,suggesting that the role of PRPS10 a and BPG2 in regulation of chloroplast development.