Molecular Mechanism Of Arabidopsis CDB1 Protein Involved In Chloroplast Biogenesis

Chloroplast is a kind of semi-autonomous organelle present in eukaryotic photosynthetic organisms and is surrounded by inner and outer membranes.It is the place for photosynthesis.Chloroplast biogenesis is a highly programmed and complicated process regulated by both nucleus and plastid encoded genes.In this process,the bacterial RNA polymerase(plastid-encodedpolymerase,PEP)encoded by the plastid gene and the phage RNA polymerase(nucleus-encodedpolymerase,NEP)encoded by the nuclear gene are responsible for the transcription of the chloroplast gene,while the proteins encoded by chloroplast genome are translated on the chloroplast 70 S ribosome.Chloroplast-encoded genes can be divided into three categories.While transcription of class I and class III genes depends on PEP and NEP,respectively,PEP and NEP can transcribe class II genes.It has been domenstrated that formation of chloroplast ribosomes is not only a simple combination of its constituent subunits and ribosomal r RNA,but also regulated by many protein factors.Up to now,many protein factors involved in chloroplast gene expression have been reported,but the specific process of ribosome biogenesis and its regulatory network are still unclear.In this thesis,we characterized two Arabidopsis thaliana proteins CDB1(Chloroplast Development and Biogenesis 1)and CDB1L(CDB1-like protein).Following results were obtained:1.CDB1 protein is a nuclear-encoded chloroplast protein.It contains a chloroplast transport peptide.No transmembrane domain and functional domain were found in CDB1.The cdb1 mutant displays white leaf phenotype and can not survive autotrophically in the soil.Transmission electron microscope observation showed that chloroplasts in cdb1 are smaller than those in WT and there is not complete and regular thylakoid structure,indicating that deletion of CDB1 leads to the developmental defect of chloroplasts.2.RNA-seq analysis showed that transcripts level of class I genes are decreased in the cdb1 mutant,which is due to the absence of the core subunits Rpo B and Rpo C2 of PEP.Western blot and RNA gel blot analysis showed that chloroplast ribosomal protein and chloroplast ribosomal r RNA are barely detected in the cdb1 mutant.Thus,our results indicate that there is no functional chloroplast ribosome were formed in cdb1,which leads to the inability to translate the proteins encoded by chloroplast genes,including Rpo B and Rpo C2.Yeast two-hybrid and Bi FC experiments showed that CDB1 interacts with RPL27 which is a subunit of 70 S ribosome.It suggesting that CDB1 regulated the biogenesis of 70 S ribosome through the interaction with RPL27.3.The homolog of CDB1 widely exists in green algae and terrestrial plants.They exhibit highly similar in sequence and structure,suggesting that they have similar functions.In addition,there is a homologous protein CDB1 L in angiosperms.The phylogenetic tree of CDB1 homologs showes that CDB1 proteins from lower photosynthetic Viridiplantae are closer to CDB1 L than to CDB1 in angiosperms.CDB1 is located in chloroplast stroma,while CDB1 L is expressed in both chloroplasts and mitochondria.Genetic analysis shows that the deletion of CDB1 L protein results in arrested embryo development,which is probably due to the defects in the mitochondria development.In addition,like in the cdb1 heterozygous plants,some of the seeds can mature but they have a pale color in cdb1 l heterozygous plants.It suggesting that CDB1 L is also required for the chloroplast development.In summary,our results show that chloroplast-located CDB1 is essential for chloroplast development.It may regulate 70 S ribosomal biogenesis by interacting with70 S ribosomal subunits RPL27.The dual-localized CDB1 L proteins in angiosperms may have a similar role as CDB1 from lower photosynthetic Viridiplantae,and operate in both chloroplasts and mitochondria.