| Functional chloroplasts are the main sites of photosynthesis,providing both relatively independent space environment and the key proteins for its successful operation.Derived from cyanobacterial ancestors,the chloroplasts belong to semi-autonomous organelles regulated not only by its own genes,but by nuclear signals to a large extent,which leads to very complex processes of chloroplast biogenesis and regulatory mechanisms.The pentatricopeptide repeat(PPR)proteins constitute one of the largest nuclear-encoded protein families in higher plants and involved in the post-transcriptional regulation of chloroplast and/or mitochondrial genes,including RNA editing,intron splicing,processing and translation initiation,and may also have the function of retrograde signal regulation.Although PPR proteins play a key role in the regulation of chloroplast gene expression and development,due to the large number of family members,the molecular mechanism of individual PPR protein remains to be further studied.In this study,the model plants Arabidopsis thaliana and Nicotiana benthamiana were chosen and the follow-up research materials were obtained through the screening and identification of the mutants and the overexpression transgenic plants.Subsequently,the function of PDM4 in gene expression and chloroplast development were investigated by mean of multiple approaches including molecular biological techniques,cell biological,physiological index detection,et al.The main results are as follows:(1)Sequence alignment and bioinformatics analysis of the PDM4 gene revealed that it belongs to P type subfamily of PPR proteins,and ubiquitous in green plants with relative conserved sequence.Quantitative real-time PCR(q RT-PCR)indicated that PDM4 was expressed in all plant tissues,and the highest expression was found in the chloroplast active parts;Confocal microscopy obseration and Western Blot analysis revealed that PDM4 was localized in the chloroplast and subcellular in the chloroplast stroma.(2)pdm4 mutant showed an albino phenotype and without photosynthetic autotrophy;the complementation analysis of the pdm4 phenotype indicated that the PDM4 is a functional protein,and PDM4 gene was responsible for the phenotype of the pdm4.(3)Loss of functions of PDM4 resulted in a significant decrease in chlorophyll content,destructive change in chloroplast morphology,and an obvious decrease in the accumulation of photosynthesis-related complexes(subunits of PSII,PSI,Cyt b6 f and ATPase).Microscope observation showed the embryo development in the pdm4 mutant was significantly delayed during the global stage compared with the wild type.These results indicated that PDM4 plays an important role in chloroplast development.(4)PDM4 involved in splicing of multiple chloroplast group II introns according to the results of reverse transcription PCR(RT-PCR)and Northern Blot analyses.Compared with wild type,the unspliced precursors of several genes accumulated to an increased level,and mature fragments decreased in the pdm4 mutant.(5)q RT-PCR and Northern Blot analyses showed that the PDM4 mutation affects NEP-dependent genes expression.The transcription level of PEP-dependent genes in pdm4 was dramatically decreased,and the transcript levels of the plastid genes were increased by varying degrees in pdm4.(6)pdm4 mutant is defective in chloroplast r RNA accumulation and processing,which proved by RNA immunoprecipitation(RIP)and Northern Blot.By RIP and q RT-PCR analysis,we found that PDM4 can specifically bind to 23 S r RNA,5S r RNA and4.5S r RNA.Therefore,we inferred that PDM4 may affect r RNA processing by binding to r RNA.In summary,this study elucidated that PDM4 protein in Arabidopsis thaliana is involved in the transcriptional regulation of chloroplast genes,and plays a key role in regulating chloroplast gene expression and development.These results laid a foundation for further studying the molecular mechanism of PPR protein affecting chloroplast development and can improve the regulatory network of plant growth. |
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