Diversity In Subcellular Location Of Plant LrgB Homologies And Its Potential Implications

The plant LrgB protein is homologous with bacterial LrgA and LrgB proteins, which is a chloroplast outer membrane protein first identified in Arabidopsis thaliana. It plays an important rule in chloroplast development and programmed cell death (PCD) regulation. Bio informatics analysis shows that LrgB homologous gene is prevalent in plant kingdom, and some plants have more than one LrgB homologies which part of them do not contain chloroplast transition peptide(CTP), suggesting that location of LrgB is various. In this study, we foucus on the diversity in the subcellular localization of plant LrgB homologies and their function on PCD regulation.In ous research, when staining leaves of atlrgB mutants using PI dye, we observed cell death, producing a powerful evidence for the role of LrgB in the regulation of cell death. In the study of diversity in subcellular location of plant LrgB homologies, we have constructed expression vectors of LrgB homologies for the localization of Ostreococcus tauri, Physcomitrella patens, Oryza sativa and Arabidopsis thaliana, and then expressed them separately in Arabidopsis thaliana, results showed that Ostreococcus tauri OtLrgB was located at endoplasmic reticulum; both Physcomitrella patens PpLrgB-1and Arabidopsis thaliana AtLrgB were located at chloroplast membrane; Oryza sativa OsLrgB-2was at plasma membrane. All the experiments above demonstated that plant LrgBs with conserved membrane spaning domain are diversity in subcellular localization, and maybe having a correlation between the localization diversity and the functions diversity. It perhaps is the key of LrgB participating in PCD regulation.Due to mitochondria having a prominent role in PCD regulation, we have constructed localization expression vectors of Arabidopsis thaliana AtLrgB with mitochondria transition peptide or without any transition peptide, results showed that they are located at mitochondria membrane and endoplasmic reticulum respectively.In order to study the functional evolution of plant LrgB, we also constructed series of localization expression vectors and overexpression vectors of bacterial CidAB/LrgAB system with chloroplast or mitochondria transition peptide or without any transition peptide. So far we have observed bacterial LrgAB fusion protein at chloroplast membrane. Furturemore, with the purpose of study the differences and similarities in subcellular location and possible interaction between LrgB homologies and PCD related protein factors, expression vectors of some PCD related genes have been constructed.Due to our hard working on Physcomitrella patens LrgB homologous gene knockout, so far we have got PpLrgB knockout clones, and then we will verify these clones on molecular level, after that we will make a further study on the functions of LrgB in Physcomitrella patens. The successful knockout of PpLrgB will greatly contribute to the study of LrgB in oher species and improves the analysis of functional conservatism and evolution greatly.Collectively, we confirmed the diversity in subcellular localization of plant LrgB homologies, and generated a variety of genetic materials. It should be helpful for exploring the mechanism of plant LrgB proteins in chloroplast development and PCD.