The Evolution Analysis And Biological Functions Of AtLrgB Gene In Arabidopsis Thaliana

Bacterial lrgA/B and cidA/B operons encode murein hydrolase regulator involved in autolysis control. The Arabidopsis genome shares a homolog of lrgB namely AtLrgB. BLAST searches of the complete Arabidopsis genome reveal that many enzymes responsible for peptidoglycan synthesis do not exist, so chloroplast peptidoglycans may not be present in Arabidopsis. BLAST searches also negate the existence of the murein hydrolase in Arabidopsis. Thus, a change in the function of the protein occurs during the evolution. In this study, we analyze the phenotypes of AtLrgB mutant and overexpressant, and deduce the possible function of the Arabidopsis AtLrgB gene. Results obtained in the present study are summarized as follows:1. An AtLrgB-EGFP fusion was constructed to analyze the subcellular localization of AtLrgB protein. Localization of AtLrgB-GFP fusion product suggested that AtLrgB was targeted to the chloroplast inner envelope. Meanwhile, PAtLrgB:GUS report lines were generated. The histochemical localization of GUS staining indicated that AtLrgB is expressed in shoots, but not in roots. GUS expression disappeared from the leaf tip to the base of the mature leaves and became restricted to the younger tissues. This expression pattern demonstrated that the AtLrgB promoter was regulated in a manner that showed resemblance to the sink-to-source transition of leaves. Investigation of accumulation of AtLrgB transcripts during a photoperiod by real-time PCR indicated that AtLrgB gene was a diurnal-regulated gene.2. AtlrgB-1 showed slightly pale-green in mesophyll of cotyledons and juvenile leaves, and so did the AtLrgB-RNAi lines. When growing on agar medium for about 8 days, necrosis started to appear on cotyledons of the atlrgB-1 mutant and then on the first true leaves. PAtLrgB:AtLrgB construct was generated to complement the abnormal phenotypes in atlrgB-1 mutant. The result demonstrated that mutant phenotype cosegregated with the T-DNA insertion.3. Transgenic 35S:AtLrgB lines were obtained to investigate the role of AtLrgB. Phenotype analysis of homozygous plants showed that overexpression of AtLrgB led to chlorotic leaves and retarded growth.4. The ultrastructure of chloroplasts on leaf sections of wild-type, atlrgB-1 mutant, and 35S:AtLrgB plants were observed. For the mesophyll cells, the thylakoid membrane organization in the chloroplasts of both the atlrgB-1 mutant and 35S:AtLrgB plants were much less abundant. AtlrgB-1 mutant plants obviously had more and larger starch grains accumulating in the chloroplasts, while starch grains in chloroplasts from 35S:AtLrgB plants vanished or became smaller than the wild-type. In the bundle sheath cells, chloroplasts from atlrgB-1 mutant plants were normal, while those from the 35S:AtLrgB plants were smaller and had fewer amount of granal membranes.5. The starch contents in leaves of the wild-type, atlrgB-1 mutant and 35S:AtLrgB plants were measured. The results demonstrated that atlrgB-1 mutant accumulated excess starch than wild-type during the day, while the 35S:AtLrgB plants had less starch accumulated. The sucrose levels in all of the three lines were also measured. In the atlrgB-1 mutant plants, sucrose content remained lower than the wild-type plants during the light period. At the dark period, the sucrose content of the mutant increased sharply, and then gradually decreased during the dark period. As for the 35S:AtLrgB plants, their sucrose levels were normal at the beginning of the light period, except a bit increase at the later light period, while during the dark period, their sucrose levels were lower than the wild-type plants.6. The effect of sucrose in the medium on the development of the atlrgB-1 mutant and 35S:AtLrgB plants were investigated. The atlrgB-1 seedlings on medium with 2% sucrose displayed alleviative phenotypes than those on medium with normal 1% sucrose, with necrosis appearing later and necrosis region smaller, while the chlorotic phenotype of the 35S:AtLrgB plants were more severe and obvious. In addition, exogenous glucose had the similar effect on the atlrgB-1 and 35S:AtLrgB plants as exogenous sucrose. These observations indicated that the abnormal phenotypes caused by the aberrant quantity of the AtLrgB protein were associated with carbodydrate availability.7. BLAST searches in the NCBI were done and the results showed that numerous homologous protein of AtLrgB existed in bacteria and plants. Amino acid sequences alignment of LrgB domains showed that they were conserved from bacteria to plants. Alignment of the N-terminal amino acids of these proteins with the LrgA gene of prokaryotes indicated that they shared some similarity with the prokaryotic LrgA domain, although this similarity was lower than that of the LrgB domain. These results demonstrated that gene fusion happened between LrgA and LrgB during plant evolution.8. To overexpress truncated form of AtLrgB,35S:AtLrgB-D1 and 35S:AtLrgB-D2 transgenic lines were generated. Phenotype analysis of homozygous plants showed that 35S:AtLrgB-Dl and 35S:AtLrgB-D2 plants exhibited chlorosis phenotypes, similar to the 35S:AtLrgB plants overexpressing the full-length gene. Meanwhile, PAtLrgB:AtLrgB-Dl and PAtLrgB:AtLrgB-D2 constructs were generated to complement the abnormal phenotypes in atlrgB-1 mutant. Both the constructs could not complement the atlrgB-1 mutation, indicating that the accurate function of AtLrgB depended on the existence of the entire protein. Thus, it was possible that the prokaryotic LrgA and LrgB evolved to become two functional domains of the AtLrgB protein in Arabidopsis.