Functional Analysis Of H Subunit Of Glycine Decarboxylase In Rice

The glycine decarboxylase complex (GDC) is a multi-protein complex, comprising P-, H-, T-and L-protein subunits. It appears to be ubiquitous in all organisms, from bacteria to eukaryotic cells. Previous proteomic study in our laboratory displayed that expression of an OsGDCH protein which was encoded by gene LOC_Os10g37180. was up-regulated in resistant rice variety B5but was unchanged in susceptible rice variety TN after feeding of brown planthopper. The specific increased expression of GDCH but not the other subunits of the GDC led us to investigate whether this finding indicated a new role for GDCH.In this study, the OsGDCH overexpression vector and the OsGDCH suppressed expression vector-RNAi (RNA interference) vector (OsGDCH-RNAi) were constructed. Transgenic rice plants were generated by introducing the constructs into indica rice variety Kasalath and japonica rice variety Hejiang19by Agrobacterium-mediated transformation. Totally, eighty-four independent OsGDCH overexpression transgenic plants were generated, in which sixty-three were generated from indica rice variety Kasalath and twenty-two were generated from japonica rice variety Hejiang19; one hundred and three independent OsGDCH-RNAi plants were generated totally, in which twenty-eight were generated from indica rice variety Kasalath and seventy-five were generated from japonica rice variety Hejiang19. We then used Southern blot to identify the single copy transgenic plants. Expression of OsGDCH in OsGDCH overexpression transgenic plants and OsGDCH-RNAi plants were identified by Northern blot and Western blot analyses, results showed that both the OsGDCH overexpression transgenic plants and the OsGDCH-RNAi plants were successfully created. According to the OsGDCH suppression degree, the OsGDCH-RNAi plants were separated into two groups:the severely suppressed plants (SSPs) and the moderately suppressed plants (MSPs). The resistance of positive OsGDCH overexpression transgenic plants to brown planthopper was tested by seedbox screening technique, however, no obvious resistance to brown planthopper was observed in the positive OsGDCH overexpression transgenic plants. Phenotype identification of OsGDCH-RN Ai plants showed that MSPs grew normally, and no substantial difference in phenotype was seen between the MSPs and wild-type plants (WT). However, SSPs displayed an obvious photorespiration-deficient phenotype:the plants are normal under high CO2conditions (0.8%; non-photorespiratory condition), but are lethal under ambient CO2conditions (0.04%; photorespiratory condition). This result suggested that the OsGDCH was a typical photorespiratory gene. Using high-performance liquid chromatography (HPLC), the content of photorespiration-related amino acid was measured in both OsGDCH-KNAi plants and wild-type plants. Results showed that glycine was largely accumulated in OsGDCH-RNAi plants, and the ratio of glycine content to serine content was higher in OsGDCH-RNAi plants under ambient CO2than that in OsGDCH-RNAi plants under high CO2. These results demonstrated that normal photorespiratory metabolism was disordered in OsGDCH-RNAi plants, and further confirmed that the OsGDCH was a typical photorespiratory gene.Southern blot analysis identified a single copy of the OsGDCH gene in the rice genome. Bioinformatics analysis showed that the OsGDCH protein has two homologues, OsGDCH2and OsGDCH3. the expression of their encoding genes was also variously suppressed in OsGDCH-RNAi plants by real-time quantitative PCR (qPCR) analysis. Tissue expression pattern analysis demonstrated that the OsGDCH was highly expressed in photosynthesis-related tissues; OsGDCH2and OsGDCH3displayed a high expression in roots and stems but the expression was seldom detected in leaves. By qPCR, expression of genes that encode the P subunit (OsGDCP) and the T subunit of GDC (OsGDCT) was also analyzed in OsGDCH-RN At plants. Results showed that the expression of OsGDCP and OsGDCT was also down-regulated in SSPs.After5days growth under ambient CO2. physiological measurement showed that chlorophyll (Chl) loss, a decrease of the Ch1a/Ch1b ratio, degradation of proteins occurred in SSPs; oxidation of membrane lipids intensively occurred, and the content of malonyldialdehyde increased in SSPs:photosynthetic rates declined, content of sucrose decreased in SSPs after growth under ambient CO2After SSPs were cultivated under ambient CO2conditions, vacuolation of mesophyll cells, chloroplast breakdown, and generation of autophagy-related structures were observed by electron microscopy. Disruption of chlorophyll complex was detected in SSPs grown under ambient CO2by blue native polyacrylamide gel electrophoresis (BN-Page). By qPCR, gene expression analysis demonstrated that senescence-associated genes (SAGs) were up-regulated and senescence down-regulated genes (SDGs) were down-regulated in SSPs after transfer from high CO2and cultivated under ambient CO2conditions. At the same time, expression of senescence-related autophagy genes was also increased in SSPs after transfer from high CO2and cultivated under ambient CO2conditions. All these results demonstrated clearly that SSPs displayed an early senescence syndrome under ambient CO2conditions.Histochemical assays showed that reactive oxygen species (ROS), including superoxide (CO2-) and hydrogen peroxide (H2O2), accumulated in the leaves of SSPs under ambient CO2. Enzyme activities measurement analysis for ROS scanvage-related enzymes, including Superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD) and Ascorbate peroxidase (APX), was carried out. Result showed that, except for POD, the antioxidant enzyme activities of SOD, CAT and APX were much lower in SSPs under ambient CO2than those in WT plants under the same conditions. The expression of SAGs, SDGs and senescence-related autophagy genes was analyzed in wild-type plants after different time periods treated with exogenous H2O2. Result showed that the expression of the SAGs and some senescence-related autophagy genes, which was up-regulated in SSPs after transfer to ambient CO2, was enhanced in WT. plants treated with H2O2. and the expression of the SDGs. which was down-regulated in SSPs after transfer to ambient CO2, was decreased in WT plants treated with H2O2. Evidences demonstrated that ROS induced senescence in SSPs under ambient CO2conditions.Results also showed that expression of senescence-related transcription factors was up-regulated in SSPs after transfer from high CO2and cultivated under ambient CO2conditions. Further analysis showed that expression of these senescence-related transcription factors was enhanced in WT plants treated with H2O2. The induced expression of transcription factors OsWRKY72was quick and rapid, which suggested that the transcription factors OsWRKY72may mediate the ROS-induced senescence in SSPs under ambient CO2conditions.