Overexpression Of Nitrogen Key Genes Modified Carbon And Nitrogen Metabolism In Rice

To make sure the food security, growing attempts have been conducted to improve crop yields by overexpressing a single key gene involved in nitrogen (N) metabolic pathway using transgenic technology. However, most of previous studies suggested that transgenic plants just accumulated gene transcripts without yield increase. To date, few studies have analyzed the effects of overexpression of these genes on carbon and nitrogen (C-N) metabolism in plant growth and development.In this study,C-N metabolism of transgenic rice plants including overexpression of GS1;1, GS1;2, AMT1;3, GS2(OX-GS1;1, OX-GS1;2, OX-AMT1;3, OX-GS2) and wild type plants (Zhonghua11) were evaluated. Through a series of studies on the growth phenotype and yield; SPAD values and photosynthesis parameters; soluble proteins and carbohydrates content; total C, N and C/N ratio; activity of NR, GS and RUBISCO; expression levels of key genes involved in C-N metabolism as well as N uptake and transport, we have studied the effect of overexpression of GS1;1, GS1;2, AMT1;3, GS2on C-N metabolism and revealed the main cause of lower biomass and yield production of transgenic plants, which will provide the useful knowedge of physiological and biochemical basis and molecular mechanisms on N efficient rice cultivation. The main results are as follows.1. C-N metabolism analysis of GS1;1, GS1;2-overexpressed plantsThe growth phenotype and C-N metabolism of OX-GSl;1, OX-GS1;2transgenic plants and wild type plants (WT, Zhonghual1) were analyzed under different N conditions (no N, low N, normal N, excessive N). Results suggested that transgenic plants had significant worse growth and yield when compared with WT. Based on the results of leaf SPAD values and photosynthesis parameters; soluble proteins and carbohydrates content; total C, N and C/N ratio; GS and RUBISCO activity analysis, we found that the overexpression of GS1;1or GS1;2had a significant effect on the C and N metabolism and the C/N balance, In addition, overexpression of GS1;1or GS1;2also changed the inorganic N uptake and transport in the transgenic plants by15N isotope tracing assay. According to the expression analysis of key genes involved in C and N metabolic pathways by qRT-PCR, genes involved in both C and N metabolic pathways were up-regulated in GS1;1-overexpressed plants only under enough N supply condition; genes involved in N metabolic pathway were up-regulated under low N condition, while genes involved in C metabolic pathway were down-regulated in OX-GS1;1plants. For G<S1;2-overexpressed plants, the expression of genes involved in C and N metabolic pathways were inhibited by the overexpression of GS1;2under no and excessive N conditions in roots. Additionally, the expression patterns of homologous genes in a family were not entirely consistent under low and normal N conditions. Overall, the overexpression of GS1;1or GS1;2caused lower N uptake capacity, changed C and N metabolites contents and altered the expression levels of key genes involved in C and N metabolic pathways, which would affect normal growth and yield production of transgenic plants.2. C-N metabolism analysis of AMT1;3-overexpressed plantsSimilarly, the growth phenotype and C-N metabolism of OX-AMT1;3transgenic plants and wild type plants (WT, Zhonghua11) were analyzed under different N conditions (no N, low N, normal N, excessive N). Results indicated that transgenic plants had significant worse growth and yield when compared with WT. OX-AMT1;3showed lower photosynthetic rate, changed soluble proteins and carbohydrates content, lower N and C contents, higher C/N ratio, reduced activity of NR and GS. Besides, the overexpression of AMT1;3led to reduced inorganic N uptake and transport by15N isotope tracing assay. According to the expression analysis for key genes involved in C and N metabolic pathways by qRT-PCR, the more nitrogen application, the more up-regulated genes in AMT1;3-overexpressed plants. Overall, the overexpression of AMT1;3caused reduced C and N metabolic levels, which would affect normal growth and yield production of transgenic plants.3. C-N metabolism analysis of GS2-overexpressed plantsThe OX-GS2transgenic plants showed higher GS2expression level, green leaves and normal growth at seedling stage; while it displayed lower GS2expression level, yellow leaves, abnormal growth at tillering stage. The OX-GS2transgenic plants and wild type plants (WT, Zhonghua11) were cultivated under different N sources (NH4NO3、 NH4NO3+Gln、Gln) until seedling and tillering stage, respevtively. The SPAD values and photosynthesis parameters; soluble proteins and carbohydrates content; total C, total N, C/N ratio; NR and GS activity and expressions of key genes involved in C, N metabolic pathways were investigated. Results suggested that OX-GS2transgenic plants showed higher soluble proteins content and lower carbohydrates content in stem and leaves at seedling stage; while lower soluble proteins content and higher carbohydrates content in stem and leaves at tillering stage when compared with wild type plants. When N source is NH4NO3, most of key genes involved in C and N metabolic pathways were down-regulated in roots and up-regulated in leaves at seedling stage,while most of key genes were up-regulated in roots and down-regulated in leaves at tillering stage. Besides, higher N content and lower C/N ratio in the stem of transgenic plants and lower inorganic N uptake and transport capacity were observed. Changes of genes expression patterns and C, N metabolic levels between seedling stage and tillering stage may caused the imbalance of carbon and nitrogen metabolism and abnormal growth in OX-GS2transgenic plants. The different physiological metabolism between seedling stage and tillering stage in OX-GS2transgenic plants is possibly the result of self-regulation of plants response to stress.