Functional Analysis Of Glutamate Synthase Genes And Cytosolic Isocitrate Dehydrogenase Genes And Genome-wide Analysis Of Amino Acid Transporter Gene Family In Rice

Nitrogen is an essential macroelement for plant growth and development, and is one of the most important limiting factors in crop production. In recent decades, luxury application of nitrogen fertilizer greatly enhanced crop production. However, it also caused severe environmental contamination. Contradictorily, demands for food are still increasing because of the increasing population in the world. To solve this problem, improving crop nitrogen use efficiency (NUE) through biotechnology is a smart choice. Rice is the staple food of China and the demand of nitrogen fertilizer in its production is huge, it is of great significance to improve the NUE of this crop.It is valuable to thoroughly identify the biological functions of key genes of nitrogen absorption and assimilation for understanding the molecular mechanism of carbon and nitrogen metabolism and improving crop NUE. The glutamine synthetase-glutamate synthase (GS-GOGAT) pathway is located at the cross-point of carbon and nitrogen metabolism, suggesting its important role in NUE improving engeering, through which inorganic nitrogen can be transformed into organic nitrogen. Researches focused on GOGAT genes are less, which lead to an indistinct understanding of its fuction in nitrogen assimilation. Another problem in GS-GOGAT pathway is that the origin of carbon skeleton (2OG, a-ketoglutarate) provided for this pathway in nitrogen assimilation is still unclear. In this research, biological functions of NADH-GOGAT in carbaon and nitrogen metabolism and the roles of cytosolic ICDH (NADP-dependent isocitrate dehydrogenase) in providing2OG for ammonium assimilation were systematically investigated, through testing the carbon and nitrogen metabolites contents, related enzyme activities and selected genes transcription levels, using NADH-GOGAT or cytosolic ICDH cosuppressed transgenic rice plants. In addition, molecular characterization, expression and functional analysis of rice amino acid transporter (OsAAT) gene family were also conducted in this research. The main results are as follows:1. Constitutive expression of OsNADH-GOGAT in japonica rice caused cosuppression of both OsNADH-GOGAT1and OsNADH-GOGAT2. In nitrogen sufficient field condition, NADH-GOGAT cosuppressed transgenic lines exhibited nitrogen deficient phenotype, such as decreased tiller number, plant height, yeild of per plant and thousand kernel weight. Metabolic analysis showed that suppression of NADH-GOGAT caused a decrese of the contents of leaf soluble sugars, total nitrogen and most of nitrogenous compounds, and an increase of the contents of2OG, isocitrate and free ammounium. The activities of some carbon and nitrogen metabolism related enzymes were also affected. These results proved that NADH-GOGAT playing an important role in rice carbon and nitrogen metabolism, and it is indispensible in the process of improving NUE in rice.2. Constitutive expression of OsICDH2in rice caused cosuppression of both OsICDH1and OsICDH2. Compared with wild type, ICDH activity in leaves and roots of cosuppression lines decreased43.5%-76.6%and19.4%-56.4%, respectively. Under sufficient nitrogen conditions, ICDH cosuppressed plants showed a retarded growth phenotype, yield of per plant in field were significantly reduced, which suggests that ICDH is essential for rice to maintaining the high production.3. When exposed to high temperature for3-6days, ICDH cosuppressed plants become chlorosis and eventurally died, while the wild type grows normally. Biochemical and physiological analysis showed that, under high temperature condition, when compared with wild type, ICDH cosuppressed lines showed decreased contents of leaf chlorophyll, nitrate, some organic acids and solulble sugars, and increased contents of leaf free ammonium, total free amino acids, asparagine and glucose-6-phosphate, increased activities of glutamate dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, fructokinase and glucokinase, and decreased GS activities, higher expression levels of OsGDHl and OsGDH2, and lower expression levels of OsGS2and OsFd-GOGAT1. We suggest that both NAD-IDHs (NAD-dependent isocitrate dehydrogenase) and cytosolic ICDH can provides2OG for GS-GOGAT cycle for nitrogen assimilation, NAD-IDHs are responsible for the major source of2OG, whereas the2OG produced by cytosolic ICDHs might act as an important compensation, especially in circumstances, such as high temperature, in which the rate of2OG production by mitochondria is inadequate. Glutamate dehydrogenase, aspartate aminotransferase, and2OG translocators may also help to supplement the inadequate supply of2OG under high temperatures.4. Totally65OsAAT genes were identified in rice genome. Among them,49were supported by full length cDNA, some of OsAAT genes exhibited tissue preferential or nitrogen starvation induced expression patterns. T-DNA insertion mutant lines of11OsAAT members were identified and characterized, some of them exhibited modified carbon and nitrogen contens, suggesting these genes play significant roles in carbon and nitrogen distribution in rice.