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Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO(2) concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.

The metabolism of vegetative organs in plants changes during the development of the reproductive organs. The regulation of this metabolism is important in the control of crop productivity. However, the complexity of the regulatory systems makes it difficult to elucidate their mechanisms. To examine these mechanisms, we constructed model experiments using Arabidopsis to analyze metabolic and gene expression changes during leaf-stage progression and after removal of the reproductive organs. Leaf gene expression levels and content of major amino acids, both of which decreased during leaf-stage progression, increased after removal of the reproductive organs. In particular, the levels of expression of cytokinin biosynthesis genes and cytokinin-responsive genes and the cytokinin content increased after removal of the reproductive organs. Analysis of plants with knockout of a cytokinin-biosynthetic gene (AtIPT3) and a cytokinin receptor gene (AHK3) indicated that glutamate dehydrogenase genes (GDH3) were regulated by cytokinin signaling. These data suggest that cytokinins regulate communication between reproductive and vegetative organs, and that GDH3 is one target of the cytokinin-mediated regulation of nitrogen metabolism.

In the photorespiratory process, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutarate and glycine. Although GGAT has been assumed to play important roles for the transamination in photorespiratory carbon cycles, the gene encoding GGAT has not been identified. Here, we report that an alanine:2-oxoglutarate aminotransferase (AOAT)-like protein functions as GGAT in peroxisomes. Arabidopsis has four genes encoding AOAT-like proteins and two of them (namely AOAT1 and AOAT2) contain peroxisomal targeting signal 1 (PTS1). The expression analysis of mRNA encoding AOATs and EST information suggested that AOAT1 was the major protein in green leaves. When AOAT1 fused to green fluorescent protein (GFP) was expressed in BY-2 cells, it was found to be localized to peroxisomes depending on PTS1. By screening of Arabidopsis T-DNA insertion lines, an AOAT1 knockout line (aoat1-1) was isolated. The activity of GGAT and alanine:glyoxylate aminotransferase (AGAT) in the above-ground tissues of aoat1-1 was reduced drastically and, AOAT and glutamate:pyruvate aminotransferase (GPAT) activity also decreased. Peroxisomal GGAT was detected in the wild type but not in aoat1-1. The growth rate was repressed in aoat1-1 grown under high irradiation or without sugar, though differences were slight in aoat1-1 grown under low irradiation, high-CO2 (0.3%) or high-sugar (3% sucrose) conditions. These phenotypes resembled those of photorespiration-deficient mutants. Glutamate levels increased and serine levels decreased in aoat1-1 grown in normal air conditions. Based on these results, it was concluded that AOAT1 is targeted to peroxisomes, functions as a photorespiratory GGAT, plays a markedly important role for plant growth and the metabolism of amino acids.

Raffinose family oligosaccharides (RFO) accumulating during seed development are thought to play a role in the desiccation tolerance of seeds. However, the functions of RFO in desiccation tolerance have not been elucidated. Here we examine the functions of RFO in Arabidopsis thaliana plants under drought- and cold-stress conditions, based on the analyses of function and expression of genes involved in RFO biosynthesis. Sugar analysis showed that drought-, high salinity- and cold-treated Arabidopsis plants accumulate a large amount of raffinose and galactinol, but not stachyose. Raffinose and galactinol were not detected in unstressed plants. This suggests that raffinose and galactinol are involved in tolerance to drought, high salinity and cold stresses. Galactinol synthase (GolS) catalyses the first step in the biosynthesis of RFO from UDP-galactose. We identified three stress-responsive GolS genes (AtGolS1, 2 and 3) among seven Arabidopsis GolS genes. AtGolS1 and 2 were induced by drought and high-salinity stresses, but not by cold stress. By contrast, AtGolS3 was induced by cold stress but not by drought or salt stress. All the GST fusion proteins of GST-AtGolS1, 2 and 3 expressed in Escherichia coli had galactinol synthase activities. Overexpression of AtGolS2 in transgenic Arabidopsis caused an increase in endogenous galactinol and raffinose, and showed reduced transpiration from leaves to improve drought tolerance. These results show that stress-inducible galactinol synthase plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions, and that galactinol and raffinose may function as osmoprotectants in drought-stress tolerance of plants.

The metabolism of vegetative organs in plants changes during the development of the reproductive organs. The regulation of this metabolism is important in the control of crop productivity. However, the complexity of the regulatory systems makes it difficult to elucidate their mechanisms. To examine these mechanisms, we constructed model experiments using Arabidopsis to analyze metabolic and gene expression changes during leaf-stage progression and after removal of the reproductive organs. Leaf gene expression levels and content of major amino acids, both of which decreased during leaf-stage progression, increased after removal of the reproductive organs. In particular, the levels of expression of cytokinin biosynthesis genes and cytokinin-responsive genes and the cytokinin content increased after removal of the reproductive organs. Analysis of plants with knockout of a cytokinin-biosynthetic gene ( AtIPT3) and a cytokinin receptor gene ( AHK3) indicated that glutamate dehydrogenase genes ( GDH3) were regulated by cytokinin signaling. These data suggest that cytokinins regulate communication between reproductive and vegetative organs, and that GDH3 is one target of the cytokinin-mediated regulation of nitrogen metabolism. [ABSTRACT FROM AUTHOR]