No abstract

The ability to coordinate carbon (C) and nitrogen (N) metabolism enables plants to regulate development and metabolic responses to different environmental conditions. The regulator(s) or sensor(s) that monitor crosstalk between biosynthetic pathways and ultimately control the flow of C or N through them have remained elusive. We used an antisense strategy to demonstrate that the putative glutamate receptor 1.1 (AtGLR1.1) functions as a regulator of C and N metabolism in Arabidopsis. Seeds from AtGLR1.1-deficient Arabidopsis (antiAtGLR1.1) lines did not germinate in the presence of an animal ionotropic glutamate receptor (iGLR) antagonist, but germination was restored upon coincubation with an iGLR agonist or the putative ligand glutamate. In antiAtGLR1.1 lines, endogenous abscisic acid (ABA) concentrations increased with iGLR antagonist treatments and decreased with coincubation with an iGLR agonist, suggesting that germination was controlled by ABA. antiAtGLR1.1 seedlings also exhibited sensitivity to increased levels of Ca 2؉ compared with wild type, and they exhibited a conditional phenotype that was sensitive to the C:N ratio. In the presence of C, specifically sucrose, but not glucose, mannitol, or sorbitol, antiAtGLR1.1 seeds did not germinate, but germination was restored upon coincubation with NO 3 ؊ , but not NH 4 ؉ . Immunoblot, isoenzyme, and RT-PCR analyses indicate that AtGLR1.1 regulates the accumulation of distinct C-and N-metabolic enzymes, hexokinase 1 (HXK1) and zeaxanthin epoxidase (ABA1), by transcriptional control. We provide a model to describe the role of AtGLR1.1 in C͞N metabolism and ABA biosynthesis, which in turn controls seed germination.

The action of ␥-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H 2 O 2 and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the crossreaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H 2 O 2 exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell.

No abstract

Two distinct cDNA clones encoding NAD(H)-dependent glutamate dehydrogenase (NAD[H]-GDH) in Arabidopsis thaliana were identified and sequenced. The genes corresponding to these cDNA clones were designated GDH1 and GDH2. Analysis of the deduced amino acid sequences suggest that both gene products contain putative mitochondrial transit polypeptides and NAD(H)- and α--ketoglutarate-binding domains. Subcellular fractionation confirmed the mitochondrial location of the NAD(H)-GDH isoenzymes. In addition, a putative EF-hand loop, shown to be associated with Ca2+ binding, was identified in the GDH2 gene product but not in the GDH1 gene product. GDH1 encodes a 43.0-kD polypeptide, designated α-, and GDH2 encodes a 42.5-kD polypeptide, designated [beta]. The two subunits combine in different ratios to form seven NAD(H)-GDH isoenzymes. The slowest-migrating isoenzyme in a native gel, GDH1, is a homohexamer composed of α- subunits, and the fastest-migrating isoenzyme, GDH7, is a homohexamer composed of [beta] subunits. GDH isoenzymes 2 through 6 are heterohexamers composed of different ratios of α- and [beta] subunits. NAD(H)-GDH isoenzyme patterns varied among different plant organs and in leaves of plants irrigated with different nitrogen sources or subjected to darkness for 4 d. Conversely, there were little or no measurable changes in isoenzyme patterns in roots of plants treated with different nitrogen sources. In most instances, changes in isoenzyme patterns were correlated with relative differences in the level of α- and [beta] subunits. Likewise, the relative difference in the level of α- or [beta] subunits was correlated with changes in the level of GDH1 or GDH2 transcript detected in each sample, suggesting that NAD(H)-GDH activity is controlled at least in part at the transcriptional level.