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The physiological role of the NADH-dependent glutamine-2-oxoglutarate aminotransferase (NADH-GOGAT) enzyme was addressed in Arabidopsis using gene expression analysis and by the characterization of a knock-out T-DNA insertion mutant (glt1-T) in the single NADH-GOGAT GLT1 gene. The NADH-GOGAT GLT1 mRNA is expressed at higher levels in roots than in leaves. This expression pattern contrasts with GLU1, the major gene encoding Fd-GOGAT, which is most highly expressed in leaves and is involved in photorespiration. These distinct organ-specific expression patterns suggested a non-redundant physiological role for the NADH-GOGAT and Fd-GOGAT gene products. To test the in vivo function of NADH-GOGAT, we conducted molecular and physiological analysis of the glt1-T mutant, which is null for NADH-GOGAT, as judged by mRNA level and enzyme activity. Metabolic analysis showed that the glt1-T mutant has a specific defect in growth and glutamate biosynthesis when photorespiration was repressed by 1% CO2. Under these conditions, the glt1-T mutant displayed a 20% decrease in growth and a dramatic 70% reduction in glutamate levels. Herein, we discuss the significance of NADH-GOGAT in non-photorespiratory ammonium assimilation and in glutamate synthesis required for plant development.

Summary In plants, the amino acid asparagine serves as an important nitrogen transport compound whose levels are dramatically regulated by light in many plant species, including Arabidopsis thaliana. To elucidate the mechanisms regulating the flux of assimilated nitrogen into asparagine, we examined the regulation of the gene family for asparagine synthetase in Arabidopsis. In addition to the previously identified ASN1 gene, we identified a novel class of asparagine synthetase genes in Arabidopsis (ASN2 and ASN3) by functional complementation of a yeast asparagine auxotroph. The proteins encoded by the ASN2/3 cDNAs contain a Pur-F type glutamine-binding triad suggesting that they, like ASN1, encode glutamine-dependent asparagine synthetase isoenzymes. However, the ASN2/3 isoenyzmes form a novel dendritic group with monocot AS genes which is distinct from all other dicot AS genes including Arabidopsis ASN1. In addition to these distinctions in sequence, the ASN1 and ASN2 genes are reciprocally regulated by light and metabolites. Time-course experiments reveal that light induces levels of ASN2 mRNA while it represses levels of ASN1 mRNA in a kinetically reciprocal fashion. Moreover, the levels of ASN2 and ASN1 mRNA are also reciprocally regulated by carbon and nitrogen metabolites. The distinct regulation of ASN1 and ASN2 genes combined with their distinct encoded isoenzymes suggest that they may play different roles in nitrogen metabolism, as discussed in this paper.

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana. Four classes of cDNAs representing four distinct AspAT genes (ASP1-ASP4) have been cloned from Arabidopsis. Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1-AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis. These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.