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Inorganic nitrogen in the form of ammonium is assimilated into asparagine via multiple steps involving glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AspAT) and asparagine synthetase (AS) in Arabidopsis. The asparagine amide group is liberated by the reaction catalyzed by asparaginase (ASPG) and also the amino group of asparagine is released by asparagine aminotransferase (AsnAT) for use in the biosynthesis of amino acids. Asparagine plays a primary role in nitrogen recycling, storage and transport in developing and germinating seeds, as well as in vegetative and senescence organs. A small multigene family encodes isoenzymes of each step of asparagine metabolism in Arabidopsis, except for asparagine aminotransferase encoded by a single gene. The aim of this study is to highlight the structure of the genes and encoded enzyme proteins involved in asparagine metabolic pathways; the regulation and role of different isogenes; and kinetic and physiological properties of encoded enzymes in different tissues and developmental stages.

Ammonium is a form of inorganic nitrogen derived from several metabolic pathways, and is assimilated into glutamine, glutamate, asparagine and carbamoylphosphate. These molecules play important roles in nitrogen assimilation, recycling, transport and storage in plants. Ammonium assimilation into asparagine is catalyzed by ammonia-dependent asparagine synthetase encoded by asnA (EC 6.3.1.1) or glutamine-dependent asparagine synthetase encoded by asnB (EC 6.3.5.4) in prokaryotes and eukaryotes. These organisms display a distinct distribution of these two forms of asparagine synthetase. Gene and primary protein structure for asparagine synthetase-A and -B from prokaryotes and eukaryotes is examined. Using nucleotide sequences, we constructed a phylogenetic tree that distinguished two major classes (classes I and II) for ASN genes from a range of organisms. Only the glutamine-dependent asparagine synthetases-B have been identified, and are encoded by a small multigene family in plants. The isoenzyme encoded by each member of the gene family provides asparagine at specific phases of development. These include the nitrogen mobilization in germinating seeds, nitrogen recycling in vegetative organs in response to stress, and nitrogen remobilization during seed embryogenesis. The expression of genes for asparagine synthetase is regulated by light and metabolites. Genetic and molecular data using mutants and transgenic plants have provided insights into the light perception by the photoreceptors, carbon and nitrogen sensing and signal transduction mechanism in the asn regulation. Global analysis of carbon and nitrogen metabolites supports the impact of asn regulation in the synthesis and transport of asparagine in plants.

This study was aimed at investigating the physiological role of ferredoxin-glutamate synthases (EC 1.4.1.7), NADH-glutamate synthase (EC 1.4.1.14) and carbamoylphosphate synthetase (EC 6.3.5.5) in Arabidopsis. Phenotypic analysis revealed a high level of photorespiratory ammonium, glutamine/glutamate and asparagine/aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate synthase, indicating that excess photorespiratory ammonium was detoxified into amino acids for transport out of the veins. Consistent with these results, promoter analysis and in situ hybridization demonstrated that GLU1 and GLU2 were expressed in the mesophyll and phloem companion cell-sieve element complex. However, these phenotypic changes were not detected in the GLU2 mutant defective in the second ferredoxin-glutamate synthase gene. The impairment in primary ammonium assimilation in the GLT mutant under nonphotorespiratory high-CO(2) conditions underlined the importance of NADH-glutamate synthase for amino acid trafficking, given that this gene only accounted for 3% of total glutamate synthase activity. The excess ammonium from either endogenous photorespiration or the exogenous medium was shifted to arginine. The promoter analysis and slight effects on overall arginine synthesis in the T-DNA insertion mutant in the single carbamoylphosphate synthetase large subunit gene indicated that carbamoylphosphate synthetase located in the chloroplasts was not limiting for ammonium assimilation into arginine. The data provided evidence that ferredoxin-glutamate synthases, NADH-glutamate synthase and carbamoylphosphate synthetase play specific physiological roles in ammonium assimilation in the mesophyll and phloem for the synthesis and transport of glutamine, glutamate, arginine, and derived amino acids.

Ferredoxin (Fd)-dependent glutamate synthase is present in green leaves, etiolated leaves, shoots and roots of Arabidopsis thaliana (ecotype Columbia). In photosynthetic green leaves and shoots, Fd-dependent glutamate synthase accounts for more than 96% of the total glutamate synthase activity in vitro with the remaining activity derived from an enzyme that uses NADH as the electron donor. In etiolated leaves and roots, Fd-dependent glutamate synthase is 3-4-fold less active than in green leaves, but represents 70-85% of the total glutamate synthase activity in these tissues. Fd-dependent glutamate synthase is detected as a single peptide of 165 kDa on a western blot of green leaf and shoot tissues, and this Fd-dependent glutamate synthase polypeptide is 3-4-fold less abundant in etiolated leaves and roots. In these non-photosynthetic tissues, there is a higher activity of NADH-dependent glutamate synthase. The A. thaliana gltS mutant (strain CS254) contains only 1.7% and 17.5% of the wild-type Fd-dependent glutamate synthase activity in leaves and roots, respectively. Western blots indicate that the Fd-dependent glutamate synthase peptide of 165 kDa is absent from leaves and roots of the gltS mutant. In contrast, NADH-dependent glutamate synthase activity in leaves and roots is unaffected. During illumination of wild-type dark-grown leaves for 72 h, the levels of Fd-dependent glutamate synthase protein and its activity increased threefold to levels equivalent to those in green leaves. In contrast, NADH-dependent glutamate synthase activity decrease twofold during illumination. The complete nucleotide sequence of the complementary DNA for A. thaliana Fd-dependent glutamate synthase has been determined. Analysis of the amino acid sequence deduced from the complete cDNA sequence (5178 bp) has revealed that A. thaliana Fd-dependent glutamate synthase is synthesized as a 1648-amino-acid precursor protein (180090 Da) which consists of a 131-amino-acid transit peptide (14603 Da) and a 1517-amino-acid mature peptide (165487 Da). The A. thaliana Fd-dependent glutamate synthase has a high similarity to maize Fd-dependent glutamate synthase (83%) and to the analogous region of NADH-dependent glutamate synthase (42%) and NADPH-dependent glutamate synthases (40-43%) from different organisms. The A. thaliana Fd-dependent glutamate synthase contains the purF-type glutamine-amido-transfer domain as well as flavin and iron-sulfur-cluster-binding domains. The deduced primary structures of A. thaliana Fd-dependent glutamate synthase and of glutamate synthases from other organisms indicate that Fd-dependent glutamate synthase may have evolved from bacterial NADPH-dependent glutamate synthase. The cDNA hybridized to RNA of about 5.3 kb from different tissues of A. thaliana. A high steady-state level of Fd-dependent glutamate synthase mRNA is found in photosynthetic green leaves and shoots, and roots contain less mRNA for Fd-dependent glutamate synthase. In the gltS mutant, there are twofold and fourfold lower levels of Fd-dependent glutamate synthase mRNA in leaves and roots, respectively, relative to those in wild-type A. thaliana. Under continuous illumination of dark-grown leaves, the Fd-dependent glutamate synthase mRNA is induced twofold to a level equivalent to that in green leaves.