In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.In higher plants, nitrogen is acquired from the environment via nitrate reduction (Crawford and Arst, 1993), ammonia uptake, or nitrogen fixation (Burris and Roberts, 1993). All inorganic nitrogen must first be reduced to ammonia by the action of nitrate and nitrite reductase before being assimilated into amino acids Miflin and Lea, 1980;Lea et al., 1990). In plants, the majority of ammonia is assimilated into organic form via the combined action of Gln synthetase and Gln 2-oxoglutarate aminotransferase. The products of Gln synthetase-Gln 2-oxoglutarate aminotransferase cycle, Gln, and Glu are highly reactive and are used in various anabolic pathways. Through the action of Asn synthetase (AS; EC 6.3.5.4), Gln will react with Asp to form Asn and Glu. Because all the substrates and products of the AS-catalyzed reaction are major nitrogen carriers in plant metabolism for transporting nitrogen in the phloem, the AS enzyme is believed to play an important role in regulating the flow of nitrogen into the organic nitrogen pool (Lam et al., 1994). Asn is an especially important nitrogen transport amino acid because it has a high nitrogen to carbon ratio (relative to the other amide amino acid Gln) and is relatively inert compared with the other nitrogen-transporting amino acids (Glu, Asp, and Gln). Therefore, Asn can be used for long-range nitrogen transport and storage, which is vital to physiological processes such as germination and nitrogen assimilation Siecie...

In Arabidopsis, asparagine (Asn) synthetase is encoded by a small gene family (ASN1, ASN2, and ASN3). It has been shown that ASN1 and ASN2 exhibit reciprocal gene expression patterns toward light and metabolites. Moreover, changes in total free Asn levels parallel the expression of ASN1, but not ASN2. In this study, we show that ASN2 expression correlates with ammonium metabolism. We demonstrate that the light induction of ASN2 is ammonium dependent. The addition and removal of ammonium exerted fast and reciprocal effects on the levels of ASN2 mRNA, specifically under light-grown conditions. NaCl and cold stress increased cellular free ammonium and ASN2 mRNA levels in a coordinated manner, suggesting that the effects of stress on ASN2 expression may be mediated via accumulation of ammonium. The correlation between ASN2 and cellular ammonium metabolism was further demonstrated by analysis of ASN2 transgenic plants. When plants were grown on Murashige and Skoog medium containing 50 mm ammonium, ASN2 overexpressors accumulated less endogenous ammonium compared with the wild-type Colombia-0 and ASN2 underexpressors. When plants were subjected to high-light irradiance, ammonium levels built up. Under such conditions, ASN2 underexpressors accumulated more endogenous ammonium than the wild-type Colombia-0 and ASN2 overexpressors. These results support the notion that ASN2 is closely correlated to ammonium metabolism in higher plants.To systematically study the differential physiological roles of different members in the asparagines synthetase (AS; the enzyme catalyzing the biosynthesis of asparagines) gene family, we previously cloned all the members of AS gene in the model plant Arabidopsis (ASN1, ASN2, and ASN3). Phylogenetic analysis revealed that although ASN1 clustered with all dicot AS genes, ASN2 and ASN3 are more closely related to monocot AS genes (Lam et al., 1998). ASN1 and ASN2 were shown to be reciprocally regulated by light and metabolites in Arabidopsis (Lam et al., 1998). Based on the phylogenetic data and gene expression data, it was suggested that ASN1 and ASN2 may play very different physiological roles in plant nitrogen metabolism (Lam et al., 1998).The expression level of ASN1 is tightly correlated with free Asn levels (Lam et al., 1994(Lam et al., , 1998(Lam et al., , 2003. In wild-type Arabidopsis, ASN1 and free Asn increase in dark-adapted plants, consistent with the idea that Asn is an important nitrogen carrier, especially under carbon-limiting conditions. In transgenic plants overexpressing the ASN1 gene, free Asn levels in source and sink tissues as well as in the phloem increase accordingly. Total amino acid pools in seeds of the ASN1 overexpressors are enhanced quantitatively and qualitatively (Lam et al., 2003). These findings suggest that ASN1 plays a major role in nitrogen assimilation, regulating nitrogen transport and storage during seed development. These data are also consistent with the notion that free Asn is an important nitrogen carrier for long-range transport and storage in...