Overexpression of the ASN1 Gene Enhances Nitrogen Status in Seeds of Arabidopsis

Abstract

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...

Significance of Light, Sugar, and Amino Acid Supply for Diurnal Gene Regulation in Developing Barley Caryopses

“…In agreement with previous reports, we found elevated amounts of Asn, which serves as an important nitrogen storage and transport compound for the allocation of nitrogen resources between source and sink organs, at the beginning of the night (Lam et al, 1995(Lam et al, , 2003.…”

A Pivotal Role of the Basic Leucine Zipper Transcription Factor bZIP53 in the Regulation ofArabidopsisSeed Maturation Gene Expression Based on Heterodimerization and Protein Complex Formation

“…Further examples of seed genes misexpressed in leaves are legumin-like CRA1 (Wang et al, 2007) and the 11-b-hydroxysteroid dehydrogenase gene (HSD1) (Li et al, 2007;see Supplemental Figure 1C online). In addition, these plants also show enhanced expression of the proline dehydrogenase (ProDH) gene, as previously reported , or the Asparagine Synthetase1 (ASN1) gene, both typical genes involved in amino acid metabolism (Lam et al, 2003) ( Figure 2B; see Supplemental Figure 1C online). Altogether, these data support the assumption that overexpression of bZIP53 in leaves triggers the misexpression of a substantial fraction of seed-specific genes.…”

“…Amino acid synthesis, transport, and SSP synthesis are linked processes that have to be coordinated in vegetative tissues as well as in the seed. In accordance, ectopic overexpression of ASN1 enhances the SSP content and nitrogen status of the seed (Lam et al, 2003). Further studies are necessary to test whether bZIP53 is coordinating other pathways in addition to SSP synthesis, such as amino acid metabolism during seed and vegetative development.…”

Heterodimers of the Arabidopsis Transcription Factors bZIP1 and bZIP53 Reprogram Amino Acid Metabolism during Low Energy Stress

“…ASN1, the key gene of the Asn biosynthesis pathway, is characterized by a complex transcriptional regulation (Lam et al, 1998(Lam et al, , 2003. Transcription of the Asn pathway genes, such as ASP3, GDH2, and ASN1, appears to be coregulated and is induced after dark treatment but repressed when sugar is available (Lin and Wu, 2004).…”

“…Asn is the major source for N/C transport in darkness (Lam et al, 1994(Lam et al, , 1998(Lam et al, , 2003. Asn biosynthesis is derived from pyruvate and requires the coordinated, transcriptional upregulation of several genes (Lin and Wu, 2004; Figure 4A).…”