Expression of asparagine synthetase genes in sunflower (Helianthus annuus) under various environmental stresses.

In sunflower, asparagine synthetase (AS; EC 6.3.5.4) is encoded by a small family of three genes (HAS1, HAS1.1 and HAS2) that are differentially regulated by light, carbon and nitrogen availability. In this study, the response of each gene to various stress conditions was examined by Northern analysis with gene-specific probes in leaves and roots. The expression of HAS1 and HAS1.1 genes was induced by osmotic stress (300 mM mannitol), salt stress (150 mM NaCl), and heavy-metal stress (20 microM CuSO(4)), more in roots than in leaves. The expression of HAS2 was not significantly altered by stress treatments. The positive response of HAS1 and HAS1.1 genes to osmotic and salt stresses occurred in the light, in contrast to that previously found in unstressed plants. Measurements of sucrose and total free amino acid contents in leaves and roots indicate that the expression of root HAS1 and HAS1.1 genes in stressed plants is not under metabolic control by the intracellular C/N ratio, suggesting the involvement of some specific stress factor(s). Growth of plants at 40 degrees C for 12h negatively affected the expression of HAS1 and HAS1.1 but not that of HAS2.     

Dark-induced and organ-specific expression of two asparagine synthetase genes in Pisum sativum.

Nucleotide sequence analysis of cDNAs for asparagine synthetase (AS) of Pisum sativum has uncovered two distinct AS mRNAs (AS1 and AS2) encoding polypeptides that are highly homologous to the human AS enzyme. The amino-terminal residues of both AS1 and AS2 polypeptides are identical to the glutamine-binding domain of the human AS enzyme, indicating that the full-length AS1 and AS2 cDNAs encode glutamine-dependent AS enzymes. Analysis of nuclear DNA shows that AS1 and AS2 are each encoded by single genes in P.sativum. Gene-specific Northern blot analysis reveals that dark treatment induces high-level accumulation of AS1 mRNA in leaves, while light treatment represses this effect as much as 30-fold. Moreover, the dark-induced accumulation of AS1 mRNA was shown to be a phytochrome-mediated response. Both AS1 and AS2 mRNAs also accumulate to high levels in cotyledons of germinating seedlings and in nitrogen-fixing root nodules. These patterns of AS gene expression correlate well with the physiological role of asparagine as a nitrogen transport amino acid during plant development.     

Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana

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.     

Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen.     

Hyaluronic acid, HAS1, and HAS2 are significantly upregulated during muscle hypertrophy

Hyaluronic acid (HA) is a component of the extracellular matrix (ECM) in most vertebrate tissues and is thought to play a significant role during development, wound healing, and regeneration. In vitro studies have shown that HA enhances muscle progenitor cell recruitment and inhibits premature myotube fusion, implicating a role for this glycosaminoglycan in functional repair. However, the spatiotemporal distribution of HA during muscle growth and repair was unknown. We hypothesized that inducing hypertrophy via synergist ablation would increase the expression of HA and the HA synthases (HAS1-HAS3). We found that HA and HAS1-HAS3 were significantly upregulated within the plantaris muscle in response to Achilles tenectomy. HA concentration significantly increased 2.8-fold after 2 days but decreased towards levels comparable to age-matched controls by 14 days. Using immunohistochemistry, we found the colocalization of HAS1-HAS3 with macrophages, blood vessel epithelia, and fibroblasts varied in response to time and/or tenectomy. At the level of gene expression, only HAS1 and HAS2 significantly increased with respect to both time and tenectomy. The profiles of additional genes that influence ECM composition during muscle repair, tenascin-C, type I collagen, the HA-degrading hyaluronidases (Hyal) and matrix metalloproteinases (MMP) were also investigated. Hyal1 and Hyal2 were highly expressed in skeletal muscle but did not change after tenectomy; however, indicators of hypertrophy, MMP-2 and MMP-14, were significantly upregulated from 2 to 14 days. These results indicate that HA levels dynamically change in response to a hypertrophic stimulus and various cells may participate in this mechanism of skeletal muscle adaptation.     

Nitrogen stress and the expression of asparagine synthetase in roots and nodules of soybean (Glycine max)

The difficulty of assaying asparagine synthetase (AS) (EC 6.3.5.4) activity in roots of soybean has been circumvented by measuring expression of the AS genes. Expression of three soybean asparagine synthetase (SAS) genes ( SAS1 , SAS2 and SAS3 ) was observed in roots of non-nodulated soybean plants cultivated on nitrate. Expression of these genes was reduced to very low levels within days after submitting the plants to a N-free medium. The subsequent return to a complete medium (containing nitrate) restored expression of all three AS genes. Roots of nodulated plants, where symbiotic nitrogen fixation was the exclusive source of N (no nitrate present), showed very weak expression of all three AS genes, but on transfer to a nitrate-containing medium, strong expression of these genes was observed within 24 h. In nodules, all three genes were expressed in the absence of nitrate. Under conditions that impair nitrogen fixation (nodules submerged in aerated hydroponics), only SAS1 expression was reduced. However, in the presence of nitrate, an inhibitor of N₂ fixation, SAS1 expression was maintained. High and low expressions of AS genes in the roots were associated with high and low ratios of Asn/Asp transported to the shoot through xylem. It is concluded that nitrate (or one of its assimilatory products) leads to the induction of AS in roots of soybean and that this underlies the variations found in xylem sap Asn/Asp ratios. Regulation of nodule AS expression is quite different from that of the root, but nodule SAS1 , at least, appears to involve a product of N assimilation rather than nitrate itself.     

Light- and carbon-signaling pathways. Modeling circuits of interactions

Here, we report the systematic exploration and modeling of interactions between light and sugar signaling. The data set analyzed explores the interactions of sugar (sucrose) with distinct light qualities (white, blue, red, and far-red) used at different fluence rates (low or high) in etiolated seedlings and mature green plants. Boolean logic was used to model the effect of these carbon/light interactions on three target genes involved in nitrogen assimilation: asparagine synthetase (ASN1 and ASN2) and glutamine synthetase (GLN2). This analysis enabled us to assess the effects of carbon on light-induced genes (GLN2/ASN2) versus light-repressed genes (ASN1) in this pathway. New interactions between carbon and blue-light signaling were discovered, and further connections between red/far-red light and carbon were modeled. Overall, light was able to override carbon as a major regulator of ASN1 and GLN2 in etiolated seedlings. By contrast, carbon overrides light as the major regulator of GLN2 and ASN2 in light-grown plants. Specific examples include the following: Carbon attenuated the blue-light induction of GLN2 in etiolated seedlings and also attenuated the white-, blue-, and red-light induction of GLN2 and ASN2 in light-grown plants. By contrast, carbon potentiated far-red-light induction of GLN2 and ASN2 in light-grown plants. Depending on the fluence rate of far-red light, carbon either attenuated or potentiated light repression of ASN1 in light-grown plants. These studies indicate the interaction of carbon with blue, red, and far-red-light signaling and set the stage for further investigation into modeling this complex web of interacting pathways using systems biology approaches.     

Hyaluronan Synthase 1 (HAS1) Requires Higher Cellular UDP-GlcNAc Concentration than HAS2 and HAS3

Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1–3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1–3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.     

<Emphasis Type="Italic">PVAS3</Emphasis>, a class-II ubiquitous asparagine synthetase from the common bean (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.     

Cloning, characterization and mRNA expression analysis of PVAS1, a type I asparagine synthetase gene from Phaseolus vulgaris

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris L.). A 2-kb cDNA clone of this gene (PVAS1) encodes a protein of 579 amino acids with a predicted molecular mass of 65,265 Da, an isoelectric point of 6.3, and a net charge of -9.3 at pH 7.0. The PVAS1 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS1 complemented an Escherichia coli asparagine auxotroph, which demonstrates that it encodes a glutamine-dependent AS. The PVAS1 protein showed the highest similarity to soybean SAS1, and piled up with other legume ASs to form an independent dendritic group of type-I AS enzymes. Northern blot analyses revealed that the expression pattern of PVAS1 resembles that of PVAS2, another AS previously described in the common bean. Unlike PVAS2, however, PVAS1 was not expressed in the nodule and was not repressed by light, suggesting different functions for these two AS genes.     

Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae.

We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.     

Correlation between AS1 gene expression and seed protein contents in different soybean (Glycine max [L.] Merr.) cultivars

In higher plants, asparagine synthetase (AS) plays an important role in regulating the nitrogen sink-source relationship. We studied the expression of AS genes in five Chinese soybean cultivars exhibiting contrasting seed protein contents. We found that only the AS2 but not the AS1 gene was induced by dark treatment. On the other hand, the expression of AS1 in leaves (especially in trifoliate leaves of young seedlings) showed a positive correlation with seed protein contents in the soybean cultivars tested. Therefore, in spite of the fact that the principle transporting compounds in soybean plants for nitrogen acquired via symbiotic fixation are ureides, AS may still play an important role in the process of nitrogen assimilation.