Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

Shi, Lei; Twary, Scott N.; Yoshioka, Hirofumi; Gregerson, Robert G.; Miller, Stephen R.; Samac, Deborah A.; Gantt, J. Stephen; Unkefer, Pat J.; Vance, Carroll P.

doi:10.1105/tpc.9.8.1339

Cited by 82 publications

(72 citation statements)

References 60 publications

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“…The relatively modest increase in free ammonium in nodules of the transformed plants in comparison with the decrease in GS activity and the fact that biological N 2 fixation releasing a constant flux of ammonium was not affected also support this hypothesis. This hypothesis also fits well with the finding that AS expression in the nodules is independent of nitrogenase activity (Shi et al, 1997). In a recent study, an increase in Asn also has been observed in leaves when photorespiratory ammonium is accumulating as the result of impaired GOGAT activity, suggesting that alternative metabolic pathways may be activated to avoid ammonium accumulation (Ferrario-Méry et al, 2002).…”

Section: Discussionsupporting

confidence: 74%

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Does Lowering Glutamine Synthetase Activity in Nodules Modify Nitrogen Metabolism and Growth of Lotus japonicus?

et al. 2003

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A cDNA encoding cytosolic glutamine synthetase (GS) from Lotus japonicus was fused in the antisense orientation relative to the nodule-specific LBC3 promoter of soybean (Glycine max) and introduced into L. japonicus via transformation with Agrobacterium tumefaciens. Among the 12 independent transformed lines into which the construct was introduced, some of them showed diminished levels of GS1 mRNA and lower levels of GS activity. Three of these lines were selected and their T 1 progeny was further analyzed both for plant biomass production and carbon and nitrogen (N) metabolites content under symbiotic N-fixing conditions. Analysis of these plants revealed an increase in fresh weight in nodules, roots and shoots. The reduction in GS activity was found to correlate with an increase in amino acid content of the nodules, which was primarily due to an increase in asparagine content. Thus, this study supports the hypothesis that when GS becomes limiting, other enzymes (e.g. asparagine synthetase) that have the capacity to assimilate ammonium may be important in controlling the flux of reduced N in temperate legumes such as L. japonicus. Whether these alternative metabolic pathways are important in the control of plant biomass production still remains to be fully elucidated.Nitrogen (N) is one of the major limiting factors for plant growth. However, an excessive external supply of N causes major problems in agriculture and the environment, mostly due to nitrate leaching into underground water (Benes et al., 1989), not only polluting the aquatic environment but also resulting in a high nitrate content in food and drinks (Moller et al., 1990). Paradoxically, dinitrogen is 80% of the atmosphere, but it can only be assimilated by prokaryotic symbiotic or free-living diazotrophic organisms possessing nitrogenase activity (Dilworth, 1974). In higher plants, nitrate and ammonium are the two major inorganic N compounds that can be directly assimilated (Beevers, 1976). They are provided either artificially by the external supply of fertilizers, bacterial nitrification, or atmospheric reduction of dinitrogen during lightning, or naturally through biological N fixation. Therefore, regulation of inorganic N assimilation and incorporation of inorganic N into organic matter is of major importance not only in maintaining a sustainable agriculture but also protecting the environment.Over the past few years, attention has been particularly focused on the enzyme Gln synthetase (GS; E.C. 6.3.1.2) because of its central role in N metabolism and its diverse metabolic and developmental regulation in different plant species and organs (Cren and Hirel, 1999). Two major isoforms exist for the GS enzyme: cytosolic GS, occurring in the cytoplasm of leaves and non photosynthetic organs, and chloroplastic GS, present only in the chloroplasts of photosynthetic tissues and the plastids of roots or etiolated plants. The relative proportions of the cytosolic and plastidial GS activity may vary within different organs of the same plant or within dif...

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Section: Discussionsupporting

confidence: 74%

“…AS protein was detected with the antibodies raised against the nodule enzyme from alfalfa (Medicago sativa; Shi et al, 1997). Proteins were visualized using peroxidaseconjugated goat anti-rabbit antibodies (Towbin et al, 1979).…”

Section: Protein Extraction Fractionation Enzymatic Assay and Westmentioning

confidence: 99%

Does Lowering Glutamine Synthetase Activity in Nodules Modify Nitrogen Metabolism and Growth of Lotus japonicus?

et al. 2003

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“…However, the levels of gene expression for the cytosolic GS1 are not significantly affected by light (37). In contrast to the situation with GS2, light was found to repress accumulation of AS1 mRNA levels in a variety of plants including Arabidopsis, Pisum, Medicago, Glycine, Triphysaria, and Phaseolus (24,26,(28)(29)(30)32). The negative effect of light on AS gene expression was also shown to be mediated, at least in part, by phytochrome in Arabidopsis and pea (30,32).…”

Section: Light and Metabolic Regulation Of Genes Involved In Nitrogenmentioning

confidence: 80%

Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway

Oliveira

Brenner

Chiu

et al. 2001

Braz J Med Biol Res

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We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogentransport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants. Key words Light and metabolic regulation of genes involved in nitrogen metabolismIn non-legume plants, nitrogen assimilation begins with the uptake of inorganic nitrogen, nitrate or ammonium, from the soil.Nitrate is subsequently reduced to ammonium (NH 4 + ) by the sequential action of nitrate reductase and nitrite reductase (1,2). The resulting ammonium is then assimilated into an organic form as glutamate and glutamine. These amino acids are the nitrogen donors in the biosynthesis of essentially all

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“…Finally, in most temperate legumes the assimilated Gln is exported out of the nodule, principally as Asn, which requires the concerted activity of a Gln-dependent Asn synthetase and Asp aminotransferase. In alfalfa, there is a nodule-enhanced Gln-dependent Asn synthetase (Shi et al, 1997) and in L. corniculatus and alfalfa there are nodule-enhanced Asp aminotransferases (P2 and AAT2, respectively), which at least in alfalfa has been located in a plastid Mett et al, 1996). In indeterminate V. faba nodules most infected cells do not make contact with uninfected cells and it has been proposed that amino acids are released by infected cells into the apoplast from which they can be actively accumulated by uninfected cells (Peiter et al, 2004).…”

mentioning

confidence: 99%

Nutrient Sharing between Symbionts

White

Prell²,

James³

et al. 2007

Plant Physiology

210

158

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In this review, we consider the exchange of nutrients between the host plant and the bacterial microsymbiont in nitrogen-fixing legume root nodules. During nodule formation, the host tissues and the bacterial microsymbiont develop in response to each other to form a specialized tissue that maintains an environment where nitrogen fixation can occur (Brewin, 2004;Mergaert et al., 2006;Prell and Poole, 2006). This complex development will not be considered here but, at the end of the process, specialized, nitrogen-fixing forms of the bacteria, known as bacteroids, reside in the plant cytosol, enclosed within plant-derived membranes. These organelle-like structures are known as symbiosomes; the plant-derived membrane that surrounds the bacteroid is the symbiosome (or peribacteroid) membrane and the space between the two is the symbiosome (or peribacteroid) space. An infected plant cell may be packed with thousands of symbiosomes. The exchange of nutrients that is fundamental to N 2 fixation therefore involves metabolism in the plant to provide carbon and nitrogen compounds to the bacteroids and to assimilate the metabolites that bacteroids release. Nutrients transferred between the symbionts must traverse both the symbiosome and the bacteroid membranes. It is clear that there is more than one pattern whereby successful nutrient exchange can take place: There are two basic types of legume nodules, determinate and indeterminate, and there are fundamental differences between the two in how they develop and in their carbon and nitrogen metabolism. This review focuses on the metabolism of carbon and nitrogen compounds in the symbionts and on the exchange of nutrients across the bacteroid and symbiosome membranes. Particular attention is paid to the movement of primary carbon and nitrogen sources and how they are utilized by both bacteroids and the plant.

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Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

Cited by 82 publications

References 60 publications

Does Lowering Glutamine Synthetase Activity in Nodules Modify Nitrogen Metabolism and Growth of Lotus japonicus?

Does Lowering Glutamine Synthetase Activity in Nodules Modify Nitrogen Metabolism and Growth of Lotus japonicus?

Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway

Nutrient Sharing between Symbionts

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