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|Glu, Gln, Asn, Gly, Ser|
|Asp, Ala, GABA|
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HORT640 - Metabolic Plant Physiology
Ammonia Assimilation and Recycling
Regulation of glutamine synthetase
There is limited evidence for repression/derepression of GS mediated by glutamine in higher plants: studies with Lemna minor show that GS level is inversely proportional to glutamine pool size (Rhodes et al, 1975; 1976).
GS from higher plant sources is subject to cumulative feedback inhibition by amino acids and nucleotides (e.g. ADP and AMP are inhibitory) [see e.g. Stewart and Rhodes (1977)].
There is limited evidence for reversible deactivation of higher plant GS: studies with Lemna minor show that GS is rapidly deactivated when plants are transferred to darkness in the presence of ammonium ions (Rhodes et al, 1979). GS can be reactivated in vitro by incubation of the enzyme with stabilizing ligands (glutathione, DTT, glutamate, Mg2+ and ATP) (Rhodes et al, 1979). The deactivated form of GS may have a different conformation than the active form of the enzyme (an -SH group at the active site is no longer accessible to titration in the deactive form of the enzyme) (Rhodes et al, 1979).
In soybean roots, GS is regulated by oxidation; oxidized GS is inactive and more susceptible to protein degradation (Ortega et al, 1999). Histidine and cysteine residues may be the site of oxidation. ATP and glutamate afford protection against oxidative inactivation (Ortega et al, 1999).
Higher plant GS is octameric, as in yeast, but is apparently not regulated by dissociation/association (Stewart et al, 1980). The subunit size is approx. 40 kDa for all isoenzymes. Different isoenzymes can be resolved by isoelectric focusing and/or ion exchange chromatography. In leaves there are 2 forms of GS: GS1 = cytosolic; GS2 = chloroplastic. GS2 contains 2 additional cysteine residues per subunit -- this may account for the higher susceptibility of this isoenzyme to sulfhydryl reagents (Stewart et al, 1980). Sequences of the chloroplastic GS genes indicate that they contain cysteines in the putative ATP and substrate binding sites, whereas in the cytosolic forms these positions are occupied by alanine residues (Baima et al, 1989).
The GS gene family in legumes is complex. In Phaseolus vulgaris leaves the GS isozymes are the result of differential expression of three members of the GS gene family:
gln-alpha and gln-beta, which encode cytosolic GS polypeptides, and gln-delta, which encodes the chloroplast-located GS (Cock et al, 1991).
The GS2 isozyme (chloroplast specific) is encoded by gln-delta. GS2 mRNA accumulates rapidly with light treatment and during photorespiration (Edwards and Coruzzi, 1989). Cock et al (1991) suggest that
regulation of gln-delta by photorespiration was the result of indirect, long-term effects on cellular metabolism; in the
short term, there was no induction of gln-delta following transfer of plants to photorespiratory conditions.
The gln-gamma gene is strongly expressed during nodule development (Forde et al, 1989).
Mutants of barley have been obtained which are deficient in GS2, the chloroplastic form of GS (Lea et al, 1989). In barley GS1 is capable of maintaining normal growth under non-photorespiratory conditions (Lea et al, 1989). Mutants of barley lacking GS2 accumulate high levels of ammonia in the leaves when placed in air (these characteristics are very similar to plants treated with inhibitors of GS; e.g. methionine sulfoximine or phosphinothricin) (Lea et al, 1989). Plant GSs are inhibited by a number of synthetic and naturally occurring compounds:
Metabolic regulation of GS expression in plants is controlled by the relative abundance of carbon skeletons versus amino acids (Oliveira and Coruzzi, 1999). In Arabidopsis the dramatic induction of mRNA for chloroplastic GS2 by light is mediated in part by phytochrome and in part by light-induced changes in sucrose levels (Oliveira and Coruzzi, 1999). In contrast, the modest induction of mRNA for cytosolic GS1 by light is primarily mediated by changes in the levels of carbon metabolites. Sucrose induction of mRNA for GS2 and GS1 occurs in a time- and dose-dependent manner (Oliveira and Coruzzi, 1999). Amino acids antagonize the sucrose induction of GS, both at the level of mRNA accumulation and that of enzyme activity; GS2 gene expression was the most dramatically regulated by metabolites (Oliveira and Coruzzi, 1999).
In the legume Lotus japonicus root cytosolic GS activity may control biomass production. Transgenic plants overexpressing GS activity in roots leads to a decrease in plant biomass production, most probably as a result of lower nitrate uptake (Limami et al, 1999).
For a discusssion of the evolution of plant GS genes see: Mathis et al (2000).
- tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae (Langston-Unkefer, 1997).
- methionine sulfoximine (MSX) (see e.g. Lea et al, 1989; Stewart et al, 1980)
- glufosinate (phosphinothricin) [the active
ingredient of "Basta" and several other herbicides used worldwide] (Hoerlein, 1994). This compound is produced as part of the tripeptide L-phosphinothricyl-L-alanyl-L-alanine by Streptomyces sp. Phosphinothricin acetyltransferase (PAT) confers "Basta" resistance in plants (Hoerlein, 1994).
Baima S, Haegi A, Stroman P, Casadoro G 1989 Characterization of a cDNA clone for barley leaf glutamine synthetase. Carlsberg Res. Commun. 54: 1-9.
Cock JM, Kim KD, Miller PW, Hutson RG, Schmidt RR 1991 A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana. Plant Mol. Biol. 17: 1023-1044.
Edwards JW, Coruzzi GM 1989 Photorespiration and light act in concert to regulate the expression of the nuclear gene for chloroplast glutamine synthetase. Plant Cell 1: 241-248.
Forde BG, Day HM, Turton JF, Wen-jun S, Cullimore JV, Oliver JE 1989 Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants. Plant Cell 1: 391-401.
Hoerlein G 1994 Glufosinate (phosphinothricin), a natural amino acid with unexpected herbicidal properties. Rev. Environ. Contam. Toxicol. 138: 73-145.
Langston-Unkefer PJ, Robinson AC, Knight TJ, Durbin RD 1987 Inactivation of pea seed glutamine synthetase by the toxin, tabtoxinine-beta-lactam. J. Biol. Chem. 262: 1608-1613.
Lea PJ, Blackwell RD, Murray AJS, Joy KW 1989 The use of mutants lacking glutamine synthetase and glutamate synthase to study their role in plant nitrogen metabolism. In (JE Poulton, JT Romeo, EE Conn eds) "Plant Nitrogen Metabolism", Recent Advances in Phytochemistry Vol 23. Plenum Press, New York, pp 157-189.
Limami A, Phillipson B, Ameziane R, Pernollet N, Jiang Q, Roy R, Deleens E, Chaumont-Bonnet M, Gresshoff PM, Hirel B 1999 Does root glutamine synthetase control plant biomass production in Lotus japonicus L.? Planta 209: 495-502.
Mathis R, Gamas P, Meyer Y, Cullimore JV 2000 The presence of GSI-like genes in higher plants: support for the paralogous evolution of GSI and GSII genes. J. Mol. Evol. 50: 116-122
Oliveira IC, Coruzzi GM 1999 Carbon and amino acids reciprocally modulate the expression of glutamine synthetase in Arabidopsis. Plant Physiol. 121: 301-310.
Ortega JL, Roche D, Sengupta-Gopalan C 1999 Oxidative turnover of soybean root glutamine synthetase. In vitro and in vivo studies. Plant Physiol. 119: 1483-1495.
Rhodes D, Rendon GA, Stewart GR 1975 The control of glutamine synthetase level in Lemna minor L. Planta 125: 201-211.
Rhodes D, Rendon GA, Stewart GR 1976 The regulation of ammonia assimilating enzymes in Lemna minor L. Planta 129: 203-210.
Rhodes D, Sims AP, Stewart GR 1979 Glutamine synthetase and the control of nitrogen assimilation in Lemna minor L. In "Nitrogen Assimilation of Plants", (EJ Hewitt, CV Cutting eds), Academic Press, London & New York, pp 59-78.
Stewart GR, Mann AF, Fentem PA 1980 Enzymes of glutamate formation: glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. In (BJ Miflin ed) "The Biochemistry of Plants", Vol 5, Academic Press, New York, pp 271-327.
Stewart GR, Rhodes D 1977 A comparison of the characteristics of glutamine synthetase and glutamate dehydrogenase from Lemna minor L. New Phytol. 79: 257-268.
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Last Update: 10/01/09