Combined agronomic and physiological aspects of nitrogen management in wheat highlight a central role for glutamine synthetase

In wheat the period of grain filling is characterized by a transition for all vegetative organs from sink to source status. To study this transition, the progression of physiological markers and enzyme activities representative of nitrogen metabolism was monitored from the vegetative stage to maturity in different leaf stages and stem sections of two wheat (Triticum aestivum) cultivars grown at high and low levels of N fertilization. In the two cultivars examined, we found a general decrease of the metabolic and enzyme markers occurred during leaf ageing, and that this decrease was enhanced when plants were N-limited. Both correlation studies and principal components analysis (PCA) showed that there was a strong relationship among total N, chlorophyll, soluble protein, ammonium, amino acids and glutamine synthetase (GS) activity. The use of a marker such as GS activity to predict the N status of wheat, as a function of both plant development and N availability, is discussed with the aim of selecting wheat genotypes with better N-use efficiency.     

小麦谷氨酰胺合成酶的原核表达特点

谷氨酰胺合成酶(GS)是植物氮同化的关键酶,为了研究小麦GS同工酶的结构及其表达特点,我们构建了小麦GS1、GSr、GSe、GS2和GS2前体GS2p的原核表达载体,并对表达条件进行了优化。结果表明,尽管小麦GS同工酶氨基酸序列同源性达70%–80%,蛋白质表达却各具特点。30℃诱导3 h后,GSr、GSe及GS2表达量达最大,诱导7 h后GS1表达量达最大,GS2p不表达,表达量依次为GS1(22%)GSr(15%)GS2(12%)GSe(5%);且GSe可溶性表达,GS1主要为可溶性表达,而GSr和GS2为包涵体。30℃诱导3 h,GS同工酶相对转录量为GSr(7.59)GS2(1.84)GS2p(1.66)GSe(1.46)GS1(1.00),酶蛋白质翻译水平与转录水平不一致。mRNA结构分析显示,GS同工酶翻译起始区稳定二级结构的自由能不同:GS1(14.4)GSr(17.2)GS2(22.6)GSe(25.4)GS2p(31.6),自由能越小,翻译起始区结构越不稳定,蛋白表达水平越高。GS1、GSr、GSe和GS2可溶性表达的最佳诱导条件不同,分别是30℃诱导5 h、16℃诱导15 h、37℃诱导5 h及25℃诱导7 h;可溶性表达量为GS1(20%)GSr(13%)GS2(10%)GSe(7%),酶活性为GS1GSeGS2,GSr无活性。可见,GS同工酶的基因序列决定了蛋白质在原核细胞中的表达量、状态及其活性。     

Direct evidence for non-enzymatic fragmentation of chloroplastic glutamine synthetase by a reactive oxygen species

Chloroplastic glutamine synthetase (GS: EC 6·3·1·2), the octamer of the 44 kDa subunit, is rapidly degraded under photo‐oxidative stress conditions in leaves, chloroplasts, and chloroplast lysates. Recent studies have suggested that chloroplastic GS might be cleaved by the hydroxyl radical under such conditions ( Thoenen & Feller 1998 ; Australian Journal of Plant Physiology 25, 279–286; Palatnik, Carrillo & Valle 1999 , Plant Physiology 121, 471–478). Herein, we present evidence which supports the above hypothesis. When the purified GS from wheat (Triticum aestivum L.) chloroplasts was exposed to the hydroxyl radical‐generating system comprising H₂O₂–FeSO₄–ascorbic acid or FeCl₃–ascorbic acid, the GS subunit was degraded into four distinct fragments having apparent molecular masses of 39, 35, 32 and 28 kDa. The apparent molecular masses and isoelectric points of these fragments were identical to those of the respective fragments found in the illuminated lysates of chloroplasts. In addition, the appearance of the GS fragments was completely suppressed in the presence of the scavenger for the hydroxyl radical, n‐propyl gallate, in the illuminated lysates of chloroplasts. These results strongly support the hypothesis that the primary cleavage of GS is directly driven by the hydroxyl radical, formed by Fenton reaction under photo‐oxidative stress conditions in chloroplasts.     

Cisgenic overexpression of cytosolic glutamine synthetase improves nitrogen utilization efficiency in barley and prevents grain protein decline under elevated CO2

Cytosolic glutamine synthetase (GS1) plays a central role in nitrogen (N) metabolism. The importance of GS1 in N remobilization during reproductive growth has been reported in cereal species but attempts to improve N utilization efficiency (NUE) by overexpressing GS1 have yielded inconsistent results. Here, we demonstrate that transformation of barley (Hordeum vulgare L.) plants using a cisgenic strategy to express an extra copy of native HvGS1‐1 lead to increased HvGS1.1 expression and GS1 enzyme activity. GS1 overexpressing lines exhibited higher grain yields and NUE than wild‐type plants when grown under three different N supplies and two levels of atmospheric CO₂. In contrast with the wild‐type, the grain protein concentration in the GS1 overexpressing lines did not decline when plants were exposed to elevated (800–900 μL/L) atmospheric CO₂. We conclude that an increase in GS1 activity obtained through cisgenic overexpression of HvGS1‐1 can improve grain yield and NUE in barley. The extra capacity for N assimilation obtained by GS1 overexpression may also provide a means to prevent declining grain protein levels under elevated atmospheric CO₂.     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

The role of glutamine synthetase in regulation of nitrogen metabolism within the soil microbial community

Recent advances in our understanding of the enzymology and regulatory systems involved in microbial metabolism of N hold promise to elucidate some of the underlying factors controlling metabolism of N in soil ecosystems. A review of recent work is used to construct a paradigm for N metabolism regulation in soil based on the central role of glutamine synthetase (GS) in such regulation within the soil microbial community. The studies involved use of GS inhibitors to elucidate the role of GS activity in regulation of soil N metabolism. Such studies have shown that the glutamine formed by microbial assimilation of NH₄ ⁺ via GS activity influences the regulatory mechanisms controlling both the production and activity of enzymes involved in N metabolism. For example, these studies showed that the inhibition of GS activity within the soil microbial community relieved the repression of urease production caused by microbial assimilation of inorganic N and blocked the short-term regulation of assimilatory nitrate reductase (ANR) by NH₄ ⁺ assimilation. Other studies have indicated that common environmental factors in soil may influence GS activity in microorganisms and thereby may influence metabolism of N within the soil microbial community. The paradigm for N metabolism regulation in soil that has emerged from such studies should lead to a better understanding of the mechanisms controlling fate of N in soil ecosystems.     

Isoforms of glutamine synthetase in asparagus spears: the cytosolic enzyme increases after harvest

Two distinct forms of glutamine synthetase (GS) have been identified in the spear tip tissues of harvested asparagus (Asparagus officinalis L. cv. Limbras 10). The GS activities were separated by anion exchange chromatography. They have distinct kinetic properties and contain polypeptides of different sizes, and the abundances of the GS isoforms change differently after harvest. Plastid GS has a 44 kD polypeptide, and during the post-harvest period the abundance of this polypeptide declined dramatically. After 5 d, the activity of plastid GS had declined to just 20% of that at harvest. Cytosolic GS has a 40 kD polypeptide and is the major constituent of the GS activity present at harvest (73% of total). After harvest, cytosolic GS activity declined by half and then, at 3 or 4 d after harvest, rose to 80% of the cytosolic GS activity present at harvest. The nitrogen metabolism of asparagus spears is significantly altered as the tissues deteriorate rapidly after harvest. We demonstrate that cytosolic GS activity increases during the post-harvest period and is likely to be a critical feature of the physiology of the tip of a harvested asparagus spear.     

Metabolomics data reveal a crucial role of cytosolic glutamine synthetase 1;1 in coordinating metabolic balance in rice

Rice plants grown in paddy fields preferentially use ammonium as a source of inorganic nitrogen. Glutamine synthetase (GS) catalyses the conversion of ammonium to glutamine. Of the three genes encoding cytosolic GS in rice, OsGS1;1 is critical for normal growth and grain filling. However, the basis of its physiological function that may alter the rate of nitrogen assimilation and carbon metabolism within the context of metabolic networks remains unclear. To address this issue, we carried out quantitative comparative analyses between the metabolite profiles of a rice mutant lacking OsGS1;1 and its background wild type (WT). The mutant plants exhibited severe retardation of shoot growth in the presence of ammonium compared with the WT. Overaccumulation of free ammonium in the leaf sheath and roots of the mutant indicated the importance of OsGS1;1 for ammonium assimilation in both organs. The metabolite profiles of the mutant line revealed: (i) an imbalance in levels of sugars, amino acids and metabolites in the tricarboxylic acid cycle, and (ii) overaccumulation of secondary metabolites, particularly in the roots under a continuous supply of ammonium. Metabolite-to-metabolite correlation analysis revealed the presence of mutant-specific networks between tryptamine and other primary metabolites in the roots. These results demonstrated a crucial function of OsGS1;1 in coordinating the global metabolic network in rice plants grown using ammonium as the nitrogen source.     

Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase

The potential role of photorespiration in the protection against salt stress was examined with transgenic rice plants. Oryza sativa L. cv. Kinuhikari was transformed with a chloroplastic glutamine synthetase (GS2) gene from rice. Each transgenic rice plant line showed a different accumulation level of GS2. A transgenic plant line, G39-2, which accumulated about 1.5-fold more GS2 than the control plant, had an increased photorespiration capacity. In another line, G241-12, GS2 was almost lost and photorespiration activity could not be detected. Fluorescence quenching analysis revealed that photorespiration could prevent the over-reduction of electron transport systems. When exposed to 150 mM NaCl for 2 weeks, the control rice plants completely lost photosystem II activity, but G39-2 plants retained more than 90% activity after the 2-week treatment, whereas G241-12 plants lost these activities within one week. In the presence of isonicotinic acid hydrazide, an inhibitor of photorespiration, G39-2 showed the same salt tolerance as the control plants. The intracellular contents of NH₄ ⁺ and Na⁺ in the stressed plants correlated well with the levels of GS2. Thus, the enhancement of photorespiration conferred resistance to salt in rice plants. Preliminary results suggest chilling tolerance in the transformant.     

高等植物谷氨酰胺合成酶研究进展

谷氨酰胺合成酶（GS）是参与高等植物氮同化过程的关键酶。介绍了高等植物谷氨酰联合成酶及其同工酶的分布、性质、生理作用及分子生物学等方面的研究进展。     

Developmentally regulated primary glucocorticoid hormone induction of chick retinal glutamine synthetase mRNA

We have characterized the glucocorticoid hormone induction of glutamine synthetase mRNA in embryonic chick retinal organ cultures by quantitative dot hybridization using a cDNA clone derived from chick retinal RNA. Hydrocortisone (Kapp = 3-4 nM) and dexamethasone (Kapp = 1-2 nM) produce an approximate 30-fold increase in glutamine synthetase mRNA after incubation of organ cultures derived from embryonic day 12 retinae with either hormone for 3 hr. Progesterone is a poor inducer. The glucocorticoid-mediated rise is rapid (t1/2 = 2-3 hr) and occurs in the presence of either of the protein synthesis inhibitors cycloheximide or puromycin, indicating that the induction is a primary or direct response to the hormone. However, the magnitude of the hormonal response observed in culture increases markedly during retinal development. These observations, coupled with the previously reported absence of a hormonal induction in embryonic liver, raise the possibility of a synergistic mechanism, involving tissue-specific regulatory molecules in addition to the glucocorticoid hormone receptor, to explain the retinal-specific primary glucocorticoid hormone induction of glutamine synthetase mRNA.     

Changes in the activities of chloroplast and cytosolic isoenzymes of glutamine synthetase during normal leaf growth and plastid development in wheat

Soluble protein extracts and chloroplasts from a serial sequence of transverse sections of a 7-d-old wheat leaf (Triticum aestivum cv. Maris Huntsman) were used to study changes in the activity of glutamine synthetase (GS; EC 6.3.1.2) during cell and chloroplast development. Glutamine synthetase activity increased more than 50-fold per cell from the base to the tip of the wheat leaf. Two isoenzymes of GS were separated using fast protein liquid chromatography (FPLC). Glutamine synthetase localized in the cytoplasm (GS1) eluted at about 0.21 M NaCl, and the isoenzyme localized in the chloroplast (GS2) eluted at about 0.33 M NaCl. The increase in GS activity during leaf development was found to be caused primarily by an increase in the activity of the chloroplast GS2. The activity of the cytoplasmic GS1 remained constant as the cells were displaced from the base to the tip of the leaf, whereas GS2 activity increased within the chloroplast throughout development. At the base of the leaf, 26% of total GS activity was cytoplasmic; the remaining 74% was in the chloroplast. At 10 cm from the base, only 4% of the activity was cytoplasmic, and 96% was in the chloroplast. The results indicate that the chloroplast GS2 is probably responsible for most of the ammonia assimilation in the mature wheat leaf, whereas cytoplasmic GS1 may serve a role in immature developing leaf cells.Abbreviations FPLC fast protein liquid chromatography - GS glutamine synthetase - GS1 cytoplasmic glutamine synthetase - GS2 chloroplast glutamine synthetase     

Effects of medium glutamine,glutamate, and ammonia on glutamine synthetase activity in cultured mouse astroglial cells

Mouse astroglial cells were grown during the last week of culture in either glutamine-free or glutamine-containing medium. The addition of cortisol to the glutamine-containing medium resulted in a doubling of astroglial glutamine synthetase (GS) activity. Withdrawal of glutamine from the medium resulted in a 50% elevation of GS and addition of cortisol to such a medium resulted in a further increase in GS which was not additive to glutamine withdrawal. Both in glutamine-free and glutamine-containing medium, the addition of glutamate resulted in a depression of both basal and cortisol induced GS activity. The simultaneous addition of ammonia plus glutamate to the culture medium ameliorated the glutamate mediated depressive effects on cortisol induced but not basal GS activity. Glutamine withdrawal from the culture medium resulted in an astroglial protein deficit. The addition of ammonia to the medium considerably reduced this deficit and the addition of glutamate completely eliminated this protein deficit.     

Purification and characterisation of glutamine synthetase fromNocardia corallina

Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold fromNocardia corallina. The apparentM _r of the GS subunit was approximately 56,000. Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000. The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine. The pH profiles assayed by the -glutamyl transferase method were similar for NH₄ ⁺-treated and untreated cell extracts and an isoactivity point was not obtained from these curves. GS activity was repressed by (NH₄)₂SO₄ and glutamate. Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity. The GS ofN. corallina cross-reacted with antisera prepared against GS from a Gram-negativeThiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positiveClostridium acetobutylicum strain.