A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

Ammonia assimilation and nitrogen fixation in Rhizobium meliloti

Summary The enzymes involved in ammonia assimilation by Rhizobium meliloti 4l and their role in the regulation of nitrogen metabolism were studied. Glutamine synthetase (GS) and glutamate synthase (GOGAT) were present at relatively high levels in cells grown in media containing either low or high concentrations of ammonia. NADP-linked glutamate dehydrogenase could not be detected.GOGAT and GS mutants were isolated and characterised. A mutant lacking GOGAT activity did not grow even on high concentrations of ammonia, it was a glutamate auxotroph and was effective in symbiotic nitrogen fixation. The GS and assimilatory nitrate reductase activities of this mutant were not repressible by ammonia but still repressible by casamino acids. A mutant with low GS activity required glutamine for optimal growth. It was ineffective and its nitrate reductase was not inducible.These findings indicate that ammonia is assimilated via the GS/GOGAT pathway in free-living R. meliloti and bacterial GOGAT is not important in symbiosis. Furthermore, GS is suggested to be a controlling element in the nitrogen metabolism of R. meliloti.     

Elevated CO2 reduces the drought effect on nitrogen metabolism in barley plants during drought and subsequent recovery

The objective of this study was to determine the response of nitrogen metabolism to drought and recovery upon rewatering in barley (Hordeum vulgare L.) plants under ambient (350 μmol mol⁻¹) and elevated (700 μmol mol⁻¹) CO₂ conditions. Barley plants of the cv. Iranis were subjected to drought stress for 9, 13, or 16 days. The effects of drought under each CO₂ condition were analysed at the end of each drought period, and recovery was analysed 3 days after rewatering 13-day droughted plants. Soil and plant water status, protein content, maximum (NR_max) and actual (NR_act) nitrate reductase, glutamine synthetase (GS), and aminant (NADH-GDH) and deaminant (NAD-GDH) glutamate dehydrogenase activities were analysed. Elevated CO₂ concentration led to reduced water consumption, delayed onset of drought stress, and improved plant water status. Moreover, in irrigated plants, elevated CO₂ produced marked changes in plant nitrogen metabolism. Nitrate reduction and ammonia assimilation were higher at elevated than at ambient CO₂, which in turn yielded higher protein content. Droughted plants showed changes in water status and in foliar nitrogen metabolism. Leaf water potential (Ψ_w) and nitrogen assimilation rates decreased after the onset of water deprivation. NR_act and NR_max activity declined rapidly in response to drought. Similarly, drought decreased GS whereas NAD-GDH rose. Moreover, protein content fell dramatically in parallel with decreased leaf Ψ_w. In contrast, elevated CO₂ reduced the water stress effect on both nitrate reduction and ammonia assimilation coincident with a less-steep decrease in Ψ_w. On the other hand, Ψ_w practically reached control levels after 3 days of rewatering. In parallel with the recovery of plant water status, nitrogen metabolism was also restored. Thus, both NR_act and NR_max activities were restored to about 75-90% of control levels when water supply was restored; the GS activity reached 80-90% of control values; and GDH activities and protein content were similar to those of control plants. The recovery was always faster and slightly higher in plants grown under elevated CO₂ conditions compared to those grown in ambient CO₂, but midday Ψ_w dropped to similar values under both CO₂ conditions. The results suggest that elevated CO₂ improves nitrogen metabolism in droughted plants by maintaining better water status and enhanced photosynthesis performance, allowing superior nitrate reduction and ammonia assimilation. Ultimately, elevated CO₂ mitigates many of the effects of drought on nitrogen metabolism and allows more rapid recovery following water stress.     

Heme oxygenase up-regulation under salt stress protects nitrogen metabolism in nodules of soybean plants

The behaviour of enzymes involved in nitrogen metabolism, as well as oxidative stress generation and heme oxygenase gene and protein expression and activity, were analysed in soybean (Glycine max L.) nodules exposed to 50, 100 and 200 mM NaCl concentrations. A significant increase in lipid peroxidation was found with 100 and 200 mM salt treatments. Moreover, superoxide dismutase, catalase and peroxidase activities were decreased under 100 and 200 mM salt. Nitrogenase activity and leghemeoglobin content were diminished and ammonium content increased only under 200 mM NaCl. At 100 mM NaCl, glutamine synthetase (GS) and NADH-glutamate dehydrogenase (GDH) activities were similar to controls, whereas a significant increase (64%) in NADH-glutamate synthase (GOGAT) activity was observed. GS activity did not change at 200 mM salt treatment, but GOGAT and GDH significantly decreased (40 and 50%, respectively). When gene and protein expression of GS and GOGAT were analysed, it was found that they were positively correlated with enzyme activities. In addition, heme oxygenase (HO) activity, protein synthesis and gene expression were significantly increased under 100 mM salt treatment. Our data demonstrated that the up-regulation of HO, as part of antioxidant defence system, could be protecting the soybean nodule nitrogen fixation and assimilation under saline stress conditions.     

Ammonia Assimilation in Canavalia ensiformis Plants under Water and Salt Stress

Nitrogen fixation and ammonia assimilation in nodules have beenthoroughly studied under stress conditions, but the behaviorof enzymes involved in ammonia assimilation to organic compoundsin plants of the Leguminosae family subjected to stress stillremains to be conclusively established. We found that understress conditions, C. ensiformis plants can switch from theirusual pathway of assimilation to an alternative one dependingon the nature of the stress and the tissue in which the processtakes place. In roots, it switches from the glutamate dehydrogenase(GDH) pathway to the glutamine synthetase (GS)/glutamate synthase(GOGAT) cycle under water stress but not under salt stress.However, in leaves under salt stress, GDH activity is maintainedbut GS activity markedly decreases (Received March 24, 1987; Accepted March 4, 1988)     

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope‐assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate‐limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS‐GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate‐free and less ammonium‐affinity GDH pathway, or alternatively the energy‐consuming GS‐GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.     

Distinct properties of two glutamine synthetase isoforms in soybean root nodules

Glutamine synthetase (GS) in nodule cytosol plays a major role in the assimilation of the ammonium produced by biological nitrogen fixation. To characterize the GS protein family in Glycine max root nodules, the catalytical properties of 2 GS1 isoenzymes (GS1β1 and GS1γ1) were compared in this study. Although, GmGS1β1 and GmGS1γ1 have very similar kinetic characteristics, they also exhibit distinct enzymatic properties in terms of thermal stability and the transferase to synthetase activity (GSt/GSs) ratios. The results demonstrated that GmGS1γ1, which displayed lower thermal stability and GSt/GSs ratios than GmGS1β1, might be considered as superior isoform to participate in the efficient assimilation of ammonia only when it is needed. Also, it is proposed that the difference of enzymatic properties between isoforms contribute to their differential roles in ammonia assimilation under variable internal and external environments.     

Response of Ammonia Assimilation in Cucumber Seedlings to Nitrate Stress

The influence of increased nitrate concentration—14 (control) and 140 mmol L⁻¹ (T)—in hydroponic culture on ammonia assimilation in cucumber (Cucumis sativus L. cv. Xintaimici) seedlings was investigated. The results showed that NH₃ accumulation in the roots and leaves of T seedlings increased significantly, indicating that NH₃ toxicity might be involved in nitrate stress. Under control conditions, GS and GOGAT activity were much higher in the leaves than in the roots, whereas GDH activity was much higher in the roots than in the leaves. Correlation analysis showed that NH₃ concentration had a strong negative linear relationship with GDH activity in the roots but had a strong negative linear relationship with GS and GOGAT activity in the leaves. These results indicate that NH₃ might be assimilated primarily via GDH reaction in the roots and via GS/GOGAT cycle in the leaves. Short-term nitrate stress resulted in the increase of GS and GOGAT activity in the roots and GDH activity in the leaves of T seedlings, indicating possible shifts in ammonia assimilation from the normal GDH pathway to GS/GOGAT pathway in the roots and from the normal GS/GOGAT pathway to the GDH pathway in the leaves under nitrate stress, but with the increase of treatment time, GS, GOGAT, and GDH activity in the roots and leaves of T seedlings decreased possibly due to low water potential and NH₃ toxicity.     

Modulations in key enzymes of nitrogen metabolism in two high yielding genotypes of mulberry (Morus alba L.) with differential sensitivity to salt stress

Effect of salinity stress on the performance of nitrogen metabolism was studied in two high yielding genotypes of mulberry with differential sensitivity to NaCl (S1 and ATP, salt tolerant and susceptible, respectively). Three-month-old healthy mulberry plants were subjected to different regimes of NaCl stress [0.0 (control), 0.5, 1.0 and 1.5% NaCl] and leaf samples were collected on 4, 8 and 12 DAT (days after treatment) for the analysis. The activities of nitrate reductase (NR: EC 1.6.6.1), nitrite reductase (NiR: EC 1.6.6.4), protease, glutamine synthetase (GS: EC 6.3.1.2) and its accumulation pattern, glutamate synthase (GOGAT: EC 1.4.1.13), glutamate dehydrogenase (NADH-GDH: EC 1.4.1.2 and NADPH-GDH: EC 1.4.1.4), aspartate aminotransferase (AAT: EC 2.6.1.1) and alanine aminotransferase (ALAT: EC 2.6.1.2) coupled with total protein content, free amino acid level and ammonia content were studied in leaves of both genotypes of mulberry. The total protein content in leaves of both genotypes declined with progressive accumulation of free amino acid levels. Further, the decrease in protein content was less in S1 than ATP, and it was correlated with protease activity, ammonia content and accumulation of free amino acid levels. Higher free amino acid levels were registered for S1 than ATP at 1.0 and 1.5% NaCl stress and on all days of sampling. Ammonia content was increased in both genotypes and comparatively higher ammonia levels were recorded for ATP. Increased NaCl concentrations lead to a decrease in the activity of NR and NiR in both the genotypes, the decrease was more pronounced in ATP than S1. The enhanced activity of GDH (NADH and NADPH) was noticed in both genotypes, whereas the NADPH-GDH activity was found relatively higher in S1. The immunoblot analysis with GS-45 antibodies revealed a specific cross-reaction with 42 and 45 kDa proteins in S1, and only 45 kDa protein in ATP genotype. However, increased GS protein accumulation pattern (both 42 and 45 kDa) was observed in S1 under high NaCl. Whereas, accumulation of 45 kDa protein was unchanged at all levels of stress and slight accumulation in 42 kDa protein at 1.5% NaCl was observed for ATP. Elevation in the enzyme activities of GS, GOGAT were coupled with AAT and ALAT observed in both the genotypes. Higher enzymatic activities of S1 than ATP under salinity stress may be due to efficient capacity of ammonia detoxification. Salt tolerance of S1 supports the higher metabolic activity under salinity leading to lesser amount of ammonia accumulation and higher levels of free amino acid in the tissue. In agreement with these results the physiological significance of enzymatic changes and ammonia assimilation during salt stress in relevance to plant nitrogen metabolism was discussed.     

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.     

Bottle gourd rootstock-grafting affects nitrogen metabolism in NaCl-stressed watermelon leaves and enhances short-term salt tolerance

The plant growth, nitrogen absorption, and assimilation in watermelon (Citrullus lanatus [Thunb.] Mansf.) were investigated in self-grafted and grafted seedlings using the salt-tolerant bottle gourd rootstock Chaofeng Kangshengwang (Lagenaria siceraria Standl.) exposed to 100 mM NaCl for 3 d. The biomass and NO₃⁻ uptake rate were significantly increased by rootstock while these values were remarkably decreased by salt stress. However, compared with self-grafted plants, rootstock-grafted plants showed higher salt tolerance with higher biomass and NO₃⁻ uptake rate under salt stress. Salinity induced strong accumulation of nitrate, ammonium and protein contents and a significant decrease of nitrogen content and the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) in leaves of self-grafted seedlings. In contrast, salt stress caused a remarkable decrease in nitrate content and the activities of GS and GOGAT, and a significant increase of ammonium, protein, and nitrogen contents and NR activity, in leaves of rootstock-grafted seedlings. Compared with that of self-grafted seedlings, the ammonium content in leaves of rootstock-grafted seedlings was much lower under salt stress. Glutamate dehydrogenase (GDH) activity was notably enhanced in leaves of rootstock-grafted seedlings, whereas it was significantly inhibited in leaves of self-grafted seedlings, under salinity stress. Three GDH isozymes were isolated by native gel electrophoresis and their expressions were greatly enhanced in leaves of rootstock-grafted seedlings than those of self-grafted seedlings under both normal and salt-stress conditions. These results indicated that the salt tolerance of rootstock-grafted seedlings might (be enhanced) owing to the higher nitrogen absorption and the higher activities of enzymes for nitrogen assimilation induced by the rootstock. Furthermore, the detoxification of ammonium by GDH when the GS/GOGAT pathway was inhibited under salt stress might play an important role in the release of salt stress in rootstock-grafted seedlings.     

NaHSO3对盐胁迫下小麦幼苗氮同化酶及脯氨酸含量的影响

以普通小麦(Triticum aestivumL.)为材料,研究了NaHSO3对不同盐度胁迫下小麦幼苗氮素同化酶和脯氨酸含量的调节。结果表明,盐胁迫降低了叶片中硝酸还原酸(NR)的活性,加入NaHSO3之后,NR活性表现出进一步的降低。谷氨酰胺合成酶(GS)在低浓度盐胁迫下活性增加,在高浓度盐胁迫下活性降低;NaHSO3加入时,即便在低盐浓度下GS活性也降低。依赖于NADH的谷氨酸脱氢酶(NADH-GDH)和依赖于NADP的异柠檬酸脱氢酶(NADP-ICDH)的变化趋势一致,在盐胁迫下它们的活性都明显增加;NaHSO3加入促进了它们活性的进一步增加,尤其对NADH-GDH活性的促进更为明显。游离脯氨酸在高浓度盐胁迫下大量积累,在低浓度盐胁迫下含量增加不明显;NaHSO3促进了盐胁迫下脯氨酸的积累,提示了NaHSO3促进了盐胁迫下小麦幼苗碳氮营养元素的贮存。     

植物内生菌促进宿主氮吸收与代谢研究进展

内生菌与植物共生能够提高宿主的氮吸收与氮代谢水平,这可能是由于内生菌在植物体内引发的多种效应的综合结果.植物内生菌能够通过促进植物根系发育和固氮作用为宿主植物提供更多的无机氮素;能够通过分泌多种胞外酶系如漆酶、蛋白水解酶等使宿主植物更好地利用有机氮素;能够提高宿主氮代谢关键酶如硝酸还原酶(NR)、谷氨酰胺合成酶(GS)等酶的活性;能够提高宿主植物激素水平和维生素含量从而促进宿主氮代谢;能够通过影响宿主植物氮代谢促进宿主植物分蘖、提高宿主植物叶绿素含量和光合速率等等.综述了国内外关于植物内生菌促进宿主氮代谢的相关报道,归纳了植物内生菌影响宿主氮素吸收与代谢的可能机制,并展望了关于植物内生菌促进宿主氮代谢机制方面的研究方向.     

Ammonium formation and assimilation in P(SARK)∷IPT tobacco transgenic plants under low N

Wild Type (WT) and transgenic tobacco plants expressing isopentenyltransferase (IPT), a gene encoding the enzyme regulating the rate-limiting step in cytokinins (CKs) synthesis, were grown under limited nitrogen (N) conditions. We analyzed nitrogen forms, nitrogen metabolism related-enzymes, amino acids and photorespiration related-enzymes in WT and P_SARK∷IPT tobacco plants. Our results indicate that the WT plants subjected to N deficiency displayed reduced nitrate (NO₃⁻) assimilation. However, an increase in the production of ammonium (NH₄⁺), by the degradation of proteins and photorespiration led to an increase in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle in WT plants. In these plants, the amounts of amino acids decreased with N deficiency, although the relative amounts of glutamate and glutamine increased with N deficiency. Although the transgenic plants expressing P_SARK∷IPT and growing under suboptimal N conditions displayed a significant decline in the N forms in the leaf, they maintained the GS/GOGAT cycle at control levels. Our results suggest that, under N deficiency, CKs prevented the generation and assimilation of NH₄⁺ by increasing such processes as photorespiration, protein degradation, the GS/GOGAT cycle, and the formation of glutamine.     

Organic acid accumulation may inhibit N2 fixation in phosphorus-stressed lupin nodules.

Nodulated lupins (Lupinus angustifolius cv. Wonga) were hydroponically grown under conditions of low phosphate (LP) or adequate phosphate (HP) to assess the effect of phosphoenolpyruvate carboxylase (PEPC)-derived organic acids on nitrogen assimilation in LP nodules. LP conditions are linked to altered organic acid metabolism, by the engagement of PEP metabolism via PEPC. In LP nodules, the enhanced organic acid synthesis may reduce the available organic carbon for nitrogen assimilation. The diversion of carbon between the organic acid- and amino acid pools was assessed through key nodular enzymes and (14)CO(2) metabolism. Under LP conditions, increased rates of organic acid synthesis via PEPC and malate dehydrogenase (MDH), coincided with reduced nitrogen assimilation via aspartate aminotransferase (AAT), aspartate synthetase (AS) and glutamine synthetase (GS)/glutamate synthase (GOGAT) activities. There was a preferential metabolism of nodular (14)CO(2) into organic acids and particularly into malate. High malate levels were associated with reduced N(2) fixation and synthesis of amino acids. These results indicate that phosphorus deficiency can enhance malate synthesis in nodules, but that excessive malate accumulation may inhibit N(2) fixation and nitrogen assimilation.