Inoculation of wheat with Bacillus sp. wp-6 altered amino acid and flavonoid metabolism and promoted plant growth.

KEY MESSAGE: Inoculation of wheat seedling with Bacillus sp. wp-6 changed amino acid metabolism and flavonoid synthesis and promoted plant growth. Plant growth-promoting rhizobacteria (PGPR), which can reduce the use of agrochemicals, is vital for the development of sustainable agriculture. In this study, proteomics and metabolomics analyses were performed to investigate the effects of inoculation with a PGPR, Bacillus sp. wp-6, on wheat (Triticum aestivum L.) seedling growth. The results showed that inoculation with Bacillus sp. wp-6 increased shoot and root fresh weights by 19% and 18%, respectively, after 40 days. The expression levels of alpha-linolenic acid metabolism-related proteins and metabolites (lipoxygenase 2, allene oxide synthase 2, jasmonic acid, 17-hydroxylinolenic acid) and flavonoid biosynthesis-related proteins and metabolites (chalcone synthase 2 and PHC 4'-O-glucoside) were up-regulated. In addition, the expression levels of amino acid metabolism-related proteins (NADH-dependent glutamate synthase, bifunctional aspartokinase/homoserine, anthranilate synthase alpha subunit 1, and 3-phosphoshikimate 1-carboxyvinyltransferase) and metabolites (L-aspartate, L-arginine, and S-glutathionyl-L-cysteine) were also significantly up-regulated. Among them, NADH-dependent glutamate synthase and bifunctional aspartokinase/homoserine could act as regulators of nitrogen metabolism. Overall, inoculation of wheat with Bacillus sp. wp-6 altered alpha-linolenic acid metabolism, amino acid metabolism, and flavonoid synthesis and promoted wheat seedling growth. This study will deepen our understanding of the mechanism by which Bacillus sp. wp-6 promotes wheat growth using proteomics and metabolomics.

Glutamate dehydrogenase mediated amino acid metabolism after ammonium uptake enhances rice growth under aeration condition.

KEY MESSAGE: Aeration stimulates the rice growth and nitrogen (N) metabolism; in which, the glutamate accumulation limited by the glutamate dehydrogenase pathway after ammonia uptake may control root N metabolism during aeration. Increasing rhizosphere oxygen content greatly improves rice growth and biomass. To study the intrinsic mechanism involved in nitrogen (N) metabolism, a hydroponic experiment was conducted by supplying two different oxygen levels to two different rice genotypes. Compared to the hypoxia-resistant cultivar (Nip; japonica rice 'Nipponbare'), the hypoxia-sensitive cultivar (U502; upland rice 'Upland 502') presented with severe oxidative damage under the lack of aeration. However, aeration significantly reduced root oxidative damage by enhancing root antioxidant capacity and leaf photosynthesis especially in U502, and significantly increased nitrate (NO3-) and ammonia (NH4+) uptake and upregulated the expression of the genes controlling these processes. Additional NO3- was mainly incorporated into amino acids in the leaves whereas NH4+ assimilation occurred mostly in the roots. The 15N gas chromatography-mass spectrometry analysis demonstrated that aeration had no influence on the compositions of the individual amino acids derived from 15NO3- in the roots, but increased labeled glutamic acid (Glu), asparagine, γ-aminobutyric acid, and alanine in 15NH4+-treated roots. Aeration inhibited root glutamate synthetase activity but this did not inhibit 15N-Glu production from 15NH4+. In contrast, aeration upregulated isocitrate dehydrogenase and glutamate dehydrogenase. These mechanisms and soluble carbohydrates may constitute an alternative pathway for Glu production in which amino acid metabolism is enhanced after NH4+ uptake during aeration. Therefore, the rice growth-enhancing effect of aeration is closely correlated with root redox equilibrium, N uptake, and amino acid metabolism. Glutamic acid accumulation is limited by the glutamate dehydrogenase pathway after NH4+ uptake and may control root N metabolism during aeration.

γ-Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings.

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH4+ ) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH4+ toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH4+ concentrations above 0.75 mM inhibited the growth of rice and caused NH4+ accumulation in both shoots and roots. Use of excessive NH4+ also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH4+ conditions, exogenous γ-aminobutyric acid (GABA) treatment limited NH4+ accumulation in rice seedlings, reduced NH4+ toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH4+ . Furthermore, we found that the activity of glutamine synthetase/NADH-glutamate synthase (GS; EC 6.3.1.2/NADH-GOGAT; EC1.4.1.14) in root increased gradually as the NH4+ concentration increased. However, when the concentration of NH4+ is more than 3 mM, GABA treatment inhibited NH4+ -induced increases in GS/NADH-GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH4+ . These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA-dependent alleviation of ammonium toxicity in rice seedlings.

Evidence supporting distinct functions of three cytosolic glutamine synthetases and two NADH-glutamate synthases in rice.

The functions of the three isoenzymes of cytosolic glutamine synthetase (GS1;1, GS1;2, and GS1;3) and two NADH-glutamate synthases (NADH-GOGAT1 and NADH-GOGAT2) in rice (Oryza sativa L.) were characterized using a reverse genetics approach and spatial expression of the corresponding genes. OsGS1;2 and OsNADH-GOGAT1 were mainly expressed in surface cells of rice roots in an NH4 (+)-dependent manner. Disruption of either gene by the insertion of endogenous retrotransposon Tos17 caused reduction in active tiller number and hence panicle number at harvest. Re-introduction of OsGS1;2 cDNA under the control of its own promoter into the knockout mutants successfully restored panicle number to wild-type levels. These results indicate that GS1;2 and NADH-GOGAT1 are important in the primary assimilation of NH4 (+) taken up by rice roots. OsGS1;1 and OsNADH-GOGAT2 were mainly expressed in vascular tissues of mature leaf blades. OsGS1;1 mutants showed severe reduction in growth rate and grain filling, whereas OsNADH-GOGAT2 mutants had marked reduction in spikelet number per panicle. Complementation of phenotypes seen in the OsGS1;1 mutant was successfully observed when OsGS1;1 was re-introduced. Thus, these two enzymes could be important in remobilization of nitrogen during natural senescence. Metabolite profiling data showed a crucial role of GS1;1 in coordinating metabolic balance in rice. Expression of OsGS1:3 was spikelet-specific, indicating that it is probably important in grain ripening and/or germination. Thus, these isoenzymes seem to possess distinct and non-overlapping functions and none was able to compensate for the individual function of another.

NADH-dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root.

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2-oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin-dependent glutamate synthase (Fd-GOGAT) and nicotinamide adenine dinucleotide (NADH)-GOGAT. While Fd-GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH-GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH-GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real-time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH-GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH-GOGAT and Fd-GOGAT did not fully overlap. Promoter-ß-glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH-GOGAT was highly accumulated in non-green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH-GOGAT transfer DNA (T-DNA) insertion lines under normal CO2 condition. The data emphasize the importance of NADH-GOGAT in the ammonium assimilation in Arabidopsis roots.

[Effects of nitrogen form on growth and quality of Chrysanthemums morifolium].

This paper is aimed to study the effects of nitrogen form on the growth and quality of Chrysanthemums morifolium at the same nitrogen level. In order to provide references for nutrition regulation of Ch. morifolium in field production, pot experiments were carried out in the greenhouse at experimental station of Nanjing Agricultural University. Five proportions of ammonium and nitrate nitrogen were set up and a randomized block design was applied four times repeatedly. The results showed that the growth and quality of Ch. morifolium were significantly influenced by the nitrogen form. The content of chlorophyll and photosynthesis rate were the highest at the NH4(+) -N /NO3(-) -N ratio of 25:75; The activities of NR in different parts of Ch. -morifolium reached the highest at the NH4(+) - N/NO3(-) -N ratio of 0: 100. The contents of nitrate nitrogen in the root and leaves reached the highest at the NH4(+) -N/NO3(-) -N ratio of 50:50. The activities of GS, GOGAT and the content of amylum increased with the ratio of NO3(-) -N decreasing and reached it's maximum at the NH4 + -N/NO3 - -N ratio of 100: 0. The content of ammonium nitrogen were the highest at the NH4 + -N /NO3 --N ratio of 75: 25, while the content of soluble sugar reached the highest at the NH4(+)-N/NO3(-) -N ratio of 25: 75. The content of flavones, chlorogenic acid and 3,5-O-dicoffeoylqunic acid were 57.2 mg x g(-1), 0.673% and 1.838% respectively, reaching the maximum at the NH4(+) -N /NO3(-) -N ratio of 25:75; The content of luteoloside increased with the ratio of NO3(-) -N increasing and reached it's maximum at the NH4(+) -N/NO3(-) -N ratio of 0: 100. The yield of Ch. morifolium reached it's maximum at the NH4(+) -N /NO3(-) -N ratio of 25:75. Nitrogen form has some remarkable influence on the nitrogen metabolism, photosynthesis and growth, Nitrogen form conducive to the growth and quality of Ch. morifolium at the NH4(+) -N /NO3(-) -N ratio of 25: 75.

Iron deficiency affects nitrogen metabolism in cucumber (Cucumis sativus L.) plants.

BACKGROUND: Nitrogen is a principal limiting nutrient in plant growth and development. Among factors that may limit NO3- assimilation, Fe potentially plays a crucial role being a metal cofactor of enzymes of the reductive assimilatory pathway. Very few information is available about the changes of nitrogen metabolism occurring under Fe deficiency in Strategy I plants. The aim of this work was to study how cucumber (Cucumis sativus L.) plants modify their nitrogen metabolism when grown under iron deficiency. RESULTS: The activity of enzymes involved in the reductive assimilation of nitrate and the reactions that produce the substrates for the ammonium assimilation both at root and at leaf levels in Fe-deficient cucumber plants were investigated. Under Fe deficiency, only nitrate reductase (EC 1.7.1.1) activity decreased both at the root and leaf level, whilst for glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.1.14) an increase was found. Accordingly, the transcript analysis for these enzymes showed the same behaviour except for root nitrate reductase which increased. Furthermore, it was found that amino acid concentration greatly decreased in Fe-deficient roots, whilst it increased in the corresponding leaves. Moreover, amino acids increased in the xylem sap of Fe-deficient plants. CONCLUSIONS: The data obtained in this work provided new insights on the responses of plants to Fe deficiency, suggesting that this nutritional disorder differentially affected N metabolism in root and in leaf. Indeed under Fe deficiency, roots respond more efficiently, sustaining the whole plant by furnishing metabolites (i.e. aa, organic acids) to the leaves.

Autophagy sustains glutamate and aspartate synthesis in Saccharomyces cerevisiae during nitrogen starvation.

Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.

Reverse genetics approach to characterize a function of NADH-glutamate synthase1 in rice plants.

Rice plants grown in anaerobic paddy soil prefer to use ammonium ion as an inorganic nitrogen source for their growth. The ammonium ions are assimilated by the coupled reaction of glutamine synthetase (GS) and glutamate synthase (GOGAT). In rice, there is a small gene family for GOGAT: there are two NADH-dependent types and one ferredoxin (Fd)-dependent type. Fd-GOGAT is important in the re-assimilation of photorespiratorily generated ammonium ions in chloroplasts. Although cell-type and age-dependent expression of two NADH-GOGAT genes has been well characterized, metabolic function of individual gene product is not fully understood. Reverse genetics approach is a direct way to characterize functions of isoenzymes. We have isolated a knockout rice mutant lacking NADH-dependent glutamate synthase1 (NADH-GOGAT1) and our studies show that this isoenzyme is important for primary ammonium assimilation in roots at the seedling stage. NADH-GOGAT1 is also important in the development of active tiller number, when the mutant was grown in paddy field until the harvest. Expression of NADH-GOGAT2 and Fd-GOGAT in the mutant was identical with that in wild-type, suggesting that these GOGATs are not able to compensate for NADH-GOGAT1 function.

CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced L-Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase.

Here, Corynebacterium glutamicum SNK118 was metabolically engineered for L-ornithine production through CRISPR-Cpf1-based genome manipulation and plasmid-based heterologous overexpression. Genes argF, argR, and ncgl2228 were deleted to block the degradation of L-ornithine, eliminate the global transcriptional repression, and alleviate the competitive branch pathway, respectively. Overexpression of CsgapC (NADP-dependent glyceraldehyde 3-phosphate dehydrogenases gene from Clostridium saccharobutylicum DSM 13864) and BsrocG (NADH-dependent glutamate dehydrogenase gene from Bacillus subtilis HB-1) resulted markedly increased ornithine biosynthesis. Eventually, the engineered strain KBJ11 (SNK118ΔargRΔargFΔncgl2228/pXMJ19-CsgapC-BsrocG) was constructed for L-ornithine overproduction. In fed-batch fermentation, L-ornithine of 88.26 g/L with productivity of 1.23 g/L/h (over 72 h) and yield of 0.414 g/g glucose was achieved by strain KBJ11 in a 10-L bioreactor. Our result represents the highest titer and yield of L-ornithine production by microbial fermentation. This study suggests that heterologous expression of CsgapC and BsrocG could promote L-ornithine production by C. glutamicum strains.

Response of nitrogen metabolism to boron toxicity in tomato plants.

Boron (B) toxicity has become important in areas close to the Mediterranean Sea where intensive agriculture has been developed. The objective of this research was to study the effects of B toxicity (0.5 mM and 2.0 mM B) on nitrogen (N) assimilation of two tomato cultivars that are often used in these areas. Leaf biomass, relative leaf growth rate (RGR(L)), concentration of B, nitrate (NO(3) (-)), ammonium (NH(4) (+)), organic N, amino acids and soluble proteins, as well as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), glutamate synthetase (GOGAT) and glutamate dehydrogenase (GDH) activities were analysed in leaves. Boron toxicity significantly decreased leaf biomass, RGR(L), organic N, soluble proteins, and NR and NiR activities. The lowest NO(3) (-) and NH(4) (+) concentration in leaves was recorded when plants were supplied with 2.0 mM B in the root medium. Total B, amino acids, activities of GS, GOGAT and GDH increased under B toxicity. Data from the present study prove that B toxicity causes inhibition of NO(3) (-) reduction and increases NH(4) (+) assimilation in tomato plants.

Effect of Dihydroartemisinin on Plasmodium NADH-Dependent Glutamate Synthase: The Implication in Malaria Management.

Artemisinin and its analogues (ARTs) are currently the most effective anti-malarial drugs, but the precise mechanism of action is still highly controversial. Effects of ARTs on Plasmodium genes expression are studied in our Lab. The overexpression of an interesting amidotransferase, NADH-dependent glutamate synthase (NADH-GltS) was found in treated by dihydroartemisinin (DHA). The increased expression occurred not only from global transcriptomics analysis on the human malaria parasite Plasmodium falciparum (P. falciparum) 3D7 and gene expression screening on all of iron-sulphur cluster proteins from P.f. 3D7 in vitro but also from Plasmodium berghei (P. berghei) ANKA in mice. Influence of DHA on NADH-GltS was specifically at trophozoite stage of P. falciparum and in a dose-dependent manner below the effective doses. L-glutamine (Gln) and L-glutamate (Glu) are the substrate and product of NADH-GltS respectively. Azaserine (Aza) is specific inhibitor for NADH-GltS. Experimental data showed that Glu levels were significantly decreasing with DHA dose increasing but NADH-GltS enzyme activities were still remained at higher levels in parasites, and appropriate amount of exogenous Glu could significantly reduce anti-malarial action of DHA but excessive amount lost the above effect. Aza alone could inhibit proliferation of P. falciparum and had an additive effect in combination with DHA. Those results could suggest that: Glutamate depletion is one of the anti-malarial actions of DHA; overexpression of NADH-GltS would be a feedback pattern of parasite itself due to glutamate depletion, but not a direct action of DHA; the "feedback pattern" is one of protective strategies of Plasmodium to interfere with the anti-malarial actions of DHA; and specific inhibitor for NADH-GltS as a new type of anti-malarial agents or new partner in ACT might provide a potential.

Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress.

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.

NADH-dependent glutamate synthase participated in ammonium assimilation in Arabidopsis root.

Higher plants have 2 GOGAT species, Fd-GOGAT and NADH-GOGAT. While Fd-GOGAT mainly assimilates ammonium in leaves, which is derived from photorespiration, the function of NADH-GOGAT, which is highly expressed in roots, (1) needs to be elucidated. The aim of this study was to clarify the role of NADH-GOGAT in Arabidopsis roots. The supply of ammonium to the roots caused an accumulation of NADH-GOGAT, while Fd-GOGAT 1 and Fd-GOGAT 2 showed no response. A promoter-GUS fusion analysis and immunohistochemistry showed that NADH-GOGAT was located in non-green tissues like vascular bundles, shoot apical meristem, pollen, stigma, and roots. The localization of NADH-GOGAT and Fd-GOGAT was not overlapped. NADH-GOGAT T-DNA insertion lines showed a reduction of glutamate and biomass under normal CO2 conditions. These data emphasizes the importance of NADH-GOGAT in the ammonium assimilation of Arabidopsis roots.

Lack of malate valve capacities lead to improved N-assimilation and growth in transgenic A. thaliana plants.

In this study we analyzed the relationship between malate valve capacities, N-assimilation, and energy metabolism. We used transgenic plants either lacking the chloroplast NADP-dependent malate dehydrogenase or mutants with a decreased transcript level of the plastid-localized NAD-dependent malate dehydrogenase. Plants were grown on nitrate or ammonium, respectively, as the sole N-source and transcripts were analyzed by qRT-PCR. We could show that the lack of malate valve capacities enhances N-assimilation and plastidial glycolysis by increasing transcript levels of Fd-GOGATs or NADH-GOGAT and plastidic NAD-GAPDHs (GapCps), respectively. Based on our results, we conclude that the lack of malate valve capacities is balanced by an increase of the activity of plastid-localized glycolysis in order to cover the high demand for plastidial ATP, stressing the importance of the plastids for energy metabolism in plant cells.

Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves.

The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ(1)-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ(1)-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.

Suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice (Oryza sativa L.).

Rice (Oryza sativa) glutamate synthase (GOGAT, EC 1.4.1.14) enzymes have been proposed to have great potential for improving nitrogen use efficiency, but their functions in vivo and their effects on carbon and nitrogen metabolism have not been systematically explored. In this research, we analyzed transcriptional profiles of rice GOGAT genes using a genome-wide microarray database, and investigated the effects of suppression of glutamate synthase genes on carbon and nitrogen metabolism using GOGAT co-suppressed rice plants. Transcriptional profiles showed that rice GOGAT genes were expressed differently in various tissues and organs, which suggested that they have different roles in vivo. Compared with the wild-type, tiller number, total shoot dry weight, and yield of GOGAT co-suppressed plants were significantly decreased. Physiological and biochemical studies showed that the contents of nitrate, several kinds of free amino acids, chlorophyll, sugars, sugar phosphates, and pyridine nucleotides were significantly decreased in leaves of GOGAT co-suppressed plants, but the contents of free ammonium, 2-oxoglutarate, and isocitrate in leaves were increased. We conclude that GOGATs play essential roles in carbon and nitrogen metabolism, and that they are indispensable for efficient nitrogen assimilation in rice.

Assimilation of excess ammonium into amino acids and nitrogen translocation in Arabidopsis thaliana--roles of glutamate synthases and carbamoylphosphate synthetase in leaves.

This study was aimed at investigating the physiological role of ferredoxin-glutamate synthases (EC 1.4.1.7), NADH-glutamate synthase (EC 1.4.1.14) and carbamoylphosphate synthetase (EC 6.3.5.5) in Arabidopsis. Phenotypic analysis revealed a high level of photorespiratory ammonium, glutamine/glutamate and asparagine/aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate synthase, indicating that excess photorespiratory ammonium was detoxified into amino acids for transport out of the veins. Consistent with these results, promoter analysis and in situ hybridization demonstrated that GLU1 and GLU2 were expressed in the mesophyll and phloem companion cell-sieve element complex. However, these phenotypic changes were not detected in the GLU2 mutant defective in the second ferredoxin-glutamate synthase gene. The impairment in primary ammonium assimilation in the GLT mutant under nonphotorespiratory high-CO(2) conditions underlined the importance of NADH-glutamate synthase for amino acid trafficking, given that this gene only accounted for 3% of total glutamate synthase activity. The excess ammonium from either endogenous photorespiration or the exogenous medium was shifted to arginine. The promoter analysis and slight effects on overall arginine synthesis in the T-DNA insertion mutant in the single carbamoylphosphate synthetase large subunit gene indicated that carbamoylphosphate synthetase located in the chloroplasts was not limiting for ammonium assimilation into arginine. The data provided evidence that ferredoxin-glutamate synthases, NADH-glutamate synthase and carbamoylphosphate synthetase play specific physiological roles in ammonium assimilation in the mesophyll and phloem for the synthesis and transport of glutamine, glutamate, arginine, and derived amino acids.

Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.).

A major source of inorganic nitrogen for rice plants grown in paddy soil is ammonium ions. The ammonium ions are actively taken up by the roots via ammonium transporters and subsequently assimilated into the amide residue of glutamine (Gln) by the reaction of glutamine synthetase (GS) in the roots. The Gln is converted into glutamate (Glu), which is a central amino acid for the synthesis of a number of amino acids, by the reaction of glutamate synthase (GOGAT). Although a small gene family for both GS and GOGAT is present in rice, ammonium-dependent and cell type-specific expression suggest that cytosolic GS1;2 and plastidic NADH-GOGAT1 are responsible for the primary assimilation of ammonium ions in the roots. In the plant top, approximately 80% of the total nitrogen in the panicle is remobilized through the phloem from senescing organs. Since the major form of nitrogen in the phloem sap is Gln, GS in the senescing organs and GOGAT in developing organs are important for nitrogen remobilization and reutilization, respectively. Recent work with a knock-out mutant of rice clearly showed that GS1;1 is responsible for this process. Overexpression studies together with age- and cell type-specific expression strongly suggest that NADH-GOGAT1 is important for the reutilization of transported Gln in developing organs. The overall process of nitrogen utilization within the plant is discussed.

Glutamate is involved in acid stress response in Bradyrhizobium sp. SEMIA 6144 (Arachis hypogaea L.) microsymbiont.

In the present study, the effect of acid stress on ammonium assimilation in Bradyrhizobium sp. SEMIA 6144 (Arachis hypogaea L.) microsymbiont was analyzed. The bacterial growth rate was decreased by 50%, and a significant increase in intracellular glutamate concentration was detected when the strain grew at acid pH (5.5). Assays of the enzymes involved in glutamate synthesis showed increased activities of glutamine synthetase (GS) and glutamate synthase (NADPH-GOGAT) under acid stress condition. This would support the contention that the GS/NADPH-GOGAT pathway contributes to the increase of glutamate synthesis as a compatible solute in response to acid stress.