Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (Km = 2450 +/- 150 microm) and glutamate (Km = 3.8 +/- 0.2 mm). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (Km for ammonium < 10 microm; Km for glutamate = 1.1 +/- 0.4 mm) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.     

The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting

Glutamine synthetase (EC 6.3.1.2) is a key enzyme of ammonium assimilation and recycling in plants where it catalyses the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, five GLN1 genes encode GS1 isoforms. GLN1;2 is the most highly expressed in leaves and is over-expressed in roots by ammonium supply and in rosettes by ample nitrate supply compared with limiting nitrate supply. It is shown here that the GLN1;2 promoter is mainly active in the minor veins of leaves and flowers and, to a lower extent, in the parenchyma of mature leaves. Cytoimmunochemistry reveals that the GLN1;2 protein is present in the companion cells. The role of GLN1;2 was determined by examining the physiology of gln1;2 knockout mutants. Mutants displayed lower glutamine synthetase activity, higher ammonium concentration, and reduced rosette biomass compared with the wild type (WT) under ample nitrate supply only. No difference between mutant and WT can be detected under limiting nitrate conditions. Despite total amino acid concentration was increased in the old leaves of mutants at high nitrate, no significant difference in nitrogen remobilization can be detected using (15)N tracing. Growing plants in vitro with ammonium or nitrate as the sole nitrogen source allowed us to confirm that GLN1;2 is induced by ammonium in roots and to observe that gln1;2 mutants displayed, under such conditions, longer root hair and smaller rosette phenotypes in ammonium. Altogether the results suggest that GLN1;2 is essential for nitrogen assimilation under ample nitrate supply and for ammonium detoxification.     

Gln49 and Ser174 residues play critical roles in determining the catalytic efficiencies of plant glutamine synthetase

Two essential residues playing critical roles in determining the substrate specificities of cytosolic glutamine synthetase (GS1) have been identified from the alignment of high-affinity (GLN1;1 and GLN1;4) and low-affinity (GLN1;2 and GLN1;3) GS1 isoenzymes in Arabidopsis, and confirmed by site-directed mutagenesis. The results indicated that either K49Q or A174S mutation is sufficient to increase the catalytic efficiencies of GLN1;3 by decreasing its Km values for ammonium. In contrast, replacement of Gln49 and Ser174 by lysine and alanine, respectively, was detrimental to glutamine synthetic activities in GLN1;4. The results suggested that Gln49 and Ser174 in the high-affinity GS1 isoenzymes are interchangeable with Lys49 and Ala174 in the low-affinity variants at the corresponding positions.     

Inhibition of ammonium assimilation restores elongation of seminal rice roots repressed by high levels of exogenous ammonium

Elongation of seminal and lateral roots of rice seedlings was markedly inhibited by high ammonium levels in growth medium. However, high exogenous nitrate concentrations had little inhibitory effect on root growth. The objective of this study was to elucidate the relationship between inhibition of rice root growth induced by high ammonium conditions and ammonium assimilation in the seedlings. Activity of glutamine synthetase (GS) was kept at a low level in the seminal roots of the seedlings grown under high nitrate levels. In contrast, high ammonium levels significantly enhanced the GS activity in the roots, so that Gln abundantly accumulated in the shoots. These results indicate that ammonium assimilation may be activated in the seminal roots under high ammonium conditions. Application of methionine sulfoximine (MSO), an inhibitor of GS, relieved the repression of the seminal root elongation induced by high ammonium concentrations. However, the elongation of lateral roots remained inhibited even under the same condition. Furthermore, MSO drastically increased ammonium level and remarkably decreased Gln level in the shoots grown under high ammonium conditions. These results show that, for rice seedlings, an assimilatory product of ammonium, and not ammonium itself, may serve as an endogenous indicator of the nitrogen status involved in the inhibition of seminal root elongation induced by high levels of exogenous ammonium.     

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–β‐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.     

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.     

Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots

Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.     

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.     

Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions

The mRNA and protein for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in root tips of rice (Oryza sativa L. cv. Sasanishiki) plants increases dramatically within 12 h of supplying a␣low concentration (>0.05 mM) of ammonium ions (T.␣Yamaya et al., 1995, Plant Cell Physiol 36: 1197–1204). To identify the specific cells which are responsible for this rapid increase, the cellular localization of NADH-GOGAT protein was investigated immunocytologically with an affinity-purified anti-NADH-GOGAT immunoglobulin G. When root tips (>1 mm) of rice seedlings which had been grown for 26 d in water were immuno-stained, signals for the NADH-GOGAT protein were detected in the central cylinder, in the apical meristem, and in the primordia of the secondary roots. Signals for ferredoxin-dependent GOGAT (Fd-GOGAT; EC 1.4.7.1) protein were also seen in the same three areas. When the roots were supplied with 1 mM ammonium ions for 24 h, there were strong signals for the NADH-GOGAT protein in two cell layers of the root surface, i.e. epidermis and exodermis, in addition to the cells giving signals in the absence of ammonium ions. The supply of ammonium ions was less effective on the profile of signals for Fd-GOGAT. Although the supply of ammonium ions had less effect on the expression of cytosolic glutamine synthetase (GS; EC 6.3.1.2), this enzyme was also found to be located in the epidermis and exodermis, as well as in the central cylinder and cortex. The results indicate that NADH-GOGAT, coupled to the cytosolic GS reaction, is probably important for the assimilation of ammonium ions in the two cell layers of the root surface. Received: 21 June 1997 / Accepted: 11 September 1997     

Evidence of ammonium assimilation via the glutamine synthetase-glutamate synthase system in rice seedling roots

When rice seedling roots were fed ¹⁵N-ammonium for 1 hr, theamide nitrogen of glutamine showed the highest ¹⁵N abundance.Moreover, glutamine amino, glutamic acid, aspartic acid andalanine showed higher ¹⁵N abundance than ammonium did. In roots whose GS activity was inhibited with MS, both the amountof ammonium and its ¹⁵N abundance were increased. In contrast,both the amount of all examined amino acids containing glutamicacid and their ¹⁵N abundance decreased in roots whose GS activitywas inhibited. From these results, it could be concluded thatthe first step of ammonium assimilation in rice seedling rootswas mainly glutamine synthesis by GS and the second was glutamicacid formation by the GOGAT system. The results of an experiment using ¹⁵N glutamine also supportedthis conclusion. (Received February 23, 1977; )     

Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl

The effects of NaCl on changes in ammonium level and enzyme activities of ammonium assimilation in roots growth of rice (Oryza sativa L.) seedlings were investigated. NaCl was effective in inhibiting root growth and stimulated the accumulation of ammonium in roots. Accumulation of ammonium in roots preceded inhibition of root growth caused by NaCl. Both effects caused by NaCl are reversible. Exogenous ammonium chloride and methionine sulfoximine (MSO), which caused ammonium accumulation in roots, inhibited root growth of rice seedlings. NaCl decreased glutamine synthetase and glutamate synthase activities in roots, but increased glutamate dehydrogenase activity. The growth inhibition of roots by NaCl or MSO could be reversed by the addition of L-glutamic acid or L-glutamine. The current results suggest that disturbance of ammonium assimilation in roots may be involved in regulating root growth reduction caused by NaCl.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSO methionine sulfoximine     

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA)

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4–10.2 times higher V _max value and 15.9–43.1 times higher K _m value for NH₄ ⁺, compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with ¹⁵NH₄ ⁺ showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH₄ ⁺ absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.     

水稻种子萌发期间谷氨酰胺合成酶和NADH-谷氨酸合酶的变化

测定了水稻种子不同萌发时期胚乳、胚芽鞘和幼根的谷氨酰胺合成酶（GS）和依赖于NADH的谷氨酸合酶（NADH－GOGAT）活性变化。胚乳和胚芽鞘的GS活性在萌发过程中升高,幼根的GS活性则有所降低。NADH－GOGAT的活性变化趋势与GS相同。Native-PAGE活性染色表明,在萌发阶段的水稻种子胚乳和幼根里,始终只观察到一种GS活性带。但是,在水稻种子萌发3d后,在胚芽鞘中除继续检测到GS1的活性外,还可以观察到GS2的活性。蛋白质印迹显示,水稻种子胚乳中的GS（GSe)和GS1和GSra一样是一种胞质型GS。实验结果提示,这些不同组织中的GS与NADH－GOGAT构成的循环途径也许是水稻种子萌发时氨同化的主要途径。     

NaCl对水稻谷氨酸合酶和谷氨酸脱氢酶的胁迫作用

在ＮａＣｌ的胁迫下,水稻幼苗根和叶的谷氨酸合酶和谷氨酸脱氢酶的活性随着营养液中的ＮａＣｌ浓度的升高而降低;游离ＮＨ４＾＋在叶中积累,在根中未见明显变化。与根相比,叶对ＮａＣｌ的胁迫作用更为敏感。叶的ＮＡＤＨ－ＧＯＧＡＴ和ＮＡＤＨ－ＧＤＨ活性在ＮａＣｌ胁迫降低的程度明显大于根。无论是否有ＮａＣｌ存在,根的ＮＡＤＨ－ＧＤＨ活性明显高于叶。ＧＳ／ＧＤＨ比值分析提示,对对照下,根中的ＮＨ４＾存在,根的ＮＡ     

GLN3 encodes a global regulator of nitrogen metabolism and virulence of C. albicans

The function of GLN3, a GATA factor encoding gene, in nitrogen metabolism of Candida albicans was examined. GLN3 null mutants had reduced growth rates on multiple nitrogen sources. More severe growth defects were observed in mutants lacking both GLN3 and GAT1, a second GATA factor gene. GLN3 was an activator of two genes involved in ammonium assimilation, GDH3, encoding NADP-dependent glutamate dehydrogenase, and MEP2, which encodes an ammonium permease. GAT1 contributed to MEP2 expression, but not that of GDH3. A putative general amino acid permease gene, GAP2, was also activated by both GLN3 and GAT1, but activation by GLN3 was nitrogen source dependent. GLN3 was constitutively expressed, but GAT1 expression varied with nitrogen source and was reduced 2- to 3-fold in gln3 mutants. Both gln3 and gat1 mutants exhibited reduced sensitivity to rapamycin, suggesting they function downstream of TOR kinase. Hyphae formation by gln3 and gat1 mutants differed in relation to nitrogen source. The gln3 mutants formed hyphae on several nitrogen sources, but not ammonium or urea, suggesting a defect in ammonium assimilation. Virulence of gln3 mutants was reduced in a murine model of disseminated disease. We conclude that GLN3 has a broad role in nitrogen metabolism, partially overlapping, but distinct from that of GAT1, and that its function is important for the ability of C. albicans to survive within the host environment.