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     

Effect of ammonium salt on enzymes of ammonium assimilation in maize seedlings

Glutamate dehydrogenase (GDH E.C. 1.4.1.2.4), glutamine synthetase (GS E.C. 6.3.1.2) and glutamate synthase (glutamine oxoglutarate amino transferase, GOGAT E.C. 2.6.1.53) activities, protein and organic nitrogen contents and growth of roots and shoots of maize seedlings raised in dark at 25±2°C in half strength Hoagland’s solution containing different ammonium salts as source of nitrogen, were determined to assess the contribution of alternate pathways in ammonium assimilation. Ammonium nitrate or in some cases ammonium chloride appeared to be the best source for both root and shoot growth and for increase in protein, total nitrogen and the enzymes of ammonium assimilation. In roots, NH₄-nitrogen appeared to be assimilated by both GDH as well as GS-GOGAT pathways specially in the dark grown seedlings, while in shoots it was primarily by GS-GOGAT pathway.     

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.     

Inhibition of maize root growth by high nitrate supply is correlated with reduced IAA levels in roots

The plant root system is highly sensitive to nutrient availability and distribution in the soil. For instance, root elongation is inhibited when grown in high nitrate concentrations. To decipher the mechanism underlying the nitrate-induced inhibition of root elongation, the involvement of the plant hormone auxin in nitrate-dependent root elongation of maize was investigated. Root growth, nitrogen and nitrate concentrations, and indole-3-acetic acid (IAA) concentrations in roots and in phloem exudates of maize grown under varying nitrate concentrations were analyzed. Total N and nitrate concentrations in shoots and roots increased and elongation of primary, seminal and crown roots were inhibited with increasing external nitrate from 0.05 to 5 mM. High nitrate-inhibited root growth resulted primarily from the reduced cell elongation and not from changes in meristem length. IAA concentrations in phloem exudates reduced with higher nitrate supply. Inhibition of root growth by high nitrate was closely related to the reduction of IAA levels in roots, especially in the sections close to root tips. Exogenous NAA and IAA restored primary root growth in high nitrate concentrations. It is concluded that the inhibitory effect of high nitrate concentrations on root growth may be partly attributed to the decrease in auxin concentrations of roots.     

Cellular compartmentation of ammonium assimilation in rice and barley

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.     

How does glutamine synthetase activity determine plant tolerance to ammonium?

The wide range of plant responses to ammonium nutrition can be used to study the way ammonium interferes with plant metabolism and to assess some characteristics related with ammonium tolerance by plants. In this work we investigated the hypothesis of plant tolerance to ammonium being related with the plants’ capacity to maintain high levels of inorganic nitrogen assimilation in the roots. Plants of several species (Spinacia oleracea L., Lycopersicon esculentum L., Lactuca sativa L., Pisum sativum L. and Lupinus albus L.) were grown in the presence of distinct concentrations (0.5, 1.5, 3 and 6 mM) of nitrate and ammonium. The relative contributions of the activity of the key enzymes glutamine synthetase (GS; under light and dark conditions) and glutamate dehydrogenase (GDH) were determined. The main plant organs of nitrogen assimilation (root or shoot) to plant tolerance to ammonium were assessed. The results show that only plants that are able to maintain high levels of GS activity in the dark (either in leaves or in roots) and high root GDH activities accumulate equal amounts of biomass independently of the nitrogen source available to the root medium and thus are ammonium tolerant. Plant species with high GS activities in the dark coincide with those displaying a high capacity for nitrogen metabolism in the roots. Therefore, the main location of nitrogen metabolism (shoots or roots) and the levels of GS activity in the dark are an important strategy for plant ammonium tolerance. The relative contribution of each of these parameters to species tolerance to ammonium is assessed. The efficient sequestration of ammonium in roots, presumably in the vacuoles, is considered as an additional mechanism contributing to plant tolerance to ammonium nutrition.     

Unusual regulatory nitrate reductase activity in cotyledons of Brassica napus seedlings: enhancement of nitrate reductase activity by ammonium supply

The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.     

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.     

Nitrate reductase,nitrite reductase and glutamate dehydrogenase levels in roots and leaves of maize seedlings

Total activities of nitrate and nitrite reductases were higher in 4 to 20 day old maize plants in the leaves than in the roots. The ratio of activities found in the leaves and in the roots respectively was much higher in the case of nitrate reductase than in the case of nitrite reductase. On the other hand higher glutamate dehydrogenase activity in the roots than in the leaves clearly indicates that the roots play a more important role in the assimilation of ammonium than in the assimilation of nitrate. When comparing the distribution of seminal and nodal adventitious roots of maize seedlings with the assimilation of inorganic nitrogen on the basis of enzyme levels, it could be deduced that during the first 20 days of seedling growth seminal roots were more involved in the assimilation of nitrate whereas nodal adventitious roots were more active in ammonium assimilation.     

Nitric oxide is involved in nitrate-induced inhibition of root elongation in Zea mays

BACKGROUND AND AIMS: Root growth and development are closely dependent upon nitrate supply in the growth medium. To unravel the mechanism underlying dependence of root growth on nitrate, an examination was made of whether endogenous nitric oxide (NO) is involved in nitrate-dependent growth of primary roots in maize. METHODS: Maize seedlings grown in varying concentrations of nitrate for 7 d were used to evaluate the effects on root elongation of a nitric oxide (NO) donor (sodium nitroprusside, SNP), a NO scavenger (methylene blue, MB), a nitric oxide synthase inhibitor (N(omega)-nitro-L-arginine, L-NNA), H(2)O(2), indole-3-acetic acid (IAA) and a nitric reducatse inhibitor (tungstate). The effects of these treatments on endogenous NO levels in maize root apical cells were investigated using a NO-specific fluorescent probe, 4, 5-diaminofluorescein diacetate (DAF-2DA) in association with a confocal microscopy. KEY RESULTS: Elongation of primary roots was negatively dependent on external concentrations of nitrate, and inhibition by high external nitrate was diminished when roots were treated with SNP and IAA. MB and L-NNA inhibited root elongation of plants grown in low-nitrate solution, but they had no effect on elongation of roots grown in high-nitrate solution. Tungstate inhibited root elongation grown in both low- and high-nitrate solutions. Endogenous NO levels in root apices grown in high-nitrate solution were lower than those grown in low-nitrate solution. IAA and SNP markedly enhanced endogenous NO levels in root apices grown in high nitrate, but they had no effect on endogenous NO levels in root apical cells grown in low-nitrate solution. Tungstate induced a greater increase in the endogenous NO levels in root apical cells grown in low-nitrate solution than those grown in high-nitrate solution. CONCLUSIONS: Inhibition of root elongation in maize by high external nitrate is likely to result from a reduction of nitric oxide synthase-dependent endogenous NO levels in maize root apical cells.     

Local NO3- or NH4+ supply modifies the root system architecture of Cedrus atlantica seedlings grown in a split-root device

To study the effects of local nitrate or ammonium supply on the architecture of the Cedrus atlantica root system, cedar seedlings were grown in split-root boxes in a growth chamber. In each box-compartment, roots were fertilized with a solution containing nitrogen, either as nitrate [Ca(NO₃)₂] or ammonium (NH₄Cl), supplied at 0.1 or 5.0 mM. For each seedling, the shoot growth was measured twice a week for 3 months. The root system architecture was also recorded twice a week by tracing the root elongation through the transparent face of the root observation boxes. The apical diameter of the tap-root relay and that of a representative sample of lateral roots were recorded once a month using a monocular magnifier.

The increase of ammonium or nitrate concentration in the nutrient solution has significantly enhanced the production of lateral roots on the tap-root relay. After 90 days of culture, percentages of short lateral roots obtained with nitrate were higher than those obtained using ammonium. A preferential carbon allocation to the shoots was also obtained with an increasing nitrogen supply. Until the 40th day of culture, the elongation of lateral roots was similar for all treatments and ranged from 0.25 to 0.5 cm day⁻¹. From the 40th day to the 95th day, significant differences were observed between the compared modes and maximum elongation rates were obtained with 5 mM NH₄⁺ (2.18 cm day⁻¹) and 5 mM NO₃⁻ (1.18 cm day⁻¹). Local applications of nitrate and ammonium at a low or a high concentration had local effects on elongation and branching of the root system in the fertilized compartment. Contrasting effects of ammonium and nitrate were observed on the apical diameter of tap-roots and lateral roots. The root-split culture device confirmed that nitrate had local effects on the architecture of the C. atlantica root system.     

Nitrate Reduction in Roots and Shoots of Barley (Hordeum vulgare L.) and Corn (Zea mays L.) Seedlings: I. N Study

Nitrate reduction in roots and shoots of 7-day-old barley seedlings, and 9-day-old corn seedlings was investigated. The N-depleted seedlings were transferred for 24 h or 48 h of continuous light to a mixed nitrogen medium containing both nitrate and ammonium. Total nitrate reduction was determined by ¹⁵N incorporation from ¹⁵NO₃⁻, translocation of reduced ¹⁵N from the roots to the shoots was estimated with reduced ¹⁵N from ¹⁵NH₄⁺ assimilation as tracer, and the translocation from the shoots to the roots was measured on plants grown with a split root system. A model was proposed to calculate the nitrate reduction by roots from these data. For both species, the induction phase was characterized by a high contribution of the roots which accounted for 65% of the whole plant nitrate reduction in barley, and for 70% in corn. However, during the second period of the experiment, once this induction process was finished, roots only accounted for 20% of the whole plant nitrate reduction in barley seedlings, and for 27% in corn. This reversal in nitrate reduction localization was due to both increased shoot reduction and decreased root reduction. The pattern of N exchanges between the organs showed that the cycling of reduced N through the plant was important for both species. In particular, the downward transport of reduced N increased while nitrate assimilation in roots decreased. As a result, when induction was achieved, the N feeding of the roots appeared to be highly dependent on translocation from the leaves.     

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.     

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.     

Understanding the differential nitrogen sensing mechanism in rice genotypes through expression analysis of high and low affinity ammonium transporter genes

Two rice genotypes, Kalanamak 3119 (KN3119) and Pusa Basmati 1(PB1) differing in their optimum nitrogen requirements (30 and 120 kg/ha, respectively) were undertaken to study the expression of both high and low affinity ammonium transporter genes responsible for ammonium uptake. Exposing the roots of the seedlings of both the genotypes to increasing (NH₄)₂SO₄ concentrations revealed that all the three families of rice AMT genes are expressed, some of which get altered in a genotype and concentration specific manner. This indicates that individual ammonium transporter genes have defined contributions for ammonium uptake and plant growth. Interestingly, in response to increasing nitrogen concentrations, a root specific high affinity gene, AMT1;3, was repressed in the roots of KN3119 but not in PB1 indicating the existence of a differential ammonium sensing mechanism. This also indicates that not only AMT1;3 is involved not only in ammonium uptake but may also in ammonium sensing. Further, if it can differentiate and could be used as a biomarker for nitrogen responsiveness. Expression analysis of low affinity AMT genes showed that, both AMT2;1 and AMT2;2 have high levels of expression in both roots and shoots and in KN3119 are induced at low ammonium concentrations. Expressions of AMT3 family genes were higher shoots than in the roots indicating that these genes are probably involved in the translocation and distribution of ammonium ions in leaves. The expression of the only high affinity AMT gene, AMT1;1, along with six low affinity AMT genes in the shoots suggests that low affinity AMTs in the shoots leaves are involved in supporting AMT1;1 to carry out its activities/function efficiently.     

The OsAMT1.1 gene functions in ammonium uptake and ammoniumepotassium homeostasis over low and high ammonium concentration ranges

Rice(Oryza sativa) grown in paddy fields is an ammonium(NH~+_4)-preferring crop;however,its AMT-type NH~+_4transporters that mediate root N acquisition have not been well characterized yet.In this study,we analyzed the expression pattern and physiological function of the OsAMT1.1 gene of the AMT1 subfamily in rice.Os AMT1.1 is located in the plasma membrane and is mainly expressed in the root epidermis,stele and mesophyll cells.Disruption of the Os AMT1.1 gene decreased the uptake of NH~+_4,and the growth of roots and shoots under both low NH~+_4and high NH~+_4conditions.Os AMT1.1 contributed to the short-term(5 min)~(15)NH~+_4influx rate by approximately one-quarter,irrespective of the NH~+_4concentration.Knockout of Os AMT1.1 significantly decreased the total N transport from roots to shoots under low NH~+_4conditions.Moreover,compared with the wild type,the osamt1.1 mutant showed an increase in the potassium(K)absorption rate under high NH~+_4conditions and a decrease under low NH~+_4conditions.The mutants contained a significantly high concentration of K in both the roots and shoots at a limited K(0.1 mmol/L)supply when NH~+_4was replete.Taken together,the results indicated that OsAMT1.1 significantly contributes to the NH~+_4uptake under both low and high NH~+_4conditions and plays an important role in Ne K homeostasis in rice.     

Accumulation of {gamma}-aminobutyric acid due to adding ammonium or glutamine to cultured rice cells

-Aminobutyric acid (GABA) was accumulated in rice cell cultureswhen ammonium was added to the medium. After the addition ofammonium, a temporary increase in the glutamine (Gln) pool wasobserved before the accumulation of GABA. GABA also was markedlyaccumulated when Gln was added to the medium in place of ammonium.When glutamic acid (Glu) was added without ammonium, no accumulationof GABA occurred. When L-methionine-DL-sulfoximine (MSO), an inhibitor of glutaminesynthetase, was added to the nitrate medium, the ammonium poolincreased with no accumulation of Gln and GABA. Even when ammoniumwas supplied to the medium, no GABA accumulated in the presenceof MSO. When azaserine (AZ), which inhibits the transamidationof Gln, was added with Gln, no GABA was accumulated, althoughthe Gln pool in the cell cultures increased significantly. The accumulation of GABA in cultured rice cells produced byammonium as the nitrogen source probably is related directlyto the Gln pool size, which is increased when ammonium is suppliedto the medium. (Received August 6, 1979; )