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     

NADH-Glutamate synthase in alfalfa root nodules. Immunocytochemical localization

In root nodules of alfalfa (Medicago sativa L.), N₂ is reduced to NH₄⁺ in the bacteroid by the nitrogenase enzyme and then released into the plant cytosol. The NH₄⁺ is then assimilated by the combined action of glutamine synthetase (EC 6.3.1.2) and NADH-dependent Glu synthase (NADH-GOGAT; EC 1.4.1.14) into glutamine and Glu. The alfalfa nodule NADH-GOGAT protein has a 101-amino acid presequence, but the subcellular location of the protein is unknown. Using immunocytochemical localization, we determined first that the NADH-GOGAT protein is found throughout the infected cell region of both 19- and 33-d-old nodules. Second, in alfalfa root nodules NADH-GOGAT is localized predominantly to the amyloplast of infected cells. This finding, together with earlier localization and fractionation studies, indicates that in alfalfa the infected cells are the main location for the initial assimilation of fixed N₂.     

Cellular localization of NADH-dependent glutamate-synthase protein in vascular bundles of unexpanded leaf blades and young grains of rice plants

Tissue and cellular localization of NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) in the unexpanced leaf blades and young grains of rice (Oryza sativa L.) was investigated using tissue-print immunoblot and immunocytological methods with an affinity-purified anti-NADH-GOGAT immunoglobulin G. Tissue-print immunoblots showed that the NADH-GOGAT protein was mostly located in large and small vascular bundles of the unexpanded blades. When the cross-sections (10μ in thickness) prepared from the paraffin-embedded blades were stained with the antibody, the NADH-GOGAT protein was detected in vascular-parenchyma cells and mestome-sheath cells. In developing grains, the NADH-GOGAT protein was detected in both phloem- and xylem-parenchyma cells of dorsal and lateral vascular bundles, and in the nucellar projection, nucellar epidermis, and aleurone cells. On the other hand, ferredoxin (Fd)-dependent GOGAT (EC 1.4.7.1) was located mainly in mesophyll cells of the leaf blade and in chloroplast-containing cross-cells of the pericarp of the grains. The spatial expression of these GOGAT proteins indicates distinct and non-overlapping roles in rice plants. In the leaf blades and young grains, NADH-GOGAT could be involved in the synthesis of glutamate from the glutamine that is transported through the vascular system from roots and senescing tissues.     

The chemistry of the lowland rice rhizosphere

Models and experimental studies of the rhizosphere of rice plants growing in anaerobic soil show that two major processes lead to considerable acidification (1–2 pH units) of the rhizosphere over a wide range of root and soil conditions. One is generation of H⁺ in the oxidation of ferrous iron by O₂ released from the roots. The other is release of H⁺ from roots to balance excess intake of cations over anions, N being taken up chiefly as NH₄ ⁺. CO₂ exchange between the roots and soil has a much smaller effect. The zone of root-influence extends a few mm from the root surface. There are substantial differences along the root length and with time. The acidification and oxidation cause increased sorption of NH₄ ⁺ ions on soil solids, thereby impeding the movement of N to absorbing root surfaces. But they also cause solubilization and enhanced uptake of soil phosphate.     

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.     

A Supply of Nitrogen Causes Increase in the Level of NADH-Dependent Glutamate Synthase Protein and in the Activity of the Enzyme in Roots of Rice Seedlings

When rice seedlings, after the growth for 26 days in water alone,were transferred to nutrient medium contained 1 mM NH₄Cl, thelevel of NADH-dependent glutamate synthase (GOGAT) protein andthe activity of the enzyme increased more than 10-fold in root,but not in shoots. Both the level of the protein and the activityreached a maximum within 24 h. NH₄Cl was effective at concentrationsas low as 50 µM. A supply of either 1 mM NaNO₃ or 0.5mM NH₄NO₃ also caused such increases, but NHa₄Cl was most effective.A supply of glutamine or glutamate was less effective. The increasewas specific to NADH-GOGAT and little change was observed inthe levels of ferredoxin-GOGAT and glutamine synthetase isoproteinsin roots. These inducible increases in the levels of NADH-GOGATprotein and in its activity were greater in the root-tip regionthan at the base of the root. Both 6-methylpurine and cycloheximidecompletely inhibited the effects of NH₄Cl. Moreover, the mRNAfor NADH-GOGAT in rice roots accumulated markedly within 12h of the start of a supply of NH₄Cl. A possible role for therapid response of NADH-GOGAT to a supply of NH₄C1 is discussed. (Received May 18, 1995; Accepted July 10, 1995)     

Nitrogen use efficiency (NUE) in rice links to NH4 + toxicity and futile NH4 + cycling in roots

Aims

Rice is known as an ammonium (NH₄ ⁺)-tolerant species. Nevertheless, rice can suffer NH₄ ⁺ toxicity, and excessive use of nitrogen (N) fertilizer has raised NH₄ ⁺ in many paddy soils to levels that reduce vegetative biomass and yield. Examining whether thresholds of NH₄ ⁺ toxicity in rice are related to nitrogen-use efficiency (NUE) is the aim of this study.

Methods

A high-NUE (Wuyunjing 23, W23) and a low-NUE (Guidan 4, GD) rice cultivar were cultivated hydroponically, and growth, root morphology, total N and NH₄ ⁺ concentration, root oxygen consumption, and transmembrane NH₄ ⁺ fluxes in the root meristem and elongation zones were determined.

Results

We show that W23 possesses greater capacity to resist NH₄ ⁺ toxicity, while GD is more susceptible. We furthermore show that tissue NH₄ ⁺ accumulation and futile NH₄ ⁺ cycling across the root-cell plasma membrane, previously linked to inhibited plant development under elevated NH₄ ⁺, are more pronounced in GD. NH₄ ⁺ efflux in the root elongation zone, measured by SIET, was nearly sevenfold greater in GD than in W23, and this was coupled to strongly stimulated root respiration. In both cultivars, root growth was affected more severely by high NH₄ ⁺ than shoot growth. High NH₄ ⁺ mainly inhibited the development of total root length and root area, while the formation of lateral roots was unaffected.

Conclusions

It is concluded that the larger degree of seedling growth inhibition in low- vs. high-NUE rice genotypes is associated with significantly enhanced NH₄ ⁺ cycling and tissue accumulation in the elongation zone of the root.     

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.     

Ammonium under solution culture alleviates aluminum toxicity in rice and reduces aluminum accumulation in roots compared with nitrate

Al stress and ammonium–nitrogen nutrition often coexist in acidic soils due to their low pH and weak nitrification ability. Rice is the most Al-resistant species among small grain cereal crops and prefers NH₄ ⁺ as its major inorganic nitrogen source. This study investigates the effects of NH₄ ⁺ and NO₃ ^? on Al toxicity and Al accumulation in rice, and thereby associates rice Al resistance with its NH₄ ⁺ preference. Two rice subspecies, indica cv. Yangdao6 and japonica cv. Wuyunjing7, were used in this study. After treatment with or without Al under conditions of varying NH₄ ⁺ and NO₃ ^? supply, rice seedlings were harvested for the determination of root elongation, callose content, biomass, Al concentration and medium pH. The results indicated that Wuyunjing7 was more Al-resistant and NH₄ ⁺-preferring than Yangdao6. NH₄ ⁺ alleviated Al toxicity in two cultivars compared with NO₃ ^?. Both NH₄ ⁺-Al supply and pretreatment with NH₄ ⁺ reduced Al accumulation in roots and root tips compared with NO₃ ^?. NH₄ ⁺ decreased but NO₃ ^? increased the medium pH, and root tips accumulated more Al with a pH increase from 3.5 to 5.5. Increasing the NO₃ ^? concentration enhanced Al accumulation in root tips but increasing the NH₄ ⁺ concentration had the opposite effect. These results show NH₄ ⁺ alleviates Al toxicity for rice and reduces Al accumulation in roots compared with NO₃ ^?, possibly through medium pH changes and ionic competitive effects. Making use of the protective effect of NH₄ ⁺, in which the Al resistance increases, is advised for acidic soils, and the hypothesis that rice Al resistance is associated with the preferred utilization of NH₄ ⁺ is suggested.     

The effects of root surface charge and nitrogen forms on the adsorption of aluminum ions by the roots of rice with different aluminum tolerances

Aim

Our objectives were to compare effects of root charge properties on Al adsorption by the roots of rice that differed in Al-tolerance, and to examine effects of different nitrogen forms on charge properties of rice roots and Al adsorption.

Methods

Streaming potential and chemical methods were used to measure root zeta potential and investigate Al chemical forms adsorbed on the roots of rice obtained from solution culture experiments.

Results

Rice roots of the Al-sensitive variety Yangdao-6 carried greater negative charge than the Al-tolerant variety Wuyunjing-7, which meant the roots of Yangdao-6 adsorbed more exchangeable and complexed Al. When both rice varieties were grown in NH₄ ⁺-containing nutrient solutions, there were less functional groups and lower negative surface charge on their roots, which reduced Al adsorption compared to the rice grown in NO₃ ^? containing nutrient solutions. The decline in nutrient solution pH due to NH₄ ⁺ uptake by rice roots was responsible for the reduced numbers of functional groups and the lower negative surface charge on the roots compared to the rice grown in NO₃ ^? containing solutions.

Conclusions

Integrated root surface charge, as expressed by zeta potential, played an important role in Al adsorption by the roots of rice with different Al-tolerance.

     

Root system architecture variation in response to different NH4 + concentrations and its association with nitrogen-deficient tolerance traits in rice

Root plasticity, a trait that can respond to different soil environments, may assist plants to scavenge the nutrients in heterogeneous soils. The objective of this paper is to understand the NH₄ ⁺-responsive root architectural changes in rice for better acquisition of Nitrogen (N). Using a root basket method, we examined the variation in root plasticity among diverse rice genotypes grown under hydroponics with different NH₄ ⁺ concentrations. Significant variation in NH₄ ⁺-responsive root architectural changes was observed among rice genotypes studied. From the hydroponic study, five contrasting genotypes with distinct rooting patterns (mono and dimorphic) were selected based on the ratio of deeper roots and rooting pattern value. These distinct genotypes were evaluated in the field to identify the role of root architecture on plant performance under different N applications. Our field results revealed that the dimorphic rooting genotypes enhance the grain yield and shoot biomass under N-deficit conditions than monomorphic root genotypes. We conclude that root architectural plasticity and dimorphic rooting pattern would be helpful to enhance the nitrogen-acquisition efficiency under N-deficit conditions in rice.     

Alteration of nitrogen metabolism in rice variety 'Nipponbare' induced by alkali stress

Aims

Alkali stress (AS) is an important agricultural contaminant and has complex effects on plant metabolism, specifically root physiology. The aim of this study was to test the role of nitrogen metabolism regulation in alkali tolerance of rice variety 'Nipponbare'.

Methods

In this study, the rice seedlings were subjected to salinity stress (SS) or AS. Growth, the contents of inorganic ions, NH ₄ ⁺ -nitrogen (free amino acids), and NO ₃ ^? -nitrogen in the stressed seedlings were then measured. The expression of some critical genes involved in nitrogen metabolism were also assayed to test their roles in the regulation of nitrogen metabolism during adaptation of rice variety 'Nipponbare' to AS.

Results

AS showed a stronger inhibiting effect on rice variety 'Nipponbare' growth than SS. AS may have more complex effects on nitrogen metabolism than SS.

Conclusions

Effects of AS on the nitrogen metabolism of rice variety 'Nipponbare' mainly comprised two mechanisms. Firstly, in roots, AS caused the reduction of NO ₃ ^? content, which caused two harmful consequences, the large downregulation of OsNR1 expression and the subsequent reduction of NH ₄ ⁺ production in roots. On the other hand, under AS (pH, 9.11), almost all the NH ₄ ⁺ was changed to NH₃, which caused a severe deficiency of NH ₄ ⁺ surrounding the roots. Both events might cause a severe deficiency of NH ₄ ⁺ in roots. Under AS, the increased expression of several OsAMT family members in roots might be an adaptative response to the reduction of NH ₄ ⁺ content in roots or the NH ₄ ⁺ deficiency in rhizosphere. Also, the down-regulation of OsNADH-GOGAT and OsGS1;2 in roots might be due to NH ₄ ⁺ deficiency in roots. Secondly, in shoots, AS caused a larger acuumulatiuon of Na⁺, which possibly affected photorespiration and led to a continuous decrease of NH ₄ ⁺ production in shoots, and inhibited the expression of OsFd-GOGAT and OsGS2 in chloroplasts.     

Altered cell wall properties are responsible for ammonium‐reduced aluminium accumulation in rice roots

The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH₄⁺) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al‐binding assay revealed that neither NH₄⁺ nor NO₃^? altered the Al‐binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH₄⁺‐supplied roots displayed lower Al‐binding capacity than those from NO₃^?‐supplied roots when grown in non‐buffered solutions. Fourier‐transform infrared microspectroscopy analysis revealed that, compared with NO₃^?‐supplied roots, NH₄⁺‐supplied roots possessed fewer Al‐binding groups (‐OH and COO‐) and lower contents of pectin and hemicellulose. However, when grown in pH‐buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al‐binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH₄⁺‐reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH₄⁺ uptake rather than direct competition for the cell wall binding sites between Al³⁺ and NH₄⁺.     

Net fluxes of ammonium and nitrate in association with H+ fluxes in fine roots of Populus popularis

Poplar plants are cultivated as woody crops, which are often fertilized by addition of ammonium (NH₄ ⁺) and/or nitrate (NO₃ ^?) to improve yields. However, little is known about net NH₄ ⁺/NO₃ ^? fluxes and their relation with H⁺ fluxes in poplar roots. In this study, net NH₄ ⁺/NO₃ ^? fluxes in association with H⁺ fluxes were measured non-invasively using scanning ion-selective electrode technique in fine roots of Populus popularis. Spatial variability of NH₄ ⁺ and NO₃ ^? fluxes was found along root tips of P. popularis. The maximal net uptake of NH₄ ⁺ and NO₃ ^? occurred, respectively, at 10 and 15 mm from poplar root tips. Net NH₄ ⁺ uptake was induced by ca. 48 % with provision of NO₃ ^? together, but net NO₃ ^? uptake was inhibited by ca. 39 % with the presence of NH₄ ⁺ in poplar roots. Furthermore, inactivation of plasma membrane (PM) H⁺-ATPases by orthovanadate markedly inhibited net NH₄ ⁺/NO₃ ^? uptake and even led to net NH₄ ⁺ release with NO₃ ^? co-provision. Linear correlations were observed between net NH₄ ⁺/NO₃ ^? and H⁺ fluxes in poplar roots except that no correlation was found between net NH₄ ⁺ and H⁺ fluxes in roots exposed to NH₄Cl and 0 mM vanadate. These results indicate that root tips play a key role in NH₄ ⁺/NO₃ ^? uptake and that net NH₄ ⁺/NO₃ ^? fluxes and the interaction of net fluxes of both ions are tightly associated with H⁺ fluxes in poplar roots.