Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Glutamate synthesis in barley roots: the role of the plastidic glucose-6-phosphate dehydrogenase

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.     

Recycling of carbon in the oxidative pentose phosphate pathway in non-photosynthetic plastids

Recycling of carbon in the oxidative pentose phosphate pathway (OPPP) of intact pea root plastids has been studied. The synthesis of dihydroxyacetone phosphate (DHAP) and evolution of CO₂ was followed in relation to nitrite reduction. A close coupling was observed between all three measured fluxes which were linear for up to 60 min and dependent upon the integrity of the plastids. However, the quantitative relationship between 1-¹⁴CO₂ evolution from glucose 6-phosphate and nitrite reduction varied with available hexose phosphate concentration. When 10 mM glucose 6-phosphate was supplied to intact plastids a stoichiometry of 1.35 was observed between ¹⁴CO₂ evolution and nitrite reduction. As exogenous glucose 6-phosphate was decreased this value fell, becoming 0.47 in the presence of 0.2 mM glucose 6-phosphate, indicative of considerable recycling of carbon. This conclusion was reinforced when using [2-¹⁴C]glucose-6-phosphate. The measured release of 2-¹⁴CO₂ was consistent with the data for 1-¹⁴CO₂, suggesting complete recycling of carbon in the OPPP. Ribose 5-phosphate was also able to support nitrite reduction and DHAP production. A stoichiometry of 2 NO ₂ ^? reduced: 1 DHAP synthesised was observed at concentrations of 1 mM ribose 5-phosphate or less. At concentrations of ribose 5-phosphate greater than 1 mM this stoichiometry was lost as a result of enhanced DHAP synthesis without further increase in nitrite reduction. It is suggested that this decoupling from nitrite reduction is a function of excess substrate entering directly into the non-oxidative reactions of the OPPP, and may be useful when the demand for OPPP products is not linked to the demand for reductant. The significance of recycling in the OPPP is discussed in relation to the coordination of nitrate assimilation with carbohydrate oxidation in roots and with the utilisation of carbohydrate by other pathways within plastids.     

Photosynthetic control of nitrate metabolism inPhormidium uncinatum,a cyanobaterium

The impact of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was studied on growth, Hill reaction, nitrate uptake, enzymes of nitrate utilization, and of oxidative pentose pathway by phototrophically growingPhormidium uncinatum and its DCMU-resistant (DCMUR) mutant. The growth-inhibitory action of DCMU was apparently the consequence of an inactivation of photosystem II (PS II) reaction and of reduction of nitrate utilization owing to an inhibition of nitrite reductase (NiR) activity. Mutation to this herbicide rendered both the processes insensitive to DCMU. Nevertheless, nitrate transport, nitrate reduction to nitrite, and ammonia assimilation of both the strains remained rather unaffected by DCMU. Photosynthetically inactive cells of the two strains exhibited higher activity levels of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) than their phototrophic cultures.These data suggest that photosynthesis regulates nitrate utilization in this cyanobacterium at nitrite reduction level and that nitrate uptake and reduction to nitrite are relaxed from this control and conditionally sustained by oxidative breakdown of reserve glycogen.     

The sources of carbon and reducing power for fatty acid synthesis in the heterotrophic plastids of developing sunflower (Helianthus annuus L.) embryos

The provision of carbon substrates and reducing power for fatty acid synthesis in the heterotrophic plastids of developing embryos of sunflower (Helianthus annuus L.) has been investigated. Profiles of oil and storage protein accumulation were determined and embryos at 17 and 24 days after anthesis (DAA) were selected to represent early and late periods of oil accumulation. Plastids isolated from either 17 or 24 DAA embryos did not incorporate label from [1-(14)C]glucose 6-phosphate (Glc6P) into fatty acids. Malate, when supplied alone, supported the highest rates of fatty acid synthesis by the isolated plastids at both stages. Pyruvate supported rates of fatty acid synthesis at 17 DAA that were comparable to those supported by malate, but only when incubations also included Glc6P. The stimulatory effect of Glc6P on pyruvate utilization at 17 DAA was related to the rapid utilization of Glc6P through the oxidative pentose phosphate pathway (OPPP) at this stage. Addition of pyruvate to incubations containing [1-(14)C]Glc6P increased OPPP activity (measured as (14)CO(2) release), while the addition of malate suppressed it. Observations of the interactions between the rate of metabolite utilization for fatty acid synthesis and the rate of the OPPP are consistent with regulation of the OPPP by redox control of the plastidial glucose 6-phosphate dehydrogenase activity through the demand for NADPH. During pyruvate utilization for fatty acid synthesis, flux through the OPPP increases as NADPH is consumed, whereas during malate utilization, in which NADPH is produced by NADP-malic enzyme, flux through the OPPP is decreased.     

Glutamate synthase activities and protein changes in relation to nitrogen nutrition in barley: the dependence on different plastidic glucose-6P dehydrogenase isoforms

In barley (Hordeum vulgare L. var. Nure), glutamate synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) are strictly correlated biochemical processes. NADH-GOGAT was the major root isoform, whose activity increased on a medium supplied with NH4+ or NO3-; by contrast, no noticeable variations could be observed in the leaves of plants supplied with nitrogen. In the leaves, the major isoform is Fd-GOGAT, whose activity increased under nitrogen feeding. G6PDH activity increased in the roots supplied with nitrogen; no variations were observed in the leaves. Moreover, an increase of the P2 isoform in the roots was measured, giving 13.6% G6PDH activity localized in the plastids under ammonium, and 25.2% under nitrate feeding conditions. Western blots confirmed that P2-G6PDH protein was induced in the roots by nitrogen. P1-G6PDH protein was absent in the roots and increased in the leaves by nitrogen supply to the plants. The changes measured in cytosolic G6PDH seem correlated to more general cell growth processes, and do not appear to be directly involved in glutamate synthesis. The effects of light on Fd-GOGAT is discussed, together with the possibility for P2-G6PDH to sustain nitrogen assimilation upon illumination.     

Sweet pepper plastids: enzymic equipment, characterisation of the plastidic oxidative pentose-phosphate pathway, and transport of phosphorylated intermediates across the envelope membrane

Chloroplasts or chromoplasts were purified from sweet-pepper (Capsicum annuum L. cv. Yolo Wonder) fruits and analysed with respect to their enzymic equipment, the transport properties across the envelope membrane, and for the presence of a functional oxidative pentose-phosphate pathway (OPPP). It was demonstrated that both types of plastid contain enzyme activities that allow glycolysis and OPPP. During the developmental conversion from chloroplasts to chromoplasts the activities of enzymes catalysing potentially rate-limiting reactions in glycolysis increased considerably. Most enzyme activities involved in the plastidic OPPP stayed constant or decreased during ripening, but transaldolase activity increased by more than 500%. To analyse whether pepper fruit chromoplasts are able to use exogenously supplied carbohydrates for the OPPP we measured the rate of ¹⁴CO₂ release after application of radioactively labelled precursors. Isolated pepper fruit chromoplasts used exogenously supplied [U¹⁴C]glucose- 6-phosphate (Glc6P) as a precursor for the OPPP. The metabolic flux through this pathway was stimulated by the presence of additional compounds which require reducing equivalents for further conversion, e.g. nitrite, or 2-oxoglutarate plus glutamine. The [¹⁴C]Glc6P-driven OPPP in isolated chromoplasts exhibited saturation with rising concentrations of Glc6P, reaching highest rates at an external concentration of about 2 mM. Exogenously given [U¹⁴C]glucose 1-phosphate (Glc1P)′ did not lead to a release of ¹⁴CO₂, indicating that this hexose phosphate is not taken up into the intact plastid. Using a proteoliposome system in which the envelope membrane proteins from sweet-pepper chromoplasts were functionally reconstituted we demonstrated that Glc6P is transported in counter-exchange with inorganic phosphate (P_i) or other phosphorylated intermediates. The Glc6P was taken up into proteoliposomes with an apparent K _m of 0.34 mM. Surprisingly, in contrast to tomato fruit plastids, isolated chromoplasts from sweet-pepper fruits do not possess a phosphate translocator allowing the uptake of Glc1P. Rising exogenous concentrations of dihydroxyacetone phosphate strongly inhibited the metabolic flux through the OPPP. This observation is discussed with respect to the presence of two phosphate translocator proteins in the envelope of sweet-pepper chromoplasts and with respect to possible metabolic changes occurring in heterotrophic tissues during development. Received: 24 April 1997 / Accepted: 16 June 1997     

Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos

The aim of this work was to investigate the partitioning of imported glucose 6-phosphate (Glc6P) to starch and fatty acids, and to CO₂ via the oxidative pentose phosphate pathway (OPPP) in plastids isolated from developing embryos of oilseed rape (Brassica napus L.). The ability of the isolated plastids to utilize concurrently supplied substrates and the effects of these substrate combinations on the Glc6P partitioning were also assessed. The relative fluxes of carbon from Glc6P to starch, fatty acids, and to CO₂ via the OPPP were close to 2∶1∶1 when Glc6P was supplied alone. Under these conditions NADPH generated via the OPPP was greater than that required by the concurrent rate of fatty acid synthesis. Fatty acid synthesis was unaffected by the presence or absence of exogenous NADH and/or NADPH and the requirement of fatty acid synthesis for reducing power is therefore met entirely by intraplastidial metabolism. When Glc6P was supplied in the presence of either pyruvate or pyruvate and acetate, the total flux from these metabolites to fatty acids was up to threefold greater than that from either Glc6P or pyruvate when they were supplied singly. In these experiments there was little competition between Glc6P and pyruvate in fatty acid synthesis and the flux to starch was unchanged. This implies that the starch and fatty acid biosynthesis pathways did not compete for the exogenously supplied ATP on which they were strongly dependent. When Glc6P and pyruvate were provided together, the NADPH generated by the OPPP pathway was less than that required by the concurrent rate of fatty acid synthesis. This suggests that the metabolism of exogenous Glc6P via the OPPP can contribute to the NADPH demand created during fatty acid synthesis but it also indicates that other intraplastidial sources of reducing power must be available under the in-vitro conditions used.     

Fatty acid synthesis and the oxidative pentose phosphate pathway in developing embryos of oilseed rape (Brassica napus L.)

The potential role of the plastidial oxidative pentose phosphate pathway (OPPP) in providing the NADPH for fatty acid synthesis in plastids from developing embryos of Brassica napus (L.) has been investigated. Measurements of distributions of enzyme activities in fractions obtained from homogenates of isolated embryos have revealed that the glucose 6-phosphate and 6-phosphogluconate dehydrogenases are present in both cytosol and plastid, as is ribose 5-phosphate isomerase. However, transketolase and transaldolase are most probably confined to the plastid, while ribulose 5-phosphate epimerase is essentially cytosolic, although a very small proportion of plastid-localized activity cannot be ruled out. The activity of the OPPP in intact plastids was measured by the release of (14)CO(2) from [1-(14)C]glucose 6-phosphate. Activity was detectable in the absence of electron sinks created by the addition of metabolites to the incubation media and was stimulated 1.3-, 3.2-, and 7.9-fold by the respective additions of glutamine plus 2-oxoglutarate, cofactors and substrates for fatty acid synthesis, or methyl viologen. An increase in OPPP activity in response to additions that are absolutely required for fatty acid synthesis in these isolated plastids provides direct evidence that these two processes are connected, most probably by NADP/NADPH metabolism. The OPPP activity with methyl viologen was more than twice that during fatty acid synthesis, suggesting that the latter is not limited by OPPP capacity. Light energy may also contribute to reductant provision and, consistent with the possibility of maintenance of a balance of NADPH from light and the OPPP, glucose 6-phosphate dehydrogenase activity in the isolated plastids was decreased by light or by DTT.     

Studies of the Enzymic Capacities and Transport Properties of Pea Root Plastids

Plastids have been isolated from pea (Pisum sativum L.) roots with a high degree of purity and intactness. In these plastids, the activity of enzymes involved in carbohydrate metabolism have been analyzed and corrected for cytosolic contamination. The results show that fructose-1,6-bisphosphatase, NAD-glyceraldehyde phosphate dehydrogenase, and phosphoglyceromutase are not present in pea root plastids. Transport measurements revealed that inorganic phosphate, dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and glucose-6-phosphate (Glc6p) are transported across the envelope in a counterexchange mode. Transport of glucose-1-phosphate was definitely excluded. The oxidation of Glc6P by intact plastids resulted almost exclusively in the formation of DHAP. The parallel measurement of DHAP formation and NO2- consumption during Glc6P-supported nitrite reduction yielded a ratio of NO2-reduced/DHAP formed of 1.6, which is relatively close to the theoretical value of 2.0. These results show that the oxidation of Glc6P, involving the uptake of Glc6P and the release of DHAP, and the reduction of NO2- are very tightly coupled to each other.     

Interaction of cytosolic and plastidic nitrogen metabolism in plants

In angiosperms, the assimilation of ammonia resulting from nitrate reduction and from photorespiration depends on the operation of the plastidic GS/GOGAT cycle. The precursor for ammonia assimilation, 2-oxoglutarate, is synthesized in the mitochondria and in the cytosol. It is imported into the plastid by a 2-oxoglutarate/malate translocator (DiT1). In turn, the product of ammonia assimilation, glutamate, is exported from the plastids by a glutamate/malate translocator (DiT2). These transport processes link plastidic and cytosolic nitrogen metabolism and are essential for plant metabolism. DiT1 was purified to homogeneity from spinach chloroplast envelope membranes and identified as a protein with an apparent molecular mass of 45 kDa. Peptide sequences were obtained from the protein and the corresponding cDNA was cloned. The function of the DiT1 protein and its substrate specificity were confirmed by expression of the cDNA in yeast cells and functional reconstitution of the recombinant protein into liposomes. Recent advances in the molecular cloning of DiT2 and in the analysis of the in vivo function of DiT1 by antisense repression in transgenic tobacco plants will be discussed. In non-green tissues, the reducing equivalents required for glutamate formation by NADH-GOGAT are supplied by the oxidative pentose phosphate pathway. Glucose 6-phosphate, the immediate precursor of the oxidative pentose phosphate pathway is generated in the cytosol and imported into the plastids by the plastidic glucose 6-phosphate/phosphate translocator.     

Nitrogen assimilation in plants: current status and future prospects

Nitrogen (N) is the driving force for crop yields; however, excessive N application in agriculture not only increases production cost, but also causes severe environmental problems. Therefore, comprehensively understanding the molecular mechanisms of N use efficiency (NUE) and breeding crops with higher NUE is essential to tackle these problems. NUE of crops is determined by N uptake, transport, assimilation, and remobilization. In the process of N assimilation, nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamine-2-oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase) are the major enzymes. NR and NiR mediate the initiation of inorganic N utilization, and GS/GOGAT cycle converts inorganic N to organic N, playing a vital role in N assimilation and the final NUE of crops. Besides, asparagine synthetase (ASN), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase (CPSase) are also involved. In this review, we summarize the function and regulation of these enzymes reported in three major crops—rice, maize, and wheat, also in the model plant Arabidopsis, and we highlight their application in improving NUE of crops via manipulating N assimilation. Anticipated challenges and prospects toward fully understanding the function of N assimilation and further exploring the potential for NUE improvement are discussed.     

Sources of reductant for nitrate assimilation in non-photosynthetic tissue: a review

Abstract. Potential sources of reductant for the pathway of nitrate assimilation in non-photosynthetic tissue are considered in terms of the intracellular distribution of the enzymes involved (relative to those of nitrate assimilation), their maximum catalytic capacity, the availability of their respective substrates, and the form in which the reductant is produced. According to these criteria, the principal sources appear to be the reactions catalysed by NAD-glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NAD-malate dehydrogenase and NADP-malic enzyme. Mitochondrial metabolism may also make a limited contribution, although the evidence is inconclusive. The capacity to generate reductant by carbohydrate oxidation within the proplastids appears insufficient to meet the demand, and an influx of reducing power from the cytoplasm in the form of dihydroxy acetone phosphate may be necessary to support nitrite reduction and the glutamate synthase reaction which occur there.     

Appearance of nitrate reductase, nitrite reductase and glutamine synthetase in different organs of the Scots pine (Pinus sylvestris) seedling as affected by light, nitrate and ammonium

Appearance of nitrate reductase (NR, EC 1.6.6.1–3), nitrite reductase (NiR, EC 1.7.7.1) and glutamine synthetase (GS, EC 6.3.1.2) under the control of nitrate, ammonium and light was studied in roots, hypocotyls and needles (cotyledonary whorl) of the Scots pine ( Pinus sylvestris L.) seedling. It was found that appearance of NiR was mainly controlled by nitrate whereas appearance of GS was strongly controlled by light. In principle, the NR activity level showed the same dependency on nitrate and light as that of NiR. In the root, both nitrate and ammonium had a stimulatory effect on GS activity whereas in the whorl the induction was minor. The level of NiR (NR) activity is high in the root and hypocotyl and low in the cotyledonary whorl, whereas the GS activity level per organ increases strongly from the root to the whorl. Thus, in any particular organ the operation of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle is not closely connected to the operation of the nitrate reduction pathway. The strong control of GS/GOGAT by light and the minor sensitivity to induction by nitrate or ammonium indicate a major role of the GS/GOGAT cycle in reassimilation of endogeniously generated ammonium.     

Sulfur starvation and restoration affect nitrate uptake and assimilation in rapeseed

We analyzed the effect of omission of sulfur (S) from the nutrient solution and then restoration of S-source on the uptake and assimilation of nitrate in rapeseed. Incubation in nutrient solution without S for 1–6 days led to decline in uptake of nitrate, activities, and expression levels of nitrate reductase (NR) and glutamine synthetase (GS). The nitrite reductase (NiR) and glutamate synthase (GOGAT) activities were not considerably affected. There was significant enhancement in nitrate content and decline in sulfate content. Evaluation of amino acid profile under S-starvation conditions showed two- to fourfold enhancement in the contents of arginine, asparagine and O-acetyl-l-serine (OAS), whereas the contents of cysteine and methionine were reduced heavily. When the S-starved plants were subjected to restoration of S for 1, 3, 5, and 7 days, activities and expression levels of NR and GS recovered within the fifth and seventh days of restoration, respectively. Exogenous supply of metabolites (arginine, asparagine, cysteine, glutamine, OAS, and methionine) also affected the uptake and assimilation of nitrate, with a maximum for OAS. These results corroborate the tight interconnection of S-nutrition with nitrate assimilation and that OAS plays a major role in this regulation. The study must be helpful in developing a nutrient-management technology for optimization of crop productivity.     

Metabolite pools during starch synthesis and carbohydrate oxidation in amyloplasts isolated from wheat endosperm

Starch synthesis and CO₂ evolution were determined after incubating intact and lysed wheat (Triticum aestivum L. cv. Axona) endosperm amyloplasts with ¹⁴C-labelled hexose-phosphates. Amyloplasts converted [U-¹⁴C]glucose 1-phosphate (Glc1P) but not [U-¹⁴C]glucose 6-phosphate (Glc6P) into starch in the presence of ATP. When the oxidative pentose-phosphate pathway (OPPP) was stimulated, both [U-¹⁴C]Glc1P and [U-¹⁴C]Glc6P were metabolized to CO₂, but Glc6P was the better precursor for the OPPP, and Glc1P-mediated starch synthesis was reduced by 75%. In order to understand the basis for the partitioning of carbon between the two potentially competing metabolic pathways, metabolite pools were measured in purified amyloplasts under conditions which promote both starch synthesis and carbohydrate oxidation via the OPPP. Amyloplasts incubated with Glc1P or Glc6P alone showed little or no interconversion of these hexose-phosphates inside the organelle. When amyloplasts were synthesizing starch, the stromal concentrations of Glc1P and ADP-glucose were high. By contrast, when flux through the OPPP was highest, Glc1P and ADP-glucose inside the organelle were undetectable, and there was an increase in metabolites involved in carbohydrate oxidation. Measurements of the plastidial hexose-monophosphate pool during starch synthesis and carbohydrate oxidation indicate that the phosphoglucose isomerase reaction is at equilibrium whereas the reaction catalysed by phosphoglucomutase is significantly displaced from equilibrium. Received: 29 March 1997 / Accepted: 5 June 1997     

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 m m and 2.0 m m 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 (NO₃⁻), ammonium (NH₄⁺), 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 NO₃⁻ and NH₄⁺ concentration in leaves was recorded when plants were supplied with 2.0 m m 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 NO₃⁻ reduction and increases NH₄⁺ assimilation in tomato plants.     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.     

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.     

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.