Regulation Of Nitrate Reductase Activity Research Articles

Light regulation of nitrate reductase by catalytic subunits of protein phosphatase 2A.

PP2A catalytic subunit C2 is of special importance for light/dark regulation of nitrate reductase activity. The level of unmethylated PP2A catalytic subunits decreases in darkness. Protein phosphatase 2A (PP2A) dephosphorylates and activates nitrate reductase (NR) in photosynthetically active tissue when plants are transferred from darkness to light. In the present work, investigation of Arabidopsis thaliana PP2A mutant lines revealed that one of the five PP2A catalytic subunit genes, e.g., C2, was of special importance for NR activation. Impairment of NR activation was, especially pronounced in the c2c4 double mutant. Though weaker, NR activation was also impaired in the c2 single mutant, and c1c2 and c2c5 double mutants. On the other hand, NR activation in the c4c5 double mutant was as efficient as in WT. The c4 single mutant had low PP2A activity, whereas the c2 single mutant possessed WT levels of extractable PP2A activity. PP2A activity was low in both c2c4 and c4c5. Differences in extracted PP2A activity among mutants did not strictly correlate with differences in NR activation, but underpinned that C2 has a special function in NR activation in vivo. The terminal leucine in PP2A catalytic subunits is generally methylated to a high degree, but regulation and impact of methylation/demethylation is barely studied. In WT and PP2A mutants, the level of unmethylated PP2A catalytic subunits decreased during 45min of darkness, but did not change much when light was switched on. In leucine carboxyl methyl transferase1 (LCMT1) knockout plants, which possess mainly unmethylated PP2A, NR was still activated, although not fully as efficient as in WT.

Nitrate reductase in detached embryos may serve as a marker of the preharvest tolerance of wheat seeds

We have observed an easily determinable parameter indicative of genetic pre-harvest sprouting (PHS) tolerance – the presence of an endosperm factor, presumably ABA, capable of inhibiting nitrate reductase (NR) induction in the embryo in the presence of NO3. This finding has importance not only for the early rapid screening of PHS tolerant of cereal cultivars but may also be an important tool to determine the mechanism of NR inhibition either by genetic repression or by post-translational down regulation of NR activity. In this work we sought a simplerelationship between ABA content and NR activity level that we assumed to be closely related to PHS susceptibility. Studied the relationship between ABA content, dry weight and ability to germinate in seeds of examined wheat varieties resistant to preharvest sprouting Lutescens 70, and unstable – Novosibirskaya 67. The data obtained are shown that the level of ABA reaching its maximum to 40-45 days . The maximum content of ABA for both varieties accounted for 40 days after flowering, when the grains reach maximum wet weight in the future, as the ripening hormone levels quickly decreased with the decrease in fresh weight of grain . The level of ABA in embryos unstable to pre-harvest sprouting wheat Novosibirskaya 67 ranged from 20 to 40% below the level of the hormone in the grains resistant Lutescens 70wheat. The results showed a significant increase in the ability to germinate, since it is a phase 40-45 DAP. Reduction of endogenous ABA content in grains at the final stage of maturation, with a relatively high percentage of germination, can be explained by the need and ensuring to the start of the germination process of the seed.

Regulation of nitrate reduction in Arabidopsis WT and hxk1 mutant under C and N metabolites

As in plants sugar sensing and signal transduction involve pathways dependent or independent on hexokinase 1 (HXK1) as a glucose sensor, research was conducted to determine which pathway is responsible for regulation of the nitrate reduction. An Arabidopsis mutant with T-DNA insertion in the AtHXK1 gene and defects in glucose signaling (hxk1) was used to determine nitrate reductase (NR) activity, NIA genes expression in leaves after 8-h treatment with sugars (glucose and sucrose), organic acids [2-oxoglutarate (2OG)] and amino acids (glutamine and glutamate). Sugars, especially sucrose, caused induction of NR actual activity accompanied by an increase of the NR activation state, indicating the posttranslational nature of the modifications. Those modifications were observed in wild-type (WT) and hxk1 leaves, suggesting that regulation of NR activity by sugars does not involve HXK1 as a glucose sensor. Moreover, sugars enhanced expression of NIA genes. However, a higher level of NIA transcripts did not lead to an increase of total NR activity in sugar-treated plants. This may suggest that posttranslational modification of NR is fundamental regulatory mechanisms controlling NR activity in response to C metabolites. Treatment of plants with 2-OG also modified NR through the posttranslational modifications. Elevation of actual NR activity and the enzyme activation state in WT and hxk1 leaves was observed. Amino acids caused a decrease of NIA gene expression and NR activities in WT and hxk1 leaves indicating that mutation in the hexokinase-dependent glucose signaling pathway did not interrupt the amino acid feedback regulation of NR.

Polyamines modulate nitrate reductase activity in wheat leaves: involvement of nitric oxide

In the present work, the effect of polyamines (PAs) on nitrate reductase (NR) activity was studied in wheat leaves exposed to exogenously added PAs while assessing the nitric oxide (NO) involvement in the regulation of the enzyme activity. A biphasic response was observed along the time of treatment using 0.1 mM of putrescine (Put), spermidine (Spd) or spermine (Spm). At 3 h, Spd and Spm significantly reduced NR activity by 29 or 35%, respectively, whereas at 6 h, the activity of the enzyme decreased by an average of 25%. At 21 h, Put increased NR activity by 63%, while Spd and Spm elevated the enzyme activity by 114%. NR activity, that was reduced by 0.1 mM Spm at 3 and 6 h, returned almost to control values when c-PTIO (an NO scavenger) was used, confirming that NO was involved in the inhibition of NR activity. Nitric oxide was also mediating the PA-increase of the enzyme activity at longer incubation times, evidenced when the raise in NR activity produced by 0.1 mM Spm at the longest incubation time returned to the value of the control in the presence of cPTIO. Neither the protein expression nor the nitrate content were modified by PAs treatments. The involvement of PAs and NO in the regulation of NR activity is discussed.

Regulation of nitrate reductase activity and its involvement in the production of nitric oxide in wheat leaves

Regulation of nitrate reductase activity and its involvement in the production of nitric oxide in wheat leaves

Loci controlling nitrate reductase activity in maize: ultraviolet-B signaling in aerial tissues increases nitrate reductase activity in leaf and root when responsive alleles are present

Environmental factors, such as ultraviolet-B (UV-B) irradiation, have the ability to affect pathways such as nitrogen metabolism. As fixed nitrogen is the keystone mineral nutrient that controls grain crop yield, any alteration in this cycle can be detrimental to plant productivity. Nitrate reductase enzyme activity is responsible for the reduction of nitrate to nitrite, and nitrate is the major form of nitrogen assimilated in plants. In maize (Zea mays L.) production, nitrate assimilation kinetics are important for both high- and low-input agricultural systems. Nitrate reductase protein activity is controlled by phosphatases and kinases. Nitrate reductase activity is responsive to environmental signals such as light-dark cycles and UV-B radiation, although the regulatory controls are not yet fully understood. We have determined the location of maize genetic factors that control nitrate reductase activity and the extent of contribution of each of these factors, both locally in the leaf tissue and via long-distance signaling loci that affect root nitrate reductase activity upon leaf UV irradiation. In the IBM94 recombinant inbred mapping population, the loci controlling regulation of nitrate reductase activity under UV-B map to different positions than the loci controlling nitrate reductase activity in unexposed plants.

Physiological responses of intertidal marine brown algae to nitrogen deprivation and resupply of nitrate and ammonium.

Intertidal macroalgae Fucus and Laminaria experience seasonally fluctuating inorganic N supply. This study examined the effects of long-term N deprivation, recovery following N resupply, and effects of elevated ammonium and nitrate exposure on N acquisition in intertidal algae using manipulations of N supply in tank culture. Over 15 weeks of N deprivation, internal N and nitrate reductase activity (NRA) declined, but maximum quantum yield of PSII was unaffected in Fucus serratus and Fucus vesiculosus. Low NRA was maintained despite no external nitrate availability and depletion of internal pools, suggesting a constitutive NRA, insensitive to N supply. Nitrate resupplied to N-starved thalli was rapidly taken up and internal nitrate pools and NRA increased. Exposure to elevated (50 microM) nitrate over 4 days stimulated nitrate uptake and NRA in Laminaria digitata and F. serratus. Exposure to elevated ammonium suppressed NRA in L. digitata but not in F. serratus. This novel insensitivity of NRA to ammonium in Fucus contrasts with regulation of NRA in other algae and higher plants. Ammonium suppression of NRA in L. digitata was not via inhibition of nitrate uptake and was independent of nitrate availability. L. digitata showed a higher capacity for internal nitrate storage when exposed to elevated ambient nitrate, but NRA was lower than in Fucus. All species maintained nitrate assimilation capacity in excess of nitrate uptake capacity. N uptake and storage strategies of these intertidal macroalgae are adaptive to life in fluctuating N supply, and distinct regulation of N metabolism in Fucus vs Laminaria may relate to position in the intertidal zone.

Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.

Regulation of nitrate reductase activity in Mycobacterium tuberculosis by oxygen and nitric oxide

Nitrate reduction by Mycobacterium tuberculosis is regulated by control of the transport of nitrate into the cell by NarK2. When oxygen was introduced into hypoxic cultures, nitrite production was quickly inhibited. The nitrate-reducing enzyme itself is relatively insensitive to oxygen, suggesting that the inhibition of nitrite production by oxygen was a result of interference with nitrate transport. This was not due to degradation of NarK2, as the inhibition was reversed by the removal of oxygen although chloramphenicol prevented new synthesis of NarK2. The oxidant potassium ferricyanide was added to anaerobic cultures to produce a positive redox potential in the absence of oxygen. Nitrite production decreased, signifying that oxidizing conditions, rather than oxygen itself, were responsible for the inhibition of nitrate transport. Nitric oxide added to cultures allowed NarK2 to be active even in the presence of oxygen. A similar result was obtained with hydroxylamine and ethanol, both of which interfere with oxygen utilization and the electron transport chain. It is proposed that NarK2 senses the redox state of the cell, possibly by monitoring the flow of electrons to cytochrome oxidase, and adjusts its activity so that nitrate is transported under reducing, but not under oxidizing, conditions.

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA110 impaired in nitrate reductase

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.

Characterization of Nitrate Reductase from Light- and Dark-Exposed Leaves (Comparison of Different Species and Effects of 14-3-3 Inhibitor Proteins).

Nitrate reductase (NR) was extracted and partially purified from leaves of squash (Curcurbita maxima), spinach (Spinacia oleracea), and three transgenic Nicotiana plumbaginifolia leaves in the presence of phosphatase inhibitors to preserve its phosphorylation state. Purified squash NR showed activation by substrates (hysteresis) when prepared from leaves in the light as well as in darkness. A 14-3-3 protein known to inhibit phosphorylated spinach NR in the presence of Mg2+ decreased by 70 to 85% the activity of purified NR from dark-exposed leaves, whereas NR from light-exposed leaves decreased by 10 to 25%. Apparent lack of posttranslational NR regulation in a transgenic N. plumbaginifolia expressing an NR construct with an N-terminal deletion ([delta]NR) may be explained by more easy dissociation of 14-3-3 proteins from [delta]NR. Partially purified [delta]NR was, however, inhibited by 14-3-3 protein, and the binding constant of 14-3-3 protein (4 x 108 M-1) and the NR-inhibiting protein concentration that results in a 50% reduction of free NR (2.5 nM) were the same for NR and [delta]NR. Regulation of NR activity by phosphorylation and binding of 14-3-3 protein was a general feature for all plants tested, whereas activation by substrates as a possible regulation mechanism was verified only for squash.

Involvement of magnesium and ATP in the regulation of nitrate reductase activity in Sinapis alba

The activity of NADH: nitrate reductase (NR, EC 1.6.6.1) was extracted from mustard ( Sinapis alba) cotyledons and leaves. When seedlings/plants were grown following light/dark cycles in the presence of nitrate, an increase in the activity of NR was observed during light periods compared to dark periods. An inhibition in NR activity was observed after treatment with Mg 2+ and ATP in extracts from both cotyledons and leaves prepared from tissue harvested during light periods. No apparent inhibition was observed in tissue extracts harvested during dark periods. Two isoforms of mustard NR (NR 1 and NR 2) were separated by the use of anion exchange chromatography. The results indicate that NR 2 (the more negatively charged isoform) corresponds to a light-inducible form, which was selectively and strongly inhibited by Mg 2+/ATP. The results presented here indicate that the modulation of NR activity by light can be attributed, at least in part, to a protein reversible phosphorylation/dephosphorylation mechanism on NR 2.

Nitrate reductase expression in maize leaves (Zea mays) during dark‐light transitions. Complex effects of protein phosphatase inhibitors on enzyme activity, protein synthesis and transcript levels

The effects of cytoplasmic protein synthesis and protein phosphatase activity on NADH:nitrate reductase (NR) activity, protein and transcript were examined in maize (Zea mays L.) seedling leaves. A rapid increase in NR activity, measured in the presence of 5 mM Mg2+, was found upon exposure of excised leaves to light. Inhibitors of protein phosphatase activity (okadaic acid [OKA] and microcystin [MC]‐LR) completely prevented the increase in NR activity. The cytoplasmic protein synthesis inhibitor, cycloheximide (CHX), did not affect Mg2+ inhibition of NR activity during the dark‐to‐light transition. Vmax NR activity, measured in the presence of 5mM Mg2+, was found upon exposure of excised leaves to light. Inhibitors of protein phosphatase activity (okadaic acid [OKA] and microcystin [MC]‐LR) completely prevented the increase in NR activity. The cytoplasmic protein synthesis inhibitor, cycloheximide (CHX), did not affect Mg2+ inhibition of NR activity during the dark‐to‐light transition. Vmax NR activity, measured in the presence of Pi and EDTA, remained constant or increased slightly in maize leaves during the first 2 h of the light period. OKA, MC‐LR or CHX treatment caused a 40 to 50% reduction in Vmax NR activity during this time. Incorporation of 35S‐Met into NR protein was reduced more than 90% by CHX and 80% by OKA. The inhibition of NR protein synthesis by CHX and OKA correlated with a 50 to 60% decrease in 35S‐Met incorporation into total soluble protein over the treatment period. The increase in NR mRNA levels early in the light period was prevented by OKA and MC‐LR, but not by CHX. OKA had a similar effect on sucrose phosphate synthase mRNA levels, but did not affect Catalasel or Catalase3 mRNA accumulation. The data suggest that light‐induced decreases in Mg2+ inhibition of NR activity and transcript levels are independent of new protein synthesis. The effects of OKA and MC‐LR indicate that protein phosphatase activities could be involved, directly or indirectly, in the regulation of NR activity, protein synthesis and transcript accumulation.

Regulation of Nitrate Reductase Activity by Nitrate during Light-Dark Transition in Detached Oat Leaves

In oat (Avena sativa L. cv. Suregrain) leaf segments, light-dark modulation of nitrate reductase (NR) activity could be observed only when segments were kept in –NO3 conditions. We present here evidence that nitrate would regulate NR activity by modulating the phosphorylation status of the enzyme.

Partial Purification and Characterization of a Calcium-Dependent Protein Kinase and an Inhibitor Protein Required for Inactivation of Spinach Leaf Nitrate Reductase

Evidence is accumulating that the activity of spinach (Spinacia oleracea L.) leaf NADH:nitrate reductase (NR) is modulated both in vitro and in vivo by protein phosphorylation. From the present study we report the partial purification of the two protein factors needed for NR inactivation. We identified NR-protein kinase (NR-PK) as a calcium-dependent and metabolite-regulated protein kinase and have provided additional evidence that phosphorylation of NR is necessary but not sufficient to inactivate the enzyme. The inhibitor protein required for inactivation of phospho-NR was purified 625-fold by polyethylene glycol fractionation and sequential column chromatography. Using partially purified inhibitor protein and NR-PK, we characterized NR inactivation (increased sensitivity to Mg2+ inhibition) in a reconstituted in vitro system. NR-PK activity was inhibited by a variety of metabolic phosphate esters including di-hydroxyacetone phosphate, glucose-6-phosphate, and fructose-1,6-bisphosphate. Light-to-dark transition experiments with a starchless tobacco (Nicotiana sylvestris) mutant, which accumulates phosphate esters during the photoperiod, indicated that NR inactivation in vivo might, indeed, be down-regulated by metabolites. Additionally, we postulate that cytosolic free calcium could play an important role in the regulation of NR activity in vivo.

The influence of cytokinins in nitrate regulation of nitrate reductase activity and expression in barley

The responses of nitrate reductase (NR) activity and levels of NR‐mRNA to environmental nitrate and exogenous cytokinins are characterised in roots and shoots of barley (Hordeum vulgare L., cv. Golf), using a chemostate‐like culture system for controlling nitrate nutrition. Experiments were mainly performed with split root cultures where nitrate‐N was supplied at a constant relative addition rate of 0.09 day−1, and distributed between the subroots in a ratio of 20%:80%. The subroot NR‐mRNA level and NR activity, as well as the endogenous level of zeatin riboside (ZR), increased when the local nitrate supply to one of the subroots was increased 4‐fold by reversing the nitrate addition ratio (i.e. from 20%:80% to 80%:20%). Also shoot levels of ZR, NR‐mRNA and NR activity increased in response to this treatment, even though the total nitrate supply remained unaltered. External supply of ZR at 0.1 μM caused an approximately 3‐fold increase in root ZR levels within 6 h. which is comparable to the nitrate‐induced increase in root ZR. External application of ZR. zeatin. isopentenyl adenine or isopentenyl adenosine at 0.1 μM caused from insignificant to 25% increases in NR‐mRNA and activity in roots and up to 100% stimulation in shoots, whereas adenine or adenosine had no effect. No synergistic effects of perturbed nitrate supply and cytokinin application were detected in either roots or shoots. The translocation of nitrate from the root to the shoot was unaffected by application of ZR or switching the nitrate distribution ratio between subroots. The data give arguments for a physiological role of cytokinins in the response of root and shoot NR to environmental nitrate availability. The nature and limitations of the physiological role of cytokinins are discussed.

The influence of cytokinins in nitrate regulation of nitrate reductase activity and expression in barley

The influence of cytokinins in nitrate regulation of nitrate reductase activity and expression in barley

Metabolite Control of Nitrate Reductase Activity in Soybean Seedlings

Summary Nitrogen nutrition was investigated as an affector of nitrate reductase activity in soybean seedlings ( Glycine max var. Williams 82). The activity of the pH 6.5 NADPH-linked, the pH 6.5 NADH-linked, and the pH 7.5 NADH-linked isoforms of nitrate reductase were measured in plants treated with nitrate, glutamine, nitrate and glutamine, and no nitrogen. Nitrate supplied to 10 day old soybeans greatly enhanced primary leaf nitrate reductase activities over a 48 hour period. Plants irrigated with Hoagland's solution without nitrogen had approximately 25 % of the pH 6.5 NADH and NADPH-linked activities of plants supplied with nitrate. The NADH-linked activity at pH 7.5 showed virtually no activity without the addition of nitrate. Plants treated with nitrate and glutamine had nitrate reductase activities intermediate to those of plants treated with nitrate alone and those lacking any nitrate treatment. The concentration of nitrate in these tissues was reduced significantly over those tissues provided only with nitrate. Glutamine supplied as a sole nitrogen source slightly enhanced the pH 6.5 linked nitrate reductase activities compared with plants given no nitrogen. These results support the concept that nitrogen nutrition and nitrogenous metabolites play key roles in the regulation of nitrate reductase activity in soybeans. In addition, they demonstrate that the pH 6.5 linked activities appear less sensitive to the flux of nitrate than the pH 7.5 linked activity.

Improved in vitro selection of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

The use of increasing knowledge on regulation of nitrate reductase activity in Nicotiana cell cultures is the basis for the described optimization of in vitro selection for nitrate reductase-deficient mutants by screening for chlorate resistance. Selection was carried out on haploid mesophyll protoplast-derived cell cultures of Nicotiana plumbaginifolia. It is demonstrated that revised selection results in high variant detectability and increased variant confirmability in comparison with the hitherto used selection scheme.

Regulation of Nitrate Reductase Activity in Cultured Spinach Cells as Studied by an Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay permitting the determination of nanogram quantities of nitrate reductase (NR) in cultured spinach cells has been developed and used for studies of the mechanism by which NR activity is regulated as a function of culture age. When 8-day old spinach cells were transferred to fresh medium, NR activity increased markedly in 2 days and thereafter decreased gradually until it became undetectable on the 10th day after the transfer. Determination of the amounts of NR by the immunosorbent assay indicated that the unique alteration of NR activity could be accounted for by the concomitant change in the amount of NR protein. Immunoblotting analysis of the subunit of NR also supported this result. It is concluded that the regulation of NR in spinach cells as a function of culture age is mediated by changes in the amount of the enzyme protein rather than by activation and inactivation of the preexisting proteins.