Nitrate Reductase Activation State Research Articles

Exogenous Putrescine Modulates Nitrate Reductase-Dependent NO Production in Cucumber Seedlings Subjected to Salt Stress.

Polyamines (PAs) are small aliphatic compounds that participate in the plant response to abiotic stresses. They also participate in nitric oxide (NO) production in plants; however, their role in this process remains unknown. Therefore, the study aimed to investigate the role of putrescine (Put) in NO production in the roots of cucumber seedlings subjected to salt stress (120 mM NaCl) for 1 and 24 h. In salinity, exogenous Put can regulate NO levels by managing NO biosynthesis pathways in a time-dependent manner. In cucumber roots exposed to 1 h of salinity, exogenous Put reduced NO level by decreasing nitrate reductase (NR)-dependent NO production and reduced nitric oxide synthase-like (NOS-like) activity. In contrast, during a 24 h salinity exposure, Put treatment boosted NO levels, counteracting the inhibitory effect of salinity on the NR and plasma membrane nitrate reductase (PM-NR) activity in cucumber roots. The role of endogenous Put in salt-induced NO generation was confirmed using Put biosynthesis inhibitors. Furthermore, the application of Put can modulate the NR activity at the genetic and post-translational levels. After 1 h of salt stress, exogenous Put upregulated CsNR1 and CsNR2 expression and downregulated CsNR3 expression. Put also decreased the NR activation state, indicating a reduction in the level of active dephosphorylated NR (dpNR) in the total enzyme pool. Conversely, in the roots of plants subjected to 24 h of salinity, exogenous Put enhanced the NR activation state, indicating an enhancement of the dpNR form in the total NR pool. These changes were accompanied by a modification of endogenous PA content. Application of exogenous Put led to an increase in the amount of Put in the roots and reduced endogenous spermine (Spm) content in cucumber roots under 24 h salinity. The regulatory role of exogenous Put on NO biosynthesis pathways may link with plant mechanisms of response to salt stress.

A low red/far-red ratio restricts nitrogen assimilation by inhibiting nitrate reductase associated with downregulated TaNR1.2 and upregulated TaPIL5 in wheat (Triticum aestivum L.)

Understanding the physiological mechanism underlying nitrogen levels response to a low red/far-red ratio (R/FR) can provide new insights for optimizing wheat yield potential but has been not well documented. This study focused on the changes in nitrogen levels, nitrogen assimilation and nitrate uptake in wheat plants grown with and without additional far-red light. A low R/FR reduced wheat nitrogen accumulation and grain yield compared with the control. The levels of total nitrogen, free amino acid and ammonium were decreased in leaves but nitrate content was temporarily increased under a low R/FR. The nitrate reductase (NR) activity in leaves was more sensitive to a low R/FR than glutamine synthetase, glutamate synthase, glutamic oxalacetic transaminase and glutamic-pyruvic transaminase. Further analysis showed that a low R/FR had little effect on the NR activation state but reduced the level of NR protein and the expression of encoding gene TaNR1.2. Interestingly, a low R/FR rapidly induced TaPIL5 expression rather than TaHY5 and other members of TaPILs in wheat, suggesting that TaPIL5 was the key transcription factor response to a low R/FR in wheat and might be involved in the downregulation of TaNR1.2 expression. Besides, a low R/FR downregulated the expression of TaNR1.2 in leaves earlier than that of TaNRT1.1/1.2/1.5/1.8 in roots, which highlights the importance of NR and nitrogen assimilation in response to a low R/FR. Our results provide revelatory evidence that restricted nitrate reductase associated with downregulated TaNR1.2 and upregulated TaPIL5 mediate the suppression of nitrogen assimilation under a low R/FR in wheat.

Okadaic acid did not change the nitrate reductase activation state in tomato seedlings

In this study, the total and actual nitrate reductase (NR) activity, and NR activation state, in tomato seedlings (Solanum lycopersicum cvs. Kmicic and Faworyt) treated with okadaic acid (OA) was evaluated. Seedlings were grown in a half-strength Murashige and Skoog (MS) medium in a growth chamber at day/night temperatures of 22/20 °C, a photon flux density of 150 µmol m-2 s-1, and a 16-h photoperiod. After 10 days, plants were transferred into MS medium with 0 (control), 0.01, 0.05, 0.1, 0.5, 1.0 µM OA. It was found that the total and actual NR activity increased in Kmicic leaves treated with 0.1, 0.5, and 1.0 µM OA compared to control. However, the NR activation state did not change in both roots and leaves of OA-treated tomato seedlings.

Response of nitrate reductase activity and NIA genes expression in roots of Arabidopsis hxk1 mutant treated with selected carbon and nitrogen metabolites

In plants sugar sensing and signal transduction involves pathways dependent or independent on HXK1 as a glucose sensor. Research was conducted to determine which pathway is responsible for regulation of the nitrate reduction. The effect of selected carbon and nitrogen metabolites on nitrate reductase (NR) activity in Arabidopsis thaliana wild type (WT) and hxk1 mutant roots was studied. Exogenously supplied sugar, sucrose (Suc) and organic acid, 2-oxoglutarate (2-OG) led to an increase in the total and actual activity of NR. It was due to both the increase in expression of NIA genes and NR activation state. The stimulatory effect of Suc and 2-OG on nitrate reduction was less pronounced in hxk1 mutant roots with T-DNA insertion in the AtHXK1 gene encoding hexokinase1 (HXK1) and characterized by reduced hexokinase activity and root level of G6P and F6P. On the other hand, it was shown that exogenous glucose did not mimic Suc-mediated NR activation in Arabidopsis roots. Taken together, this data suggest that the Suc signaling pathway might be independent from hexose's sensor dependent mechanism.

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.

Study of the senescence process in primary leaves of sunflower (Helianthus annuus L.) plants under two different light intensities

Different parameters that vary during leaf development may be affected by light intensity. To study the influence of different light intensities on primary leaf senescence, sunflower (Helianthus annuus L.) plants were grown for 50 days under two photon flux density (PFD) conditions, namely high irradiance (HI) at 350 μmol(photon) m-2 s-1 and low irradiance (LI) at 125 μmol(photon) m-2 s-1. Plants grown under HI exhibited greater specific leaf mass referred to dry mass, leaf area and soluble protein at the beginning of the leaf development. This might have resulted from the increased CO2 fixation rate observed in HI plants, during early development of primary leaves. Chlorophyll a and b contents in HI plants were lower than in LI plants in young leaves. By contrast, the carotenoid content was significantly higher in HI plants. Glucose concentration increased with the leaf age in both treatments (HI and LI), while the starch content decreased sharply in HI plants, but only slightly in LI plants. Glucose contents were higher in HI plants than in LI plants; the differences were statistically significant (p<0.05) mainly at the beginning of the leaf senescence. On the other hand, starch contents were higher in HI plants than in LI plants, throughout the whole leaf development period. Nitrate reductase (NR) activity decreased with leaf ageing in both treatments. However, the NR activation state was higher during early leaf development and decreased more markedly in senescent leaves in plants grown under HI. GS activity also decreased during sunflower leaf ageing under both PFD conditions, but HI plants showed higher GS activities than LI plants. Aminating and deaminating activities of glutamate dehydrogenase (GDH) peaked at 50 days (senescent leaves). GDH deaminating activity increased 5-fold during the leaf development in HI plants, but only 2-fold in LI plants. The plants grown under HI exhibited considerable oxidative stress in vivo during the leaf senescence, as revealed by the substantial H2O2 accumulation and the sharply decrease in the antioxidant enzymes, catalase and ascorbate peroxidase, in comparison with LI plants. Probably, systemic signals triggered by a high PFD caused early senescence and diminished oxidative protection in primary leaves of sunflower plants as a result.

Virus-induced gene silencing of 14-3-3 genes abrogates dark repression of nitrate reductase activity in Nicotiana benthamiana

In order to study the effect of repression of 14-3-3 genes on actual activity of the nitrate reductase (NR) in Nicotiana benthamiana leaves, Nb14-3-3a gene was silenced by virus-induced gene silencing (VIGS) method using potato virus X (PVX). Expression of Nb14-3-3a as well as Nb14-3-3b genes was altogether repressed in the leaves of PVX-14-3a-infected plants. Furthermore, two-dimensional gel electrophoresis and immunoblot analysis with anti-14-3-3 antiserum suggested that the expressions of Nb14-3-3a and Nb14-3-3b proteins are accordingly repressed in PVX-14-3a-infected plants. It is well known that binding of 14-3-3 proteins to phosphorylated NR leads to substantial decrease in NR activity of leaves under darkness. Therefore, we studied the changes in NR activity in response to light/dark transitions in the leaves of PVX-14-3a-infected plants. NR activation state was kept at a high level under darkness in PVX-14-3a-infected plants, but not in PVX-green fluorescent protein (GFP)-infected and control plants. This result suggests that Nb14-3-3a and/or Nb14-3-3b proteins are indeed involved in the inactivation of NR activity under darkness in N. benthamiana.

Modulation of nitrate reductase activity in rice seedlings under aluminium toxicity and water stress: role of osmolytes as enzyme protectant

Nitrate reductase (NR) activity in the presence of Mg2+ (NR act) representing the non-phosphorylated NR state and the activity in the presence of EDTA (NR max) representing maximum NR activity was measured in roots and shoots of 15 d grown aluminium and water stressed rice seedlings to examine changes in NR activation state due to these stresses. Seedlings subjected to a moderate water stress level of -0.5 MPa for 24 h or grown in presence of 80 microM Al3+showed decreased level of NR max but resulted in higher NR act and NR activation state. However, seedlings grown in presence of a higher level of 160 microM Al3+ showed a decline in NR act as well as NR max. With a higher water stress Level of -2.0MPa a marked decline in the levels of both NR act and NR max was observed, whereas NR activation state remained almost unaltered with severe water stress. NR activity appeared to be sensitive to H2O2, PEG-6000, NaCl and various metal salts. Incorporation of these components in the enzyme assay medium led to decreased affinity of enzyme towards its substrate with increase in Km and decrease in Vmax values. Addition of each of the osmolytes i.e. 1 mol/L proline, 1 mol/L glycine betaine or 1 mol/L sucrose in the enzyme assay medium caused a considerable protection to the enzyme against the damaging effects of stressful components. An enhanced level of proline and glycine betaine was observed in Al-stressed seedlings and sucrose in Al as well as water stressed seedlings. Results suggest that Al toxicity and water stress decrease total amount of functional NR in rice seedlings and the osmolytes proline, glycine betaine and sucrose appear to have a direct protective action on enzyme NR under stressful conditions

Activation of nitrate reductase of cashew leaf by exogenous nitrite

Nitrate reductase (NR) plays a central role in plant primary metabolism and exhibits complex regulation mechanisms for its catalytic activity. There is controversy in the literature concerning the possible direct effect of NO2- on the stimulation and/or activation of NR activity. The influence of NO2- was studied on the NR activity in the leaves of 30-day-old cashew (Anacardium occidentale L.) seedlings. Addition of NO2- to the reaction mixture containing leaf enzymatic extract resulted in a remarkable increase in NR activity. A trace amount (5 mumol.L-1) of NO2- was required to achieve full NR activity. The in vitro NR-activity showed a steady time-dependent increase when incubated in the presence of only NO3- + NO2-. In contrast, in vitro NR activity was practically absent in a NO2- -free reaction medium, even in the presence of a saturating NO3- concentration. No oxidation of NO2- to NO3- was detected during the experiment. Although NO2- clearly activated the in vitro NR activity, it had no effect on the in vivo leaf-NR activity determined under absence of oxygen. NADH at concentrations greater than 0.5 mmol.L-1 decreased the rates of in vitro NR activity. These results indicated, at least partially, a strong cashew leaf NO2- dependency of NR activation and/or activity. Finally, based on these results, it is suggested that this singular NR activity model induced by exogenous NO2- in cashew leaf extracts is caused by changes in the NR activation state by NO2- itself.

Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers.

Assimilatory nitrate reductase (NR) of higher plants is a most interesting enzyme, both from its central function in plant primary metabolism and from the complex regulation of its expression and control of catalytic activity and degradation. Here, present knowledge about the mechanism of post-translational regulation of NR is summarized and the properties of the regulatory enzymes involved (protein kinases, protein phosphatases and 14-3-3-binding proteins) are described. It is shown that light and oxygen availability are the major external triggers for the rapid and reversible modulation of NR activity, and that sugars and/or sugar phosphates are the internal signals which regulate the protein kinase(s) and phosphatase. It is also demonstrated that stress factors like nitrate deficiency and salinity have remarkably little direct influence on the NR activation state. Further, changes in NR activity measured in vitro are not always associated with changes in nitrate reduction rates in vivo, suggesting that NR can be under strong substrate limitation. The degradation and half-life of the NR protein also appear to be affected by NR phosphorylation and 14-3-3 binding, as NR activation always correlates positively with its stability. However, it is not known whether the molecular form of NR in vivo affects its susceptibility to proteolytic degradation, or whether factors that affect the NR activation state also independently affect the activity or induction of the NR protease(s). A second and potentially important function of NR, the production of nitric oxide (NO) from nitrite is briefly described, but it remains to be determined whether NR produces NO for pathogen/stress signalling in vivo.

Modulation of nitrate reductase activity in cucumber ( Cucumis sativus) roots

Nitrate reductase (NR) (EC 1.6.6.1) activity and NR activation state, i.e. activity in the presence of Mg 2+ relative to activity in the absence of Mg 2+, in cucumber ( Cucumis sativus) leaves increased in the light and decreased in the dark. In contrast to leaves, NR activation state in the roots did not show light/dark-dependent changes. Root NR was activated by anoxia or by addition of uncoupler (CCCP) or mannose. These treatments decreased ATP levels in root tissue. On the contrary, high oxygen supply promoted some NR inactivation. When an extract from anoxic roots was preincubated with ATP, NR was gradually inactivated. Subsequent addition of 5′-AMP resulted in a remarkable reactivation of the enzyme. NR extracted from hyperoxygenated roots was activated by preincubation with 5′-AMP, and the process was reversed by ATP. These results suggest the participation of adenine nucleotides on the in vivo modulation of NR activity in cucumber roots. NR was activated in vivo by cellular acidification and inactivated by alkalinisation. The acid-induced activation of NR was greatly prevented by okadaic acid, a protein phosphatase inhibitor. Our data indicate that, as in barley roots, anoxia, uncouplers, and mannose feeding activate cucumber root NR, at least partly, by enhancing NR dephosphorylation via a decrease in the internal level of ATP and a concomitant cellular acidification.

Nitrate reductase activation state in leaves of molybdenum-deficient winter wheat

Summary Nitrate reductase (NR, EC 1.6.6.1) activity, NR activation state, and in vivo nitrate reduction were measured in leaves of winter wheat ( Triticum aestivum L. cv. Sadovo 1) in order to study the effect of molybdenum (Mo) deficiency on light-induced changes in NR activation. It was established that dark to light transition of the plants resulted in a fast increase in actual and maximum NR activities (NRA act and NRA max ) and vice versa; the deactivation of NR occurred soon after light to dark transition. The diurnal rhythm of NRA act and NRA max was similar in phase in Mo-deficient and Mo-supplied plants. However, the absolute values of NRA act and NRA max were about two to three times higher in Mosupplied compared to Mo-deficient plants, i.e. only the amplitude of the rhythm was affected by Mo deficiency. The activation state of NR, given as percentage ratio of NRA act to NRA max , was higher in Mo-deficient plants, reaching a maximum of 60-66% in the light, independent of the light intensity over 210 μmol m -2 s -1 . In both Mo-deficient and Mo-supplied plants, the level of NRA act — thought to reflect the in situ NR activity — exceeded by several times the rate of in vivo nitrate reduction. The results indicate that Mo deficiency itself does not alter the timing of light/dark changes in NR activity, but it reduces significantly the levels of NRA max and NRA act and results in a higher NR activation state in Mo-deficient plants. A possible mechanism of this effect is discussed.

Increased nitrate reductase activity in leaf tissue after application of the fungicide Kresoxim-methyl

Spinach (Spinacia oleracea L.) leaf discs floating on buffer solution were treated with Kresoxim-methyl (KROM), an inhibitor of respiratory electron transport. In the leaf tissue, actual and maximal nitrate reductase (NR) activities, nitrite content and ATP levels were determined. In darkened leaf discs incubated without KROM (control) actual NR activity decreased to 20% after 6 h in the dark. Treatment with 10 μg ml−1 (corresponding to 32 μM) KROM totally prevented inactivation of NR in the dark and also diminished NR-protein degradation during prolonged darkness. Due to restricted nitrite reduction in darkened leaf tissues, nitrite accumulated in KROM-treated discs. Inhibition of respiration decreased ATP and increased AMP levels in KROM-treated discs. In illuminated leaf discs, NR was highly activated to 65%. Nevertheless, KROM-treatment caused an additional activation of NR (activation state 76%) in the light. Possible side-effects of KROM on nitrite reduction and photosynthesis were also checked in the leaf-disc system. Neither nitrite reduction nor photosynthesis were altered in KROM-treated discs. The extent of KROM-induced activation of NR was dependent on the applied concentration and on the pH of the external medium. The highest activation of NR was achieved at an external pH of 4.8, confirming previous results (Kaiser and Brendle-Behnisch, 1995, Planta 196: 1–6) that cytosolic acidification might play an important role in the modulation of NR activity.

A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding.

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611-621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.

Drought-induced effects on nitrate reductase activity and mRNA and on the coordination of nitrogen and carbon metabolism in maize leaves

Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3- reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.

Modulation of carbon and nitrogen metabolism, and of nitrate reductase, in untransformed and transformed Nicotiana plumbaginifolia during CO 2 enrichment of plants grown in pots and in hydroponic culture

Transformed plants of Nicotiana plumbaginifolia Viv. constitutively expressing nitrate reductase (35S-NR) or β-glucuronidase (35S-GUS) and untransformed controls were grown for two weeks in a CO2-enriched atmosphere. Whereas CO2 enrichment (1000 μl · l−1) resulted in an increase in the carbon (C) to nitrogen (N) ratio of both the tobacco lines grown in pots with vermiculite, the C/N ratio was only slightly modified when plants were grown in hydroponic culture in high CO2 compared to those grown in air. Constitutive nitrate reductase (NR) expression per se did not change the C/N ratio of the shoots or roots. Biomass accumulation was similar in both types of plant when hydroponic or pot-grown material, grown in air or high CO2, were compared. Shoot dry matter accumulation was primarily related to the presence of stored carbohydrate (starch and sucrose) in the leaves. In the pot-grown tobacco, growth at elevated CO2 levels caused a concomitant decrease in the N content of the leaves involving losses in NO− 3 and amino acid levels. In contrast, the N content and composition were similar in all plants grown in hydroponic culture. The 35S-NR plants grown in air had higher foliar maximum extractable NR activities and increased glutamine levels (on a chlorophyll or protein basis) than the untransformed controls. These increases were maintained following CO2 enrichment when the plants were grown in hydroponic culture, suggesting that an increased flux through nitrogen assimilation was possible in the 35S-NR plants. Under CO2 enrichment the NR activation state in the leaves was similar in all plants. When the 35S-NR plants were grown in pots, however, foliar NR activity and glutamine content fell in the 35S-NR transformants to levels similar to those of the untransformed controls. The differences in NR activity between untransformed and 35S-NR leaves were much less pronounced in the hydroponic than in the pot-grown material but the difference in total extractable NR activity was more marked following CO2 enrichment. Foliar NR message levels were decreased by CO2 enrichment in all growth conditions but this was much more pronounced in pot-grown material than in that grown hydroponically. Since β-glucuronidase (GUS) activity and message levels in 35S-GUS plants grown under the same conditions of CO2 enrichment (to test the effects of CO2 enrichment on the activity of the 35S promoter) were found to be constant, we conclude that NR message turnover was specifically accelerated in the 35S-NR plants as well as in the untransformed controls as a result of CO2 enrichment. The molecular and metabolic signals involved in increased NR message and protein turnover are not known but possible effectors include NO3 −, glutamine and asparagine. We conclude that plants grown in hydroponic culture have greater access to N than those grown in pots. Regardless of the culture method, CO2 enrichment has a direct effect on NR mRNA stability.

Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status.

The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal. NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO 3 - , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO 3 - on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg2+. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO 3 - did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO 3 - content and the NR activation state. Similarly, the NR activation state did not respond to NO 3 - . Since the NR activation state determined from the degree of Mg2+-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO 3 - -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO 3 - . We conclude that NO 3 - does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C3 species) or maize (a C4 species). The short-term regulation of NR therefore differs from the NO 3 - -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.