Metabolite control of squash nitrate reductase

Nitrate reductase (EC 1.6.6.1) catalyzes the pyridine nucleotide-linked reduction of nitrate to nitrite in higher plants. We have shown that in squash (Cucurbita maxima Duchesne var. Buttercup), exogenous nitrate increases nitrate reductase activity by increasing steady-state levels of nitrate reductase protein, while glutamine diminishes nitrate reductase activity both by decreasing steady-state levels of nitrate reductase protein and by decreasing cellular nitrate concentrations in plant cells. Other amino acids affect nitrate reductase similarly to glutamine; other metabolites tested including nitrate did not cause major perturbations in the synthesis of other cellular proteins. Thus, it appears that the effects of nitrate and reduced nitrogen compounds on enzymes of the nitrate assimilatory pathway are highly specific for these enzymes, and have little effect on other cellular proteins.     

Intact plastids are required for nitrate- and light-induced accumulation of nitrate reductase activity and mRNA in squash cotyledons

Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids.     

Light and nitrate induction of nitrate reductase in kinetin-and gibberellic acid-treated mustard cotyledons

Nitrate reductase activity in gibberellic acid and kinetin treated mustard (Brassica juncea Coss. cv. T-59 ‘Varuna’) seedlings, grown in the presence or absence of light and/or NO₃ was investigated. While both light and NO₃, alone could induce NR activity, their combination showed additive effects. Kinetin treatment significantly promoted both light- and NO₃- induced NR activities, assayed by either in vivo or in vitro techniques, whereas, gibberellic acid was almost ineffective. In the absence of both light and NO₃, however, phytohormones alone could not induce NR activity. Both light-induced and NO₃ induced NR fractions had a pH optima of 7.5, preferred NADH as an electron donor (NADH: NADPH ratio 2.5) and K_m values for NO₃ was 0.2 mM. Actinomycin D, cycloheximide and tungstate were equally effective in suppressing the development of NR activity after exposure to light or NO₃. These results indicate that two independent NR fractions operate, with apparently identical properties but separate control mechanisms.     

Induction of nitrate reductase by chloramphenicol in detached cucumber cotyledons

Summary Chloramphenicol (CAP) induced nitrate reductase activity (NRA) in detached, etiolated cucumber (Cucumis sativus L.) cotyledons. The effect was reduced by cycloheximide. Light was not necessary for induction of the enzyme but potentiated the effect of CAP as an inducer of NRA, despite the fact that the antibiotic inhibited chlorophyll accumulation.CAP at suboptimal concentrations (2.5–5 mM) acted synergistically with succinic acid-2,2-dimethylhydrazide (B-Nine) and benzylaminopurine (BAP) in respect to induction of NRA, the effect being especially marked in darkness. The highest NRA was found in cotyledons treated for 24 h with a mixture of CAP+BAP+B-Nine. The effects of KNO₃ and CAP on NRA were neither synergistic nor additive.KNO₃ and BAP induced NADH-NR (EC 1.6.6.1). The same was true for CAP and B-Nine, but the latter two compounds induced, in addition, some activity of another NR that could utilize NADPH as an electron donor.     

Induction of nitrate reductase, nitrite reductase, and glutamine synthetase isoforms in sunflower cotyledons as affected by nitrate, light, and plastid integrity

Summary We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO ₃ ^– , NO ₂ ^– or NH ₄ ⁺ ), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO ₂ ^– or NH ₄ ⁺ the synthesis of NiR and chloroplastic GS (GS₂) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO ₃ ^– concentration indicate that NO ₃ ^– -dependent enzyme induction is elicited by NO ₃ ^– per se and not by a product of its assimilatory reduction, e.g., NO ₂ ^– or NH ₄ ⁺ . In the presence of NO ₃ ^– , light remarkably enhanced the appearance of NR, NiR, and GS₂, while the activity of the cytosolic GS isoform (GS₁) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light- and NO ₃ ^– -dependent increase of GS₂ activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO ₃ ^– on the appearance of NR, NiR, and GS₂ isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO ₃ ^– and/or for the accumulation of newly formed enzymes in the organelle.Abbreviations CAP chloramphenicol - CHX cycloheximide - GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic (chloroplastic) GS - NF norflurazon - NiR nitrite reductase - NR nitrate reductase     

Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

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 μ M 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 μ M 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.     

Effect of cinnamate on nitrate reductase activity in isolated cucumber cotyledons

The effect of cinnamic acid on in vivo nitrate reductase activity and protein content in cucumber cotyledons was studied. Cinnamate increased in vivo nitrate reductase activity and also the total protein content at lower concentrations (0.01–0.1 mM). Higher concentration, however, proved inhibitory. The effect of cinnamate on nitrate reductase activity has been discussed.     

Immunochemical characterization and localization of nitrate reductase in norflurazon-treated soybean cotyledons

Immunochemical procedures were used to characterize and localize NADH:nitrate reductase (NR; EC 1.6.6.1) in cotyledons of norflurazon-treated soybeans [ Glycine max (L.) Merr. cv. 'Hill']. Antiserum prepared to NR isolated from Chlorella strongly reacted against NR from norflurazon-treated cotyledons. This serum inhibited the NR activity in crude extracts of norflurazon-treated soybean cotyledons by 98% even at a 1:2000 dilution of crude serum. Pre-immune serum had no effect on the activity. These data indicate that there are similar antigenic determinants at the active site of both Chlorella and norflurazon-treated soybean NR. Whole cotyledons were homogenized in lithium dodecyl sulfate-containing buffer, electrophoretically separated and blotted to nitrocellulose. When the blots were reacted with the anti-NR serum only a single protein (M_r= 98 kdalton) was visualized. Immunofluorescence studies on fixed tissue sections revealed intense fluorescence in the cytoplasm. Weaker reactions were associated with organelles tentatively identified as plastids. Pre-immune serum controls were completely unstained using immunocytochemical procedures.     

Phytochrome regulation of nitrate reductase in wheat

In excised wheat leaves, the activity of nitrate reductase was enhanced by a brief pulse of red light and this increase was reversed by far-red light irradiation. Even under continuous far-red light, nitrate reductase activity increased by 258% after 18 h. When leaves were kept in distilled water during exposure to red light and then transferred to potassium nitrate, there was no difference in endogenous nitrate concentration. The nitrate reductase activity was the same whether leaves were floated in potassium nitrate or in distilled water during irradiation. Partial to complete inhibition of enzyme activity was observed when leaves were incubated in actinomycin-D and cycloheximide respectively, following 4 h of red light irradiation.In vitro irradiation of extract had no significant effect on nitrate reductase activity     

In vitro assay and light regulation of nitrate reductase in red alga Gracilaria chilensis

Nitrate reductase (NR) is the first enzyme in the nitrogen assimilation pathway. The in vitro NR activity of Gracilaria chilensis was assayed under different conditions to reveal its stability and biochemical characteristics, and an optimized in vitro assay is described. Maximal NR activities were observed at pH 8.0 and 15 degrees C. The apparent Km value for NADH was 8 microM and for nitrate 680 microM. Crude extracts of G. chilensis stored at 4 degrees C showed a 50% decrease of NR activity after 24 h. The highest NR activity value (253.20+/-2.60 x 10(-3) U g(-1)) was obtained when 100% von Stosch medium (500 microM NO3-) was added before extraction of apical parts. Algae under light:dark cycles of 12:12h exhibited circadian fluctuation of NR activity and photosynthesis with more than 2 times higher levels in the light phase. No evidence of endogenous diel rhythm controlling NR activity or photosynthesis was observed. Light pulses lasting 10 or 60 min during the darkness increased the NR activity by 30% and 45%, respectively. The results indicate that NR and photosynthesis are regulated mainly by light and not by a biological clock.     

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

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

The inhibition of nitrate reductase induction by spermine in excised radish cotyledons

The induction of nitrate reductase in excised cotyledons of radish seedlings was inhibited by the polyamines, spermidine and spennine, in light and dark, but putrescine had no effect. Spermine had no effect on the uptake of nitrate or the stability of the enzyme, but inhibited the synthesis of the enzyme.