Reversible Inactivation of Nitrate Reductase by NADH and the Occurrence of Partially Inactive Enzyme in the Wheat Leaf

Soybean (Glycine max IL.I Merr.) leaves have been shown to contain three forms of nitrate reductase (NR). Two of the forms, which are present in leaves of wild-type (cv. Williams) plants grown in the absence of NO3-, are termed constitutive and designated c,NR and c2NR. The third form, which is present in N03-grown mutant (nrj) plants lacking the constitutive forms, is termed inducible and designated iNR. Samples of cjNR, c2NR, and iNR obtained from appropriately treated plants were analyzed for the presence of partial activities, response to inhibitors, and ability to complement a barley NR which lacks the molybdenum cofactor (MoCo) but is otherwise active.The three forms were similar to most assimilatory NR enzymes in that they (a) exhibited NADH-cytochrome c reductase, reduced flavin mononucleotide-NR, and reduced methyl viologen-NR partial activities; (b) were inhibited by p-hydroxymercuribenzoate at the site of initial electron transport through each enzyme; (c) were more inhibited by CN-in their reduced enzyme state as compared with their oxidized state; and (d) complemented a MoCo-defective NR ( e.g. contained cofactors with characteristics similar to the MoCo found in barley NR and commercial xanthine oxidase). However, among themselves, they showed dissimilarities in their response to treatment with HC03-and CN-, and in their absolute ability to complement the barley NR. The site of effect for these treatments was the terminal cofactor-containing portion of each enzyme. This indicated that, although a terminal cofactor (presumably a MoCo) was present in each form, structural or conformational differences existed in the terminal cofactor-protein complex of each form.Assimilatory NR3 from higher plants has been characterized as a flavin-and heme-containing molybdoenzyme which catalyzes several partial reactions (NADH-Cyt c reductase, reduced flavin-NR, MV°-NR) in addition to the NAD(P)H-NR reaction (7,9). The NADH-Cyt c reductase (diaphorase) activity, associ-

Section: Discussionsupporting

confidence: 75%

Nitrate Reductases from Wild-Type and nr₁-Mutant Soybean (Glycine max [L.] Merr.) Leaves

Nelson¹,

Streit²,

Harper³

1986

“…Therefore, absence of nitrate reduction in leaves with closed stomata cannot be explained satisfactorily by a lack of reducing equivalents nor by "overreduction" of NR (2). As reported for Chlorella, it seems possible that cyanide or a related compound formed in leaves at low CO2 concentrations is also involved in an activation/inactivation process of NR in higher plants (8,16 …”

Section: Discussionmentioning

confidence: 99%

“…Other reactions which appear to be sensitive to drought include nitrate reduction. NR2 activity in leaves decreased under drought (10,20,22,27), and in one investigation the enzyme configuration was in an inactive form under water stress (2). Some plants accumulate nitrate under conditions of drought (15), which might also indicate an inhibition of nitrate reduction.…”

mentioning

confidence: 99%

Low CO₂ Prevents Nitrate Reduction in Leaves

Kaiser¹,

Förster²

1989

132

The correlation between CO2 assimilation and nitrate reduction in detached spinach (Spinacia oleracea L.) leaves was examined by measuring light-dependent changes in leaf nitrate levels in response to mild water stress and to artificially imposed CO2 deficiency. The level of extractable nitrate reductase (NR) activity was also measured. The results are: (a) In the light, detached turgid spinach leaves reduced nitrate stored in the vacuoles of mesophyll cells at rates between 3 and 10 micromoles per milligram of chlorophyll per hour. Nitrate fed through the petiole was reduced at similar rates as storage nitrate. Nitrate reduction was accompanied by malate accumulation. (b) Under mild water stress which caused stomatal closure, nitrate reduction was prevented. The inhibition of nitrate reduction observed in water stressed leaves was reversed by external CO2 concentrations (10-15%) high enough to overcome stomatal resistance. (c) Nitrate reduction was also inhibited when turgid leaves were kept in C02-free air or at the C02-compensation point or in nitrogen. (d) When leaves were illuminated in C02-free air, activity of NR decreased rapidly. It increased again, when CO2 was added back to the system. The half-time for a 50% change in activity was about 30 min. It thus appears that there is a rapid inactivation/activation mechanism of NR in leaves which couples nitrate reductase to net photosynthesis. (10,20,22, 27) 14, 23, 28, 29). This might lead to the suspicion that nitrate reduction in leaves should respond to intercellular CO2 concentrations, and thereby to stomatal aperture. Therefore, we examined the response of nitrate reduction to water stress and to CO2 availability in detached spinach leaves. It is shown that nitrate reduction and net CO2 assimilation in leaves are tightly coupled, and that under mild water stress nitrate reduction decreases as a result of stomatal closure. MATERIALS AND METHODS Plant MaterialSpinach (Spinacia oleracea cv 'Yates') was grown in a greenhouse under additional artificial illumination providing an average photon flux density of 400 ,uE m-2 s-' (PAR), with a day length of 11 h at about 22°C. Nitrate Reduction in Whole Leaves and Leaf SegmentsFor measuring nitrate reduction, four to eight freshly harvested detached leaves (with the petiole in distilled water or in various salt solutions) were kept in a large glass cuvette (about 4 L gas volume) and were supplied with humidified air, C02-free air or N2 at a flow rate of about 4 L min-'. The cuvette was mounted in a temperature-controlled growth chamber at a photon flux density of 400 ,uE m-2 s-' PAR (HQi-lamps 400 W, Schreder, Winterbach, FRG) and a temperature of 20°C. Determination of Nitrate LevelsFor determination of nitrate levels (and levels of other anions), leaf samples were taken at the appropriate time intervals over an 8 h period. The cuvette was opened without stopping the gas flow, and leaf discs were cut out with a cork borer (10 mm diameter). Discs from four leaves of the same treatment were usually pooled...

“…1 and 2). While in vivo inactivation and reactivation ofNR in some algae, especially (1,4,7,22). In rice a protein inhibitor having no proteinase activity was isolated bound to NR and might be involved in regulation of NR activity (15).…”

Section: Nr Enhancement With Light and K-nitrate Simultaneouslymentioning

confidence: 99%

Regulation of Corn Leaf Nitrate Reductase

Remmler¹,

Campbell²

1986

106

ABSTRACIThe appearance and disappearance of NADH:nitrate reductase (NR) in the leaves of corn (Zea mays L. W64A x W182E) were studied using activity assays, an enzyme-linked immunosorbent assay (ELISA) and western blotting. N-starved, etiolated corn plants were treated with nutrients containing either 35 millimolar NH4-nitrate or K-nitrate and immediately thereafter given light. The curve for enhancement of NR activity had three phases: I hour lag, 5 hour rapid increase, and steady state. The pattern for NR protein, as measured with the ELISA, also had three phases, but the increase was more rapid and the steady state was established earlier. To differentiate the effects of N nutrition from those of light, N-starved etiolated plants were given N nutrients 4 hours before light. During the dark pretreatment, NR activity and protein increased. When the light was turned on the NR activity and protein increased very rapidly without a lag. Western blots of polyacrylamide gels of native and denatured crude extracts showed that NADH:NR polypeptide was absent prior to treatment with N nutrients, but appeared after nitrate was given in dark or light. A low level of NR activity was found in N-starved, etiolated plants and it was shown by western blotting to be an NR form with a different electrophoretic mobility in nondenaturing gels. Since this minor NR form was not influenced by either nitrate or light, it was designated a constitutive NR. Dark decay of NR activity and protein was also studied. After the plants which had been in light with N nutrients for 24 hours were transferred to dark, the NR activity dropped by 30% within 1 hour, but the NR protein did not decrease. This inactivation of NR was further supported by returning the plants to the light after 1.5 hours of dark and finding the activity restored without change in NR protein. After the initial activity drop, a parallel decrease in NR activity and protein was observed, which was likely due to irreversible degradation by proteolysis.Hageman and Flesher (10) first studied NR2 activity in corn seedlings as affected by light and nitrate. They found rates of NR activity appearance greatly influenced by the previous N nutrition of plants when etiolated plants were greened by exposure to white light. The appearance of NR activity was co-dependent on light and nitrate in the 8-d-old, etiolated seedlings (10). From their study and others that have followed, a more or less standard response pattern for "induction" of NR activity by nitrate can