Nitric Oxide and Nitrous Oxide Production by Soybean and Winged Bean during the in Vivo Nitrate Reductase Assay

This study was conducted to determine by gas chromatography (GC) and mass spectrometry (MS) the identity and the quantity of volatile N products produced during the helium-purged in vivo NR assay of soybean (Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identification and quantitation was collected by cryogenic trapping of volatile compounds carried in the He-purge gas stream. As opposed to an earlier report, acetaldehyde oxime production was not detected by our GC method, and acetaldehyde oxime was shown to be much more soluble in water than the compound(s) evolved from soybean leaflets. Nitric oxide (NO) and nitrous oxide (N₂O) were identified by GC and GC/MS as the main N products formed. NO and N₂O produced from soybean leaflets were both labeled with ¹⁵N when ¹⁵N-nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Other compounds co-trapped with NO and N₂O were identified as air (N₂, O₂), CO₂, methanol, acetaldehyde, and ethanol. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N₂O (13.9 and 0.37 micromole per gram fresh weight per 30 minutes, respectively) than did the soybean cv Williams (1.67 and 0.09 micromole per gram fresh weight per 30 minutes, respectively). In both species NO production was dominant. In contrast, with similar assays, NO and N₂O were not evolved from leaves of the nr₁ soybean mutant which lacks the constitutive NR enzymes. In addition to soybean cv Williams, six other Glycine sp. examined evolved significant quantities of NO_(x) (NO and NO₂). Other species including Neonotonia wightii (Arn.) Lackey comb. nov., Pueraria montana (Lour.) Merr., and Pueraria thunbergiana Benth. evolved lower levels of NO_(x).     

Evolution of Nitrogen Oxide(s) during In Vivo Nitrate Reductase Assay of Soybean Leaves

Studies were conducted to quantitate the evolution of nitrogen oxides (NO_(x)) from soybean [Glycine max (L.) Merr.] leaves during in vivo nitrate reductase (NR) assays with aerobic and anaerobic gas purging. Anaerobic gas purging (N₂ and argon) consistently resulted in greater NO_(x) evolution than did aerobic gas purging (air and O₂). The evolution of NO_(x) was dependent on gas flow rate and on NO₂⁻ formation in the assay medium; although a threshold level of NO₂⁻ appeared to exist beyond which the rate of NO_(x) evolution did not increase further.     

NADH-Nitrate Reductase Inhibitor from Soybean Leaves

A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.

The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO₃. The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in V_max.

The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.

Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.

     

The Nitric Oxide Production in the Moss Physcomitrella patens Is Mediated by Nitrate Reductase

During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.     

Regulation of Nitrate Reductase Activity in Leaves of Soybean Bragg and Its Nodulated Mutants

The extracts from leaves of nodulated soybean (Glycine max (L.) Merr. ) cv. Bragg and its nodulated mutants i. e. non-nodulated Nod 49, supernodulated nts 382 and nts 246 contained inhibitors of activities iNR, c1NR and c2NR in vitro. Both white light illumination of 300 μE · m-2 · s-1 and inoculation with strain USDAll0 were essential conditions for ac- cumulating these inhibitors in leaves. Comparing inhibiting activities of the extracts from different varieties indicated that Nod 49 extract showed stronger inhibition than Bragg extract did, but nts 382 extract had only weakest inhibitory effect. The inoculated Bragg root extract possessed the same inhibitory activity as its leaf extract. The inoculated nts 382 root extract, like its leaf extract, showed only a little inhibitory activity. However inoculated Nod 49 root extract lead to an inhibition of leaf c2NR activity, which was different from its leaf extract that inhibited three kinds of NR activities. The above results suggested that both leaf and root extracts contained common inhibitory factor which was accumulated after inoculation.     

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-darkmodulation of nitrate reductase (NR) activity could be observedonly when segments were kept in –NO₃ conditions. We presenthere evidence that nitrate would regulate NR activity by modulatingthe phosphorylation status of the enzyme. (Received June 19, 1995; Accepted August 14, 1995)     

Developmental and Biochemical Regulation of 'Constitutive' Nitrate Reductase Activity in Leaves of Nodulating Soybean

The developmental profile of ‘constitutive’ nitratereductase activity (cNRA) in leaves of soybean (Glycine max(L.) cv. Bragg) plants at different ages is described. The youngestleaves had most cNRA and the activity dropped off as a newerleaf developed above it. Each leaf had its distinct active periodof in vivo cNRA. This pattern was different in urea-grown andsymbiotically-grown plants (inoculated with Bradyrhizobium japonicumstrain USDA 110), where the latter had no detectable in vivocNRA in older leaves. Urea-grown plants maintained considerablein vivo NRA in such older leaves. When symbiotically-grown plantshad their nodules removed, in vivo cNRA reappeared in olderleaves within 1 d of removal, nearly reaching levels of youngleaves at 3 d after nodule excision. Allantoic acid (ALL), oneof the known transport ureides of soybeans, was implicated asa possible signal molecule from nodules to leaves. Allantoicacid (100 µM) inhibited in vitro c₁ NRA significantly,with 400 µM ALL resulting in complete inhibition. In contrast,allantoin (ALN) had no inhibitive effect on NRA. Inhibitionof c₁NRA by ALL was by a competitive process, judging from Lineweaver-Burkeplots against nitrate. Kinetics showed a constant Vmax of around105 nmol NO₂ mg^–1 protein h^–1 and a K_m for nitrateof 15 mM, which increased to 60 mM in the presence of 200 µMallantoic acid. Non-specific (ionic and pH-related) influenceswere eliminated. Allantoic acid also had a slight stimulatingeffect of in vitro NRA (up about 25% at 400 µM). Thesefindings suggest that c1NRA may be involved in ureide metabolism,rather than in vivo nitrate metabolism. Key words: Root-shoot interaction, nitrogen metabolism, nodulation, symbiosis     

Soyabean Stem In Vivo Nitrate Reductase Activity

Stem from three- and four-week-old Soyabean [Glycine max (L.)Merr. cv. Tracy] plants reduced from 0.3 to 0.7 µmol nitrateh^–l g^–l f. wt. Leaf activity was 4.7–7.6 µmolnitrate h^–l g^–l f. wt. Outer stem was two to fourtimes more active at reducing nitrate than was inner stem. Plantnitrate nutrition had a strong effect upon the ratio of activitypresent in stem and leaf. More nitrate increased the proportionpresent in leaves. Glycine max L., soyabean, nitrate assimilation, nitrogen metabolism, Rhizobium japonicum     

Limitations on Leaf Nitrate Reductase Activity during Flowering and Podfill in Soybean

The objective of this study was to identify factors which limit leaf nitrate reductase (NR) activity as decline occurs during flowering and beginning seed development in soybean (Glycine max [L.] Merr. cv Clark). Level of NR enzyme activity, level of reductant, and availability of NO₃⁻ as substrate were evaluated for field-grown soybean from flowering through leaf senescence. Timing of reproductive development was altered within one genotype by (a) exposure of Clark to an artificially short photoperiod to hasten flowering and podfill, and (b) the use of an early flowering isoline. Nitrogen (N) was soil-applied to selected plots at 500 kilograms per hectare as an additional variable. Stem NO₃⁻ concentration and in vivo leaf NR activity were significantly correlated (R² = 0.69 with nitrate in the assay medium and 0.74 without nitrate in the medium at P = 0.001) across six combinations of reproductive and soil N-treatment. The supply of NO₃⁻ from the root to the leaf tissue was the primary limitation to leaf NR activity during flowering and podfill. Levels of NR enzyme and reductant were not limiting to leaf NR activity during this period.     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Triadimenol Increases Nitrate Levels and Nitrate Reductase Activity in Canola Leaves

Nitrate reductase activity in the first true leaves of canola(Brassica napus L.) seedlings grown in one-quarter strengthHoagland's solution from seeds pretreated with triadimenol (0.3or 30 g (a.i.) kg^–1 of seed) was higher than controlsduring the growth period of 15 to 25 d after planting. Triadimenolalso increased chlorophyll levels, the increase being more pronouncedat its lower concentration. The treatment also increased theweight and nitrate content of the leaves. When seedlings weregrown in nutrient solution containing 1 to 20 mM nitrate, theincrease in nitrate reductase activity by triadimenol was higherat lower rather than at higher nitrate concentrations. The nitratelevels and Kjeldahl nitrogen in the triadimenol-treated leaveswas higher than the controls at concentrations of added nitrateabove 2 mM. Addition of nitrate to plants grown in ammonium,increased nitrate reductase activity more in plants grown fromtriadimenol-treated seeds than controls. However, addition of10µM triadimenol for 24 h to ammonium-grown plants hadlittle effect on enzyme activity, both in the absence as wellas the presence of nitrate. This study demonstrates that triadimenolincreases nitrate reductase activity and nitrate accumulationin the leaves and at least part of the increased enzyme activityis independent of nitrate accumulation. Key words: Triazoles, nitrate content, nitrate reductase activity     

Comparison of in Vivo and in Vitro Assays of Nitrate Reductase in Wheat (Triticum aestivum L.) Seedlings

The effectiveness of the in vivo and in vitro assays for nitrate reductase (NR) in estimating the amounts of reduced N made available to plants was tested against the daily increases in reduced N (Nesslerization) actually accumulated by the plant. With growth-chamber-grown wheat seedlings, the average ratio values (input of reduced N as estimated by the in vitro assay to actual accumulation of N by the plant) were 3.9 for shoots, 3.7 for the roots, and 4.1 for the entire plant, over a 10-day period. With the in vivo assay, the average ratio values were 0.7 for the shoot, 1.8 for the root, and 0.9 for the entire plant. Although the linear regressions between the accumulated N in the plant and the estimated N input (by both in vitro and in vivo assays) were significant and positive, the in vivo assay provided the closest approximation of the actual amount of N accumulated.     

Ex Vivo Measurement of Brain Tissue Nitrite and Nitrate Accurately Reflects Nitric Oxide Synthase Activity In Vivo

Abstract: The ex vivo tissue concentration of nitrite and nitrate (NO_x) was found to correlate closely with the activity of nitric oxide synthase (NOS; EC 1.14.13.39) in various brain regions. Systemic administration of the nonselective NOS inhibitor N ^ω-nitro- l -arginine ( l -NA) at doses that completely inhibited both central and peripheral NOS, depleted whole-brain and CSF NO_x by up to 75% but had no effect on plasma NO_x. Selective inhibition of central NOS by intracerebroventricular administration of l -NA methyl ester produced similar decreases in levels of whole-brain NO_x. A residual concentration of NO_x of 10–15 µ M remained in all brain regions even after complete inhibition of brain NOS. Brain NO_x content decreased rapidly and in parallel with the inhibition of brain NOS. The ex vivo measurement of levels of brain NO_x was found to reflect the in vivo efficacy of several different types of NOS inhibitor: l -NA, N ^ω-monomethyl- l -arginine, and 7-nitroindazole. Intraperitoneal administration of the NOS substrate l -arginine increased brain NO_x concentrations by up to 150% of control values. These results demonstrate that the ex vivo measurement of levels of brain tissue NO_x is a rapid, reliable, and straightforward technique to determine NOS activity in vivo. This method can be used to assess both the regional distribution and the degree of inhibition of NOS activity in vivo.     

Accumulation of Demolybdo Nitrate Reductase during Induction and Its Separation from Active Nitrate Reductase in Chlorella

During induction of nitrate reductase in Chlorella vulgaris,synthesis of the precursor, demolybdo cytochrome c reductase,exceeds the synthesis of active enzyme. Evidence is also presentedwhich shows that the purification procedure of Funkhouser etal. [(1980) Plant Physiol. 65: 939] separates demolybdo cytochromec reductase from active nitrate reductase. ¹Supported in part by a grant to B. V. from the Deutsche Forschungsgemeinschaftand a contribution of the Texas Agricultural Experiment Station. (Received July 27, 1983; Accepted September 13, 1983)     

油菜叶片硝酸还原酶同功酶的纯化与特性

通过对硝酸还原酶（ＮＲ）亲和层析洗脱过程的部分改进,从油菜叶片中分离纯化到诱导型（ｉＮＲ）及组成型（ｃＮＲ）两种硝酸还原酶同功酶。电泳分析表明两者均达到银染单带纯。ｃＮＲ分子量（ＭＷ）为４５０ｋＤ．ｉＮＲＭＷ为２２０ｋＤ;两者亚基ＭＷ均为１１０ｋＤ,但亚基数目不一样,氨基酸组成也有差异。ｉＮＲ与ｃＮＤ的等电点ｐＨ不同,分别为４．４及６．０,免疫交叉反应显示ｃＮＲ的抗原性为ｉＮＲ的７５％。     

Comparative Induction of Nitrate Reductase by Nitrate and Nitrite in Barley Leaves

The comparative induction of nitrate reductase (NR) by ambient NO₃⁻ and NO₂⁻ as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum valgare L.) seedlings was determined. The dynamic interaction of NO₃⁻ influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO₃⁻. As the ambient concentration of NO₃⁻ increased, the relative influences imposed by influx and reduction on NO₃⁻ accumulation changed with influx becoming a more predominant regulant. Significant levels of NO₃⁻ accumulated in NO₂⁻-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO₂⁻, about 60% more NO₃⁻ accumulated in the leaves than in the absence of the inhibitors. In NO₃⁻-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mm. No NR induction occurred in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 mm. In fact, NR induction from NO₂⁻ solutions was not seen until NO₃⁻ was detected in the leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NR as did equivalent amounts of NO₃⁻ accumulating from NO₃⁻-fed leaves. In all cases the internal concentration of NO₃⁻, but not NO₂⁻, was highly correlated with the amount of NR induced. The evidence indicated that NO₃⁻ was a more likely inducer of NR than was NO₂⁻.     

In Vivo Blue-Light Activation of Chlamydomonas reinhardii Nitrate Reductase

Chlamydomonas reinhardii cells, growing photoautotrophically under air, excreted to the culture medium much higher amounts of NO₂⁻ and NH₄⁺ under blue than under red light. Under similar conditions, but with NO₂⁻ as the only nitrogen source, the cells consumed NO₂⁻ and excreted NH₄⁺ at similar rates under blue and red light. In the presence of NO₃⁻ and air with 2% CO₂ (v/v), no excretion of NO₂⁻ and NH₄⁺ occurred and, moreover, if the bubbling air of the cells that were currently excreting NO₂⁻ and NH₄⁺ was enriched with 2% CO₂ (v/v), the previously excreted reduced nitrogen ions were rapidly reassimilated. The levels of total nitrate reductase and active nitrate reductase increased several times in the blue-light-irradiated cells growing on NO₃⁻ under air. When tungstate replaced molybdate in the medium (conditions that do not allow the formation of functional nitrate reductase), blue light activated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue light. It appears that under high irradiance (90 w m⁻²) and low CO₂ tensions, cells growing on NO₃⁻ or NO₂⁻ may not have sufficient carbon skeletons to incorporate all the photogenerated NH₄⁺. Because these cells should have high levels of reducing power, they might use NO₃⁻ or, in its absence, NO₂⁻ as terminal electron acceptors. The excretion of the products of NO₂⁻ and NH₄⁺ to the medium may provide a mechanism to control reductant level in the cells. Blue light is suggested as an important regulatory factor of this photorespiratory consumption of NO₃⁻ and possibly of the whole nitrogen metabolism in green algae.     

硝酸还原酶也是植物体内的NO合成酶

一氧化氮(N0)是一种广泛存在于植物体内的氧化还原信号分子和毒性分子。文章介绍了近年来有关植物硝酸还原酶具有催化亚硝酸盐单电子还原合成NO功能及其调节机制和生理意义的研究进展。