硝酸盐对硝酸还原酶活性的诱导及硝酸还原酶基因的克隆

硝酸盐在植物体内的积累过多已成为影响蔬菜品质并影响人类健康的重要因素。硝酸还原酶（NR）是硝酸盐代谢中的关键酶,提高其活性有利于硝酸盐的降解。为了解植物不同组织中NR的活性,用活体测定法检测了经50mmol/L的KNO₃诱导不同时间后的油菜、豌豆和番茄幼苗根茎叶中NR活性,同时为了明确外源诱导剂浓度与植物体内NR活性的关系,检测了经不同浓度KNO₃诱导2h后的矮脚黄、抗热605、小白菜和番茄叶片中的NRA。结果表明,不同植物组织NR活性有很大差异,叶中NR活性较高,根其次,茎最低;不同植物的NR活性随诱导时间呈不同的变化趋势,相同植物不同组织的NR活性变化趋势相似;不同植物叶片NRA为最高时KNO₃浓度不同。用30mmol/L的KNO3诱导番茄苗2h后,从番茄根和叶中提取总RNA,用RT-PCR方法获得NR cDNA,全长2736bp,编码911个氨基酸。为进一步利用该基因提高植物对硝酸盐的降解能力打下基础。     

水稻硝酸还原酶的RNA干涉及其酶活性分析

分析水稻硝酸还原酶（NR）基因生物信息学的结果显示：水稻基因纽中有2个NR基因成员：一个为NR[NADH]（NR1）：另一个为NR[NAD（P）H]（NR2）。两者的蛋白序列相似性为70％。用RT—PCR技术从水稻cDNA中获得了NR1和NR2的cDNA片段,其大小分别为1086bp和892bp。构建RNA干涉载体（称pRNAi—NR1和pRNAi-NR2）转化水稻愈伤组织后检测转基因后代酶活性的结果表明：两种干涉植株的根叶中的NR活性均大幅度下降,并且根叶中的活性变化呈线性正相关关系。表明2个基因可能均有调控根叶中NR活性的作用。     

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

植物中硝酸还原酶和亚硝酸还原酶的作用

植物通过硝酸盐同化途径以硝酸盐和氨的形式吸收氮元素。硝酸盐的同化是一个受到严格控制的过程,其中两个先后参加反应的酶——硝酸还原酶(NR)和亚硝酸还原酶(NiR)对初级氮的同化起主要调控。在高等植物中,NR和NiR基因的转录及转录后加工受到各种内在和外在因素的影响,翻译后调控是消除亚硝酸盐积累的重要机制。随着分子生物学技术的发展,可以更容易地通过突变体和转基因方式来研究NR和NiR基因的调控。     

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)     

Nitrate Reductase mRNA Regulation in Nicotiana plumbaginifolia Nitrate Reductase-Deficient Mutants

Light and substrate regulation of nitrate reductase (NR) expression were compared in wild type and mutant lines of Nicotiana plumbaginifolia. Mutants affected in the NR structural gene (nia) or in the biosynthesis of the NR molybdenum cofactor (cnx) were examined. nia mutants expressing a defective apoenzyme, as well as cnx mutants, overexpressed NR mRNA, whereas nia mutants devoid of detectable NR protein had reduced or undetectable NR mRNA levels. Diurnal fluctuations of NR mRNA were specifically abolished in nia and cnx mutants, suggesting that the integrity of NR catalytic activity is required for the expression of diurnal oscillations. Unlike some fungal mutants, the nia and cnx mutants examined retained nitrate inducibility of NR expression. The possibility of autogenous control of NR expression in higher plants is discussed.     

Chlorate Reducing Activity of Spinach Nitrate Reductase

The activities of 2-oxoaldehyde-metabolizing enzymes (glyoxalase I, glyoxalase II, methyl- glyoxal reductase, methylglyoxal dehydrogenase and lactaldehyde dehydrogenase) were found to be widely distributed among microorganisms. One of the enzymes, methylglyoxal reductase, which catalyzes the reductive conversion of methylglyoxal into lactaldehyde, was purified from Escherichia coli cells. The enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis and was a monomer with a molecular weight of 43000. The enzyme was most active at pH 6.5 and 45°C. The enzyme utilized both NADPH and NADH for the reduction of 2- oxoaldehydes (glyoxal, methylglyoxal, phenylglyoxal and 4,5-dioxovalerate) and some aldehydes (glycolaldehyde, D,l-glyceraldehyde, propionaldehyde and acetaldehyde). The Km values of the enzyme for methylglyoxal, NADPH and NADH were 4.0 mm, 1.7 fiM and 2.8 /¿m, respectively. The product of methylglyoxal reduction was identified as lactaldehyde. The enzyme from E. coli cells was different from the yeast and goat liver enzymes in both molecular structure and substrate specificity.     

Regulation of Nitrate Reductase in Chlorella vulgaris

When excised barley roots (Hordeum distichum L.) are appropriately pretreated, the level of nitrate reductase in the roots increases upon exposure to nitrate. Relatively low levels of nitrate (10 μm) gave maximum induction of nitrate reductase. This increase was inhibited by inhibitors of protein and RNA synthesis, indicating that de novo protein synthesis is probably involved. Induction of nitrate reductase by nitrate is partially prevented by the inclusion of ammonium, an eventual product of nitrate reduction, in the incubation medium. Under the experimental conditions used, ammonium did not inhibit the uptake of nitrate by excised barley roots. It is concluded, therefore, that ammonium, or a product of ammonium metabolism, has a direct effect on the synthesis of nitrate reductase in this tissue.     

Monoclonal Antibodies Specific for NADH-Ferricyanide Reductase Domain of Spinach Leaf Nitrate Reductase

Two monoclonal antibodies, 17(3)9 and 36(79)4, were preparedagainst nitrate reductase from Spinacia oleracea L. leaves.An enzyme-linked immunosorbent assay showed that 17(3)9, butnot 36(79)4, reacted more strongly to heat-denatured than nativeantigen. These antibodies inhibited NADH-nitrate reductase aswell as its various partial activities including reduced methylvilogen-nitrate reductase, reduced flavin mononucleotide-nitratereductase and NADH-cytochrome c reductase activities, but notNADH-ferricyanide reductase activity. Immunoblotting after electrophoreticseparation of nitrate reductase fragments obtained by Staphyrococcusaureus V8 protease digestion of native enzyme revealed thatthe two monoclonal antibodies bind to different epitopes locatedon the 28 kDa of the NADH-ferricyanide reductase domain. (Received October 2, 1987; Accepted June 9, 1988)     

Regulation of Nitrate Reductase in Acetabularia mediterranea

Nitrate reductase (NR) activity has been characterized on cell-freeextracts of the marine unicellular green alga, Acetabulariamediterranea. Reduced methyl viologen and bromophenol blue aswell as NAD(P)H were effective electron donors for the enzyme. NADH-NR activity increased during cell development reachingits maximum level when the cells approached their maximum length.A substantial increase was also seen in enucleated cells. Theenzyme activity was only present in the green parts of the celland became diminished as the stalks bleached in ageing cells.Under a 12 h light: 12 h darkness photoperiod, NADH-NR activityexhibited pronounced daily fluctuations reaching its maximumon the first hour of illumination (this increase was impairedby cycloheximide) and a minimum in the middle of the dark period. In N-starved algal cultures, NR was constitutively present atappreciable levels and new synthesis took place in the presenceof either NO₃ or low concentrations of NH⁺₄ . However, in thepresence of this cation the enzyme remained mostly in its inactiveform, and could be reactivated in vitro with ferrycyanide. Key words: Nitrate reductase, Acetabularia, daily rhythms, nitrate, ammonium     

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     

Variation in Nitrate Reductase Activity in Lolium

Nitrate reductase activity was studied in the leaves and rootsof Lolium perenne. Growth temperatures of 8, 15, or 20 °Cdid not affect activity, but the same temperatures during assayhad differential effects on the nitroso couple used to measureenzyme activity. Activity increased with increasing light intensity,reaching a high plateau value at around 40 W m^–2. Nitratecontent of leaves, also measured in this experiment, did notvary significantly with different light intensities. Increasingnitrate in the nutrient solution up to 0.5 mM N also increasedactivity. Adding ammonium chloride at similar levels to thenitrate caused no marked repression of activity. Removal ofnitrate from the nutrient solution decreased enzyme activitywithin 24 h. Marked diurnal fluctuations occurred in activity,apparently in response to light intensity, since the nitratelevel in the plant varied little. The enzyme activity of rootswas much less than that of leaves. In the parents and progeny from a half diallel cross, the parentalgenotypes differed significantly in activity, but the numberof families involved was too small for the regression of progenyon parents (b = 1.74) and the correlation coefficient (r = 0.44NS) to achieve significance. In this experiment a significantpositive regression was obtained between nitrate reductase activityand dry matter yield.     

Sunflower Nitrate Reductase: Purification and Characterization

A single isoform, NADH: nitrate reductase (NR), was purified 500 folds from sunflower leaves by affinity chromatography on Blue Sepharose CL-6B. Purified NR had a pH optima of 7.25 and a molecular weight of 210 kD. In SDS-PAGE, two bands of 47 and 56 kD were obtained. NADH: ferric citrate reductase activity was copurified with NR with a specific activity of 2. The Vmax of NADH: ferric citrate reductase was 8.69 units mg^-1 protein and the apparent Km for ferric citrate was 0.435 mM.     

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⁻.     

Nitrate Absorption by Barley: II. Influence of Nitrate Reductase Activity

The influence of protein synthesis and nitrate reductase activity on nitrate absorption by barley (Hordeum vulgare L.) was investigated. Cycloheximide decreased nitrate absorption. Pretreatment studies showed that cycloheximide affects either energy transfer or nitrate reductase activity or both.