Influences of Mo on Nitrate Reductase, Glutamine Synthetase and Nitrogen Accumulation and Utilization in Mo-Efficient and Mo-Inefficient Winter Wheat Cultivars

The objective is to study whether the accumulation and utilization of plant N are controlled by Mo status in winter wheat cultivars. Mo-efficient cultivar 97003 （eft） and Mo-inefficient cultivar 97014 （ineff） were grown in severely Mo-deficient acidic soil （Tamm-reagent-extractable Mo 0.112 mg kg^-1） with （＋Mo） and without （-Mo） the application of 0.13 mg kg^-1 Mo. The accumulation and use efficiency of plant total N were significantly higher in ＋Mo than that in -Mo and in eft than that in ineff under Mo deficiency. N use efficiency was remarkably higher in maturity but it was forwarded to jointing stage after Mo supply, thus indicating that Mo supply promoted the N use efficiency besides N uptake and eff was efficient in N uptake and utilization. The overall activity of nitrate reductase （NR, EC 1.6.6.1） was significantly higher in ＋Mo than in -Mo and ratio of ＋Mo/-Mo was even to 14.8 at filleting stage for ineff. Activity of glutamine synthetase （GS, EC 6.3.1.2） was significantly lower in ＋Mo than in -Mo. Concentration of nitrate and glutamate were also significantly lower in ＋Mo than in -Mo, thus provided evidences for enhancing N use efficiency by Mo supply. Activities of NR and GS were significantly higher and concentrations of nitrate and glutamate were significantly lower in eff than ineff under Mo deficiency, thus indicated eff was more efficient in N reduction and utilization. It is therefore concluded that Mo could promote N accumulation and utilization in winter wheat which was directly related to NR and feedback regulated by GS. Higher Mo status also results in higher accumulation and utilization of plant N in eft.

Changes of Nitrate Reductase Activity in Cucumber Seedlings in Response to Nitrate Stress

In China, nitrogen fertilizer application rates in intensive agricultural systems have increased dramatically in recent years, especially in protected vegetable production systems. This excessive use of nitrogen fertilizer has resulted in soil secondary salinization, which has become a significant environmental stress for crops such as cucumber, in the protected farmland of China. So it is necessary to illuminate how crops respond to nitrate stress. The objective of this work was to examine the effects of increased nitrate concentration [14 （CK） and 140 mmol L^-1 （T）] on NO3- concentration, and in vitro and in vivo nitrate reductase activities in the roots and leaves of cucumber （Cucumis sativus L. cv. Xintaimici） seedlings with hydroponic culture. The results showed that the NO3- concentration in the roots and leaves of T seedlings significantly increased over treatment course, and at 12 d increased by 1.08 and 1.72 times with respect to CK seedlings, respectively; in vitro nitrate reductase activity of T was increased dramatically to 1.74 times of CK in the roots at 2 d and 1.56 times of CK in the leaves at 6 d, and then decreased. At 12 d, in vitro activity was still 24.3% higher in the roots and only 9.9% lower in the leaves than CK. Compared with in vitro nitrate reductase activity, in vivo activity responded differently to the increase of treatment time. At the beginning, in vivo nitrate reductase activity in the roots and leaves of T had no significant difference from CK, whereas with the increase of treatment duration, the activity decreased. At 12 d, in vivo activity in the roots and leaves of T lowered by 20.1 and 52.8% with respect to CK, respectively. This evidence suggests that posttranslational activation of nitrate reductase in cucumber seedlings may be seriously inhibited by nitrate stress.

Effect of Nitrate on Activities and Transcript Levels of Nitrate Reductase and Glutamine Synthetase in Rice

Real-time polymerase chain reaction analysis was used to compare the effect of NO-3on the activities of nitrate reductase(NR)and glutamine synthetase(GS),and the transcript levels of two NR genes,OsNia1 and OsNia2,two cytosolic GS1 genes,OsGln1;1 and OsGln1;2,and one plastid GS2 gene OsGln2,in two rice(Oryza sativa L.)cultivars Nanguang(NG)and Yunjing(YJ).Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH+ 4nutrition.Supply of NO -3increased NR activity in both leaves and roots.Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNia1.NO-3also caused an increase in GS activity,but had a complex effect on the expression of the three GS genes.In roots,the OsGln1;1 transcript increased,but OsGln1;2 decreased.In leaves,NO-3had no effect on the GS1 expression,but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N.These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes. In addition,the NO-3 induced increase of biomass,NR activity,GS activity,and the transcript levels of NR and GS genes were proportionally higher in NG than in YJ,indicating a stronger response of NG to NO-3nutrition than YJ.

Studies on some characteristics ofnitrate reductase from sugar beet（BetavulgarisL．） leaves

Some characteristics of nitrate reductase from sugar beet leaves shown in this paper were as follows:The nitrate reductase from sugar beet leaves required NADH as an electron donor.Accordingly,the nitrate reductase was classified as NADH-dependent(E.C.1 .6.6.1).The Km value of the nitrate reductase for NADH and NO-3 were 0.86 m mol and 0.18μp mol respectively.The optimum pH in reaction mixture solution for nitrate reduction activity was 7.5.The effect of variable concentrations of inorganic phosphorus in the reaction buffer on nitratereductase activity was investigated.When the inorganic phosphorus concentration was below35 mmol,the nitrate reductase activity was increased with increase of inorganic phosphorus concentration.Conversely,when the inorganic phosphorus concentration was Over 35 mmol.the nitrate reductase activity was inhibited.The nitrate reductase activity assayed in vitro was 3.2 and 5.6 times of that assayed in vivo under the condition of exogenous and endogenous ground substance respectively.

Inhibitive Determination of Heavy Metal Ions by Conductometric Nitrate Reductase Biosensor

A conductometric nitrate biosensor based on methyl viologen/Nafion/nitrate reductase（NR） interdigitated electrode for heavy metal determination was proposed.The activity of NR（EC 1.6.6.2） from Asper gillusniger（A.niger） was assayed as a function of metal concentration in the presence of Cu^2＋,Zn^2＋,Cd^2＋ and Pb^2＋.Parameters influencing the performance of the biosensor were optimized for the application of it in the inhibition determination of heavy metal ions.Detection limits for Cu^2＋,Zn^2＋,Cd^2＋ and Pb^2＋ were about 0.05,0.5,0.1 and 1.0 μmol/L,respectively.The results show that NR activity could not be regained after exposure to Cu^2＋,but could be partially recovered after exposure to Zn^2＋,Pb^2＋ and Cd^2＋.

Eff ects of temperature on photosynthetic performance and nitrate reductase activity in vivo assay in Gracilariopsis lemaneiformis (Rhodophyta)

Gracilariopsis lemaneiformis is an economically-valued species and widely cultured in China at present.After being acclimated to diff erent growth temperatures(15,20,25,and 30°C)for 7 days,the relative growth rate(RGR),nitrate reductase activity,soluble protein content and chlorophyll a fl uorescence of G.lemaneiformis were examined.Results show that RGR was markedly aff ected by temperature especially at 20°C at which G.lemaneiformis exhibited the highest eff ective quantum yield of PSII[Y(II)]and lightsaturated electron transport rate(ETR max),but the lowest non-photochemical quenching.Irrespective of growth temperature,the nitrate reductase activity increased with the incubation temperature from 15 to 30°C.In addition,the greatest nitrate reductase activity was found in the thalli grown at 20°C.The value of temperature coeffi cient Q10 of alga cultured in 15°C was the greatest among those of other temperatures tested.Results indicate that the optimum temperature for nitrate reductase synthesis was relatively lower than that for nitrate reductase activity,and the relationship among growth,photosynthesis,and nitrate reductase activity showed that the optimum temperature for activity of nitrate reductase in vivo assay should be the same to the optimal growth temperature.

Reducing phosphorylation of nitrate reductase improves nitrate assimilation in rice

Nitrate reductase(NR) is an important enzyme for nitrate assimilation in plants, and post-translational phosphorylation regulates NR activity. To evaluate the impact of the dephosphorylation of nitrate reductase 1(NIA1) protein on NR activity,nitrogen metabolism and plant growth, NIA1 phosphorylation site directed mutant lines(S532 D and S532 A) and an OsNia1 over-expression line(OE) were constructed, and the phenotype, NIA1 protein and its phosphorylation level, NR activity,nitrate metabolism and reactive oxygen metabolism of the transgenic lines were analysed. Exogenous NIA1 protein was not phosphorylated in S532 D and S532 A mutant lines, and their NR activities, activity states of NR and assimilation efficiencies of NO3–-N were higher than those in Kitaake(WT) and OE. The changes in these physiological and biochemical indexes in the OE line were less than in S532 D and S532 A compared to WT. These results suggest that the removal of transcriptional level control had little effect on nitrogen metabolism, but the removal of post-translational modification had a profound effect on it. With the removal of NIA1 phosphorylation and the improvement in the nitrate assimilation efficiency, the plant height and chlorophyll content of S532 D and S532 A decreased and the hydrogen peroxide and malondialdehyde contents of rice seedlings increased, which may be related to the excessive accumulation of nitrite as an intermediate metabolite. These results indicated that the phosphorylation of NR may be a self-protection mechanism of rice. The reduced phosphorylation level of nitrate reductase improved the assimilation of nitrate, and the increased phosphorylation level reduced the accumulation of nitrite and prevented the toxic effects of reactive oxygen species in rice.

Direct Electrochemistry with Nitrate Reductase in Chitosan Films

Stable films made from chitosan (CS) on pyrolytic graphite electrode (PGE) gave direct electrochemistry for incorporated enzyme nitrate reductase (NR). Cyclic voltammetry of CS / NR films showed a pair of well-defined and nearly reversible redox peaks at about -0.430 V vs. SCE at pH 7.0 phosphate buffers.

The removal of nitrate reductase phosphorylation enhances tolerance to ammonium nitrogen deficiency in rice

Nitrate reductase(NR) is a key enzyme for nitrogen assimilation in plants,and its activity is regulated by posttranslational phosphorylation.To investigate the effects of dephosphorylation of the NIA1 protein on the growth and the physiological and biochemical characteristics of rice under different forms of nitrogen supplies,the phenotypes,nitrogen metabolism and reactive oxygen metabolism were measured in NIA1 phosphorylation site-directed mutant lines(S532 D and S532 A),an Os Nia1 over-expression line(OE) and Kitaake(wild type,WT).Compared with WT and OE,S532 D and S532 A have stronger nitrogen assimilation capacities.When ammonium nitrate served as the nitrogen source,the plant heights,dry weights of shoots and chlorophyll(Chl) contents of S532 D and S532 A were lower than those of the WT and OE,whereas hydrogen peroxide(H_(2)O_(2)),malondialdehyde(MDA) and nitrite contents were higher.When potassium nitrate served as the nitrogen source,the plant heights,dry weights of shoots and Chl contents of S532 D and S532 A were higher than those of the WT and OE,there were no significant differences in the contents of H_(2)O_(2) and MDA in the leaves of the test materials,and the difference in nitrite contents among different lines decreased.When ammonium sulfate served as the nitrogen source,there were no significant differences in the physiological indexes of the test materials,except NR activity.Compared with ammonium nitrate and ammonium sulfate,the content of NH_(4)^(+)-N in the leaves of each plant was lower when potassium nitrate was used as the nitrogen source.The q PCR results showed that Os GS and Os NGS1 were negatively regulated by downstream metabolites,and Os Nrt2.2 was induced by nitrate.In summary,when ammonium nitrate served as the nitrogen source,the weak growth of NIA1 phosphorylation site-directed mutant lines was due to the toxicity caused by the excessive accumulation of nitrite.When potassium nitrate served as the nitrogen source,the assimilation rates of nitrate,nitrite and ammonium salt were accelerated in NIA1 phosphorylation site-directed mutant lines,which could provide more nitrogen nutrition and improve the tolerance of rice to ammonium nitrogen deficiency.These results could provide a possible method to improve the efficiency of nitrogen utilization in rice under low-nitrogen conditions.

New Rice Mutants(NR676 and NR827) Low in Nitrate Reduction

Mutants deficient in nitrate reductase(NR) were classfied into two groups,nia types are deficient in apoprotein and cnx types are lack of Mo-Co.This paper reports the characteristics of low NR mutants,which are not deficient in NR activity but 40%～60% of the NR activity of the wild type,Nipponbare.Mutants NR676 and NR827 were selected as seedings showing poor growth with nitrate as sole mitrogen source from M 2 population.They exhibited chlorate resistance.Genetic analysis in the F 1 and F 2 indicated that chlorate resistance in the mutants was transmitted by a single recessive nuclear gene and that NR676 and NR827 were induced by a mutation at a single locus.In the F2 population,all of the seedings showing yellow green leaves were resistant to chlorate and low in NR activity.Photosynthetic rate and mRNA expression of NR676 and NR827 were lower than wild type.These results suggested that low NR activity and chlorate resistance of NR676 and NR827 were caused by a defect in photosynthetic process.

Nitrate Reductase: Properties and Regulation

Nitrate Reductase (NR) is a rating-limit and key enzyme of nitrate assimilation in plants,so,NR activity is important for growth,development and the dry matter accumulation of plants.The regulation of NR activity appears to be rather complex and many studies have been devoted to the description of regulation and properties,but in this paper we focus on the properties and regulation of NR in higher plants.

Effects of allelochemicals on activity of nitrate reductase

Study on the effects of allelochemicals such as trans \|ferulic acid ( t \|FA), benzoic acid (BA) and p \|hydroxybenzoic acid ( p \|HA), isolated from decomposed wheat straw on the activity of nitrate reductase at different concentrations of allelochemicals and different pH is described. t \|FA (0.26, 2.58 and 5.15 mmol/L) and BA (4.09, 8.19 mmol/L) showed a certain inhibition to the activity of nitrate reductase. The highest inhibition rate was 18.40%, but BA (0.41 mmol/L) and p \|HA (0.36, 1.81 and 3.62 mmol/L) showed stimulation, the more strong stimulation rate was 15.80%. At pH 6 condition, the activity of nitrate reductase was stronger inhibited than pH 7 and pH 8, but the mixture of 3 allelochemicals at pH 6 showed a stimulation. The mixture, however, at pH 7 and pH 8 showed some inhibition. It was found that there was a relationship between production of NO\+-\-2 and transformation of NO\+-\-3.

高粱种质资源成熟期氮效率评价及筛选

氮素作为影响作物生长发育的重要元素,是限制作物高产的重要限制因素之一。硝酸还原酶(Nitrate Reductase,简称NR)作为高等植物氮素吸收利用的限速酶,直接调节氮代谢。因此,叶片硝酸还原酶活性作为影响高粱产量的重要生理指标被用于评价氮素吸收、利用效率。本研究选取了97份不同来源的高粱材料,通过对97份高粱材料进行成熟期倒二叶和倒三叶的硝酸还原酶活性测定,为筛选高粱氮高效材料奠定基础。

不同类型燕麦种质蛋白质形成的生理机制分析

为探究不同燕麦种质资源蛋白质含量的生理差异,以高蛋白燕麦种质A34和低蛋白燕麦种质B23为材料,采用大田试验分析了籽粒蛋白质含量及花后相关代谢酶活性的动态变化规律。结果显示,种质资源A34和B23籽粒蛋白质含量在开花后的变化规律相似。与B23相比,A34的旗叶谷氨酰胺合成酶活性在花后5~15 d显著降低,花后20~30 d显著升高;花后5~30 d,A34旗叶的硝酸还原酶、丙酮酸磷酸二激酶、谷氨酸合成酶、天冬酰胺合成酶活性以及游离氨基酸含量显著升高,籽粒谷氨酸丙酮酸转氨酶活性和可溶性糖含量显著降低。这表明种质资源A34通过调控叶片和籽粒蛋白质相关代谢酶活性促进籽粒蛋白质的积累。

不同蛋白质含量小麦品种叶片NRA与氮素积累关系的研究

以鲁麦５号和昌乐５号两种不同蛋白质含量小麦品种为材料，研究了各生育期叶片ＮＲＡ与氮素积累的关系。其结果是，在不同施氮量下，各生育期叶片ＮＲＡ、ＮＯ－３－Ｎ、ＮＨ２－Ｎ、还原Ｎ含量皆随施氮量增加而增加，但生育前期昌乐５号的皆大于鲁麦５号，而生育后期则相反，鲁麦５号的皆大于昌乐５号，籽粒蛋白质含量亦为鲁麦５号高于昌乐５号。

外源硝酸还原酶(NR)抑制剂对油菜植株内NR活性的影响及其与硝酸盐含量的关系

为进一步揭示硝酸还原酶(nitrate reductase,NR)活性的调控机制及其与植株体内硝酸盐含量的关系。本试验在正常供氮(15 mmol L^(–1)NO_3~–)和缺氮(7.5 mmol L^(–1)NO_3~–)条件下,以氮高效(H1:742和H2:Xiangyou 15)和氮低效(L1:814和L2:H8)油菜基因型为研究材料,通过NR活性的专性抑制剂处理,研究NR活性和硝酸盐含量的基因型和氮水平差异。结果表明,NR专性抑制剂处理可以显著降低叶片NR活性,正常供氮和缺氮条件下分别降低53.0%和57.6%,但对叶片硝酸盐的含量没有显著影响。正常供氮条件下的NR活性和硝酸盐含量比缺氮条件下分别高46.9%和16.4%。氮高效油菜基因型的硝酸盐含量显著低于氮低效基因型。H2的NR活性(NRA_(act))显著高于氮低效基因型的本质原因是其主效基因(nia2)的相对表达量高于氮低效基因型。本研究充分表明NR活性和硝酸盐含量存在明显的基因型和氮水平差异,一定程度的NR活性变化对植株体内硝酸盐的含量并没有显著的影响。

枫香种源生物量和NRA对供氮水平的响应差异

为研究不同纬度枫香种源经过无营养胁迫生长后对供氮水平的响应差异,按种源与氮2因素裂区设计砂培试验,以N水平为主处理,枫香种源为副处理,3次重复,随机排列(水平内种源间)。结果表明:低氮胁迫会刺激枫香叶片提前形成花青素变红,且低纬区种源变红早于高纬区种源;另外低纬区种源与高纬区种源硝酸还原酶活性(NRA)存在各自的变化规律。以最高生物量为对照,在0 N水平下福建延平种源生物量降低最少(54.54%),且具有较高的生物量,但其在2N水平生物量迅速降低,耐肥性较差;其它种源生物量变化趋势一致。各种源根冠比从0 N到1/2 N水平均迅速降低:广西南丹>福建延平>浙江永康>安徽泾县,即随着纬度的升高,枫香种源根冠比受N的影响越来越小。统计分析发现,福建延平种源在无N及低N水平时NRA极显著高于其它种源,且具有较高生物量,受环境影响较小(主要是棚内重复间光照的差异),可以认为福建延平是优良的耐低N型种源。

不同氮肥运筹对超高产小麦NR活性和产量影响的研究

通过三种氮肥处理试验,研究了氮肥运筹对各生育期NR活性及产量、产量性状的影响,阐述了NR活性与产量和产量性状的关系。结果表明:从分蘖期始,小麦叶片NR活性表现出逐渐上升的趋势,至开花期达到最高,其后又逐渐下降。不同氮肥处理的NR活性在分蘖至孕穗差异不大,在孕穗之后,氮肥一次底施的处理一的NR活性明显低于药隔期追施的处理二和处理三。药隔期追施氮肥可明显促进成穗数、穗长、结实小穗数、穗粒数、千粒重、穗粒重等产量性状的增加,对提高产量具有重要作用。对两种类型品种,普通型的豫麦41号宜采用药隔期重施追肥,而大粒型的兰考86(79)则以药隔期补施氮较好。NR活性与产量、产量性状的相关关系,在分蘖至拔节期间表现不密切,但孕穗至成熟期间与产量的相关系数均达高度正相关,与穗粒数、穗粒重的相关关系也较为密切,反映出通过药隔期追施氮肥,增加小麦中后期NR活性对提高产量是非常重要的。

有机无机肥配施对水稻光合特性及NR与SPS活性的影响

采用田间小区试验,研究了有机肥(稻草、猪粪、猪粪堆肥和沼渣沼液)与化肥配施对水稻光合特性、硝酸还原酶(NR)及蔗糖磷酸合成酶(SPS)活性的影响。结果表明,猪粪堆肥与化肥配施处理在提高水稻功能叶净光合速率、水稻碳氮代谢关键时期NR和SPS酶活性方面较其他类型的配施处理有较明显优势,并能提高水稻产量和改善品质。与施纯化肥相比,以20%的猪粪堆肥氮配施80%的化肥氮处理的早稻孕穗期功能叶NR酶活性提高了65.06%,蜡熟期SPS活性提高了7.70%,游离氨基酸含量提高43.87 mg/kg,产量增加19.65%。