Nitrate reductase activity and its diurnal variation rhythm for Camptotheca acuminata seedlings

Nitrate reductase activity (NRA) in different plant organs and leaves in different positions of Camptotheca acuminata seedlings was determined by an In vivo assay, the diurnal variation rhythm of NRA in leaves of different positions was observed,and the correlations between leaf NRA, leaf area and lamina mass per unit area (LMA) were also examined. The results showed that NRA in the leaf was significantly highest, compared with that in other organs such as roots, stems and leaves. In this experiment, the 10 leaves were selected from the apex to the base of the seedlings in order. The different NRA occurred obviously in leaves of different positions of C. acuminata seedlings from the apex to the base, and NRA was higher in the 4th-6th leaves.The diurnal change rhythm of leaf NRA showed a one peak curve, and maximum NRA value appeared at about midday (at 12:30 or so). No obvious correlations between NRA and leaf area or lamina mass per unit area were observed. This study offered scientific foundation for the further research on nitrogen metabolism of C. acuminata.

Dynamic Changes of Nitrate Reductase Activity within 24 Hours

[Objective] The research aimed to study the circadian rhythm of nitrate re- ductase activity （NRA） in plant. [Method] The wheat plants at heading stage were used as the materials for the measurement of dynamic changes of nitrate reductase activity （NRA） within 24 h under the conditions of constant high temperature. [Resulti The fluctuation of NRA in wheat changed greatly from 20：00 pm to 11：00 am. The enzyme activity remained constant, but at 14：00 the enzyme activity was the high- est, higher than all the other time points except the enzyme activity measured at11：00. The enzyme activity was the lowest of 17：00, which was lower than all the other time points except the enzyme activity measured at 2：00. [Conclusion] There were autonomous rhythm changes of NRA in wheat in a certain degree.

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.

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 NO3^- on the activities of nitrate reductase （NR） and glutamine synthetase （GS）, and the transcript levels of two NR genes, OsNial 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 （Y J）. Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH4^＋ nutrition. Supply of NO3^- increased 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 OsNial. NO3 also 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, NO3^- had 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 NO3-induced increase of biomass, NR activity, GS activity, and the transcript levels of NR and GS genes were proportionally higher in NG than in Y J, indicating a stronger response of NG to NO3^- nutrition than YJ.

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.

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.

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.

Effects of Nitrogen Sources on Nitrate Reductase Activity and Some Physiological Parameters in Psyllium （Plantago ovata F.） under Salinity Stress

The effects of three nitrogen sources （ammonium, nitrate and ammonium＋nitrate） and three salt treatments （0, 100 and 200 mM NaCl） on nitrate reductase activity, proline, soluble protein and carbohydrate contents in psylliom （Plantago ovata） plants were Studied. The nitrate reductase activity tended to increase when NO3- was included in the root-zone solution, but decrease as salinity increased. All N sources stimulated plant growth and nutrient uptake. Shoot and root dry matter tended to decrease as salinity increased, but less so when both NH4＋ and NO3- were present. Shoot biomass accumulated to significantly greater quantities under the mixed-N treatments than when produced using either NH4＋ or NO3- N-form alone. Although not statistically significant, the root biomasses showed a similar tendency. Generally, leaf proline and soluble shoot carbohydrate contents increased with increasing salinity in contrast to soluble protein which decreased regardless of the N source. Under salinity conditions, the concentration of Na＋ in shoot and root tissues was highest in ammonium-N treatment, while that of K＋ was highest in the mixed-N treatment.

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.

Response of Nitrate Metabolism in Seedlings of Oilseed Rape(Brassica napus L.) to Low Oxygen Stress

In order to understand the response of nitrate metabolism in seedlings of oilseed rape （Brassica napus L.） to low oxygen stress （LOS）, two cultivars were studied at different light, LOS time and exogenous nitrate concentrations under hydroponic stress. Results show that N-uptake and dry matter of rape seedlings were decreased after LOS stress while nitrate accumulation （NA） under LOS was induced by darkness. Nitrate accumulation peaked at 3 d while root activity （RA, deifned as dehydrogenase activity） decreased with prolonged waterlogging exposure. Exogenous nitrate signiifcantly elevated NA and RA. Tungstate （TS） and LOS inhibited nitrate reductase （NR） activity while NR transcription and activity were enhanced by exogenous nitrate. Low oxygen stress stimulated the activity of superoxide dismutase （SOD） and peroxidase （POD） slightly, but inhibited that of catalase （CAT）. B. napus L. Zhongshuang 10 （ZS10）, a LOS tolerant cultivar, displayed smaller decrease upon dry matter under LOS, higher NA in darkness and lower NA in light than B. napus L. Ganlan CC （GAC）, a LOS sensitive variety. ZS10 had lower NA and higher RA after waterlogging and exogenous nitrate treatment, and higher NR activity under TS inhibition than GAC, but the activity of antioxidant enzymes did not change under LOS. The results indicate that nitrate metabolism involved tolerance of rape seedlings to LOS, with lower accumulation and higher reduction of nitrate being related to higher LOS tolerance of rape seedlings exposed to waterlogging.

Effect of Nitrate on Root Development and Nitrogen Uptake of Suaeda physophora Under NaCl Salinity

The effects of NaCl salinity and NO^-3 on growth, root morphology, and nitrogen uptake of a halophyte Suaeda physophora were evaluated in a factorial experiment with four concentrations of NaCl （1, 150, 300, and 450 mmol L^-1） and three NO^-3 levels （0.05, 5, and 10 mmol L^-1） in solution culture for 30 d. Addition of NO^-3 at 10 mmol L^-1 significantly improved the shoot （P 〈 0.001） and root （P 〈 0.001） growth and the promotive effect of NO^-3 was more pronounced on root dry weight despite the high NaCl concentration in the culture solution, leading to a significant increase in the root：shoot ratio （P 〈 0.01）. Lateral root length, but not primary root length, considerably increased with increasing NaCl salinity and NO^-3 levels （P 〈 0.001）, implying that Na^＋ and NO3^- in the culture solution simultaneously stimulated lateral root growth. Concentrations of Na^＋ in plant tissues were also significantly increased by higher NaCl treatments （P 〈 0.001）. At 10 mmol L^-1 NO^-3, the concentrations of NO^-3 and total nitrogen and nitrate reductase activities in the roots were remarkably reduced by increasing salinity （P 〈 0.001）, but were unaffected in the shoots. The results indicated that the fine lateral root development and effective nitrogen uptake of the shoots might contribute to high salt tolerance of S. physophora under adequate NO^-3 supply.

Nitrate Uptake of Rice as Affected by Growth Stages and Ammonium

Solution culture experiments were carried out to study the NO3- uptake kinetics by rice roots and the nitrate reductase activity (NRA) in leaves of four typical rice genotypes (conventional indica, conventional japonica, hybrid indica, and hybrid japonica) at different growth stages. The effects of NH4+ on the NO3- uptake kinetics was also studied. The results indicated that the four genotypes responded differently to NO3- and NH4+. Comparing indica rice with japonica rice, hybrid rice with conventional rice, formers absorbed NO3-more rapidly than laters. The superiority of indica rice and hybid rice for NO3- uptake was mainly due to the higher Vmax at the early growth stage (20 days) and the higher affinity of nitrate transporters for NO3- at the middle growth stage (50 days). At both growth stages NH4+ significantly inhibited NO3- uptake for japonica rice, but showed little effect for indica rice. Conversely, NH4+ stimulated the uptake of NO3- for hybrid rice. NH4+ could repress NRA in leaves of all four genotypes with a sensitive order as following: hybrid japonica > hybrid indica > conventional indica > conventional japonica.

The δ^15N response and nitrate assimilation of Orychophragmus violaceus and Brassica napus plantlets in vitro during the multiplication stage cultured under different nitrate concentrations

Natural nitrogen isotope composition(δ^(15)N) is an indicator of nitrogen sources and is useful in the investigation of nitrogen cycling in organisms and ecosystems. δ^(15)N is also used to study assimilation of inorganic nitrogen. However, the foliar δ^(15)N of intact plants, which is a consequence of nitrate assimilation occurring in the roots and shoots, is not suited for studying nitrate assimilation in cases where nitrate is the sole nitrogen source. In this study, Orychophragmus violaceus(Ov) and Brassica napus(Bn) plantlets, in which nitrate assimilation occurred in the leaves, were used to study the relationship between foliar δ^(15)N and nitrate assimilation.The plantlets were grown in vitro in culture media with different nitrate concentrations, and no root formation occurred for the plantlets during the multiplication stage.Nitrogen isotope fractionation occurred in both the Ov and the Bn plantlets under all treatments. Furthermore, the foliar nitrogen content of both the Ov and Bn plantlets increased with increasing nitrate concentration. Foliar nitrogen isotope fractionation was negatively correlated with foliar nitrogen content for both the Ov and Bn plantlets. Our results suggest that the foliar nitrogen isotope fractionation value could be employed to evaluate nitrate assimilation ability and leaf nitrate reductase activity.Moreover, high external nitrate concentrations couldcontribute to improved foliar nitrogen content and enhanced nitrate assimilation ability.

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。