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.

A Modified Method for Measuring Root Iron Reductase Activity Under Normal Laboratory Conditions

Based on the strong chelating property of bathophenanthroline disulfonic acid (BPDS) with root chelate reductase activity is usually measured with a spectrophotometer using MES (2-morpholinoethanesulfonic acid) or HEPES (2-(4-(2-Hydroxyethyl)-1-piperazinyl) ethanesulfonic acid) buffer in the dark because of high autoreduction rate of in the presence of light. However, the exclusion of light is inconvenient, especially when analyzing a large number of samples. The objective of this study was to develop a new method for determination of root reductase activity under normal laboratory conditions using a suitable buffer composition and concentration to eliminate the autoreduction of A modified method using a Tris (2-amino-2-hydroxymethyl-1,3-propanediol) buffer at pH 7.5 instead of MES or HEPES buffer and a decreased FeEDTA (Fe ethylene diamine tetraacetic acid) concentration of 50 μmol L-1 was developed. The autoreduction of using the Tris buffer was undetectable for temperatures at 4 and 28 °C and was also much lower than that using the other buffers even with sunlight during measurement of reduction. Furthermore, the differences in reductase activity among 5 plant species and 14 red clover cultivars (Trifolium pratense L.) could be easily detected with the modified method. The method developed in this study to measure root Fe chelate reductase activity was not only effective and reliable but also easily managed under normal laboratory light conditions.

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.

Effect of Repetitive Bleeding on NADH-Cytochrome b_5 Methemoglobin Reductase Activity and Molybdenum Content in Erythrocytes of Rats

Reticulocytosls in rats was induced by repetitive bleeding. Both the in vitro and the in vivo studies showed that the detected reductive speed of methemoglobin of the bleeding group was faster than that of the control group at all time intervals. At the same time, the NADH-cytochrome b5 methemoglobin reductase activity and the molybdenum content in erythrocytes of the bleeding group were significantly increased. Regressional analysis showed that there was a significantly positive correlation between the enzyme activity and the molybdenum content. It is proposed that molybdenum might be required for the enzyme activity

AtECB1/MRL7, a Thioredoxin-Like Fold Protein with Disulfide Reductase Activity, Regulates Chloroplast Gene Expression and Chloroplast Biogenesis in Arabidopsis thaliana

Plastid-encoded RNA polymerase （PEP） is closely associated with numerous factors to form PEP complex for plastid gene expression and chloroplast development. However, it is not clear how PEP complex are regulated in chloroplast. Here, one thioredoxin-like fold protein, Arabidopsis early chloroplast biogenesis 1 （AtECB1）, an allele of MRL7, was identified to regulate PEP function and chloroplast biogenesis. The knockout lines for AtECB1 displayed albino phenotype and impaired chloroplast development. The transcripts of PEP-dependent plastid genes were barely detected, suggesting that the PEP activity is almost lost in atecbl-1. Although AtECB1 was not identified in PEP complex, a yeast two-hybrid assay and pull-down experiments demonstrated that it can interact with Trx Z and FSD3, two intrinsic subunits of PEP complex, respectively. This indicates that AtECB1 may play a regulatory role for PEP-dependent plastid gene expression through these two subunits. AtECB1 contains a βαβαββα structure in the thioredoxin-like fold domain and lacks the typical C-X-X-C active site motif. Insulin assay demonstrated that AtECB1 harbors disulfide reductase activity in vitro using the purified recombinant AtECB1 protein. This showed that this thioredoxin-like fold protein, AtECB1 also has the thioredoxin activity. AtECB1 may play a role in thioredoxin signaling to regulate plastid gene expression and chloroplast development.

Effects of Nitrogen Fertilization on the Growth and N Uptake of Late-sowing Winter Oilseed Rape (Brassica napus L.)

[Objective] The aim of this work was to analyze the N fertilization on the vegetative growth and N uptake of different winter rapeseed varieties at wintering stage. [Method] In two consecutive years （2009-2011）, two winter rapeseed varieties （B. napus L.）, an early maturity variety Zhongyou 116 （ZY116） and a middle-late application maturity variety Zhongyouza 12 （ZYZ12） were employed. Field experiments with different N levels （0, 90, 180, 270, 360 kg N/hm 2 ） were designed. At the wintering stage, the dry matter weight, the nitrogen content and concentration of plants, leaf nitrate reductase activity （NRA） and seed yields were investigated. [Result] The shoot dry matter of ZY116 increased rapidly when N rate ranged from 0 to 180 kg/hm 2 , and it raised slightly when N rate ranged from 180 to 360 kg/hm 2 . The shoot dry matter of ZYZ12 were changed in a single peak curve; the peak of shoot dry matter appeared at 270 kg N/hm 2 . The N concentration and N content in shoot and root increased rapidly when the N rate changed from 90 to 180 kg/hm 2 . Moreover, the N concentration and N content root of in ZYZ12 were much higher than that of ZY116. Present study revealed that the changed trend of leaf nitrate reductase activities （NRA） were significantly increased at the N rate of 180 kg/hm 2 in ZY116 and ZYZ12 compared with the N rate of 90 kg/hm 2 in two years. [Conclusion] Optimal nitrogen application significantly increased the dry weights and N uptake at wintering stage as well as increasing the yield of winter oilseed rape.

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.

Sequence Diversity and Enzyme Activity of Ferric-Chelate Reductase LeFRO1 in Tomato

Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron ho- meostasis in strategy I plants. LeFROI is a major ferric-chelate reductase involved in iron uptake in tomato. To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity, we cloned the coding sequences of LeFRO1 from 16 tomato varieties collected from different regions, and detected three types of LeFRO1 （LeFRO1MM, LeFRO1Ailsa and LeFRO1Monita） with five amino acid variations at the positions 21, 24, 112, 195 and 582. Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity （LeFRO1AiISa 〉 LeFRO1MM 〉 LeFRO1M^nita）. The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase, because modification of this amino acid resulted in a significant reduction of enzyme activity. Further, we showed that the combination of the amino acid residue lie at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFRO1. In conclusion, the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress, and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato.

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.

Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth,Physiological Activities,and Alkaloids Production in Catharanthus roseus L.

Sodium alginate is a polysaccharide that is largely obtained from the brown algae （Sargassum sp.）.It has been used as a wonderful growth promoting substance in its depolymerized form for various plants.The aim of this study was to find out the effects of various concentrations of γ-irradiated sodium alginate （ISA）,viz.,deionized water （control,T0）,20 （T1）,40 （T2）,60 （T3）,80 （T4）,and 100 ppm （T5） on the agricultural performance of Catharanthus roseus L.（Rosea） in terms of growth attributes,photosynthesis,physiological activities,and alkaloid production.The present work revealed that ISA applied as leaf-sprays at concentrations from 20 to 100 ppm might improve growth,photosynthesis,physiological activities,and alkaloid production in C.roseus L.significantly.Of the various ISA concentrations,80 ppm proved to be the best one compared to other concentrations applied.

Nodule study in Albizia chinensis in relation to nitrogen metabolism,morphology and biomass

Morphology,biomass,nitrate reductase(NR)and nitrogenase activity in Albizia chinensis(Osb.)Merr.nodules were assessed on monthly and seasonal basis for 1 year.Average NR and nitrogenase activity was higher during the rainy season,reaching a maximum in August.Thereafter,activity decreased through autumn and reached a minimum value during winter.Fresh and dry biomass of nodules increased gradually from summer to the rainy season and then started decreasing with the onset of winter as nodules began to senesce.Among four developmental stages of the nodules that correspond to their increasing age,NR and nitrogenase activity remained low in stage 1 nodules and peaked in stage 2.The activity of both enzymes further decreased with increasing age(stage 2 to stage 4).Morphological features such as shape,diameter and color varied considerably among the developmental stages.Stage 1 nodules were cream-colored,oval to heartshaped with smallest average diameter whereas at stage 2,they became bilobed to tetralobed.On the other hand,stage 3 nodules had the largest average diameter and were multilobed in structure.Stage 4 nodules that correspond to the senescing stage were dark brown to black,multilobed,flattened and hollow due to degeneration of nodular tissue.

Synthesis and evaluation of 8-deaza-5,6,7,8-tetrahydromethotrexate derivatives as dihydrofolate reductase inhibitors

A series of N5-substituted 8-deaza-5,6,7,8-tetrahydromethotrexate derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase（DHFR）.The results indicated that modification of the pteridine ring of methotrexate（MTX） rendered poor activity against human DHFR.

Effect of Nitrogen Starvation on the Responses of Two Rice Cultivars to Nitrate Uptake and Utilization

Ammonium(NH+4) is the main nitrogen(N) form for rice crops, while NH+4near the root surface can be oxidized to nitrate(NO-3)by NH+4-oxidizing bacteria. Nitrate can be accumulated within rice tissues and reused when N supply is insufficient. We compared the remobilization of NO-3stored in the tissue and vacuolar between two rice(Oryza sativa L.) cultivars, Yangdao 6(YD6, indica)with a high N use efficiency(NUE) and Wuyujing 3(WYJ3, japonica) with a low NUE and measured the uptake of NO-3, expression of nitrate reductase(NR), NO-3transporter genes(NRTs), and NR activity after 4 d of N starvation following 7-d cultivation in a solution containing 2.86 mmol L-1NO-3. The results showed that both tissue NO-3concentration and vacuolar NO-3activity were higher in YD6 than WYJ3 under N starvation. YD6 showed a 2- to 3-fold higher expression of OsNRT2.1 in roots on the 1st and 4th day of N starvation and had significantly higher values of NO-3uptake(maximum uptake velocity, Vmax) than the cultivar WYJ3.Furthermore, YD6 had significantly higher leaf and root maximum NR activity(NRAmax) and actual NR activity(NRAact) as well as stronger root expression of the two NR genes after the 1st day of N starvation. There were no significant differences in NRAmax and NRAact between the two rice cultivars on the 4th day of N starvation. The results suggested that YD6 had stronger NRA under N starvation, which might result in better NO-3re-utilization from the vacuole, and higher capacity for NO-3uptake and use, potentially explaining the higher NUE of YD6 compared with WYJ3.

Promotive effect of pyridine on indole degradation by activated sludge under anoxic conditions

Batch experiments were carried out to investigate the promotive effect of pyridine on indole degradation under denitrifying conditions.The seed sludge was obtained from a local coal-coking wastewater treatment facility and was acclimated in the laboratory.Indole and pyridine were supple-mented to the synthetic wastewater at different ratios.The optimum ratio of chemical oxygen demand(COD)to nitrate(C/N)was 8.4-8.9 for both denitrification and indole and pyridine degradation.At a temperature of 28°C and pH of 7.0-7.5,the nitrate reductase activity(NRA)was in the best state.The addition of pyridine could promote NRA and the degradation of indole.When the initial concentration of indole was 150 mg/L,the concentration ratio of indole to pyridine was in the range of 1-10.Under optimum C/N conditions,the degradation of indole could be described with pseudo-zero-order kinetics.There was no accumulation of nitrite during the reaction.When the concentration ratio of pyridine to indole was less than 0.25 with an increase in the pyridine proportion,there were more significant augment rates for NRA and the degradation of indole than the situation when the concentration ratio was more than 0.25.