Seedling Nitrate Reductase Activity and Nitrate Partitioning in Maize (Zea mays L.) Strains Divergently Selected For Post-anthesis Leaf-Lamina Nitrate Reductase Activity

Two experiments were conducted to evaluate the effects of phenotypicrecurrent selection for high and low post-anthesis leaf-laminain vivo NRA on nitrate uptake, nitrate partitioning and in vitroNRA of seedling roots and leaves. In Experiment 1, intact plantsof cycle 0, 4, and 6 of the high and low NRA strains were grownon NH₄-N for 11 d, then exposed to 1.0 mol m^–3 KNO₃, andcultures sampled at 6 h and 28 h (induction and post-inductionperiods). Nitrate uptake, tissue nitrate concentration and invitro NRA were determined. The pattern of response to selectionin seedling leaf NRA was similar to that observed for in vivoNRA of field grown plants. Leaf NRA increased between 6 h and28 h. Root NRA was not affected by selection or sampling time.Treatments differed in total fresh weight but not in reductionor uptake of nitrate per unit weight, indicating a lack of correspondencebetween NRA and reduction and supporting the idea that concomitantreduction by NR is not obligatorily linked to nitrate influxin the intact plant. In Experiment 2, dark-grown plants of cycle 0, and 6 of thehigh and low NRA strains were cultured without N, detopped onday 6, transferred the following day to 0-75 mol m^–3 KNO₃and sampled at 6 h and 28 h. In contrast to Experiment 1, selectionpopulations differed in nitrate reduction and root NRA, whichby 28 h reached higher average levels than root NRA of intactplants. Translocation and reduction were inversely related amongstrains within each sampling time. The high level of translocationin detopped plants of the low NRA strain was difficult to reconcilewith its low leaf NRA level of Experiment 1. It is suggestedthat nitrate transport in detopped roots is altered relativeto the intact system in a way which permits greater NRA inductionand nitrate reduction. The results indicate that nitrate partitioningby detopped root systems should be interpreted with caution. Key words: Zea, nitrate reductase activity, nitrate uptake, nitrate reduction, nitrate partitioning, selection     

Effects of Root Temperature and Form of Nitrogen Nutrition on Nitrate Reductase Activity in Oilseed Rape (Brassica napus L.)

The effect of root temperature and form of inorganic nitrogensupply on in vitro nitrate reductase activity (NRA) was studiedin oilseed rape (Brassica napus L. cv. bien venu). Plants weregrown initially in flowing nutrient solution containing 10 µMNH₄NO₃ and then supplied with either nitrate or ammonium for15 d at root temperatures of 3, 7, 11 or 17 °C. Shoot temperatureregime was similar for all plants; 20/15 °C, day/night.Root NRA was highest when roots were grown at 3 and 7 °C.In laminae and petioles NRA was highest when roots were 11 or17 °C. The plants supplied with ammonium had much lowerlevels of NRA in roots after 5 d than the plants supplied onlywith nitrate. NRA in the laminae of plants supplied with ammoniumwas low relative to that in plants supplied with nitrate onlywhen root temperature was 11 or 17 °C. Values of the apparent activation energy (E_a) of NR, calculatedfrom the Arrhenius equation, in laminae and petioles were differentfrom roots suggesting difference in enzyme conformation. Evidencethat the temperature at which roots were growing affected E_awas equivocal. Oilseed rape, Brassica napus L., activation energy, ammonium, Arrhenius equation, nitrate, root temperature, nitrate reductase     

Nitrate reductase activity in chicory roots following excision

In young chicory plantlets (Cichorium intybus L. Witloof cv.Flash), nitrate assimilation takes place mainly in the roots.Nitrate reductase activity (NRA) was measured in roots deprivedof shoot control by excision and transferred into a sucrose-containingmedium. Such a treatment resulted in a drop of about 60% ofNRA within 3 h. The level of NR protein decreased after 12 hand the level of NR-mRNA after several days. This adaptationof nitrate assimilation to excision was affected by a phosphorylation-dephosphorylationmechanism as shown by increased sensitivity to magnesium ofin vitro NRA. Okadaic acid, a serinethreonine protein phosphatasesinhibitor, enhanced the decrease of NRA. Conversely, staurosporine,a serine-threonine protein kinases inhibitor, antagonized theinhibition of NRA. This suggests that excision caused a rapidinactivation of NRA in roots of chicory by modifying the phosphorylationbalance towards a phosphorylated NR form which could enter aninactive complex. Key words: Chicory, nitrate reductase, staurosporine     

A Response Surface Analysis of the Effects of Cyclohexanecarboxylic Acid and 2,4-Dichlorophenoxy Acetic Acid on Nitrogen Metabolism in Phaseolus vulgaris L.

Primary leaves of 2-week-old bush bean plants (Phaseolus vulgariscv ‘Top Crop’) were treated with one of 36 combinationsof cyclohexanecarboxylic acid (CHCA) and 2,4-dichlorophenoxyacetic acid (2,4-D), each at six equally spaced concentrationsFive, 7, 9 and 11 days after treatment, one plant from eachcombination was harvested, and primary leaves were monitoredfor nitrate reductase activity (NRA), nitrate nitrogen (N) concentration,organic N concentration, and d wt NRA was assayed by an in vivoprocedure Data from the resulting 6 x 6 x 4 factorial configuration foreach of the four responses were analysed using the method oforthogonal polynomials, which is described in detail This method,based on equally spaced levels, produced independent polynomialexpressions in CHCA concentration, 2,4-D concentration, and/ortime-to-harvest after treatment, which were then fitted to thedata on each response by simple linear regression analyses.A graphical procedure was used to compare predicted responsesfrom the best fit equations with the actual data. Examination of computer-generated plots of the three-dimensionalresponse surfaces revealed that NRA, organic N concentration,and d wt decreased with increasing 2,4-D concentration, whereasnitrate N content increased Increasing CHCA concentration generallyhad the opposite effect on NRA and nitrate N content, althoughinteraction between the two chemicals was evident in the latterresponse Organic N content remained unchanged as CHCA concentrationincreased, while d wt first increased then decreased Possibleexplanations for these results are discussed, as are the advantagesof using factorial designs with equally-spaced levels in plantgrowth studies. Phaseolus vulgaris L, bush bean, nitrogen metabolism, plant growth regulators, cyclohexanecarboxylic acid, 2,4-dichlorophenoxy acetic acid, regression analysis, orthogonal polynomials, response surfaces analysis     

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     

Seasonal variations in nitrate content,total nitrogen,and nitrate reductase activities of macrophytes from a chalk stream in Upper Bavaria

Summary 11 macrophytic species from a groundwater influenced chalk stream in Upper Bavaria were investigated during a period of one year in order to determine differences in the endogenous nitrate content, in total nitrogen content and in nitrate reductase activity (NRA). Nitrate concentrations of different plants taken from the same site of the river varied by a factor of approximately 10³. A maximum of 1,958 mol NO ₃ ^- g^-1 dry w. could be measured in the petioles of Nasturtium officinale, which accounts for 12% of plant dry w. Very high values were also found in Callitriche obtusangula and Veronica angallis-aquatica. In comparison to the ambient water, mean accumulation rates of up to 131 could be found. In Fontinalis antipyretica, the plant poorest in nitrate, the ratio was only 1.24:1. Elodea canadensis belonged to a group of plants having very low nitrate concentrations. Since NRA was very low too, it is assumed that nitrogen nutrition of this species depends rather on ammonia than on nitrate. With a few exceptions nitrate content of different plant organs varied markedly. In general they were lowest in leaves and highest in shoot axes. Appreciable amounts of nitrate were also found in the roots of plants. No correlation could be found between endogenous nitrate content and NRA. In contrast to endogenous nitrate content and NRA, total nitrogen concentrations of the plants did not differ significantly.     

小白菜光合性能与硝酸还原酶活性关系的研究

对营养液培养条件下的6个不同基因型小白菜幼苗在不同光照度和硝酸还原酶抑制剂Na_2WO_4处理下的光合参数、硝酸盐含量和硝酸还原酶活性(NRA)进行分析,以明确小白菜光合性能与NRA的关系.结果发现:随着光照强度的增强,小白菜NRA极显著增强,其硝酸盐含量则显著降低,并且基因型间有显著差异;Na_2WO_4处理能使小白菜的NRA和净光合速率显著降低,而对其叶绿素含量、气孔导度、胞间CO_2浓度和蒸腾速率无显著影响.研究表明,光照度是制约小白菜幼苗硝酸还原酶活性和硝酸盐含量的重要因素之一,小白菜幼苗的硝酸还原酶活性与其净光合速率有密切关系,但对其他光合参数无直接影响.     

Unusual regulatory nitrate reductase activity in cotyledons of Brassica napus seedlings: enhancement of nitrate reductase activity by ammonium supply

The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.     

Characterization of the 'In Vivo' Nitrate Reductase Activity in the Roots and Leaves of Eichhornia crassipes

Studies of in vivo nitrate reductase activity (NRA) in Eichhorniacrassipes showed that adding 5% isopropanol gave highest NRAin all parts of the plant, but it was also necessary to flushthe assay media containing leaves with N₁ to obtain the maximalactivity. There were differences in the pH optima for NRA, theroot optimum being 6·5 leaf 7·5–8·0,and the petiole showing a major peak at 6·5 and a minorpeak at 7·.5–8·0. Possible significancesof these differences are discussed. Nitrate reductase activity, Eichhornia crassipes     

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。     

Uptake of 13C and 15N (ammonium, nitrate and urea) by Microcystis in Lake Kasumigaura

The uptake rate of carbon and nitrogen (ammonium, nitrate andurea) by the Microcystis predominating among phytoplankton wasinvestigated in the summer of 1984 in Takahamaira Bay of LakeKasumigaura. The V_max values of Microcystis for nitrate (0.025–0.046h^–1) and ammonium (0.15–0.17 h^–1) were considerablyhigher than other natural phytoplankton. The ammonium, nitrateand urea uptake by Microcystis was light dependent and was notinhibited with nigh light intensity. The K₁ values were farlower than the I_k values. The carbon uptake was not influencedby nitrogen enrichment. Microcystis accelerated the uptake rateby changing V_max/K _s value when nitrogen versus carbon contentin cells declined. Nitrate was scarcely existent in TakahamairiBay during the summer, when Microcystis usually used ammoniumas the nitrogen source. However, the standing stock of ammoniumin the water was far lower than the daily ammonium uptake rates.Therefore, the ammonium in this water had to be supplied becauseof its rapid turn-over time (–0.7–2.6 h).     

Nitrate reductase activity in vegetation below an arctic bird cliff,Svalbard, Norway

Abstract. Vegetated sites below bird-nesting cliffs are uniquely nutrient-rich habitats in the otherwise nutrient-poor arctic environment. Plants from six distinct vegetation zones below such a cliff at 79° N, Svalbard, Norway, were collected for analysis under greenhouse conditions. Leaf nitrate reductase activity (NRA) was analysed in 42 species representing 25 % of the Svalbard vascular flora. The species mean NRA values ranged from 0.37 to 8.34 μmols of nitrite ions formed per gram of plant fresh weight per hour. Species in the vegetated zone growing closest to recent guano deposits had the highest NRA values, (mean = 4.47) whereas plants growing farther below the cliff had significantly lower values (mean = 0.55). A similar pattern was detected in a duplicate set of plants induced with 15 mM KNO₃; vegetation zone means for NRA ranged from 5.08 to 0.98 μmols of nitrite ions formed per gram of plant fresh weight per hour. Maximally induced species NRA values were highest in the first zones below the cliff and decreased downslope. This gradient paralleled the steep soil nitrate gradient, which decreased from 13.84 mg/l at the cliffbase to 1.03 mg/l downslope. Correspondingly, soil ammonium ions in the vegetation zones ranged between 1.96 mg/l at the cliff-base to 0.03 mg/l downslope. Correlations between NRA and soil nitrate provide a systematic basis for assigning scalar ‘nitrogen figures’ as indicators of habitat preference, here for the first time applied to arctic species.     

小麦叶内硝酸还原的代谢库

随营养液中No_3~-浓度升高,叶片内No_3~-总量、代谢库大小(NIPS)及硝酸还原酶(NR)活性均升高,其中MPS与NK活性呈同步变化;No_3~-浓度达2.0mmol/L时,两者趋于稳值;若再增加NO_3~-浓度,则被吸收的NO_3~-积累于液泡中,而代谢库中NO_3~-含量(MPS)与NO_3~-总量之比有一定程度降低。低氮(NO_3~-浓度为1.0 mmol/L)情况下,反应液中无NO_3~-时,叶片内NR活性品种间有差异,但在50 mmol/L NO_3~-反应液中则品种间无差异;NK活性高的品种鲁麦8号及品种321叶内有大的NO_3~-代谢库,反应液中NO_3~-对NR活性刺激程度低,代谢库NO_3~-含量与叶NO_3~-总量之比高,而叶组织长时间反应过程中其NR活性衰减速率低。     

Nitrate assimilation in coexisting vascular plants in mire and swamp forest habitats in Central Sweden

Summary In order to monitor the nitrate assimilation capability of mire plants, in vivo current and maximally induced nitrate reductase activity (NRA) were investigated in 14 species of vascular plants from four different sites in a central Swedish mire. One of the sites was a swamp forest. The plants studied included species with both wide and restricted ecological ranges, and the mire sites selected covered a wide range of plant productivity. At the most productive site, current NRA differed among coexisting species. This differentiation in the use of nitrate as a source of nitrogen suggested the possibility of resource partitioning with regard to nitrogen acquisition. Maximally induced NRA, measured 3 days after an addition of nitrate, was highest at the most productive sites and differed among coexisting species. Plant species characteristic of rich fens had the highest maximally induced NRA. In all species, there was a positive correlation between the ability to assimilate peaks of available nitrate and total leaf nitrogen concentration.     

Distribution of nitrate reductase activity in nodulated lucerne plants

Nitrogen fixing plants of lucerne (Medicago sativa L. cv. Aragón) were grown in a glasshouse for three months in the absence of nitrate, and then supplied with 5 mM KNO₃ for a week. In control (non-nitrate fed) plants, nitrate reductase activity (NRA EC 1.6.6.1) was detected only in nodules. After nitrate supply, root NRA showed a transient increase. Shoot NRA increased with time, paralleling changes in nitrate distribution; stem NRA represented nearly 50% of total NRA in plant tissues. Total nitrogen, expressed on a dry weight basis, tended to decrease in shoots upon nitrate supply. Bacteroid NRA (EC 1.7.99.4) showed a great variation depending on Rhizobium meliloti strains, ranging from 5 to 40% of total plant NRA. However, different Rhizobium strains did not give different results in terms of plant growth parameters, nitrate or organic nitrogen content.     

A study on nitrate reductase activity (NRA) of geophytes from Mediterranean environment

Nitrate reductase activity (NRA) in different compartments of 14 Mediterranean geophytes (bulbous, tuberous and rhizomatous) and actual mineral nitrogen (NO₃⁻ and NH₄⁺) in their soils were investigated. The nitrate reduction capacities of each species were determined as NRA per total plant material. Differences among compartments for NRA were significant in all species. The highest NRA was found in leaves of tuberous species (Anemone coronaria, Cyclamen coum) and of most bulbous species (Allium flavum, Allium guttatum, Bellevelia sarmatica, Galanthus plicatus, Leucojum aestivum, Ornithogalum nutans, Tulipa sylvestris). Therefore, in this group of species the contribution of the leaves to total plant NRA was the highest. The other bulbous species (Allium scorodoprasum, Crocus chrysanthus, Fritillaria bithynica, Muscari neglectum) and one rhizomatous taxon (Iris suaveolens) have a different NRA distribution within the plants. In these species the highest values of NRA were found in different organs. For example, in Allium scorodoprasum the highest NRA was in tunics, and in flowers in M. neglectum. Although leaves are the main compartments reducing nitrate in most of the studied geophytes, other compartments also contribute to total plant nitrate reduction.Our results show that the nitrate reduction capacity is different among geophyte species. Even if it roughly reflects the nitrogen supply in a habitat, differences in nitrate reduction capacities of different species collected from same sites indicate that the nitrate reducing capacity is species-specific.     

Influence du d?bit d'eau de mer dans un dispositif de culture ? fibres creuses dialysantes sur la croissance phytoplanctonique et 1'extraction des nitrates du milieu

It is demonstrated that the productive capacity of mass dialysisculture of phytoplankton is intimately related to the rate offlow of nutrient medium along the membrane. Comparison of culturesof Phaeodactylum tricornutum, subject to different medium flowrates (92 versus 250 ml.min^–1) during a 17-day growthperiod, reveals increased productivity (in terms of cell numbers,chlorophyll a, dry weight, cell nitrogen, cell carbon, ATP)at the higher flow rate. Differences are most pronounced inthe latter stages of growth (i.e., linear and stationary) attainingratios between 2 and 3 for cell nitrogen, cell carbon and ATP.Lower C/N ratios for the greater yielding culture translatesas enhanced osmotic diffusion as well as cellular assimilationof nitrates. The positive effects of increasing medium flowrate on phytoplankton growth result from improved dialyzer performancewith respect to low molecular weight solutes. This is evidencedin the proportionately greater values obtained for mass transferof nitrate-nitrogen. Accordingly, measures of nitrate clearanceindicate a progression from 29 to 85 ml.min^–1 in responseto augmenting sea water flow from 32 to 292 ml.min ^–1.The culture apparatus recently developed consists of a separatedialysis unit (a hollow-fiber cartridge used in hemodialysis)coupled to a temperature-controlled growth chamber. Culturesreceive a continuous flow of natural sea water and are grownin the batch dialysis mode. The main advantages of this methodrelate to its potential for large-scale, axenic algal culturebased on the abundant source of cheap nutrients that constitutesnatural sea water.     

Effect of Plant Growth Regulators on Nitrate Utilization by Roots of Nitrogen-Depleted Dwarf Bean

We examined the effect of pretreatments (18 h at 5 µmoldm^–3) with abscisic acid, the ethylene-releasing substance‘Ethephon’, gibberellic acid, indoleacetic acid,kinetin and zeatin on nitrate uptake and in vivo nitrate reductaseactivity (NRA) in roots of nitrogen-depleted Phaseolus vulgarisL. Nitrate uptake showed an apparent induction pattern witha steady state after about 6 h, in all treatments. The nitrateuptake rate after 6 h was unaffected or at most 30% lower aftertreatments with the plant growth regulators. Gibberellic acid, kinetin and zeatin induced substantial NRAin roots in the absence of nitrate, whereas Ethephon enhancedNRA only during nitrate nutrition. Kinetin-induced NRA (Ki-NRA)was maximal after a pretreatment at 1 µmol dm^–3,and showed a lag phase of 6–8 h. Ki-NRA was additive tonitrate-induced NRA (NO^–₃-NRA) for at least 24 h, independentof the induction sequence. After full induction, Ki-NRA approximated20% of NO-₃-NRA. Abscisic acid counteracted the developmentof Ki-NRA, but not of NO^–₃-NRA. Cycloheximide and tungstatewere equally effective to suppress the development of nitratereductase activity after supply of kinetin or NO^–₃. Our data are consistent with the operation of two independentenzyme fractions (Ki-NRA and NO^–₃-NRA) with apparentlyidentical properties but with separate control mechanisms. Theabsence of major effects of plant growth regulators on the time-courseand rate of nitrate uptake suggests that exogenous regulators,and possibly endogenous phytohormones are of minor importancefor initial nitrate uptake. The differential effect of someregulators on nitrate uptake and root NRA furthermore indicatesthat the processes of uptake and reduction of NO^–₃ arenot obligatory or exclusively coupled to each other.     

Nitrate concentration and nitrate reductase activity in the leaves of three legumes and three cereals*

Nitrate concentration and nitrate reductase activity (NRA) were studied in the leaves of soybean (Glycine max), groundnut (Arachis hypogaea and cowpea (Vigna unguiculata) and sorghum (Sorghum bicolor), pearl millet (Pennisetum americanum) and maize (Zea mays) at three nitrogen fertiliser levels in two field experiments. Higher nitrate concentrations were detected in the leaves of groundnut, cowpea and pearl millet than in sorghum and maize. Nitrate content in the leaves and leaf NRA were not related across crop species, nor was a generalised pattern of leaf NRA and leaf nitrate observed within legumes or within cereals. Nitrogen application resulted in higher nitrate availability in the leaves, with varied leaf NRA.