Nitrate Reductase Activity in Maize (Zea mays L.) Leaves: II. Regulation by Nitrate Flux at Low Leaf Water Potential

Experiments were conducted to determine whether the nitrate flux to the leaves or the nitrate content of the leaves regulated the nitrate reductase activity (NRA) in leaves of intact maize (Zea mays L.) seedlings having low water potentials (ψ_w) when other environmental and endogenous factors were constant. In seedlings that were desiccated slowly, the nitrate flux, leaf nitrate content, and NRA decreased as ψ_w decreased. The decrease in nitrate flux was caused by a decrease in both the rate of transpiration and the rate of nitrate delivery to the transpiration stream. Upon rewatering, the recovery in NRA was correlated with the nitrate flux but not the leaf nitrate content.     

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     

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.     

Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress

The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO₃⁻. Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO₃⁻. During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO₃⁻, while with rewatering, leaf NRA recovery was quite important especially in the NO₃⁻-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO₃⁻ and in those without NO₃⁻ contrary to the behavior of the leaves. Beyond −15.10⁵ pascal, nodular NRA began to decrease in plants watered with NO₃⁻. This phenomenon was not observed in nodules of plants given water only.     

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。     

Time-course of Nitrate Uptake and Nitrate Reductase Activity in Nitrogen-depleted Dwarf Bean

The rate of nitrate uptake by N-depleted French dwarf bean (Phaseolus vulgaris L. cv. Witte Krombek) increased steadily during the first 6 h after addition of NO₃ -After this initial phase the rale remained constant for many hours. Detached root systems showed the same time-course of uptake as roots of intact plants. In vivo nitrate reductase activity (NRA) was assayed with or without exogenous NO₃^- in the incubation medium and the result ing activities were denoted potential and actual level, respectively. In roots the difference between actual and potential NRA disappeared within 15 min after addition of nitrate, and NRA increased for about 15 h. Both potential and actual NRA were initially very low. In leaves, however, potential NRA was initially very high and was not affected by ambient nitrate (0.1–5 mol m^-3) for about 10 h. Actual and potential leaf NRA became equal after the same period of time. In the course of nitrate nutrition, the two nitrate reductase activities in leaves were differentially inhibited by cycloheximide (3.6 mmol m^-3) and tungstate (1 mol m^-3). We suggest that initial potential NRA reflects the activity of pre-existing enzyme, whereas actual NRA depends on enzyme assembly during NO₃^- supply. Apparent induction of nitrate uptake and most (85%) of the actual in vivo NRA occurred in the root system during the first 6 h of nitrate utilization by dwarf bean.     

Effect of water deficit and sulphur dioxide on total soluble proteins,nitrate reductase activity and free proline content in sunflower leaves

Sunflower (Helianthus annus L. cv. PSH-7) plants were subjected to different osmotic potentials, using polyethylene glycol-6000 (PEG-6000), after, prior to and during SO₂ fumigation. Total soluble proteins and nitrate reductase activity (NRA) decreased, and free proline content increased with the increasing water stress. These biochemical parameters were more adversely affected in fumigated plants than in non-fumigated ones, when mild water stress was provided prior to and during fumigation. When severe water stress was given prior to and during fumigation, total soluble proteins, NRA and free proline content were nearly the same in fumigated and non-fumigated water-stressed plants; it is because the stomatal closure was observed in water-stressed plants. The leaf water potential decreased with the increasing water stress; however, it was not significantly affected due to SO₂ fumigation.     

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 the forage legume Lotus japonicus L.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.     

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.     

Nitrate reduction and nitrate content in ash trees (Fraxinus excelsior L.): distribution between compartments,site comparison and seasonal variation

Summary Nitrate reductase activity (NRA), nitrate content and biomass components of leaflets, leaf stalks, old stem, current-year stem and roots of ash trees (Fraxinus excelsior L.) growing in their natural habitats were investigated. In addition, NRA, total nitrogen and nitrate concentration were analyzed in the leaves and roots of ash trees from four different field sites. The highest NRA per gram biomass and also per total compartment biomass was found in the leaflets, even though root biomass was much higher than total leaflet biomass. The highest nitrate concentrations were found in the leaf stalks. Correlations between nitrate availability in the soil and NRA in leaves were not significant due to high variability of the actual soil nitrate concentrations. The seasonal variation in foliar NRA, nitrate concentration and total nitrogen concentration is much smaller in F. excelsior than reported for herbaceous species and is mainly caused by changes in the actual soil nitrate availability and by senescence of the leaves.     

Nitrate reductase activity in leaves and roots ofAlnus glutinosa (L) Gaertner

Summary Thein vivo nitrate reductase activity (NRA) was determined inAlnus glutinosa plants grown nonsymbiotically on ammonium, nitrate, a combination of both, or symbiotically with atmospheric nitrogen as the only nitrogen source. Root NRA was absent when ammonium or atmospheric nitrogen was the nitrogen source. With nitrate in the culture solution the roots showed a high NRA. However, the leaf NRA behaved quite differently: with negligible activities on all nitrogen sources except atmospheric nitrogen. The foliar NRA measured, however, is likely not due to the activity of the plant but of microbial origin. Methods commonly used to facilitate produced nitrite to leak out of the tissue, such as addition of propanol and cutting the plant material, did not increase the nitrite release from the leaves. A turbidity developed when testing the samples for nitrite which was positively correlated with the NRA. Populations of microorganisms in the phyllosphere did not differ between the nutritional treatments. Bacteria, able to grow on a low-nitrogen medium, were present on the leaves. Nitrifiers could not be detected. The bacteria on the leaves appear to produce nitrite when incubated with leaf material. Grassland Species Research Group, Publication no. 106     

Effect of Shoot Removal and Malate on the Activity of Nitrate Reductase Assayed in Vivo in Barley Roots (Hordeum vulgare cv. Midas)

There is a diurnal variation of nitrate reductase activity (NRA) measured in vivo in barley roots (Hordeum vulgare cv. Midas). In intact plants receiving a 16-hour photoperiod, NRA increases when the light is switched on, reaches a maximum value after 7 to 8 hours, and thereafter declines. Shoot removal (detopping) at the start of the photoperiod prevents the rise in NRA; detopping after 5 hours light leads to a rapid fall in NRA. The inclusion of 10 millimolar malate in the external medium causes a rise in NRA in plants detopped at the beginning of the photoperiod and thus seems to substitute partially for the illuminated shoot. Oxalate, fumarate, and tartrate did not have this effect. Preincubation of the roots of intact plants with 10 millimolar malate for 3 hours, prior to detopping, causes an increase in the flux of amino acids into the xylem sap of detopped roots.     

Nitrate nutrition ofDeschampsia flexuosa (L.) Trin. in relation to nitrogen deposition in Sweden

Summary Current and maximally induced nitrate reductase activity (NRA), total-N, nitrate, K, P, Ca, Mg, Mo and sucrose in leaves ofDeschampsia flexuosa was measured three times during the vegetation period in forests along a deposition gradient (150 km) in south Sweden, in north Sweden where the nitrogen deposition is considerably lower, and at heavily N-fertilized plots. In addition, the interaction between nitrogen nutrition and light was studied along transects from clearings into forest in both south and north Sweden. Plants from sites with high nitrogen deposition had elevated current NRA compared to plants from less polluted sites, indicating high levels of available soil nitrate at the former. Current NRA and total N concentration in grass from sites with high deposition resembled those found at heavily N-fertilized plots. Under such circumstances, the ratio current NRA: maximally induced NRA as well as the concentration of nitrate was high, while the concentration of sucrose was low. This suggests that the grass at these sites was already utilizing a large portion of its capacity to assimilate nitrate. Light was found to play an important role in the assimilation of nitrate; leaf concentration of sucrose was found to be negatively correlated with both nitrate and total N. Consequently, grass growing under dense canopies in south Sweden is not able to dilute N by increasing growth. The diminished capacity of the grass to assimilate nitrate will increase leaching losses of N from forests approaching N saturation.     

施氮和去除子叶对大叶桃花心木幼苗叶片硝酸还原酶活性的影响

研究了热带落叶乔木大叶桃花心木(Swietenia macrophylla)在施氮和去除子叶后幼苗叶片的硝酸还原酶活性(NRA)变化。结果表明,在非施氮(对照)条件下,NRA随着幼苗叶片的发育先升高后降低;施氮后幼苗叶片NRA在各取样时期(除35 d外)均显著高于非施氮处理(P<0.05),并随着取样时期的延续,叶片NRA逐渐降低。在幼苗发育的不同时期去除子叶,4周后,叶片NRA均显著升高(P<0.05)。     

Development of Nitrate Reductase Activity in Expanding Leaves of Nicotiana tabacum in Relation to the Concentration of Nitrate and Potassium

Up to 80% of the total nitrate reductase activity (NRA) determined in vivo in different parts of vegetative tobacco plant (Nicotiana tabacum) was located in the leaves. The NRA reached a peak when a leaf had expanded to 27% of its final weight and 33% of its final area. Thereafter, with advancing expansion and age of the leaf, the activity declined. This pattern of development of NRA during the ontogenesis of leaves was not influenced by raising the supply of NO₃⁻ from 3 to 6 milliequivalent per cubic decimeter in the substrate solution. The concentration of NO₃⁻ in leaves, stem and root was inversely related to NRA at both NO₃⁻ levels. Raising the supply of K⁺ from 1 to 6 milliequivalent per cubic decimeter at either concentration of NO₃⁻ slowed down the development of NRA in the initial stages of expansion, but promoted it subsequently. The peak of the activity which developed in a leaf of 62% of its final area was higher at the higher supply of K⁺. The higher activity was maintained thereafter in the expanding and in matured and older leaves. It was concluded that NRA and the pattern of its development in expanding leaves is related to the availability of metabolites and their incorporation into enzyme proteins. Both these processes are influenced by: (a) the vertical profile of concentration of K⁺ in the shoot and (b) the concentration of K⁺ in a leaf, which depend upon its supply.     

Rapid Modulation of Spinach Leaf Nitrate Reductase by Photosynthesis : II. In Vitro Modulation by ATP and AMP

Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO₂ or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg²⁺ or Ca²⁺, kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg²⁺ and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO₂ compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (β, γ-imido ATP, β, γ-methyl-ATP) had no effect on NR, whereas γ-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.     

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.     

Proline accumulation and nitrate reductase activity in contrasting sorghum lines during mid-season drought stress

Six lines of sorghum ( Sorghum bicolor L. Moench) with differing drought resistance (IS 22380, ICSV 213, IS 13441 and SPH 263, resistant and IS 12739 and IS 12744, susceptible) were grown under field conditions in the semi-arid tropics and analysed for proline and nitrate reductase activity (NRA; EC 1.6.6.1) during a mid-season drought. The resistant lines accumulated high levels of proline, while the susceptible lines showed no significant proline accumulation. Most of the proline was accumulated after growth of the plants had ceased. In a separate greenhouse experiment, most of the proline was found in the green rather than the fired portions of leaves. The levels returned to that of irrigated controls within 5 days of rewatering. Proline levels increased as leaf water potential and relative water content fell, and there was no apparent difference among the different sorghum lines with change in plant water status. Susceptible lines accumulated less proline than resistant lines as leaf death occurred at higher water potentials. Proline accumulation may, however, contribute to the immediate recovery of plants from drought. Leaf NRA reached high levels at about 35 days after sowing in both the stressed and irrigated plants, after which it declined. The decline in NRA was more pronounced in the stressed than in the irrigated plants and closely followed changes in the growth rate. Upon rewatering, NRA increased several-fold in all the lines and, in contrast to proline accumulation, genotypic differences in NRA were small, both during stress and upon rewatering. The high sensitivity of NRA to mild drought stress was reflected in the rapid decline of activity with small changes in leaf water potential and relative water content. The results are discussed in the light of a possible role for proline during recovery from drought, and the maintenance of NRA during stress and its recovery upon rewatering.