Interrelationships of Nitrate Uptake, Nitrate Reductase, and Nitrogen Use Efficiency in Selected Kentucky Bluegrass Cultivars

The effect of nitrogen form (NH(4)-N, NH(4)-N + NO(3) (-), NO(3) (-)) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO(3) (-) increases, and NH(4) (+) and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO(3) (-), NH(4)-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH(-) and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH(-) or by the synthesized organic anions rather than by nitrate itself. Addition of HCO(3) (-) to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO(3) (-), like OH(-), increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO(3) (-), NH(4)-N, and amide-N.

Nitrate Uptake and Reduction by Durum Wheat (Triticum turgidum) and Tritordeum (Hordeum chilense x Triticum turgidum)

Half or whole root systems of micropropagated `Gala' apple ( Malus ×domestica Borkh.) plants were subjected to drought stress by regulating the osmotic potential of the nutrient solution using polyethylene glycol (20% w/v) to investigate the effect of root drying on NO 3 - content and metabolism in roots and leaves and on leaf photosynthesis. No significant difference in predawn leaf water potential was found between half root stress (HRS) and control (CK), while predawn leaf water potential from both was significantly higher than for the whole root stress (WRS) treatment. However, diurnal leaf water potential of HRS was lower than CK and higher than WRS during most of the daytime. Neither HRS nor WRS influenced foliar NO 3 - concentration, but both significantly reduced NO 3 - concentration in drought-stressed roots as early as 4 hours after stress treatment started. This reduced NO 3 - concentration was maintained in HRS and WRS roots to the end of the experiment. However, there were no significant differences in NO 3 - concerntation between CK roots and unstressed roots of HRS. Similar to the effect on root NO 3 - concentration, both HRS and WRS reduced nitrate reductase activity in drought-stressed roots. Moreover, leaf net photosynthesis, stomatal conductance and transpiration rate of HRS plants were reduced significantly throughout the experiment when compared with CK plants, but the values were higher than those of WRS plants in the first 7 days of stress treatment though not at later times. Net photosynthesis, stomatal conductance and transpiration rate were correlated to root NO 3 - concentration. This correlation may simply reflect the fact that water stress affected both NO 3 - concentration in roots and leaf gas exchange in the same direction.

Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum).

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO3 −–N, NH4 +–N, NH4NO3). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO3 −–N) or mixed (NH4NO3) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO3 −–N or NH4NO3. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO3 −-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH4 +–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH4 +–N the enzyme activity in roots was even higher than in those supplied with NO3 −–N.

Effects of Irradiation with Light of Different Wavelengths on Nitrate Reductase Activity in Roots of Zea mays L.

We examined the effect of pretreatments (18 h at 5 //mol dm-3) with abscisic acid, the ethylene-releasing substance 'Ethephon', gibberellic acid, indoleacetic acid, kinetin and zeatin on nitrate uptake and in vivo nitrate reductase activity (NRA) in roots of nitrogen-depleted Phaseolus vulgaris L. Nitrate uptake showed an apparent induction pattern with a steady state after about 6 h, in all treatments. The nitrate uptake rate after 6 h was unaffected or at most 30% lower after treatments with the plant growth regulators. Gibberellic acid, kinetin and zeatin induced substantial NRA in roots in the absence of nitrate, whereas Ethephon enhanced NRA only during nitrate nutrition. Kinetin-induced NRA (Ki-NRA) was maximal after a pretreatment at 1 //mol dm~3, and showed a lag phase of 6-8 h. Ki-NRA was additive to nitrate-induced NRA (NOj-NRA) for at least 24 h, independent of the induction sequence. After full induction, Ki-NRA approximated 20% of NO7-NRA. Abscisic acid counteracted the development of Ki-NRA, but not of NOj-NRA. Cycloheximide and tungstate were equally effective to suppress the development of nitrate reductase activity after supply of kinetin or NO3. Our data are consistent with the operation of two independent enzyme fractions (Ki-NRA and NO7-NRA) with apparently identical properties but with separate control mechanisms. The absence of major effects of plant growth regulators on the time-course and rate of nitrate uptake suggests that exogenous regulators, and possibly endogenous phytohormones are of minor importance for initial nitrate uptake. The differential effect of some regulators on nitrate uptake and root NRA furthermore indicates that the processes of uptake and reduction of NOj are not obligatory or exclusively coupled to each other.

Nitrate reductase activity in roots and shoots of aquatic macrophytes

According to the ability to assimilate nitrate all plants are usually divided into three groups, including those with high nitrate reductase activity in plant root, low nitrate reductase activity in plant root and approximately equal nitrate reductase activity in root and leaves.The authors studied the assimilation of nitrates by different organs in plants of Brassicaceae ( Raphanus sativus convar. oleifera, Raphanus sativus loba, Raphanus sativus, garden radish of the Zhara, Saksa, Ledyanaya sosul’ka varieties), Poaceae (spring wheat, winter wheat, barley, maize) and Fabaceae ( Pisum sativum, Soja hispida, Lupinus perennis, garbanzo) in controlled conditions of development. The nitrate reductase activity, the content of nitrates in organs, and also the mass of dump matter were determined in 15-days plants. The most activity of nitrate reductase in all organs was revealed in Raphanus sativus convar. oleifera. This activity was highest in leaves almost in all studied species, which carry out the basic role in process of reduction of nitrates. So grouping the plants on their ability to reduce nitrates did not have clear boundaries. The species differ in this index even within the range of one family.

Irradiated seeds of Phaseolus vulgaris cv. Rajmah using Synchrotron X Ray Beam (BL-07) in RRCAT, Indore at doses of 1, 10 and 20 Gy were used to raise the seedling and the effects on growth and biochemical constituents in 4 - 8 days, old seedlings were analyzed. The seed irradiation effect on seedling development up to about 4 - 5 days, % germination, seedling length and seedling vigor are significantly decreased at 10 and 20 Gy doses with strong -ve correlation. Other parameters, like relative water content, electrical conductivity and acid phosphatase activity are also decreased. Decrease in various biochemical constituents, like, protein and proline has shown significant reduction at 10 and 20 Gy and phenol at 1 - 20 Gy. However, peroxidase activity is increased at 1 and 10 Gy. Amongst the antioxidant enzymes, only superoxide dismutase activity has shown significant increase at 10 and 20 Gy. For seed irradiation (1 and 10 Gy) effect on seedling development up to 8 days involving transfer to hydroponic culture after 4 days, in shoot tissue, decrease in nitrate reductase activity and pigment content is observed, while nitrate reductase activity in root tissue is increased. The results demonstrate adverse effects on growth as well as biochemical constituents along with increased antioxidant effect in bean seedlings with irradiation of seeds at high dose of synchrotron X radiations. Also the nitrate assimilation and photosynthetic activity are reduced in shoot tissue with seed irradiation, however, increased nitrate reductase activity in roots suggests the involvement of NO signaling.

Nitrate reductase activity (NRA) in turfgrass roots should play an important role in the N metabolism of the whole plant because a major part of the shoots is removed by mowing. However, preliminary experiments demonstrated a lack of in vivo NRA in roots of Kentucky bluegrass grown in Hoagland's nutrient solutions, which were constantly aerated. We hypothesized that O2 inhibited NRA by oxidizing carbohydrate available for NO3– reduction in roots. To test this hypothesis, Kentucky bluegrass (Poa pratensis L.), cultivar Merit, was grown in nutrient solutions containing 0.1 mM NO3–. Grass cultures were treated with +O2 (solution aerated), –O2 (not aerated), +Suc (sucrose added to solution, final concentration = 25 mM) or –Suc (not added) for 12 days. Plants were harvested at 5:00 pm, separated into shoots and roots, and analyzed for in vivo NRA. The results partly supported the above hypothesis because the –Suc–O2 roots showed significantly higher NRA than –Suc+O2 roots. However, +Suc roots did not exhibit greater NRA than –Suc roots, possibly because of a decreased pH in the solutions. Oxygen increased root growth and hence the growth of the whole plant, while sucrose decreased leaf N content and leaf NRA but did not improve growth.

The effect of nitrate availability on characteristics of the nitrate assimilatory system was investigated in N‐limited barley (Hordeum valgare L. cv. Golf), grown with the seminal root system split into initially equal‐sized halves. The cultures were continuously supplied with nitrate‐N at a relative addition rate (RA) of 0.09 day−1, which resulted in relative growth rates (RG) that were ca 85% of those observed under surplus nitrate nutrition. The total N addition was divided between the subroots in ratios of 100:0, 80:20, 70:30, 60:40, and 50:50. For comparison, standard cultures were grown at RAs ranging from 0.03 to 0.18 day−1. Initially, biomass and N partitioning to the subroots responded strongly and proportionally to the nitrate distribution ratio. After 12‐14 days no further effect was observed. The Vmax for net nitrate uptake and in vitro nitrate reductase (NR) activity were measured in acclimated plants, i.e., after > 14 days under a certain nitrate regime. In subroots fed from 20 to 100% of the total N addition, Vmax for net nitrate uptake increased slightly, whereas NR activity was unaffected. Uptake and NR activities were insignificant in the 0%‐subroot. Uneven nitrate supply to individual subroots had negligible effect on the whole‐plant ability for nitrate uptake, and the relative Vmax (unit N taken up per unit N in whole plant tissue and time) remained about 7‐fold in excess of the demand set by growth. Balancing nitrate concentrations (the resulting external nitrate concentrations at a certain RA) generally ranged between 2 and 10 μM at growth‐limiting RA, both when predicted from uptake kinetics and when actually measured. When comparing split root and standard cultures when acclimated, it appears that uptake and NR activities in roots respond more strongly to over‐all nitrate availability than to nitrate availability to individual subroots.

Bean (Phaseolus vulgaris L.) plants were cultured for 19 d on complete or on phosphate deficient culture media. Low inorganic phosphate concentration in the roots decreased ATP level and nitrate uptake rate. The mechanisms which may control nitrate uptake rate during phosphate deficiency were examined. Plasma membrane enriched fractions from phosphate sufficient and phosphate deficient plants were isolated and compared. The decrease in total phospholipid content was observed in plasma membranes from phosphate deficient roots, but phospholipid composition was similar. No changes in ATPase and proton pumping activities measured in isolated plasma membrane of phosphate sufficient and phosphate deficient bean roots were noted. The electron microscope observations carried out on cortical meristematic cells of the roots showed that active ATPases were found in plasma membrane of both phosphate sufficient and phosphate deficient plants. The decrease in inorganic phosphate concentration in roots led to increased nitrate accumulation in roots, accompanied by a corresponding alterations in NO3 distribution between shoots and roots. Nitrate reductase activity in roots of phosphate deficient plants estimated in vivo and in vitro was reduced to 50–60% of the control. The increased NO3 concentration in root tissue may be explained by decreased NR activity and lower transport of nitrate from roots to shoots. Therefore, the reduction of nitrate uptake during phosphate starvation is mainly a consequence of nitrate accumulation in the roots.

The effectiveness of reforestation programs on degraded soils in the Mediterranean region is frequently limited by a low soil availability and a poor plant uptake and assimilation of nutrients. While organic amendments can improve the nutrient supply, inoculation with mycorrhizal fungi can enhance plant nutrient uptake. A pot experiment was conducted in 2004 to study the influence of inoculation with an arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck & Smith) or with a mixture of three AM fungi (G. intraradices, G. deserticola Trappe, Bloss. & Menge, and G. mosseae (Nicol & Gerd.) Gerd. & Trappe) and of an addition of composted sewage sludge or Aspergillus niger–treated dry‐olive‐cake residue on plant growth, nutrient uptake, mycorrhizal colonization, and nitrate reductase (NR) activity in shoot and roots of Juniperus oxycedrus L. Six months after planting, the inoculation of the seedlings with G. intraradices or a mixture of three AM fungi was the most effective treatment for stimulating growth of J. oxycedrus. There were no differences between the two mycorrhizal treatments. All treatments increased plant growth and foliar N and P contents compared to the control plants. Mycorrhizal inoculation and organic amendments, particularly fermented dry olive cake, increased significantly the NR activity in roots.

The effect of exogenous NH4+ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8‐day‐old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO3+ in the presence of 0, 1 or 10 mM NH4+, Exogenous NH4+ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO3+, In contrast, the induction of NiRA was inhibited by NH4+ at all NO3+ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH4+ Pre‐treatment with NH4+ had no effect on the subsequent induction of NRA in the absence of additional NH4+ whereas the induction of NiRA in NH4+‐pretreated roots was inhibited in the absence of NH4+ At 10 mM NO3+ L‐methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH4+ was present. In contrast, the induction of NiRA was inhibited by L‐methionine sulfoximine irrespective of NH4+ supply. During the postinduction phase, exogenous NH4+ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH3+ whereas, NiRA was unaffected by NH4+ at either substrate concentration. The results indicate that exogenous NH4+ regulates the induction of NRA in roots by limiting the availability of NO3+. Conversely, it has a direct effect, independent of the availability of NO3+, on the induction of NiRA. The lack of an NH4+ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.