Nitrate reductase activity of radish cotyledons as affected by phytohormones and different nitrogen sources

Radish (Raphanus sativus L.) seedlings pretreated with different hormones viz. kinetin, gibberellic acid and abscisic acid were subjected to different N-forms. The seedlings were treated with different concentrations of KNO₃, NH₄Cl and NH₄NO₃ and the changes in nitrate reductase activity were seen in light and dark conditions in the cotyledons. Nitrate reductase activity was affected differently by hormone application. Nitrate increased and ammonia decreased nitrate reductase activity; in both light and dark-grown seedlings KNO₃ induced more in vitro nitrate reductase activity. NH ₄ ⁺ when combined with NO ₃ ⁻ , however, could level up to some extent, with KNO₃ in light, except in kinetin. A transient response of induction of NR activity was evident with decreased levels after a certain specific ambient N-concentration, despite the presence of high N in the medium. However, the pattern of transition varied with the hormones applied. Further, hormones are found to affect induction of different isoforms of nitrate reductase by NH ₄ ⁺ and NO ₃ ⁻ . NH ₄ ⁺ induced isoform was prominently promoted by kinetin treatment in dark. The data documents a particular kind of interaction between controlling factors (light, N-source and phytohormones) which affect nitrate reductase levels.     

The occurrence of nitrate reductase in apple leaves

Nitrate reductase utilizing NADH or reduced flavin mononucleotide (FMNH₂) as electron donor was extracted from the leaves, stems and petioles, and roots of apple seedlings. Successful extraction was made possible by the use of insoluble polyvinylpyrrolidone (Polyclar AT) which forms insoluble complexes with polyphenols and tannins. The level of nitrate reductase per gram fresh weight was highest in the leaf tissue although the nitrate content of the roots was much higher than that of the leaves. Nitrite reductase activity was detected only in leaf extracts and was 4 times higher than nitrate reductase activity. Nitrate was found in all parts of young apple trees and trace amounts were also detected in mature leaves from mature trees. Nitrate reductase was induced in young leaves of apple seedlings and in mature leaves from 3 fruit-bearing varieties. An inhibitor of polyphenoloxidase, 2-mercaptobenzothiazole was used in both the inducing medium and the extracting medium in concentrations from 10⁻³ to 10⁻⁵m with no effect upon nitrate reductase activity.     

Comparison of diurnal changes in nitrate and potassium contents in lucerne shoots

During 195-min light exposure following 5 d in dark, nitrate content was studied in different organs of lucerne plants in early bud stage. Nitrate content varied considerably especially in stems. Rapid diurnal variations in nitrate content were found in lower and upper halves of stems, in petioles and in leaf blades. The results reflected discontinuous nitrate movement in lucerne shoots. The positive correlation between the diurnal course of the nitrate and potassium contents in different plant organs showed that the K⁺ transport followed the NO₃ ^? transport. Similar diurnal changes were found also in Na⁺ and Ca²⁺ contents. Discontinuous salt movements occurring in xylem sap flow were in contrary to continuous transpiration stream and could be a consequence of temporary adsorption or binding of salts in xylem vessels.     

Seed germination in Chenopodium album L.: further evidence for the dependence of the effects of growth regulators on nitrate availability

Abstract Chenopodium album L. plants, grown under controlled environmental conditions on different levels of soil nitrate, produced seeds with proportionately different NO^?₃ contents. Regardless of the endogenous NO^?₃ content, few seeds germinated in water or upon treatment with KNO₃. Ethylene promoted germination, and the extent of germination was positively correlated with the endogenous seed NO^?₃ content. Combined application of ethylene and KNO₃ in the dark had a synergistic effect on NO^?₃ -deficient seed. The synergism between ethylene and KNO₃ was attributable to the NO^?₃ moiety of the nitrate salt. Ethylene and light showed moderate synergism in seeds with low or high endogenous nitrate. Addition of nitrate, however, masked the interaction between ethylene and light. Gibberellic acid₄₊₇ (GA₄₊₇) or red light, each alone or combined with KNO₃, had little effect on germination. When applied together in the dark, ethylene and GA₄₊₇ synergistically enhanced the germination of NO^?₃-deficient seed. The combined effects of the two hormones on this seed were further enhanced by the addition of KNO₃. There was no synergism between ethylene and GA₄₊₇ in NO^?₃-rich seed. These interactions among GA₄₊₇, ethylene and KNO₃ were not affected by light. The results confirm and further elaborate our earlier finding that the sensitivity of C. album seeds to ethylene may depend on nitrate availability.     

Effects of NO3– and BAP on organogenesis in tissue-cultured shoot primordia induced from shoot apices of Utricularia praelonga St. Hil.

The effects of NO₃ ^– and BAP on organogenesis in shoot primordia of Utricularia praelonga subcultured in B5 liquid medium were studied. In B5 liquid basal medium supplemented with 24.73 mM KNO₃ and 2.0 mg/l BAP the subcultured shoot primordia continuously multiplied into numerous small, globular masses, while with dilution of the KNO₃ to 3 mM organogenesis was promoted. Pulse treatment of the shoot primordia with 3 mM KNO₃ in B5 liquid medium for 72 h and then transplantation to the B5 basal liquid-medium induced meristemoids in this tissue. When the shoot primordia regenerated meristemoids, they never reverted back into the proliferation cycle. The addition of BAP in the B5 liquid medium with 3 mM KNO₃ regulated the differentiation rate of the stems and leaves in the meristemoids induced in the masses of shoot primordia. The control produced 3 parts stems to 1 part leaves; medium with 0.02 mg/l BAP regenerated approximately 2 parts stems and 1 part leaves; that of 0.20 mg/l BAP 1 part stems and 2 parts leaves; and medium with 2.00 mg/l BAP regenerated leaves only. Received: 17 September 1997 / Revision received: 30 October 1997 / Accepted: 18 November 1997     

Induction of a high-capacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism

Roots of nitrate-starved and nitrate-pretreated seedlings of Hordeum vulgare were used to investigate the induction of a high-capacity uptake mechanism for nitrate. When exposed to 0.2 mmol·l^-1KNO₃, nitrate-starved roots took up nitrate at a rate of approx. 1 mol·(g FW)^-1·h^-1; K⁺ was absorbed at a rate ten-times higher. Nitrate uptake accelerated after a lag of about 1 h, until it matched the rate of K⁺ uptake about 4 h later. p-Fluorophenylalanine (FPA), which prevents the synthesis of functioning proteins, suppressed the development of the high-capacity mechanism. Pretreatment of the roots with 0.2 mmol·l^-1 Ca(NO₃)₂ for 24 h established the high-capacity mechanism. Pretreated roots were able to absorb nitrate at high rates immediately upon exposure to 0.2 mmol·l^-1KNO₃, in the absence or presence of FPA. The high-capacity mechanism, once established, appeared to have a protein turnover as slow as that of the low-capacity mechanism or that of the mechanism involved in the uptake of K⁺. In contrast, the mechanisms for the transport of nitrate and K⁺ into the xylem vessels were completely blocked by FPA within 1 h of application, confirming earlier evidence for a rapid turnover of the transport proteins in the xylem parenchyma.Nitrate reduction proceeded at rates which were roughly one-tenth as large as the rates of the respective nitrate-uptake processes, indicating that nitrate-reductase activity was determined by the rate of nitrate uptake and not vice versa.We conclude that the formation of a high-capacity nitrate-uptake mechanism in barley roots occurs in response to nitrate uptake through a constitutive mechanism of low capacity which appears to function as a sensing mechanism for nitrate in the environment of the roots.Abbreviation FPA p-fluorophenylalanine     

Role of potassium and malate in nitrate uptake and translocation by wheat seedlings

Wheat seedlings (Triticum vulgare) treated with 1 mm KNO₃ or NaNO₃, in the presence of 0.2 mm CaSO₄, were compared during a 48-hour period with respect to nitrate uptake, translocation, accumulation and reduction; cation uptake and accumulation; and malate accumulation. Seedlings treated with KNO₃ absorbed and accumulated more nitrate, had higher nitrate reductase levels in leaves but less in roots, accumulated 17 times more malate in leaves, and accumulated more of the accompanying cation than seedlings treated with NaNO₃. Within seedlings of each treatment, changes in nitrate reductase activity and malate accumulation were parallel in leaves and in roots. Despite the great difference in malate accumulation, leaves of the KNO₃-treated seedlings had only slightly greater levels of phosphoenolpyruvate carboxylase than leaves of NaNO₃-treated seedlings. NADP-malic enzyme levels increased only slightly in leaves and roots of both KNO₃- and NaNO₃-treated seedlings. The effects of K⁺ and Na⁺ on all of these parameters can best be explained by their effects on nitrate translocation, which in turn affects the other parameters. In a separate experiment, we confirmed that phosphoenolpyruvate carboxylase activity increased about 2-fold during 36 hours of KNO₃ treatment, and increased only slightly in the KCl control.     

Effect of form and level of applied nitrogen on nitrogenase and nitrate reductase activities in faba beans

The effects of nitrogen applied at increasing levels of 0, 4, 8, 16 and 32 mM N (KNO₃ or NH₄Cl) were studied in faba bean (Vicia faba) nodulated byRhizobium leguminosarum bv.viceae RCR lool. Nitrogenase activity was higher at 4 and 8 mM N than the zero N treatment (control), but 16 and 32 mM N significantly reduced the efficiency of nodule functions. Nitrate reductase activities (NRA) of leaves, stems, roots, nodules and nodule fractions (bacteroid and cytosol) were increased with rising the NO₃ ^? or NH₄ ⁺ levels. NRA decreased in the order of nodules>leaves>stems>roots. Cytosolic NR was markedly higher than that recorded in the bacteroid fractions. Nitrate levels were linearly correlated to NRA of nodules. Accumulation of NO₂ ^? within nodules suggests that NO₂ ^? inhibits nodule’s activity after feeding plants with NO₃ ^? or NH₄ ⁺.     

Nitrate content and nitrate reductase activity in Rumex obtusifolius L.

Summary With Rumex obtusifolius L., the influence of some environmental conditions on nitrate uptake and reduction were investigated. Nitrate concentrations of plant material were determined by HPLC, the activity of nitrate reductase by an in vivo test. As optimal incubation medium, a buffer containing 0.04 M KNO₃; 0.25 M KH²PO₄; 1.5% propanol (v/v); pH 8.0 was found. Vacuum infiltration caused an increase of enzyme activity of up to 40%.High nitrate concentrations were found in roots and leaf petioles. Nitrate reductase activity of these organs, however, was low. On the other hand, the highest nitrate reductase activity was observed in leaf laminae, which contained lowest nitrate concentrations.In leaves, nitrate content and nitrate reductase activity exhibited inverse diurnal fluctuations. During darkness, decreasing activities of the enzyme were followed by increasing nitrate concentrations, while during light the contrary was true. In petioles diurnal fluctuations in nitrate content were observed, too. No significant correlations with illumination, however, could be found.Our results prove that Rumex obtusifolius is characterized by an intensive nitrate turnover. Theoretically, internal nitrate content of the plant would be exhausted within a few hours, if a supply via the roots would be excluded.     

Influence of Ethephon on Nitrate Reductase, Protein Amino Nitrogen and Nitrate Content of Solanum tuberosum L.

Nitrate reductase activity was stimulated in roots and stems,but suppressed in leaves of potato plants grown in nutrientculture by 30 mg.liter^–1 ethephon applied to the culturesolution. In stems, nitrate reductase activity was stimulatedafter 5 h and by 24 h it was more than two fold that of thecontrol. The magnitude of stimulation by ethephon was less inroots compared to stems. Ethephon treatment enhanced ethyleneproduction by roots, stems and leaves but the level of productionwas not significantly different in these organs. The stimulationof nitrate reductase activity was prevented by cycloheximideand cordycepin suggesting the involvement of new protein synthesis. Ethephon enhanced TCA precipitable protein levels in both rootsand stems while that in leaves was not significantly affected.Amino nitrogen content increased in parallel with protein contentin response to ethephon, with roots exhibiting substantial stimulation.Nitrate accumulation in stem tissues was not affected by ethephontreatment but was increased in roots at 24 and 48 h. Leaf NO³content declined with time in both ethephon-treated and controlplants and after 24 h significantly less NO³ accumulated intreated leaves. These results are explained in terms of ethephonstimulated protein synthesis and increase in cellular metabolismand permeability. (Received August 21, 1984; Accepted January 7, 1985)     

Nitrate Uptake by Roots as Regulated by Nitrate Reduction Products of the Shoot

A mechanism is proposed by which secondary products of nitrate reduction in the shoot control the uptake of nitrate by the roots. KNO₃ enters the roots and is translocated to the shoot where nitrate is reduced and, at the same time, malate is produced. The reduction of nitrate is stoichiometric to the synthesis of malate (1). Part of the K-malate moves down to the root system in which malate is oxidized, yielding KHCO₃ which exchanges for KNO₃. Nitrate reduction in the shoot promotes the synthesis of malate which, after its translocation to the root, allows the preferential uptake of nitrate. Thus, plants reducing large amounts of nitrate may take up the anion without a superfluous accumulation of the cation. Furthermore, the utilization of nitrate by the shoot regulates its uptake by the root.     

外源K+和水杨酸在缓解融雪剂对油松幼苗生长抑制中的效应与机理

随着融雪剂在国内外寒冷地区的广泛应用及其在城市使用量的逐年增加,融雪剂对城市生态环境的危害引起了广泛的重视。其中,融雪剂在城市道路土壤中的积累对植物生长的影响已日益凸现。以油松幼苗为材料,通过分析0.2%浓度融雪剂胁迫下外源钾(K+)和水杨酸(salicylic acid,SA)对油松幼苗各生长生理指标的影响,探讨外源K+和SA在缓解融雪剂对油松幼苗生长抑制中的机理与剂量效应关系。结果表明,0.2%浓度的融雪剂处理对油松生长有明显的抑制作用,而20 mmol/L KNO3和2 mmol/L SA能明显诱导过氧化物酶(peroxidase,POD)活性的增强,缓解膜脂过氧化作用,降低丙二醛(malondialdehyde,MDA)在叶片中的积累,维持细胞膜的稳定性。虽然外源K+和SA对油松幼苗叶片胞间CO2浓度(intracellular CO2concentrations,Ci)和气孔导度(stomatal conductance,Gs)的缓解作用并不显著,但其可通过提高叶绿素含量促进光合作用的进行,缓解融雪剂胁迫对油松幼苗生长的抑制,分别增加生物量24.9%和63.6%。可见,20 mmol/L KNO3和2 mmol/L SA处理能有效缓解融雪剂对油松幼苗的伤害,为城市化学融雪剂的污染防治提供科学依据。     

Nitrate reductase activity and nitrate accumulation in in vitro produced axillary shoots,plantlets and seedlings of Pinus pinaster

The initial and induced in vivo Nitrate Reductase Activity, the nitrate accumulation by in vitro-produced axillary shoots and plantlets of Pinus pinaster were compared respectively with those of shoots collected from seedlings and whole plants.The usefulness of the nitrate of the medium used for in vitro axillary shoot formation is demonstrated by the occurrence of initial NR activity in the explants. When fed in a non in vitro situation with a 50 mM KNO₃ solution, they have the same induced capacity to reduce nitrate as do shoots from seedlings, even though the latter accumulate less nitrate. Plants regenerated in vitro exhibit an ability to reduce nitrate similar to that of seedlings. In both types of plants, the Nitrate Reductase potential is greater in roots than in shoots.Abbreviation NR Nitrate Reductase - BA 6-Benzyladenine     

STIMULATION OF NITRATE NET UPTAKE AND REDUCTION BY RED AND BLUE LIGHT AND REVERSION BY FAR-RED LIGHT IN THE GREEN ALGA ULVA RIGIDA1

The steady-state levels of nitrate, nitrite, and ammonium were estimated in the green alga Ulva rigida C. Agardh in darkness after addition of 0.5 mM KNO₃ and irradiation with red (R) and blue (B) light pulses of different duration (5 and 30 min). The net uptake of nitrate was very rapid. Seventy-five percent of the nitrate added was consumed after 60 min in darkness. Although uptake was stable after R or B, efflux of nitrate occurred within 3 h in the dark control and when R or B were followed by far-red (FR) irradiation. The internal nitrate concentration after 3 h in darkness was similar after R and B light pulses; however, the intracellular ammonium was higher after R than after B. The intracellular nitrate and ammonium decreased when FR tight pulses were applied immediately after R or B. Thus, the involvement of phytochrome in the transport of nitrate and ammonium is proposed. Nitrate reductase activity, measured by the in situ method, was increased by both R and B light pulses. The effect was partially reversed by FR light. Nitrate reductase activity was higher after 5 min of R light than after 5 min of B. However, after 30-min light pulses, the relative increase in activity was reversed for R and B. We propose that phytochrome and a blue-light photoreceptor are involved in regulation of nitrogen metabolism. Nitrate uptake and reduction correlates with previously detected light-regulated accumulation of protein in Ulva rigida under the same experimental conditions.     

Nitrate Uptake during Recovery from Nitrogen Deficiency

Two-week-old nitrogen-deficient wheat plants attained a high rate of nitrate uptake on the first day of exposure to nutrient solutions supplemented with KNO₃. Ammonium uptake from similar solutions supplemented with NH₄NO₃ was also high during the first day of exposure, but nitrate uptake from this solution was lower than from the KNO₃ treatment. During the next two to three days there was a progressive decrease in uptake of both nitrogen ions. A steady increase in uptake then occurred as the plants fully recovered from the nitrogen-deficient state. The transient low nitrate uptake after three or four days of exposure to KNO₃ was not due to an excessive accumulation of nitrate in the tissue, nor to a failure in nitrate reduction as indicated by the rate of nitrate accumulation relative to the uptake rate. Nitrogen supplied as ¹⁵N-nitrite during the low uptake period was effectively incorporated into organic forms and effectively translocated to the shoots. Failure of the root tissue to increase in soluble carbohydrates during illumination was characteristic of the low uptake period. This contrasted with an increase in root soluble carbohydrates in the light during rapid uptake associated with full recovery from the nitrogen-deficient state. It is concluded that carbohydrate translocation to the root system was insufficient during the intermediate recovery period for optimal nitrate uptake, although it was sufficient for effective reduction and translocation of nitrate and reduced nitrogen. Ammonium uptake from NH₄NO₃ was restricted during darkness by the third day whereas there was little difference between light and dark periods in nitrate uptake from KNO₃ until about the sixth day of recovery. The extent to which ammonium restricted nitrate uptake increased progressively for two or three days following which a lessening influence seemed evident, and the effects were not directly associated with the rate of ammonium uptake.     

Effect of Previous Nitrate Deprivation on 15N-nitrate Absorption and Assimilation by Wheat Seedlings

Nitrate uptake and assimilation were examined in intact 18 days old wheat (Triticum aestivum, cv Capitole) seedlings either permanently grown on nitrate (high-N seedlings) or N-stressed by transfer to an 0 N-solution for the final 7 days (low-N seedlings). The N-stressed seedlings were characterized by a lower organic N content (2.5 mg instead of 4.9 mg per seedling) and an increased root dry weight.The seedlings received ¹⁵NO₃K for 7 h in the light. Nitrate uptake was 2.8 times higher in low-N than in high-N seedlings. The assimilation rate was 35 and 16 μmol NO₃^?·^h?1· g^?1 dry weight respectively. Partitioning of NO₃^? to reduction and assimilation was the very same in both kinds of seedlings. The results support the view that 50 % of the nitrate reduction in Triticum aestivum, cv Capitole could be achieved in the roots.The present observations are interpreted as evidence that factors closely associated with the seedling N-status may have a major role in regulating NO₃^? uptake and assimilation. In low-N seedlings, the high amount of carbohydrates in roots may add its stimulus to the specific inducing effect of nitrate whereas in high-N seedlings, excess of nitrate or amino-acids may set the pace by negative feedback control.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

Nitrate and ammonium accumulation in bermudagrass in relation to nitrogen fertilization and season

Seasonal changes in nitrate and ammonium concentrations were studied inCynodon dactylon (L.) Pers. plants grown for one year in the field in a Mediterranean area. Plants cultivated in a sandy loam soil were fertilized with nitrate-N or ammonium-N at two application rates (250 and 1000 kg N ha⁻¹ year⁻¹) and compared to controls with no added N. Plots were harvested every three weeks from May to November. Shoots were separated into leaves and stems and analyses carried out in both fractions. Nitrogen applications generally led to elevated nitrate concentrations both in leaves and stems at all sampling dates but had little influence on the ammonium concentrations of the tissues. Higher nitrate and ammonium concentrations were found in stems than in leaves, although no levels higher than 0.22% NO ₃ ⁻ −N and 0.10% NH ₄ ⁺ −N were detected in either fraction. Nitrate tended to accumulate mostly in autumn and spring whereas low accumulations were found in summer. Ammonium showed both in leaves and stems a progressive but limited accumulation throughout the period with a peak in October, followed by a strong decrease in November.     

Nitrate reductase activity (NRA) in Asphodelus aestivus Brot. (Liliaceae): distribution among organs,seasonal variation and differences among populations

Nitrate reductase activity (NRA) in different compartments (leaves, inflorescence stalks, flowers and tuberous roots) of Asphodelus aestivus Brot. (Liliaceae) and actual mineral nitrogen (NO₃⁻-N and NH₄⁺-N) in soil surrounding the roots were investigated over one year. Although the highest NRA was found in the leaves, the other plant compartments, such as flowers and tuberous roots, also have nitrate assimilation capacity. High nitrate assimilation capacity under suitable conditions is considered to be a good strategy for development and dominance of this species in Mediterranean environments. There was a seasonal variation in nitrate assimilation in leaves and actual NO₃⁻-N content of soils. Depending on actual nitrate content of soils, nitrate assimilation increased in winter.