The Response of Nitrate Reductase Activity and Nitrate Assimilation in Maize Roots to Growth Regulators at Acidic pH

Nitrate and total nitrogen contents, and nitrate reductase (NR) activity of the excised maize roots in buffered or unbuffered nitrate solution (at pH 6.5 or 4.5) as affected by putrescine (PUT), abscisic acid (ABA) and salicylic acid (SA) were investigated. In unbufferred solution, the NR activity was lower at pH 4.5 as compared to that at pH 6.5, but in bufferred solution the activity was higher at lower pH. Supply of 100 µM PUT or 500 µM SA, promoted NR activity and 50 µM ABA inhibited the activity at pH 6.5. However, at pH 4.5, PUT and SA inhibited NR activity and ABA had no effect. In most cases, the increase in NR activity was positively correlated with total organic nitrogen and a negatively with nitrate content. A reverse situation was found when NR activity was inhibited by the growth regulators.     

Growth and development of seminal and crown root systems in N-limited barley,and their contributions to nitrate acquisition during vegetative and generative growth

Barley (Hordeum vulgare L., cvs Golf and Laevigatum) was grown under nitrogen limitation, controlled by the relative rate of nitrate-N addition (RA), in solution culture. The seminal and crown root systems were kept apart, but in contact with the same nutrient solution throughout culturing. Growth, nitrate uptake, and in vitro nitrate reductase (NR) activity in the different root parts were studied at plant ages from 40 (late vegetative stage) to 110 (mid grain-filling) days. The RA was during this time interval stepwise decreased from 0.08 day^–1 to 0.005 day^–1. The ratio between seminal root dry weight and total plant dry weight decreased drastically during post-anthesis growth, whereas the contribution by crown roots remained unchanged. Tissue nitrogen concentrations in seminal roots did not change with time, but decreased in crown roots after day 80. The NR activity decreased with age in both seminal and crown roots. The V_max for net nitrate uptake decreased throughout the experiment in the seminal root system, but not in the crown root system. The kinetic properties (V_max and K_M) were used to calculate the nitrate concentration required to maintain a relative rate of nitrate-N uptake that equals the relative addition rate. These concentrations (2 to 5 mmol m^–3) were found to closely match actually measured nitrate concentrations in the nutrient solution (1 to 6 mmol m^–3). From uptake kinetics, it was deduced that the contribution by seminal roots to total nitrate uptake at these concentrations decreased from more than 50% in vegetative plants, to about 20% just after main shoot anthesis, and to less than 5% during grain-filling. ei]Section editor: H Lambers     

The effect of chloride on nitrate reductase level,on anaerobic nitrite production,and on nitrate content in excisedPisum sativum L. roots

The effect was studied of chloride ions, added in the form of different salts, on nitrate reductase (NR) level in excised pea roots, on anaerobic nitrite production in an assay medium lacking both nitrate and n-propanol, on nitrate content in the roots, and on in vivo NR activity determined in an assay medium containing 5% n-propanol. The presence of Cl in nitrate containing nutrient solutions resulted in lower NR levels, however counterions supplied together with Cl tended to modify slightly this general trend. The negative effect of Cl ions was also apparent, when Cl ions were applied before nitrate ions. Anaerobic nitrite production in the medium lacking both nitrate and n-propanol was not influenced by chloride ions. Nitrate content in the roots was reduced in the presence of chloride both at 3 mM and 15 mM NO₃ in nutrient solutions; however, at 16 mM NO₃, nitrate content in the roots exoeeded even in the presence of 15 mM Cl nitrate content in those root segments which were cultivated in a nutrient solution with 6 mM nitrate, which is the concentration at which NR reaches the level of saturation in excised pea roots. The results obtained suggest that a special induction nitrate pool exists in plant cells besides the storage and metabolic nitrate pools.     

Modifications of the activity of nitrate reductase from cucumber roots

The regulatory properties of NADH-dependent nitrate reductase (NR) in desalted root extracts from hydroponically grown cucumber (Cucumis sativus L.) seedlings were examined. The lowest activity of NR was detected in extracts incubated with Mg²⁺ and ATP. An inhibitory effect of Mg-ATP was cancelled in the presence of staurosporine (the protein kinase inhibitor) and completely reversed after addition of ethylenediaminetetraacetate (EDTA) as well as AMP into reaction mixture. Reactivation of enzyme due to AMP presence, contrary to the chelator-dependent NR activation, was sensitive to microcystin LR (the protein phosphatase inhibitor). Above results indicated that the nitrate reductase in cucumber roots was regulated through reversible phosphorylation of enzyme protein. A drop in the activity of NR was also observed after incubation of enzyme at low pH. At low pH, the presence of ATP alone in the incubation medium was sufficient to inactivate NR, indicating that H⁺ can substitute the Mg²⁺ in formation of an inactive complex of enzyme. ATP-dependent inactivation of NR at low pH was prevented by staurosporine and reversed by AMP. However, AMP action was not altered by microcystin LR suggesting that in low pH the nucleotide induced reactivation of NR is not limited to the protein phosphorylation.     

Nitrate-independent expression of plant nitrate reductase in Lotus japonicus root nodules

Nitrate-independent nitrate reductase (NR) activity is generally found in legume root nodules. Therefore, the effects of nitrate on plant NR activity and mRNA were investigated in the root nodules of Lotus japonicus (L. japonicus). Both NR activity and mRNA levels in roots and root nodules were up-regulated by the addition of nitrate. In the absence of nitrate, NR activity and mRNA were detected in root nodules but not in roots. Southern blotting analysis indicates that NR is encoded by a single gene in L. japonicus. No nitrate was detected in the root nodules or roots of plants grown in the absence of nitrate, while its accumulation was observed in plants supplied with exogenous nitrate. These results indicate that inducible-type NR can be expressed in root nodules in the absence of nitrate. The activation state of the nitrate-independent activity of NR was as high as that of NR activity induced by nitrate. NR mRNA expressed independently of nitrate in root nodules without nitrate was localized in the infected regions of the root nodules. Thus, the expression could be related to the specific structure and environment of root nodules.     

Nitrate regulation of nitrate uptake and nitrate reductase expression in barley grown at different nitrate:ammonium ratios at constant relative nitrogen addition rate

Barley (Hordeum vulgare L. cv. Golf) was cultured using the relative addition rate technique, where nitrogen is added in a fixed relation to the nitrogen already bound in biomass. The relative rate of total nitrogen addition was 0.09 day^?1 (growth limiting by 35%), while the nitrate addition was varied by means of different nitrate: ammonium ratios. In 3- to 4-week-old plants, these ratios of nitrate to ammonium supported nitrate fluxes ranging from 0 to 22 μmol g^?1 root dry weight h^?1, whereas the total N flux was 21.8 ± 0.25 μmol g^?1 root dry weight h^?1 for all treatments. The external nitrate concentrations varied between 0.18 and 1.5 μM. The relative growth rate, root to total biomass dry weight ratios, as well as Kjeldahl nitrogen in roots and shoots were unaffected by the nitrate:ammonium ratio. Tissue nitrate concentration in roots were comparable in all treatments. Shoot nitrate concentration increased with increasing nitrate supply, indicating increased translocation of nitrate to the shoot. The apparent V_max for net nitrate uptake increased with increased nitrate fluxes. Uptake activity was recorded also after growth at zero nitrate addition. This activity may have been induced by the small, but detectable, nitrate concentration in the medium under these conditions. In contrast, nitrate reductase (NR) activity in roots was unaffected by different nitrate fluxes, whereas NR activity in the shoot increased with increased nitrate supply. NR-mRNA was detected in roots from all cultures and showed no significant response to the nitrate flux, corroborating the data for NR activity. The data show that an extremely low amount of nitrate is required to elicit expression of NR and uptake activity. However, the uptake system and root NR respond differentially to increased nitrate flux at constant total N nutrition. It appears that root NR expression under these conditions is additionally controlled by factors related to the total N flux or the internal N status of the root and/or plant. The method used in this study may facilitate separation of nitrate-specific responses from the nutritional effect of nitrate.     

Evidence for the nitrate-dependent spatial regulation of the nitrate reductase gene in chicory roots

Young chicory plants (Cichorium intybus L. var. Witloof) show a tenfold higher nitrate reductase NR activity in roots compared to leaves. Northern analysis revealed, besides the nitrate inducibility of the nitrate reductase gene (nia), a higher level of expression in the roots. By modifying the external nitrate concentration the NR activity in the leaves remained negligible whereas a maximal activity was observed in the roots when grown in the presence of 5 mM nitrate. Surprisingly, variation of the external nitrate concentration induced changes in the spatial regulation of nia within the root. In-situ hybridization mainly localized nia mRNA in the cortical cells of roots grown at low nitrate concentrations (0.2 mM). At high nitrate concentrations (5 mM), nia mRNA was more abundant in the vascular tissues. The root apex revealed a strong signal under both conditions. The isolation and characterization of the NR structural gene from chicory is also presented. Southern blot analysis revealed the presence of a single nia gene per haploid genome of chicory.     

Foliar and fine root nitrate reductase activity in seedlings of four forest tree species in relation to nitrogen availability

Summary Seedlings of red maple, white pine, pitch pine and red pine were fertilized with nutrient solutions containing 4 levels of nitrate or ammonium additions. These levels corresponded to approximately 0.5, 1, 2 and 4 times normal availability of nitrogen in northeastern forests. Nitrate reductase (NR) activity was assayed in roots and leaves. Red maples treated with nitrate showed much higher leaf activities and higher ratios of leaf NR activity to root NR activity than any other species. Ammonium additions to red maple and white pine appeared to inhibit NR activity in leaves. With high nitrate additions, NR activity was induced in roots and leaves of pine species, but activity in roots remained much higher than in leaves.     

Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.     

Involvement of nitrate reduction in the tolerance of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.) plants to prolonged root hypoxia

The putative role of nitrate and nitrate reductase in the tolerance to prolonged hypoxia was investigated in tomato plants. Nitrogen nutrition has been modified either by deprivation of nitrate or by addition of tungstate—an inhibitor of nitrate reductase (NR)—in the culture medium. In the absence of nitrate as well as in the presence of tungstate, plant growth was significantly disturbed. In the presence of nitrate, the growth of hypoxic plants maintained, nitrate absorption and NR activity increased and a significant release of nitrite into the medium was observed. This mechanism of nitrate reduction, called nitrate respiration, could be an alternative pathway to oxygen-dependent respiration during root hypoxia and a transient adaptation of tomato roots to hypoxic conditions.     

Effects of cadmium on activity of nitrate reductase and on other enzymes of the nitrate assimilation pathway in bean

We examined the influence of cadmium (Cd) exposure on nitrate assimilation in bean (Phaseolus vulgaris L. cv Morgane). Bean plants were submitted to either a short- (24 h) or long-term (7 d) supply of Cd in the nutrient solution. Addition of Cd decreases very significantly both the water and nitrate uptake of the treated plants when compared to untreated plants. Cadmium also induces a decrease in nitrate reductase (NR, EC 1.6.6.1) activation state after 24 h of exposure whereas, after 7 d, NR activation state was similar to that of the control bean plants. On the other hand, the level of NR protein was decreased by about 80 % after 7 d of Cd exposure and by only 15 % 1 d after Cd addition. We then investigated the in vitro effect of Cd on NR catalytic activities and inactivation by phosphorylation. The molybdenum cofactor-binding domain of NR seemed to be the most affected by Cd which did not interfere with the in vitro inactivation process of NR by MgATP. Glutamate synthase and NR activities were more inhibited by supply of Cd in the long-term experiment than the activities of nitrite reductase and glutamine synthetase. Conversely, an increase in glutamate dehydrogenase activity was observed in parallel with an increase in ammonium concentration. It thus appears that Cd treatment induces both rapid and long-term changes in the activity of the enzymes involved in nitrate assimilation, partly in response to reduced water and nutrient uptake. Moreover we have also shown that Cd itself can have a direct effect on the activities of these enzymes.     

The effect of some ammonium salts on nitrate reductase level,onin vivo nitrate reduction and on nitrate content in excisedpisum sativum roots

The effect of some ammonium salts on nitrate reductase (NR) level, onin vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excisedPisum sativum roots, and their effect was compared with that of KNO₃, Ca(NO₃)₂ and NaNO₃ at 15 mM NO₃ ^- concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH₄ ⁺ at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH₄H₂PO₄, (NH₄)₂SO₄ and NH₄NO₃). However their positive effect is independent of the presence of NH₄ ⁺ ions, and it is obviously the result of an increased concentration of H⁺ ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH₄NO₃ solution where NH₄ ⁺ is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH₃). (H₂O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence. Thein vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO₃ ^- pool (i.e. NO₂ ^- production under anaerobic conditions), nor between NR level and the total nitrate content in the roots. One-salt solutions of NaNO₃, Ca(NO₃)₂ and KNO₃ exert different effects on the level of nitrate reductase and on the content of NO₃ ^- in the roots, but the in vivo NO₃ ^- reduction shows the same trend as NR level in the roots influenced by these salts. Cl^- ions, supplied in NH₄C1, depress both NR level and NO₃ ^- content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level,in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other.     

镉胁迫下紫花苜蓿幼苗内源一氧化氮和活性氧的生成

以"甘农三号"紫花苜蓿幼苗为材料,在水培条件下,研究了不同浓度镉(Cd)胁迫下紫花苜蓿根、茎和叶内源一氧化氮(NO)和活性氧(ROS)的生成机制以及根系活力的变化.结果表明:在0~2.0 mmol·L-1范围内,随着Cd浓度的增加,幼苗内NO含量呈现先升高后降低的趋势,最后可维持在略高或持平于对照的水平.幼苗内一氧化氮合成酶(NOS)活性、硝酸还原酶(NR)活性、亚硝酸根离子(NO2-)含量和类胡萝卜素(Car)含量的变化与NO含量变化规律相似却又不全相同.NOS和NR是影响幼苗茎中NO含量的主要因素,NOS、NO2-和NR则是影响叶中NO含量的主要因素,而根中NO含量主要与NOS活性和NO2-含量有较大相关性.随着Cd浓度的增加,幼苗内过氧化氢(H2 O2)含量、丙二醛(MDA)含量、超氧阴离子(O-2·)含量和相对电导率(REC)呈现显著升高趋势,说明高浓度的Cd处理会使ROS大量积累,细胞膜遭破坏,细胞质外流,进而引发膜脂过氧化.随着Cd浓度的增加,紫花苜蓿根系活力的变化为先升高后降低,指示了低浓度Cd处理会促进植物代谢,增强其生命力;而高浓度Cd会致使植株代谢受抑制,细胞受损害.NO和ROS的相关性不大,说明二者虽同为自由基,但它们产生和变化方式大有差别.     

Influence of Different Concentrations of NO-3 and NH+4 on the Activity of Glutamine Synthetase and Other Relevant Enzymes of Nitrogen Metabolism in Wheat Roots

Influence of different concentrations of NO3 and NH+ on the activity of glutamine synthetase (GS), asparagine synthetase (AS), glutamate dehydrogenase (GDH), nitrate reductase (NR) and the changes of GS-mRNA in wheat roots have been studied with enzymes activity assay and Northern blot. The results showed that the higher GS activity was found in roots of wheat when NH+4-N was the sole nitrogen source than when NO3-N was the sole nitrogen source. GS-mRNA of Northern blot was simillar to GS activity. 3 mmol/L NO3- promoted the activity of AS. The change of AS was independent of the change of GS. GDH activity was not been detected, and change in regulation of NR activity was not found.     

The terminal reductases for selenate and nitrate respiration in Thauera selenatis are two distinct enzymes.

A number of approaches have been used to show that a recently isolated selenate-respiring bacterium, Thauera selenatis, is able to synthesize both a selenate reductase (SR) and a nitrate reductase (NR). (i) The pH optimum of the SR was found to be 6.0; that of the NR was 7.0. (ii) The presence of nitrate did not inhibit selenate reduction in selenate-grown cells. (iii) In cell extracts, the highest SR or NR activity was observed in cells grown with the respective electron acceptor. (iv) Mutants that were unable to grow with nitrate as the terminal electron acceptor and lacked NR activity were isolated; these mutants grew normally with selenate and synthesized SR. (v) The SR was found in the periplasmic space of the cell, whereas the NR was present in the cytoplasmic membrane. A hypothetical electron transport system involving the SR is described.     

Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution

The effect of nitrogen form (NH₄-N, NH₄-N + NO₃⁻, NO₃⁻) 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 NO₃⁻ increases, and NH₄⁺ and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO₃⁻, NH₄-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 HCO₃⁻ to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO₃⁻, 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 NO₃⁻, NH₄-N, and amide-N.     

Partitioning of Sugar between Growth and Nitrate Reduction in Cotton Roots

The level of endogenous sugars was inversely related to nitrate availability in young cotton (Gossypium hirsutum L.) plants, with high nitrate causing a greater decline in sugar content of roots than of shoots. High nitrate (low sugar) plants also displayed relatively more shoot growth and less root growth than low nitrate (high sugar) plants. These data are consistent with the theory that roots are poor competitors for sugar, and that sugar supply is a major factor limiting root growth in vivo.

The effects of endogenous sugar level on root growth and on nitrate reductase activity in the root were different. When root sugar level was experimentally controlled by varying nitrate concentration in the nutrient solution, root growth was less sensitive than nitrate reductase activity to sugar deficiency. Also, in sterile root tips cultured on media containing a wide range of sucrose concentrations, growth rate was considerably less sensitive to endogenous sugar deficiency than was nitrate assimilation rate. Similarly, in plants which were detopped or girdled, nitrate reductase activity in the roots declined more rapidly than did root sugars, especially glucose and fructose. These results suggest that when sugar is deficient, cotton roots preferentially use it for growth at the expense of nitrate reduction.

     

Iron availability in plant tissues-iron chlorosis on calcareous soils

The article describes factors and processes which lead to Fe chlorosis (lime chlorosis) in plants grown on calcareous soils. Such soils may contain high HCO₃ ^- concentrations in their soil solution, they are characterized by a high pH, and they rather tend to accumulate nitrate than ammonium because due to the high pH level ammonium nitrogen is rapidly nitrified and/or even may escape in form of volatile NH₃. Hence in these soils plant roots may be exposed to high nitrate and high bicarbonate concentrations. Both anion species are involved in the induction of Fe chlorosis.Physiological processes involved in Fe chlorosis occur in the roots and in the leaves. Even on calcareous soils and even in plants with chlorosis the Fe concentration in the roots is several times higher than the Fe concentration in the leaves. This shows that the Fe availability in the soil is not the critical process leading to chlorosis but rather the Fe uptake from the root apoplast into the cytosol of root cells. This situation applies to dicots as well as to monocots. Iron transport across the plasmamembrane is initiated by Fe^III reduction brought about by a plasmalemma located Fe^III reductase. Its activity is pH dependent and at alkaline pH supposed to be much depressed. Bicarbonate present in the root apoplast will neutralize the protons pumped out of the cytosol and together with nitrate which is taken up by a H⁺/nitrate cotransport high pH levels are provided which hamper or even block the Fe^III reduction.Frequently chlorotic leaves have higher Fe concentrations than green ones which phenomenon shows that chlorosis on calcareous soils is not only related to Fe uptake by roots and Fe translocation from the roots to the upper plant parts but also dependent on the efficiency of Fe in the leaves. It is hypothesized that also in the leaves Fe^III reduction and Fe uptake from the apoplast into the cytosol is affected by nitrate and bicarbonate in an analogous way as this is the case in the roots. This assumption was confirmed by the highly significant negative correlation between the leaf apoplast pH and the degree of iron chlorosis measured as leaf chlorophyll concentration. Depressing leaf apoplast pH by simply spraying chlorotic leaves with an acid led to a regreening of the leaves.     

Effect of mycorrhiza and nitrate nutrition on nitrate reductase activity in Scots pine seedlings

In vivo nitrate reductase (EC 1.6.6.1) activity was measured in seedlings of Scots pine ( Pinus sylvestris L.) inoculated with Cenococcum geophilum (Sow.) Ferd. & Winge, Paxillus involutus (Batsch:Fr) Fr, Piloderma croceum Erikss, & Hjortst, and Suillus variegatus (Fr.) O. Kuntze. The activity was higher in the mycorrhizal pine roots than was previously found in the fungus symbiont alone, but lower than in the roots of nonmycorrhizal pine seedlings. The differences observed in a previous study between the fungal species under pure culture conditions were not found in the present work for mycorrhiza synthezised with the same fungal species. An increase in the nitrate concentration of the nutrient solution increased the proportion of the nitrate reductase activity in the needles. The mycorrhizal root tips had higher nitrate reductase activity than nonmycorrhizal root tips in the same root system.