Nitrate reduction and compartmentation in tomato leaves. II. Nitrate loss by leaf sections in a gaseous atmosphere and the size of the metabolic nitrate pool

Nitrate disappearance in tomato ( (ycopersicon esculentum Mill. cv. Azes) leaf sections kept under a stream of gas (nitrogen or air) has been studied, using leaf sections from plants supplied with low (7.5 mM) or high (17.5 mM) nitrate levels in their nutrient solution. Cessation of nitrate loss occurred in leaf sections taken from plants irrigated with low (7.5 mM) nitrate-containing nutrient solution. Resumption of nitrate disappearance occurred upon addition of exogenous nitrate by vacuum infiltration to leaf sections, suggesting that cessation of nitrate loss was due to exhaustion of the metabolic pool. We estimated that 53% of the total nitrate in leaf sections from low nitrate plants was located in a storage pool, probably the vacuole. The remainder was located in a pool, readily available for reduction (the metabolic pool). This pool is composed of nitrate in the free space as well as in the cytoplasm which was estimated to contain about 20% of the total nitrate.
Either under air or nitrogen, less nitrite was accumulated than nitrate assimilated suggesting that nitrite accumulation was not an adequate parameter for the estimation of nitrate utilization. Anaerobic conditions inhibited nitrite reduction whereas nitrate assimilation was not blocked. Nitrate loss from endogenous pool in leaf sections placed under aerobic conditions is suggested as an adequate method for the estimation of the metabolic pool of nitrate.     

Endogenous nitrate loss as an assay for nitrate reduction in vivo

An in vivo assay method for nitrate reduction is proposed, based on the use of endogenous nitrate rather than on the accumulation of nitrite. Loss of endogenous nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaves. Leaf sections were incubated in the dark in a gaseous environment of air or N₂. Nitrate disappeared under both conditions, the highest loss being observed in tissue under anaerobiosis. Nitrite accumulated only in leaf sections under anaerobiosis, but the amount of nitrite accumulated was much lower than the amount of nitrate lost. A comparative study of the capacity of barley leaf sections to use endogenous nitrate and accumulate nitrite showed that both activities were dependent on temperature in a manner characteristic of enzymatic reactions. Disappearance of endogenous nitrate increased with increasing levels of nitrate in the tissue.     

Nitrate metabolism in soybean root nodules

The nitrate metabolism in nodules induced by Bradyrhizobium japonicum strain PJ17 on roots of soybean [ Glycine max (L.) Merr. cv. Hodgson] has been characterized by the nitrate reductase (NR; EC 1.6.6.1 and EC 1.6.6.3) activity of both partners of the symbiosis. NR activities of bacteroids and nodular cytosol were comparable and significantly higher than those of the roots. Nitrate reduction led to nitrite accumulation in root nodules, which was maximum after pod filling. The nodule had the capacity to metabolize nitrite via nitrite reductase (NiR; EC 1.6.6.4), at least in the cytosolic fraction. This activity was partly inhibited by the low content of free O₂ in the nodule. Indeed, nitrite accumulation decreased in the presence of an increased external pressure of O₂.     

Nitrate uptake and nitrite release by tomato roots in response to anoxia

Excised root systems of tomato plants (early fruiting stage, 2nd flush) were subjected to a gradual transition from normoxia to anoxia by seating the hydroponic root medium while aeration was stopped. Oxygen level in the medium and respiration rate decreased and reached very low values after 12 h of treatment, indicating that the tissues were anoxic thereafter. Nitrate loss from the nutrient solution was strongly stimulated by anoxia (after 26 h) concomitantly with a release of nitrite starting only after 16 h of treatment. This effect was not observed in the absence of roots or in the presence of tungstate, but occurred with whole plants or with sterile in vitro cultured root tissues. These results indicate that biochemical processes in the root involve nitrate reductase. NR activity assayed in tomato roots increased during anoxia. This phenomenon appeared in intact plants and in root tissues of detopped plants. The stimulating effect of oxygen deprivation on nitrate uptake was specific; anoxia simultaneously entailed a release of orthophosphate, sulfate, and potassium by the roots. Anoxia enhanced nitrate reduction by root tissues, and nitrite ions were released into xylem sap and into medium culture. In terms of the overall balance, the amount of nitrite recovered represented only half of the amount of nitrate utilized. Nitrite reduction into nitric oxide and perhaps into nitrogen gas could account for this discrepancy. These results appear to be the first report of an increase in nitrate uptake by plant roots under anoxia of tomato at the early fruiting stage, and the rates of nitrite release in nutrient medium by the asphyxiated roots are the fastest yet reported.     

Variation in Nitrate Reductase, Nitrite, and Nitrite Reductase in Some Grasses and Cereals

Nitrite accumulation may result from unbalance between nitratereductase which produces nitrite and nitrite reductase whichremoves it. In the first experiment, using three light levelsand three nitrate levels, on Lolium, maize, and oats, both enzymesresponded to increased light, though not always significantly.The effect of nitrate was more variable. Nitrate reductase activityincreased to the intermediate or highest level of nitrate, butthere was no clear response in nitrite reductase activity orin nitrite concentration. In the second experiment, using fournitrate levels but only one, high, light intensity on Loliumand barley, the results were clearer. With increasing nitratesupply, nitrate reductase activity increased more than nitritereductase activity. This was particularly marked in Lolium,in which nitrite accumulated at the highest nitrate supply.Thus high nitrate supply unbalances the two enzymes in the directionleading to nitrite accumulation.     

Nitrate entry and nitrite formation in the infected region of soybean nodules

The entry of nitrate into the infected region of soybean nodules and the possibility of a subsequent nitrite accumulation was studied. Nitrate was observed to gain access to the infected region in the short-term and significant amounts could be measured within 2 d of nitrate supply. The availability of nitrate in the bacteroid-containing region did not cause free-nitrite accumulation for at least 8d. However, to avoid the artefactual production of nitrite during extraction it was necessary to disrupt nodules in the presence of zinc acetate and ethanol, to prevent bacteroid nitrate reductase activity. Nitrite rapidly accumulated if nodules were extracted without prior enzyme-inactivation, or if bacteroids were allowed access to nitrate, or, more significantly, if nodules were not extracted immediately following detachment. Nitrate accumulation in detached nodules was mediated by oxygen concentration within the nodule; in the presence of pure N2 gas, nitrite accumulation was three times greater than in air and, conversely, it was prevented by exposure to pure O2. Furthermore, nitrite produced in detached nodules under atmospheric conditions was scavenged by transferring these nodules into 100% oxygen. However, measurements of apparent functional leghaemoglobin, using a nodule oximeter, suggested that after 8 d nitrate exposure up to 83% of Lb activity was lost, possibly due to interactions with nitrite produced in the nodule interior leading to the formation of nitrosylleghaemoglobin.Key words: Glycine max, cortex, infected region, leghaemoglobin, nitrate, nitrite, nodules, soybean      

植物中硝酸还原酶和亚硝酸还原酶的作用

植物通过硝酸盐同化途径以硝酸盐和氨的形式吸收氮元素。硝酸盐的同化是一个受到严格控制的过程,其中两个先后参加反应的酶——硝酸还原酶(NR)和亚硝酸还原酶(NiR)对初级氮的同化起主要调控。在高等植物中,NR和NiR基因的转录及转录后加工受到各种内在和外在因素的影响,翻译后调控是消除亚硝酸盐积累的重要机制。随着分子生物学技术的发展,可以更容易地通过突变体和转基因方式来研究NR和NiR基因的调控。     

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O₂ (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 g· ml^-1) or chloramphenicol (50 g·ml^-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N₂ fixation.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Nitrate Utilization by the Diatom Skeletonema costatum: I. Kinetics of Nitrate Uptake

Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skeletonema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kinetics, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed-transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carrier-mediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased.     

The Effect of Inhibitors of Photosynthetic and Respiratory Electron Transport on Nitrate Reduction and Nitrite Accumulation in Excised Z. mays L. Leaves

The assimilation of nitrate and nitrite under dark and lightconditions in Zea mays L. leaves was investigated. Nitrate wasassimilated under dark-aerobic conditions. Anaerobiosis stimulatednitrate reduction and nitrite accumulation under dark conditions.Vacuum infiltration of inhibitors of respiratory electron transport,antimycin A and rotenone, stimulated nitrate reduction and nitriteaccumulation under dark-aerobic conditions. Vacuum infiltrationof low concentrations of PCP, DNP and mCCCP depressed nitratereduction and nitrite accumulation under dark-aerobic conditions,whereas, infiltration of higher concentrations stimulated nitratereduction and nitrite accumulation. The greatest level of nitrateand nitrite reduction occurred under light conditions. The inhibitorof photosynthetic electron transport, DCMU, stimulated the accumulationof nitrite in the light, but decreased nitrate reduction. Whenthe inhibitors of respiratory electron transport antimycin Aand rotenone, were supplied together with DCMU in the light,nitrite accumulation was enhanced. Low concentrations of mCCCPdecreased both nitrate reduction and nitrite accumulation underlight conditions when supplied with DCMU. Key words: Nitrate reduction, Nitrite accumulation, Leaves     

硝酸还原酶抑制剂钨酸钠对油菜硝态氮积累的影响

采用溶液培养方法,选取硝酸盐积累差异明显的两个油菜品种（低硝态氮积累品种‘红油3号’和高硝态氮积累品种‘中双6号’,研究苗期根系硝酸还原酶（NR）活性被抑制以后两个油菜品种叶片、叶柄和根系中NR活性和硝态氮含量的变化。结果表明：1.0mmol.L-1的NR活性抑制剂Na2WO4对两个油菜品种的根系NR活性抑制效果最佳;根系NR活性被抑制以后,两个油菜品种的根系NR活性、硝态氮吸收速率均显著下降,而硝态氮含量却显著上升;且Na2WO4对‘中双6号’硝态氮吸收的抑制程度强于其对‘红油3号’的抑制。叶片和叶柄的NR活性变化不显著,但叶柄硝态氮含量显著下降,叶片硝态氮含量稳定,且这一趋势在低积累品种‘红油3号’中表现得更为明显。     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate I. Nitrite Accumulation and Formation of Nitrosylleghemoglobin in Nodules

The accumulation of nitrite in nodules was investigated to elucidatethe mechanism of inhibition of nitrogen fixation in nodulesof soybean (Glycine max. [L.] Merr.) plants supplied with nitrate.Acetylene-reducing activity (ARA) in nodules fell within 24h as a result of the supply of exogenous nitrate, accompaniedby an increase in the accumulation of nitrite in the cytosolbut not in the bacteroids of nodules. Nitrate reductase (NR)activity in the nodule cytosol remained high, irrespective ofthe supply of nitrate. Nitrosylleghemoglobin (LbNO) was detectedspectrophotometrically in the extract from nodules in whichnitrogen fixation was inhibited by nitrate. In experiments invitro, it was found that LbNO was easily formed from leghemoglobinin the presence of nitrite and dithionite. Thus, it is suggested that nitrogen fixation was inhibited primarilyby a decrease in the function of leghemoglobin, attributableto the formation of LbNO, which was caused by the accumulationof nitrite generated from nitrate by NR in the nodule cytosol. (Received August 22, 1989; Accepted January 24, 1990)     

Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula

The coordinate appearance of the bispecific NAD(P)H-nitrate reductase (NR; EC 1.6.6.2) and nitrite reductase (NiR; EC 1.7.7.1) was investigated in leaves and roots from European white birch seedlings (Betula pendula Roth). Induction by nitrate and light of both enzymes was analyzed by in vitro assays and by measuring NR- and NiR-encoding mRNA pools with homologous cDNAs as probes. When birch seedlings were grown on a medium containing ammonium as the sole nitrogen source, low constitutive expression of NR and NiR was observed in leaves, whereas only NiR was significantly expressed in roots. Upon transfer of the seedlings to a nitrate-containing medium, mRNA pools and activities of NR and NiR dramatically increased in leaves and roots, with a more rapid induction in leaves. Peak accumulations of mRNA pools preceded the maximum activities of NR and NiR, suggesting that the appearance of both activities can be mainly attributed to an increased expression of NR and NiR genes. Expression of NR was strictly light-dependent in leaves and roots and was repressed by ammonium in roots but not in leaves. In contrast with NR, constitutive expression of NiR was not affected by light, and even a slight induction following the addition of nitrate was found in the dark in roots but not in leaves. No effect of ammonium on NiR expression was detectable in both organs. In leaves as well as in roots, NiR was induced more rapidly than NR, which appears to be a safety measure to prevent nitrite accumulation.     

Nitrate metabolism in roots and nodules of Vicia faba in response to exogenous nitrate

Activities of nitrate reduction enzymes, nitrate reductase activity (NRA) and nitrite reductase activity (NiRA) from roots and nodules of 5 mutant genotypes and one commercial cultivar (Alameda) of faba bean ( Vicia faba L. var. minor) grown in the presence of N₂ alone or with additional NO⁻₃ in the medium have been studied. A naturally occurring mutant (VFM109) with impaired ability to reduce nitrate in its nodules is described. All the other cultivars of V. faba showed nodule NRA, although the range was very wide, from almost negligible (VFM72) up to 2 μmol h⁻¹ (g FW)⁻¹. This activity was entirely of plant origin. Root NRA also ranged widely accross cultivars. However, the level of activity expressed as well as the response of NRA to nitrate followed a pattern opposite to that observed in nodules. Roots and nodules of all cultivars showed very high rates of NiRA, respectively 50 and 150-fold higher than NRA, thus precluding accumulation of nitrite in these tissues. Root enzymes were significantly stimulated by nitrate while negative (NRA) or little effect (NiRA) was found for nodules. Nitrate and nitrite reduction are carried out by inducible enzymes in roots of V. faba and by constitutive enzymes in nodules, indicating that there may be different forms of these enzymes in each tissue. Differences in the plant genotype were a major cause of the variability in nitrate and nitrite reduction by nodulated root systems of V. faba .     

Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia

The mechanism of nitrate reductase (NR) regulation under long-term anoxia in roots of whole plants and the putative role of nitrate in anoxia tolerance have been addressed. NR activity in tomato roots increased significantly after 24 h of anaerobiosis and increased further by 48 h, with a concomitant release of nitrite into the culture medium. Anoxia promoted NR activation through dissociation of the 14-3-3 protein inhibitor and NR dephosphorylation. After 24 h of anoxia, the total amount of NR increased slightly up to 48 h. However, NR-mRNA levels remained constant between 0 h and 24 h of root anoxia and decreased after 48 h. This is probably due to the inhibition of NR degradation and the accumulation of its native form. NR was slightly dephosphorylated in the absence of oxygen and nitrate. Under anoxia, NR dephosphorylation was modulated by nitrate-controlled NR activity. In addition, the presence of nitrate prevents anoxic symptoms on leaves and delays wilting by 48 h during root anoxia. In the absence of nitrate, plants withered within 24 h, as they did with tungstate treatment, an inhibitor of NR activity. Thus, anoxia tolerance of tomato roots could be enhanced by nitrate reduction.     

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.     

Main centers of nitrate and nitrite reduction in young and nodulated yellow lupine (<Emphasis Type="Italic">Lupinus luteus</Emphasis>)

Nitrate and nitrite reduction centers in non-nodulated and symbiotic yellow lupine were analyzed. In young seedlings, nitrate was exclusively accumulated in roots, which also was shown as the main nitrate reduction center. In contrast, leaves were shown to play a key role in nitrite reduction. A similar distribution of nitrate reductase (NR) and nitrite reductase was found in nodulated plants. However, in field conditions characterized by low nitrate content, a disproportionately high level of NR activity in nodules was also observed during all stages of symbiotic growth. This feature was confirmed in nitrate-fed hydroponic cultures. Nodule NR activity was one order of magnitude higher than in roots, in spite of the small stored nitrate pool found inside nodules. This suggests that nodule NR activity had been induced not by nitrate itself but indirectly. Since bacteroids were shown to be responsible for the vast majority of nodule NR activity, the plausible explanation of this effect seems to be a dissimilatory nature of rhizobial NR. Considering that environmental nitrate could cause hypoxia inside nodules, this is the proposed way of the observed nodule NR induction.     

Critical evaluation of the in vivo nitrate reductase assay for detection of two nitrate pools in wheat leaves

The usefulness of the nitrate-free in vivo nitrate reductase assay for the study of nitrate pools in wheat leaves was investigated. Leaf sections from 7-day-old wheat seedlings, exposed 24 h before harvest to 1.5, 3.0 or 5.0 m M KNO₃ were used. After 2 to 4 h of incubation nitrite production ceased, reaching a plateau. The time required to reach the plateau and the level of the plateau increased with increasing endogenous nitrate content. At nitrite plateau the amount of nitrate left in the tissue was independent of the original nitrate content in the tissue. Addition of nitrate at plateau caused resumed nitrite production. It is concluded that nitrate was the limiting factor in nitrite production.
Oxygen inhibited nitrate reduction and stimulated further assimilation of nitrite. A considerable initial leakage of nitrate from tissue to the assay medium, followed by a slower continuous leakage, was observed throughout the incubation. N₂-flushing or inclusion of Triton X-100 in the assay medium increased nitrite production by making more nitrate available for reduction. These treatments also increased the leakage of nitrate. At plateau levels the amount of nitrate left in the tissue was dependent on the oxygen tension in the assay medium. Under low oxygen tension nearly all nitrate in the tissue was available for reduction. Nitrite production at plateau is not a useful index for a metabolic nitrate pool and nitrate left in the tissue is not a useful index for a nitrate storage pool because both parameters are highly dependent on the oxygen tension in the assay medium. Further, in view of the considerable leakage, the nitrate-free in vivo nitrate reductase assay cannot be used to detect two separate nitrate pools in wheat leaves.