Effect of nitrates and sucrose on the induction of nitrate reductase in etiolated seedling leaves from selected barley genotypes in darkness

The parental genotypes, cv. Aramir and R567 line, as well as the selected DH lines C23, C47/1, C41 and C55, growing in darkness differed significantly in the level of NR activity in crude leaf extracts independently of nitrate concentration in the medium. The highest activity of the enzyme was found in the line C23. When plants grew on the medium with 0.5 mM KNO₃, NR activity in that genotype was almost 10-fold higher than in the parents and lines C41, C55 and also 3.5-fold higher than in the line C47/1. An increase of nitrate concentration in the medium to 10 mM caused a significant increase of NR activity in all the genotypes under study. In the line C23 this enzyme activity was only 20% lower than that found previously in the green leaves of that genotype in light. NR from the leaves of C23 and C41 lines was thermally unstable under in vitro conditions. This enzyme in the leaf extracts from the line C23 was characterized by a considerably lower unstability. The lines DH C23 and C41 growing in the dark on the medium with 0.5 mM KNO₃ did not differ in nitrate accumulation in leaves, whereas a larger nitrate content was found in the leaves of the line C41 when it grew on the medium with 10 mM KNO₃. Independently of nitrate concentration in the medium, leaves of the line C23 were found to have a higher sucrose content than those of the line C41. Excised, etiolated leaves of barley treated with 0.5 and 10 mM KNO₃ in dark under conditions favorable to transpiration had a low NR activity. Leaf treatment with a solution containing 10 mM KNO₃ + 0.2 M sucrose caused, on the average, a 13-fold increase of NR activity in comparison to leaves treated only with 10 mM KNO₃ and about a 6-fold increase of this enzyme in comparison to leaves treated with 0.5 mM KNO₃ + 0.2 M sucrose.     

Iron Mediated Nitrate Reductase Activity in Different Parts of Young Maize Seedlings

Incubation of 5-d-old maize seedlings in the half-strength Hoagland's nutrient solution containing 10 mM KNO₃ with FeCl₃ or FeSO₄ (0.5 or 2.0 mM) caused a significant increase in nitrate reductase (NR) activity and slightly increased total protein content in root, shoot and scutellum. In case of root, NADPH:NR activity was inhibited contrary to the NADH:NR activity. In spite of NR activity, nitrate uptake was inhibited from 13 to 37 % by the iron. The results presented demonstrate an isoform specific, organ specific, and to some extent salt specific responses of NR to iron.     

Effect of light and nitrates on nitrate reductase activity and stability in seedling leaves of selected barley genotypes

Parental genotypes (cv. Aramir and line R567) and the selected doubled haploid (DH) lines C23, C47/1, C41, C55 did not differ in NR activity when they grew on a nutrient solution containing 10 mM KNO₃ and were illuminated with light at 124 μmol·m⁻²·s⁻¹ intensity. A decrease of nitrate content in the nutrient medium to 0.5 mM at 44 μmol·m⁻²·s⁻¹ light intensity caused a significant reduction of NR activity in the parental genotypes as well as in the lines C41 and C55. An increase in light intensity to 124 μmol·m⁻²·s⁻¹ raised NR activity in the leaf extracts of these genotypes. However, independently of light intensity, a high level of this enzyme activity was maintained in the line C23 growing on the nutrient medium with 10 mM and 0.5 mM KNO₃. The NR activity in that line dropped only when nitrate content in the medium decreased to 0.1 mM. NR in the leaves of the line C23, as compared to C41, was characterized by a higher thermal stability in all experimental combinations. An increase in light intensity had no significant influence on NR thermal stability in the leaves of the line C41, but induced a significant increase of this enzyme stability in the line C23. The lines C23 and C41 growing on the nutrient medium with 0.5 mM KNO₃ differed appreciably by nitrate concentration in leaves. A higher accumulation of nitrates was detected in the leaves of the line C41.     

Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO₃. Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO₃, subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO₃-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.     

Light and nitrate induction of nitrate reductase in kinetin-and gibberellic acid-treated mustard cotyledons

Nitrate reductase activity in gibberellic acid and kinetin treated mustard (Brassica juncea Coss. cv. T-59 ‘Varuna’) seedlings, grown in the presence or absence of light and/or NO₃ was investigated. While both light and NO₃, alone could induce NR activity, their combination showed additive effects. Kinetin treatment significantly promoted both light- and NO₃- induced NR activities, assayed by either in vivo or in vitro techniques, whereas, gibberellic acid was almost ineffective. In the absence of both light and NO₃, however, phytohormones alone could not induce NR activity. Both light-induced and NO₃ induced NR fractions had a pH optima of 7.5, preferred NADH as an electron donor (NADH: NADPH ratio 2.5) and K_m values for NO₃ was 0.2 mM. Actinomycin D, cycloheximide and tungstate were equally effective in suppressing the development of NR activity after exposure to light or NO₃. These results indicate that two independent NR fractions operate, with apparently identical properties but separate control mechanisms.     

Factors affecting nitrate reductase activity in some monocot and dicot species

Activity of nitrate reductase (NR), the first enzyme in the nitrate-assimilation pathway, was estimated in the cotyledons of the sunflower( Helianthus annuus) using a standardized in-vivo method. Seedlings were grown in the light on a nitrate medium. Various factors that affect NR activity were optimized, including the molarity and pH of the reaction buffer, nitrate concentration, and use of a surfactant. We also determined whether NADH was required for nitrate reduction. The surfactant propanol (2%) gave the best results, and no NADH supplement was necessary: In a separate study, we compared the effect of various culturing components on in-vivo NR activity among monocot and dicot species, and found that Triton X-100 was the best surfactant for monocots whereas dicots performed better with n-propanol. Monocot species also required additional NADH as an external energy source. Moreover, specific purification procedures were needed to enhance NR activity in dicotyledons. Finally, we also assessed the efficacy of in-vivo versus in-vitro procedures for assaying monocots versus dicots.     

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.     

Light dependent reduction of nitrate by pea chloroplasts in the presence of nitrate reductase and C4-dicarboxylic acids

In the presence of purified nitrate reductase (NR) and 1 mM NADH, illuminated pea chloroplasts catalysed reduction of NO₃^? to NH₃ with the concomitant evolution of O₂. The rates were slightly less than those for reduction of NO₂^? to NH₃ and O₂, evolution by chloroplasts in the absence of NR and NADH (ca 6 μg atoms N/mg Chl/hr). Illuminated chloroplasts quantitatively reduced 0.2 mM oxaloacetate (OAA) to malate. In the presence of an extrachloroplast malate-oxidizing system comprised of NAD-specific malate dehydrogenase (NAD-MDH), NAD, NR and NO₃^?, illuminated chloroplasts supported OAA-dependent reduction of NO₃^? to NH₃ with the evolution of O₂. The reaction did not proceed in the absence of any of these supplements or in the dark but malate could replace OAA. The results are consistent with the reduction of NO₃^?by reducing equivalents from H₂O involving a malate/OAA shuttle. The ratios for O₂, evolved: C₄-acid supplied and N reduced: C₄-acid supplied in certain experiments imply recycling of the C₄-acids.     

Substrates regulate the phosphorylation status of nitrate reductase

The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7-day-old oat (Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg²⁺. NR sensitivity to Mg²⁺ remained unchanged when 10 mM nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg²⁺ inhibition of NR, which was strongly diminished in the presence of 10 mM NO₃^?+ 100 µM NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg²⁺ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0-40% saturation fraction (F1). After incubation of F1 with ATP, the sensitivity of the enzyme to Mg²⁺ inhibition remained low, but it strongly increased after mixing F1 with a 45-60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho-NR, would be required to keep the phosphorylated enzyme inactive in a +Mg²⁺ medium. Addition of 10 mM NO₃^?+ 100 µM NADH together with desalted F2 did not prevent Mg²⁺ inhibition suggesting that NO₃^? did not interfere with IP binding to phospho-NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg²⁺, After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 mM NO₃^? at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho-NR induced by the substrates. The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase- or the kinase-proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.     

Protective effect of exogenous nitrate on the mitochondrial ultrastructure ofOryza sativa coleoptiles under strict anoxia

Summary The effect of exogenous KNO₃, the terminal acceptor of electrons in oxygen-free medium, on mitochondrial ultrastructure and on the growth rate of 4-day-old rice coleoptiles under strictly anoxic conditions was studied. Exogenous nitrate (10 mM) did not exert any significant effect on the growth rate of coleoptiles of intact seedlings compared to their growth in KNO₃-free medium. Anaerobic incubation of detached coleoptiles in KNO₃-free medium for 48 h resulted in the complete destruction of mitochondrial and other cell membranes. In the presence of KNO₃, no mitochondrial-membrane destruction was observed even after 48 h anoxia although the mitochondrial ultrastructure was modifed. Cristae were arranged in parallel rows and elongated dumbbell-shaped mitochondria appeared in some cells. The data obtained indicate a protective role of exogenous nitrate as electron acceptors in oxygen-free medium. The results of the present investigation are discussed and compared with reports of either markedly damaging or favorable effects of exogenous nitrate on the growth, metabolism, and energetics of rice and other plants under hypoxic and anoxic conditions.     

Two differentially regulated nitrate reductases required for nitrate-dependent,microaerobic growth of Bradyrhizobium japonicum

Native PAGE of Triton x-100-solubilized membranes from Bradyrhizobium japonicum strain PJ17 grown microaerobically (2% O₂, v/v) in defined nitrate-containing medium resolved two catalytically active nitrate reductase (NR) species with apparent molecular masses of 160 kDa (NR_I) and 200 kDa (NR_II). NR_I and NR_II were also found in membranes from cells of strain PJ17 that were first grown in defined medium with glutamate and further incubated microaerobically in the presence of 5 mmol/l KNO₃. However, only NR_I was detected in cell membranes of strain PJ17 when nitrate was omitted from the microaerobic incubation medium. Four mutants unable to grow at low O₂ tension in the presence of nitrate were isolated after transposon Tn5 mutagenesis. Membranes from mutants GRF110 and GRF116 showed mainly NR_I, while the other two mutants, GRF3 and GRF4, expressed mostly NR_II. These results indicate that the ability of B. japonicum PJ17 to grow under microaerobic conditions depends upon the presence of two membrane-bound NR enzymes whose synthesis seem to be independently induced by microaerobiosis (NR_I) or by both microaerobiosis and nitrate (NR_II).Abbreviations NR Nitrate reductase - M _r Relative molecular mass - PMSF Phenylmethylsulfonyl fluoride     

Influence of fruit load and environmental factors on nitrate reductase activity and on concentration of nitrate and carbohydrates in leaves of eggplant (Solanum melongena)

Both the in vivo (+ nitrate) nitrate reductase (NR) activity (leaf disks incubated in the presence of KNO₃) and the in vivo (? nitrate) NR activity (leaf disks incubated without KNO₃) in leaves of eggplant (Solanum melongena L. cv. Bonica) were affected by rapidly growing fruits. Plants with a fruit load showed more pronounced diurnal variation in (+ nitrate) NR activity and higher (? nitrate) NR activity than plants without fruit. The higher (? nitrate) NR activity was accompanied by higher nitrate and lower sucrose and starch contents of leaves. The more pronounced diurnal changes in (+ nitrate) NR activity were paralleled by more pronounced diurnal variation in carbohydrate content of leaves. Fruit removal led to a decrease in both (? nitrate) NR activity and nitrate concentration in leaves, while the carbohydrate content increased. Plants supplied with ammonium instead of nitrate showed only slightly lower (+ nitrate) but no (? nitrate) NR activity. As for plants treated with nitrate, diurnal changes in (+ nitrate) NR activity were most pronounced in leaves of plants with fruit and this again was paralleled by a more pronounced diurnal variation in the carbohydrate concentration in the leaves. Increasing the oxygen level of the atmosphere to 50% led to a dramatic decrease in the (+ nitrate) NR activity and to an increase in both (? nitrate) NR activity and nitrate concentration, which was accompanied by decreasing carbohydrate contents of the leaves. Low light intensities and extended dark periods caused similar changes in NR activity and nitrate and carbohydrate concentrations in leaves. Increasing the nitrate concentration in the nutrient solution led to a rise in (+ nitrate) and (? nitrate) NR activity, but only the (? nitrate) NR activity paralleled the nitrate concentration in the leaves. This increase in the nitrate concentration was accompanied by a decrease in the carbohydrate content of the leaves. It is concluded that the level of and the diurnal changes in both (+ nitrate) and (? nitrate) NR activity and the concentration of nitrate in the leaves are dependent upon their carbohydrate status.     

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.     

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     

Pyridine nucleotide specificity and other properties of purified nitrate reductase from Chlorella variegata

Nitrate reductase (NR) (EC 1.6.6.2) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise NADH or NADPH for nitrate reduction with apparent K _m values of 11.5 M and 14.5 M, respectively. Apparent K _m values for nitrate are 0.13 mM (NADH-NR) and 0.14 mM (NADPH-NR). The diaphorase activity of the enzyme is inhibited strongly by parachloromercuribenzoic acid; NADH or NADPH protects the enzyme against this inhibition. NR proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of NAD(P)H and cyanide causes a reversible inactivation of the NR proper activity although preincubation with either NADH or NADH and ADP has no significant effect.Abbreviations NR Nitrate reductase - FAD Flavin-adenine dinucleotide - FMN Riboflavin 5-phosphate - p-CMB para-Chloromercuribenzoic - BV Benzyl viologen     

Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves

An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 0.2 m KNO₃ (with or without phosphate buffer, pH 7.5) and incubation of the infiltrated tissue and medium under essentially anaerobic conditions in the dark. Nitrite production, for computing enzyme activity, was determined on aliquots of the incubation media, removed at intervals.     

Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A x W182E) : Two Molecular Forms of the Enzyme

The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N₂ and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0°C and for several months at −80°C. The enzyme was purified using (NH₄)₂SO₄ fractionation, brushite-hydroxyl-apatite chromatography and blue-sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO₃. About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO₂⁻ per minute per milligram protein). A sequential elution with NADH followed by KNO₃ (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO₃⁻-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed.     

Regulation of nitrate reductase by chloramphenicol in the leaves ofVigna mungo (L.)Hepper (Black-gram)

Chloramphenicol has been found to inhibit nitrate reductase activity in black-gram leaves. It inhibitsin vivo nitrate reductase activity up to 50–67%, and the catalytic property of the enzyme up to a maximum of 70–98%. Modulators, such as KNO₃, NADH and HCO₃ could not protect enzyme inhibition by chloramphenicol. It is suggested that the chloramphenicol inhibition is mainly through its effect on the catalytic process of the enzyme.     

Factors affecting in vivo nitrate reductase activity in triticale

Factors influencing in vivo nitrate reductase activity in triticale (×Triticosecale Wittmack) primary leaves were investigated. Nitrate reductase activity was found to be a function of reaction time or tissue weight. In the range of 1–10 mm, the optimum slice width for nitrate reductase activity in triticale was found to be 1–2 mm. The optimum exogenous nitrate concentration is 300 mM. Substantial nitrite production was obtained even when exogenous nitrate was omitted from the assay. Of the five low molecular weight organic solvents tested, n-propanol is the most effective in enhancing enzyme activity. The optimum n-propanol concentration is 1% (v/v). The concentration of phosphate buffer (pH 6) does not affect nitrate reductase activity. Enzyme activity drops significantly below or above pH 6. In our system, nitrite production is enhanced by incubating under nitrogen, instead of air. The highest level of in vivo activity of nitrate reductase was found to be 10–15 cm from tip, which is close to the basal meristem of triticale primary leaves. Younger but physiologically mature leaves have higher nitrate reductase activity than old leaves.     

Effect of nitrate on water transfer across roots of nitrogen pre-starved maize seedlings

The addition of 10 mM KNO₃ to the solution bathing the roots of young nitrogen-starved seedlings of Zea mays L. enhanced root water transfer within 15 h, compared with 10 mM KCl addition. The free exudation flux was 2.2–3.9 times higher in excised KNO₃-treated roots than in KCl-treated ones. Cryo-osmometry data for xylem sap suggested that, compared with chloride, nitrate treatment increased the steady solute flux into the xylem, but did not modify the osmotic concentration of sap. Root growth was not significantly modified by nitrate within 15 h. Root hydraulic conductances were measured by using either hydrostatic-pressure or osmotic-gradient methods. During hydrostatic experiments, the conductance (k_p), which is thought to refer mainly to the apoplasmic pathway, was 1.6 times larger in KNO₃-than in KCl-treated plants. From experiments in which polyethylene glycol (PEG) 8000 was used as external osmolyte, osmotic conductances (k_s) were found to be smaller by 5–20 times than k_p for the two kinds of plants. The KCl-treated roots were characterized by a low k_s which was the same for influx or efflux of water. By contrast, KNO₃-treated roots exhibited two distinct conductances k_s1 and k_s2, indicating that influx of water was easier than efflux when the water flow was driven by the osmotic pressure gradient. Infiltration of roots with KNO₃ solution supported the idea that nitrate might enhance the efficiency of the cell-to-cell pathway. The low k_s value of KCl-treated roots and the existence of two contrasting k_s values (k_s1 and k_s2) for KNO₃-treated roots are discussed in terms of reversible closing of water channels.