In Vivo Determination of Parameters of Nitrate Utilization in Wheat (Triticum aestivum L.) Seedlings Grown with Low Concentration of Nitrate in the Nutrient Solution

Six genotypes of winter wheat (Triticum aestivum L.) differing in grain protein concentration were grown on a nutrient solution containing low concentrations of NO₃⁻ (2 millimolar). Total NO₃⁻ uptake varied between genotypes but was not related to grain protein content. An in vivo nitrate reductase assay was used to determine the affinity of the enzyme for NO₃⁻, and large phenotypic variations were observed. In vivo estimations of the concentration and size of the metabolic pool were variable. However, the three genotypes with the higher ratios of metabolic pool size to leaf total NO₃⁻ concentration were the high protein varieties. It is proposed that a high affinity of nitrate reductase for nitrate might be a biochemical marker for the capacity of the plant to continue assimilating NO₃⁻ for a longer period during the last stage of growth.     

Increase in nitrate reductase activity in the presence of sucrose in bean leaf segments

The supply of sucrose to leaf segments from light-grown bean seedlings caused a substantial increase in substrate inducibility of in vivo and in vitro nitrate reductase activity but only a small increase in total protein. Cycloheximide and chloramphenicol inhibited the increase in enzyme activity by nitrate and sucrose. The in vivo decline in enzyme activity in nitrate-induced leaf segments in light and dark was protected by sucrose and nitrate. The supply of NADH also protected the decline in enzyme activity, but only in the light. In vitro stability of the extracted enzyme was, however, unaffected by sucrose. The size of the metabolic nitrate pool was also enhanced by sucrose. The experiments demonstrate that sucrose has a stimulatory effect on activity or in vivo stability ' of nitrate reductase in bean leaf segments, which is perhaps mediated through increased NADH level and/or mobilization of nitrate to the metabolic pool.     

Differential effect of irradiance and nutrient nitrate on the relationship of in vivo and in vitro nitrate reductase assay in chlorophyllous tissues

Growth at increasing continuous irradiance (at high nutrient nitrate) and nutrient nitrate concentrations (at high continuous irradiance) furnished increases in the in vivo and in vitro nitrate reductase activities of corn (Zea mays L.), field peas (Pisum arvense L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and globe amaranth (Gomphrena globosa L.) leaves and of marrow (Cucurbita pepo L.) cotyledons. Ratios of in vivo to in vitro activity declined exponentially in all species with increasing nitrate reductase levels promoted by nutrient nitrate. The ratios were more nearly independent of nitrate reductase levels generated by adjusting the irradiance; major exceptions were marrow and wheat at low (1.5 klux and less) irradiances and peas throughout the irradiance range, where decreases in the ratio were accompanied by increases in in situ nitrate concentration. The ratio also increased at the highest irradiance (39.2 klux) in wheat and barley, associated with a decline of in vitro nitrate reductase. These differences in response to irradiance and nutrient nitrate indicate that the in vivo assay does not provide a simple measure of nitrate reductase but rather yields a more composite measure of nitrate reduction, possibly related both to nitrate reductase level and to the supply of reductant for in vivo activity.     

A Re-evaluation of the Nitrate Reductase Content of the Maize Root

The standard procedure for the in ritro extraction of nitrate reductase from the tip region (0-2 cm) of the primary root of the maize (Zea mays L.) seedling indicated an activity of the enzyme approximately 5-fold higher than that obtained with an in vivo assay. In more mature regions of the primary root the ratio of in vitro to in vivo activity was much lower and in older seedlings was less than unity. The mature root extracts had a more labile nitrate reductase and a higher level of an inactivating enzyme. The use of phenylmethylsulphonyl fluoride in the extraction medium gave only a partial protection of the nitrate reductase from the old root samples. Casein (3%) resulted in a greatly increased yield of nitrate reductase (36-fold with one sample) and a more constant in vitro-in vivo activity ratio for all root samples. With casein in the extraction medium, much higher levels of nitrate reductase were recovered from the mature root zone, and the root content of this enzyme was now shown to be quite a significant proportion of the total in the maize seedling. Casein was shown to inhibit the action of the inactivating enzyme on nitrate reductase. Evidence is also presented for a nitrate reductase inactivating enzyme in the maize scutella and leaf tissues and in the roots and shoots of pea seedlings.     

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.     

In vivo Characterization of Nitrate Reductase Activity in Cotton

The in vivo nitrate reductase activity in leaf tissue of cotton (Gossypium hirsutum L.) was characterized. Enzymatic activity was linear with time up to 60 min. The assay for nitrate reductase activity was optimized in leaf slices 400 μm wide incubated in an anaerobic system at 30°C, in a 0.02 M KNO₃ medium at pH 7.0 with 1 % propanol. In vivo activity was highest in recently matured leaves at the top of the plant. Both light and nitrate enhanced in vivo enzymatic activity. The activity was highest after 9 hours in the light and then decreased steadily for several more hours even in the presence of light. The nitrate reductase activity was more strongly correlated to the levels of NO₃-N in the culture solution than to the NO₃-N level in the tissue. The utility of this technique in nitrate reductase assay in a tissue containing large amounts of phenolic compounds is discussed.     

In vivo nitrate reductase activity in dark- and light-grown sugarcane callus

The in vivo nitrate reductase activity in 8 day old dark-grown sugarcane callus was over three fold that of the light-grown callus. NADH (0.3 mM) in the reaction system, increased the in vivo nitrate reductase activity by more than two fold both in the dark- and the light-grown callus tissues. The NADH dependence of nitrate reductase activity followed Michaelian kinetics. The apparent Km values for NADH were 0.083 mM and 0.20 mM, respectively, for the dark- and the light-grown callus. In vivo nitrate reductase activity in green sugarcane leaves (field grown) was unaffected by NADH in the reaction system. Under the standard conditions of assay up to 60% of the NADH penetrated into the sugarcane callus within 2 min. No penetration of NADH into the sugarcane leaf discs was, however, recorded under identical conditions.NCL Communication No. 3454     

Differential Carbon Monoxide Sensitivity of Cytochrome-Oxidase in the Leaves of Tall and Dwarf Wheat Cultivars

Differential response in the leaves of tall and dwarf wheat to CO, an inhibitor of cytochrome oxidase and to SHAM, an inhibitor of alternative oxidase appears to be correlated with presence of Rht dwarfing genes. This was detected by in vivo nitrate reductase assay after CO treatment and direct O₂ uptake in presence of SHAM. Pretreatment of the leaves with Triton X-100 at a concentration which specifically inhibits the accessibility of exogenous NAD(P)H to alternative oxidase, Significantly enhanced the CO response as assessed by in vivo NR assay. This supports the hypothesis that the competition for NADH between NR and mitochondrial respiration is regulated by NADH-dehydrogenase located on the outer surface of inner mitochondrial membrane.     

Limitations to the Measurement of In Vitro Nitrate Assimilation by Exogenous Additives and Endogenous Interference Factors in the Leaves of Zea mays L. Seedlings

An evaluation of existing assay procedures for the measurementof nitrate assimilation in the leaves of Zea mays L. has highlightedlimitations in established in vitro assay techniques. Both exogenouslyadded compounds and endogenous leaf components affected theresults of an in vitro nitrate reductase (NADH: oxido-reductase,EC 1.6.6.1 [EC] .) assay. Reducing agents employed as enzyme protectantswere excluded from the assay in order to accurately measurethe concentration of nitrogen compounds by colorimetric andHPLC analysis. Endogenous nitrate levels in a leaf extract asmeasured by these two analytical techniques indicated significantinterference in the colorimetric method due to the presenceof various organic compounds. This interference was most apparentat low nitrate concentrations, however, changes in nitrate concentrationappeared to be more closely comparable between the two techniques.In addition, endogenous leaf components also interfered withthe precise determination of nitrite that had accumulated duringan in vitro nitrate reductase assay. These endogenous factorsacted directly upon the colorimetric assay of nitrite by a concentration-dependentreaction with the diazotizing reagent sulphanilamide. The interferingcomponents were of low molecular weight ( 5000 daltons) andeasily separable from nitrate reductase by molecular sieve chromatography.Their interference in the nitrite assay could only be partiallyprevented by heating or storage, while other treatments studied,including those frequently used to terminate an in vitro assaysuch as zinc acetate precipitation or chloroform extraction,had less effect in alleviating the interference. Similar endogenouscomponents which affected the colorimetric assay of nitritewere also found in leaf extracts from wheat, pea, soybean andsunflower seedlings. Zea mays L., nitrate reductase, reducing agents, plant interference factors     

Glycine supports in vivo reduction of nitrate in barley leaves

Glycine, a photorespiratory intermediate, enhanced the in vivo reduction of nitrate in barley (Hordeum vulgare L.) leaf slices, when included in the assay medium. Isonicotinyl hydrazide, an inhibitor of glycine oxidation, partially reduced NO₂⁻ production. The enhancement caused by glycine treatment was reversed by isonicotinyl hydrazide when both were present together in the medium. Similar effects were observed when the excised leaves were preincubated with the metabolite and the inhibitor. Glycine also partially relieved the inhibition of nitrate reduction caused by malonate, an inhibitor of the tricarboxylic acid cycle. The results support the hypothesis that glycine decarboxylation activity is a source of NADH for nitrate reductase activity.     

Seasonal Fluctuations of Nitrate Reductase Activity in Ditelosomic Stocks of Wheat

In vivo nitrate reductase activity was measured over the seasonin individual organs of the main tiller of the euploid and fourditelosomics of the wheat variety Chinese Spring. A generalbiphasic profile of leaf activity was obtained in vivo and invitro, the peaks corresponding to emergence of leaf 7 (the pre-flagleaf) and the ear. A wide range of seedling nitrate reductaseactivity was exhibited by these stocks and a significant positivecorrelation was obtained between seedling activity and the dailymean of activity integrated over the season. Seasonal euploid-ditelosomicdifferences in nitrate reductase activity reflected differencesover the vegetative stage, but no significant inter-stock differencesin activity were found over the reproductive stage. The highseasonal nitrate reductase activity of ditelo-7B^L and –7B^Sappeared to be due largely to high mean activities of individualleaves, while that of ditelo-4B^L depended on the longer durationof active tissue than was exhibited by the euploid. Significantactivity was assayed in non-leaf organs, especially the rachisand awns, and constituted an important proportion of the totaltiller activity late in the season.     

The Occurrence of Nitrate Reduction in the Leaves of Woody Plants

Nitrate reductase activities greater than 02 µmol h^–1g^–1 f. wt, measured by an in vivo assay, occurred in 41per cent of a large sample (555 species) of woody plants. Ifseveral taxonomic groups (Gymnosperms, Ericaceae and Proteaceae)with consistently low activities were discounted activitiesgreater than 02 µmol h^–1 g^–1 f. wt occurredin 73 per cent of the species. This compares with 93 per centin herbaceous species, suggesting that leaf nitrate reductionis of common occurrence in woody plants. In a small sample ofspecies leaf nitrate reductase activity correlated with nitrateconcentration in the xylem sap. Low activities occurred consistentlyin the Gymnosperms, Ericaceae and Proteaceae. Feeding cut shootsof representatives of these groups with nitrate caused inductionof leaf nitrate reductase activity in the Gymnosperms and Proteaceae,but only limited induction in the Ericaceae. The Ericaceae,with the exception of two species, had low activities and lownitrate reductase inducibility. Root assimilation may predominatein the Gymnosperms and Proteaceae. It is suggested that nitratereduction generally occurs in the leaves of trees from a varietyof plant communities and that this may be related to the lowerenergy cost of leaf, as opposed to root, nitrate assimilation. Nitrate reductase, trees and shrubs, leaves, nitrate assimilation, nitrate translocation, nitrate reductase induction, energy cost, plant ecology     

Involvement of Reversible Inactivation in the Regulation of Nitrate Reductase Enzyme Levels in Chlamydomonas reinhardtii

All nitrate reductase-related activities of Chlamydomonas reinhardtii wild-type and mutant 305 cells were degraded in vivo under conditions in which the reversible inactivation could take place. When the enzyme was in the inactive form, half-lives of all nitrate reductase-related activities in wild and mutant 305 strains decreased significantly. The only nitrate reductase-related activity present in mutant 104, nitrate reductase-diaphorase, was incapable of undergoing reversible inactivation and was not degraded under any of the conditions tested. Addition of nitrate to inactive nitrate reductase of mutant 305 caused the in vivo reactivation of the enzyme and halted its degradation. Our results indicate that reversibly inactivated nitrate reductase from C. reinhardtii is the main target for a degradation system, and that nitrate reductase related diaphorase must be integrated in a reversibly inactive nitrate reductase complex to undergo degradation. A physiological role for the interconversion process of nitrate reductase can be understood on the basis of these facts.     

Reevaluation of anaerobic nitrite production as an index for the measurement of metabolic pool of nitrate

The use of anaerobic nitrite production as an index for the measurement of metabolic pool of nitrate was reevaluated using primary leaves of 7-day-old barley and 10-day-old soybean seedlings. The seedlings were grown in nutrient solutions containing 5 to 15 millimolar nitrate. The nitrate-free in vivo assay system of nitrate reductase was used for measuring the production of nitrite. Both the duration and extent of nitrite production were dependent on the level of endogenous nitrate in the tissue. At cessation of nitrite production, 30 to 50% of the endogenous nitrate was reduced to nitrite. Nitrate from the tissue leaked continuously into the surrounding medium so that, at cessation of nitrite production, nitrate supply from the tissue was exhausted. The cessation of nitrite production, therefore, may have been caused by the depletion of endogenous nitrate from the tissue. It is concluded that anaerobic nitrite production is not a valid index for the measurement of the size of the metabolic pool of nitrate.     

Nitric Oxide Emissions from Soybean Leaves during in Vivo Nitrate Reductase Assays

Recent work identified acetaldehyde oxime as the predominant product purged by inert gases from anaerobic in vivo nitrate reductase (NR) assays of soybean (Glycine max [L.] Merr.) leaves. Another recent study supported earlier research findings which identified the primary product evolved from soybean leaves as nitric oxide (NO). This paper provides evidence that eliminates acetaldehyde oxime and confirms that NO is the primary nitrogenous product purged from the in vivo NR assay system. A portion of the evidence is based on the high water solubility of acetaldehyde oxime. Other evidence presented is the failure by chemical and spectrophotometric means to detect oximes in gases emitted in the purging of the reaction medium or in the leaf tissues. The gaseous product from the in vivo NR assay system reacted identically to NO standards and did not resemble acetaldehyde oxime standards. It was concluded that the predominant N product within the leaves was nitrite and that the predominant gaseous N product evolved from the assay was NO.     

Comparison of in Vivo and in Vitro Assays of Nitrate Reductase in Wheat (Triticum aestivum L.) Seedlings

The effectiveness of the in vivo and in vitro assays for nitrate reductase (NR) in estimating the amounts of reduced N made available to plants was tested against the daily increases in reduced N (Nesslerization) actually accumulated by the plant. With growth-chamber-grown wheat seedlings, the average ratio values (input of reduced N as estimated by the in vitro assay to actual accumulation of N by the plant) were 3.9 for shoots, 3.7 for the roots, and 4.1 for the entire plant, over a 10-day period. With the in vivo assay, the average ratio values were 0.7 for the shoot, 1.8 for the root, and 0.9 for the entire plant. Although the linear regressions between the accumulated N in the plant and the estimated N input (by both in vitro and in vivo assays) were significant and positive, the in vivo assay provided the closest approximation of the actual amount of N accumulated.     

Limitations on Leaf Nitrate Reductase Activity during Flowering and Podfill in Soybean

The objective of this study was to identify factors which limit leaf nitrate reductase (NR) activity as decline occurs during flowering and beginning seed development in soybean (Glycine max [L.] Merr. cv Clark). Level of NR enzyme activity, level of reductant, and availability of NO₃⁻ as substrate were evaluated for field-grown soybean from flowering through leaf senescence. Timing of reproductive development was altered within one genotype by (a) exposure of Clark to an artificially short photoperiod to hasten flowering and podfill, and (b) the use of an early flowering isoline. Nitrogen (N) was soil-applied to selected plots at 500 kilograms per hectare as an additional variable. Stem NO₃⁻ concentration and in vivo leaf NR activity were significantly correlated (R² = 0.69 with nitrate in the assay medium and 0.74 without nitrate in the medium at P = 0.001) across six combinations of reproductive and soil N-treatment. The supply of NO₃⁻ from the root to the leaf tissue was the primary limitation to leaf NR activity during flowering and podfill. Levels of NR enzyme and reductant were not limiting to leaf NR activity during this period.     

In vivo nitrate reduction in relation to nitrate uptake, nitrate content, and in vitro nitrate reductase activity in intact barley seedlings

A study was done to relate the in vivo reduction of nitrate to nitrate uptake, nitrate accumulation, and induction of nitrate reductase activity in intact barley seedlings (Hordeum vulgare L. var. `Numar'). The characteristics of nitrate uptake in response to both time and ambient concentration of nitrate regulated reduction and accumulation. Uptake, accumulation, and in vivo reduction achieved steady state rates in 3 to 4 hours, whereas extractable (in vitro) nitrate reductase activity was still increasing at 12 hours. In vivo reduction of nitrate was better correlated exponentially than linearly over time with in vitro activity of nitrate reductase. A similar relationship occurred over increasing concentration of nitrate in the ambient solution. The results suggest that the rate of in vivo reduction of nitrate in barley seedlings may be regulated by the rate of uptake at the ambient concentrations of nitrate employed in the study.     

Control and variation ofin vivo nitrate reductase activity inLolium perenne L. cv S24

Summary Variation in nitrate reductase activity associated with vegetative development and morphological differences inLolium perenne L. cv S24 was examined using anin vivo technique of enzyme assay. Little difference in enzyme activity per unit weight of leaf laminar tissue was found between tillers, between and within leaf laminae but activity in leaf laminae, was much higher than that of leaf sheaths; the activity of shoot material was greater than the apparent root activity. Senescence of leaf material was not accompanied by marked changes invivo nitrate reductase activity.     

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.