Nitrogen—Inorganic Forms

An analysis is described for the rapid determination of nitrate‐N in plant extracts. The complex formed by nitration of salicylic acid under highly acidic conditions absorbs maximally at 410 nm in basic (pH>12) solutions. Absorbance of the chromophore is directly proportional to the amount of nitrate‐N present. Ammonium, nitrite, and chloride ions do not interfere.

Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid

The Determination of Nucleic Acids

The mineral nutrient needed in greatest abundance by plants is nitrogen. Plants, however, must compete for nitrogen in the soil with abiotic and biotic processes such as erosion, leaching, and microbial consumption. Soil nitrogen is also lost when crops are harvested and plant material is removed from the soil. To be competitive, plants have evolved several mechanisms to acquire nitrogen at low concentrations and to use a variety of forms of nitrogen. Plants can assimilate inorganic forms, such as nitrate and ammonia, and organic forms, such as urea. Some plants, including legumes, can fix dinitrogen gas in association with symbiotic bacteria (see Mylona et al., 1995, this issue). This review focuses on the assimilation of nitrate, a transient yet critical form for plants. Two key questions arise when considering nitrate assimilation. First, how do plants acquire optimal quantities of nitrate when the soil nitrate concentration can vary from 10 pM to 100 mM? Second, how is nitrate assimilation integrated into the overall metabolic program of plants so that optimal amounts of energy and carbon are provided? What is known about nitrate assimilation at the cellular leve1 is summarized in Figure 1. Once taken up, nitrate is either stored in the vacuole or reduced to nitrite by nitrate reductase (NR). Nitrite enters the chloroplast (or plastid in the root) and is then reduced to ammonja by nitrite reductase (NiR). Ammonia can then be fixed into carbon by the action of glutamine synthetase (see Lam et al., 1995, this issue). Reducing energy is provided in the form of NAD(P)H for NR and reduced ferredoxin for NiR. Glutamate provides the immediate carbon skeleton. Because nitrate assimilation produces several hydroxide ions, organic acids are also required for pH homeostasis. Coordinating these steps is a regulatory network that is responsive to both interna1 and external signals, some of which are diagrammed in Figure 1. Ultimately, it is the understanding of this regulatory network that will answer the aforementioned key questions. Genetic analysis of nitrate assimilation has been possible because mutants blocked in the pathway can be rescued by providing ammonia as a source of nitrogen. Such mutants are obtained either by screening directly with an in vitro assay for plants defective in nitrate reduction or by selecting plants that are resistant to chlorate, the chlorine analog of nitrate. When chlorate is taken up and reduced by NR, toxic chlorite is produced. In higher plants, chlorate-resistant mutants are impaired in nitratelchlorate reduction because they have either a defective nitrate reductase structural gene (NIA) or a defective molybdenum cofactor gene (CNX). The one exception is the chll mutant of Arabidopsis, which is defective in nitrate/chlorate uptake. Only in fungi and algae have regulatory mutants been descri bed. There are many excellent recent reviews of the nitrate assimilation field (Pelsy and Caboche, 1992; Warner and Kleinhofs, 1992; Cove, 1993; Crawford and Arst, 1993; Marzluf, 1993; Crawford, 1994; Hoff et al., 1994; Huppe and Turpin, 1994). This review provides an update on recent molecular advances that have uncovered genes and mechanisms responsible for nitrate uptake, reduction, and regulation.

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Nitrate: nutrient and signal for plant growth.

AGPase, ADP glucose pyrophosphorylase
GS, glutamine synthetase
GOGAT, glutamate : oxoglutarate amino transferase
NADP-ICDH, NADP-dependent isocitrate dehydrogenase
NR, nitrate reductase
OPPP, oxidative pentose phosphate pathway
3PGA, glycerate-3-phosphate
PEPCase, phosphoenolpyruvate carboxylase
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase
SPS, sucrose phosphate-synthase

This review first summarizes the numerous studies that have described the interaction between the nitrogen supply and the response of photosynthesis, metabolism and growth to elevated [CO2]. The initial stimulation of photosynthesis in elevated [CO2] is often followed by a decline of photosynthesis, that is typically accompanied by a decrease of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), an accumulation of carbohydrate especially starch, and a decrease of the nitrogen concentration in the plant. These changes are particularly marked when the nitrogen supply is low, whereas when the nitrogen supply is adequate there is no acclimation of photosynthesis, no major decrease in the internal concentration of nitrogen or the levels of nitrogen metabolites, and growth is stimulated markedly. Second, emerging evidence is discussed that signals derived from nitrate and nitrogen metabolites such as glutamine act to regulate the expression of genes involved in nitrate and ammonium uptake and assimilation, organic acid synthesis and starch accumulation, to modulate the sugar-mediated repression of the expression of genes involved in photosynthesis, and to modulate whole plant events including shoot–root allocation, root architecture and flowering. Third, increased rates of growth in elevated [CO2] will require higher rates of inorganic nitrogen uptake and assimilation. Recent evidence is discussed that an increased supply of sugars can increase the rates of nitrate and ammonium uptake and assimilation, the synthesis of organic acid acceptors, and the synthesis of amino acids. Fourth, interpretation of experiments in elevated [CO2] requires that the nitrogen status of the plants is monitored. The suitability of different criteria to assess the plant nitrogen status is critically discussed. Finally the review returns to experiments with elevated [CO2] and discusses the following topics: is, and if so how, are nitrate and ammonium uptake and metabolism stimulated in elevated [CO2], and does the result depend on the nitrogen supply? Is acclimation of photosynthesis the result of sugar-mediated repression of gene expression, end-product feedback of photosynthesis, nitrogen-induced senescence, or ontogenetic drift? Is the accumulation of starch a passive response to increased carbohydrate formation, or is it triggered by changes in the nutrient status? How do changes in sugar production and inorganic nitrogen assimilation interact in different conditions and at different stages of the life history to determine the response of whole plant growth and allocation to elevated [CO2]?

https://www.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-3040.1999.00386.x

The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background

An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 02 m KNO 3 (with or without phosphate buffer, pH 75) 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 By adding, separately, various metabolites of the glycolytic, pentose phosphate, and citric acid pathways to the infiltrating media, it was possible to use the in vivo assay to determine the prime source of reduced nicotinamide adenine dinucleotide (NADH) required by the cytoplasmically located NADH-specific nitrate reductase It was concluded that sugars that migrate from the chloroplast to the cytoplasm were the prime source of energy and that the oxidation of glyceraldehyde 3-phosphate was ultimately the in vivo source of NADH for nitrate reduction This conclusion was supported by experiments that included: inhibition studies with iodoacetate; in vitro studies that established the presence and functionality of the requisite enzymes; and studies showing the effect of light (photosynthate) and exogenous carbohydrate on loss of endogenous nitrate from plant tissue The level of nitrate reductase activity obtained with the in vitro assay is higher (25- to 20-fold) than with the in vivo assay for most plant species The work done to date would indicate that the in vivo assays are proportional to the in vitro assays with respect to ranking genotypes for nitrate-reducing potential of a given species The in vivo assay is especially useful in studying nitrate assimilation in species like giant ragweed from which only traces of active nitrate reductase can be extracted

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

Five-or six-day old seedlings of corn ( Zea mays L) were exposed to 025 mm Ca(NO 3 ) 2 , 10 mm sodium 2-[N-morpholino]-ethanesulfonate, 5 μg Mo per liter and 50 μg of chloramphenicol per ml at pH 6 Nitrate uptake was determined from depletion of the ambient solution The pattern of nitrate uptake was characterized, after the first 20 minutes, by a low rate which increased steadily to a maximal rate by 3 to 4 hours Transfer of nitrate to the xylem did not totally account for the increase Development of the maximal accelerated rate did not occur at 3 C with excised roots nor with seedlings whose endosperm had been removed Use of CaCl 2 rather than Ca(NO 3 ) 2 resulted in a linear rate of chloride uptake during the first 4 hours, and chloride uptake was not as restricted by endosperm removal as was nitrate uptake Nitrite pretreatments or the addition of cycloheximide (2 μg ml −1 ), puromycin (400 μg ml −1 ) and 6-methylpurine (05 mm) restricted maximal development of the accelerated nitrate uptake rate Actinomycin D (20 μg ml −1 ) inhibited the rate only after about three hours exposure The RNA and protein synthesis inhibitors also restricted nitrate reductase induction in the apical segments of the root tissue The data suggest that development of the maximal accelerated rate of nitrate uptake depended upon continuous protein synthesis, and the hypothesis that synthesis of a specific nitrate transport protein must occur is advanced But the alternative hypothesis, ie , that induction of nitrate reductase (and/or a consequence of the act of nitrate reduction) provided the required stimulus, remains tenable

Nitrate Uptake by Dark-grown Corn Seedlings: Some Characteristics of Apparent Induction.

Use of a dissimilatory nitrate reductase from Escherichia coli and formate as a reductive system for nitrate assays

RECENT work (3, 5) has shown that when cauliflower or corn plants are placed in the dark they rapidly lose their nitrate reductase activity. Corn plants lose approximately 50 and 90% of their nitrate reductase activity within 24 and 48 hours, respectively, after being placed in the dark. The variations in nitrate reductase activity are consistent with the diurnal variation in nitrate content of various plant species (1, 4). These observations would permit the prediction that corn plants will undergo a diurnal variation in nitrate reductase activity under field conditions. Miller (10) grew corn plants to maturity under a series of artificial shade structures that reduced the light intensity, 0, 30, 60, 70, 80, and 90%. The effects of artificial shade on the growth, morphology, and ear set were comparable to the effects of competitive (self) shading obtained increasing plant population. Hybrid Hy2 X Oh7 (recommended for high density planting) is superior in yield to hybrid WF9 )< C103 (not recommended for high density planting) when grown under the artificial shade structures. In subsequent experiments with artificial shade, Knipmeyer (7) found that the nitrate content of young (2 to weeks) corn plants increased in proportion to the decreased light intensity. He also found that the corn plants grown at high planting rates (28,000 plants per acre) tended to have a higher concentration of nitrate (significantly different at the 8 to 10% level) than plants grown at the low (7,000 plants per acre) rate. The accumulation of nitrate by the crowded plants was attributed to the depressing effects of the low light intensity (competitive plane shade) on the nitrate reductase activity (5) and to an adequate (high) soil nitrogen content and sufficient soil moisture to permit free movement of nitrates (7). Precise information concerning the effect of either artificial or competitive plant shading on the level of nitrate reductase activity of corn plants grown under field conditions is not known. The objectives of the work reported herein were to determine (a) if corn plants grown under field conditions would show a diurnal variation in nitrate reductase, nitrate, and water soluble protein content; and (b) if reduction of light intensity by either artificial competitive plant shading would affect nitrate reductase activity.

Diurnal Variation and Other Light Effects Influencing the Activity of Nitrate Reductase and Nitrogen Metabolism in Corn 1

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

R. H. Hageman

Papers

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

Nitrate Uptake by Dark-grown Corn Seedlings: Some Characteristics of Apparent Induction.

Use of a dissimilatory nitrate reductase from Escherichia coli and formate as a reductive system for nitrate assays

Diurnal Variation and Other Light Effects Influencing the Activity of Nitrate Reductase and Nitrogen Metabolism in Corn 1

The Occurrence of Nitrate Reductase in Apple Leaves