Effects of nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration and nitrate reductase activity in three leafy vegetables

S-Adenosylmethionine synthetase from Escherichia coli.

Nitrogen metabolism, translocation, and recycling in apple trees

Publisher Summary This chapter describes nitrate and nitrite reduction. Nitrate is the predominant form of nitrogen available to most cultivated plants grown under normal field conditions. Although ammoniacal fertilizers are used almost exclusively, the ammonia derived from these fertilizers is oxidized to nitrate by soil organisms. Nitrification is extremely rapid when the soil is well aerated, moist, and above 7°–10°C. Certain bacteria can utilize nitrate nitrogen as the sole nitrogen source for the synthesis of all nitrogen containing compounds of the cell. This nitrate assimilation can occur under both aerobic and anaerobic conditions. Nitrate reduction by partially purified enzyme preparations is frequently stimulated by phosphate. Nitrate reduction, mediated by either reduced pyridine nucleotides or reduced viologen dyes, is inhibited by cyanide and azide; however, pyridine-nucleotide-mediated reduction of cytochrome c is insensitive to these inhibitors. A reactivation of an inactive nitrate reductase apoenzyme extracted from molybdenum-deficient plants can be achieved by the addition of acid-treated nitrate reductase or by addition of phosphate buffer washes of nitrate reductase absorbed on adenosine monophosphate -Sepharose.

Nitrate and Nitrite Reduction

The relationships between photosynthesis, carbohydrate metabolism and respiration in leaves of C3 plants are reviewed. We first provide an overview of how mitochondrial respiration relies on, and responds to, the supply of photosynthetic products in the light. The pathways by which the various substrates (glycine, oxaloacetate, malate and/or pyruvate) enter the mitochondria and are oxidized, are discussed. We also provide an overview of the pathways of mitochondrial electron transport, with particular attention being paid to the non-phosphorylating alternative oxidase (AOX). We then discuss what is known about leaf respiration rates in light versus darkness (both O2 consumption and CO2 release). The extent to which mitochondrial O2 consumption continues in the light is highly variable, being inhibited, not affected or even stimulated in various reports. On the other hand, non-photorespiratory mitochondrial CO2 release (R) is invariably inhibited by light (5–80% inhibition). R is sensitive to the lowest irradiance values, and is inhibited rapidly. Three methods via which R in the light is measured are outlined and mechanisms via which light might inhibit R are discussed. The effect that light to dark transitions have on respiration are also discussed: we distinguish the initial, photorespiratory post-illumination burst (PIB) from the post-illumination rise in respiration (LEDR, light-enhanced-dark-respiration) which occurs following the PIB. The chapter also considers the demand for mitochondrially-derived ATP for photosynthesis and carbohydrate metabolism and the potential role of respiration during over-reduction of the chloroplast is highlighted. Mitochondrial respiration appears to be critical for the provision of ATP necessary for energy demanding processes in the light. Moreover, there is growing evidence that respiration helps a plant cope with excess photosynthetic redox equivalents, which otherwise can result in photo-oxidative stress.

Photosynthesis, carbohydrate metabolism and respiration in leaves of higher plants.

15N-labelled nitrate was used to show that nitrate reduction by leaf discs in darkness was suppressed by oxygen, whereas nitrite present within the cell could be reduced under aerobic dark conditions. In other experiments, unlabelled nitrite, allowed to accumulate in the tissue during the dark anaerobic reduction of nitrate was shown by chemical analysis to be metabolised during a subsequent dark aerobic period. Leaves of intact plants resembled incubated leaf discs in accumulating nitrite under anaerobic conditions. Nitrate, n-propanol and several respiratory inhibitors or uncouplers partly reversed the inhibitory effect of oxygen on nitrate reduction in leaf discs in the dark. Of these nitrate and propanol acted synergistically. Reversal was usually associated with inhibition of respiration but some concentrations of 2,4-dinitrophenol (DNP) and ioxynil reversed inhibition without affecting respiratory rates. Respiratory inhibitors and uncouplers stimulated nitrate reduction in the anaerobic in vivo assay i.e. in conditions where the respiratory process is non-functional. Freezing and thawing leaf discs diminished but did not eliminate the sensitivity of nitrate reduction to oxygen inhibition.

Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves

The possible source of NADH, the energy donor for nitrate reductase (EC 1.6.6.1), has been studied using an in vivo assay involving freezing the material (leaves of Spinacea oleracea L.) in liquid nitrogen in order to render the tissue permeable to added substrates. Glycolysis and the pentose phosphate pathway were capable of generating NADH through glyceraldehyde-3-phosphate dehydrogenase. Malate and isocitrate were also capable of generating NADH white other organic acids tested were not, including glycolate which was ineffective even under anaerobic conditions.

D. P. Hucklesby

Papers

Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves

Sources of reducing power for nitrate reduction in spinach leaves.

A modified Hanes and Isherwood spray for improved detection of phosphate esters in thin layer chromatography