Abstract
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.
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Abbreviations
- DNP:
-
2,4-dinitrophenol
- HOQNO:
-
8-hydroxyquinoline-N-oxide
- DCPIP:
-
2,6-dichlorophenolindophenol
- CCCP:
-
Carbonyl cyanide m-chlorophenylhydrazone
- TES:
-
N-tris(hydroxymethyl)methyl-2-amino ethanesulphonic acid
- HEPES:
-
N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid
References
Atkins, C.A., Canvin, D.T.: Nitrate, nitrite and ammonia assimilation by leaves: Effects of inhibitors. Planta 123, 41–51 (1975)
Bould, C., Bradfield, E.G., Clarke, G.M.: Leaf analysis as a guide to the nutrition of fruit crops. I — General principles, sampling techniques and analytical methods. J. Sci. Food Agric. 11, 229–242 (1960)
Butt, V.S., Beevers, H.: The regulation of pathways of glucose catabolism in maize roots. Biochem. J. 80, 21–27 (1961)
Canvin, D.T., Atkins, C.A.: Nitrate, nitrite and ammonia assimilation by leaves: Effect of light, carbon dioxide and oxygen. Planta 116, 207–224 (1974)
Chaparro, A., Maldonado, J.M., Diez, J., Relimpio, A.M. Losada, M.: Nitrate reductase inactivation and reducing power and energy charge in Chlorella cells. Plant Sci. Lett. 6, 335–343 (1976)
Ducet, G., Hewitt, E.J.: Relation of molybdenum status and nitrogen supply to respiration in cauliflower leaves. Nature (London) 173, 1141–1143 (1954)
Ferrari, T.E., Varner, J.E.: Control of nitrate reductase activity in barley aleurone layers. Proc. Nat. Acad. Sci. USA 65, 729–736 (1970)
Ferrari, T.E., Varner, J.E.: Intact tissue assay for nitrite reductase in barley aleurone layers. Plant Physiol. 47, 790–794 (1971)
Ferrari, T.E., Yoder, O.C., Filner, P.: Anaerobic nitrite production by plant cells and tissues: Evidence for two nitrate pools. Plant Physiol. 51, 423–431 (1973)
Harold, F.M.: Antimicrobial agents and membrane function. Adv. Microb. Physiol. 4, 45–104 (1970)
Hewitt, E.J.: Sand and water culture methods used in the study of plant nutrition. 2nd ed. Commonwealth Bureau of Horticulture, Tech. Commun. 22, 1966
Hewitt, E.J.: Assimilatory nitrate-nitrite reduction. Ann. Rev. Plant Physiol. 26, 73–100 (1975)
Hewitt, E.J., Hucklesby, D.P., Betts, G.F.: In: Recent aspects of nitrogen metabolism in plants, pp. 47–81, Hewitt, E.J., Cutting, C.V., ed. London: Academic Press 1968
Jackson, W.A., Volk, R.J.: Photorespiration. Ann. Rev. Plant Physiology. 21, 385–432 (1970)
Jaworski, E.G.: Nitrate reductase assay in intact plant tissues. Biochem. Biophys. Res. Commun. 43, 1274–1279 (1971)
Jones, R.W., Sheard, R.W.: Accumulation and stability of nitrite in intact aerial leaves. Plant Sci. Lett. 11, 285–291 (1978)
Klepper, L.A.: A mode of action of herbicides: Inhibition of the normal process of nitrite reduction. Univ. of Nebraska. Lincoln, Coll. of Agric. Bull. No. 259 (1974)
Klepper, L.A., Flesher, D., Hageman, R.H.: Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves. Plant Physiol. 48, 580–590 (1971)
Lloyd-Jones, C.P., Hudd, G.A., Hill-Cottingham, D.G.: The determination of nitrogen-15 in plant material with an emission spectrometer. Analyst 99, 580–587 (1974)
Mann, A.F., Hucklesby, D.P., Hewitt, E.J.: Sources of reducing power for nitrate reducing in spinach leaves. Planta 140, 261–263 (1978)
Radin, J.W.: In vivo assay of nitrate reductase in cotton leaf discs. Effect of oxygen and ammonium. Plant Physiol. 51, 332–336 (1973)
Slater, T.F., Sawyer, B.: A colorimetric method for estimating the pyridine nucleotide content of small amounts of animal tissue. Nature (London) 193, 454–456 (1962)
Sloan, C.H., Sublett, B.J.: Colorimetric method of analysis for nitrates in tobacco. Tobacco Science 10, 121–125 (1966)
Umbreit, W.W., Burris, R.H., Stauffer, J.F.: Manometric Techniques, 4th Ed Minneapolis: Burgess Publishing 1964
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Mann, A.F., Hucklesby, D.P. & Hewitt, E.J. Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves. Planta 146, 83–89 (1979). https://doi.org/10.1007/BF00381259
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DOI: https://doi.org/10.1007/BF00381259