Abstract
Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L. cv. Polka F1) leaves showed reversible modulation, being activated in the light and inactivated in the dark (t/2 = 20–30 min). The large changes in enzyme activity during light-dark transients were observed only when assayed in buffers containing free Mg2+. In the presence of EDTA (5 mM), the enzyme activity was high and the light modulation was barely evident.
The inactivation of NR in the dark could be totally prevented by anaerobiosis, or by feeding mannose or 2,4-dinitrophenol through the leaf petiole. All these treatments drastically decreased ATP levels and increased AMP levels in leaf extracts, thus pointing to a close correlation between adenine-nucleotide levels and NR activity. Treatment of leaves in the dark with 2,4-dinitrophenol or with anaerobiosis brought about an accumulation of nitrite, thus confirming that under these conditions NR remained active also in vivo. The in-vivo dark-inactivated enzyme was reactivated in vitro by preincubating a leaf extract with AMP in the presence of the myokinase inhibitor p1,p5-di(adenosine 5′)pentaphosphate. It is suggested that NR responds to artificially induced drastic changes in cytosolic adeninenucleotide levels, being active when ATP is low and AMP is high. Adenine nucleotides also appear to participate in the light-dark modulation of NR, but additional regulatory factors have to be postulated.
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Abbreviations
- DNP:
-
2,4-dinitrophenol
- NR:
-
nitrate reductase
- NRA:
-
nitrate-reductase activity
- AP5A:
-
p1,p5-di(adenosine-5′)pentaphosphate
References
Budde, J.A., Chollet, R. (1988) Regulation of enzyme activity in plants by reversible phosphorylation. Physiol. Plant. 72, 435–439
Canvin, D.T., Woo, K.C. (1979) The regulation of nitrate reduction in spinach leaves. Can. J. Bot. 57, 1155–1160
Goller, M., Hampp, R., Ziegler, H. (1982) Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat. Planta 156, 255–263
Gardeström, P., Wigge, B. (1988) Influence of photorespiration on ATP/ADP ratios in the chloroplasts, mitochondria and cytosol, studied by rapid fractionation of barley (Hordeum vulgare) protoplasts. Plant Physiol. 88, 69–76
Hagemann, R.H., Reed, A.J. (1980) Nitrate reductase from higher plants. Methods Enzymol. 69, 270–280
Hampp, R., Goller, M., Ziegler, H. (1982) Adenylate levels, energy charge, and phosphorylation potential during dark-light and light-dark transition in chloroplasts, mitochondria, and cytosol of mesophyll protoplasts from Avena sativa L. Plant Physiol. 69, 448–455
Heineke, D., Riens, B., Grosse, H., Hofrichter, P., Peter, U., Flügge, U.I., Heldt, H.W. (1991) Redox transfer across the inner chloroplast envelope membrane. Plant Physiol. 95, 1131–1137
Kaiser, W.M., Brendle-Behnisch, E. (1991) Rapid modulation of spinach leaf nitrate reductase activity by photosynthesis. I. Modulation in vivo by CO2 availability. Plant Physiol. 96, 363–367
Kaiser, W.M., Förster, J. (1989) Low CO2 prevents nitrate reduction in leaves. Plant Physiol. 91, 970–974
Kaiser, W.M., Spill, D. (1991) Rapid modulation of spinach leaf nitrate reductase activity by photosynthesis. II. In vitro modulation by ATP and AMP. Plant Physiol. 96, 368–375
Mann, A.F., Hucklesby, D.P., Hewitt, E.J. (1979) Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves. Planta 146, 83–89
Reed, V.C., Canvin, D.T. (1982) Light and dark controls of nitrate reduction in wheat (Triticum aestivum L.) protoplasts. Plant Physiol. 69, 508–513
Remmler, J.L., Campbell, W.H. (1986) Regulation of corn leaf nitrate reductase. Plant Physiol. 80, 442–447
Stitt, M., McC. Lilley, R., Heldt, H.W. (1982) Adenine nucleotide levels in the cytosol, chloroplasts and mitochondria of wheat leaf protoplasts. Plant Physiol. 70, 971–977
Wyn Jones, R., Sheard, R.W. (1978) Accumulation and stability of nitrite in intact aerial leaves. Plant Sci. Lett. 11, 285–291
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This work was supported in part by the Deutsche Forschungsgemeinschaft, SFB 251. We are grateful to G. Harris (Wellesley College, Department of Biological Sciences, Wellesley, Mass., USA) and D. Walker (Robert Hill Institute, University of Sheffield, UK) for a critical reading of the manuscript.
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Kaiser, W.M., Spill, D. & Brendle-Behnisch, E. Adenine nucleotides are apparently involved in the light-dark modulation of spinach-leaf nitrate reductase. Planta 186, 236–240 (1992). https://doi.org/10.1007/BF00196253
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DOI: https://doi.org/10.1007/BF00196253