Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

Abstract

In leaves of spinach plants (Spinacia oleracea L.) performing CO2 and N03-assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.In a green plant cell, photosynthetic nitrate assimilation involves two reductive steps, located in different subcellular compartments. In the cytosol, nitrate is reduced to nitrite via NADH-dependent NR2 (27), and after transport into the chloroplasts (3), nitrite is further reduced to ammonia by nitrite reductase located in the stroma (2). Because this reaction needs reduced ferredoxin, which is provided by PSI, it is essentially light dependent, although a minor amount of nitrite reduction may also occur during darkness at the expense of reducing equivalents generated by the oxidative pentose phosphate pathway (1, 24) and starch breakdown (13 (27) are too slow to account for a rapid decrease of enzyme activity during darkening. Although a number of metabolites such as ADP and ATP (5, 17), Pi (20), NADH and cyanide (7,26), and amino acids (18,19) were reported to affect NR activity, a systematic study of the putative regulatory role of these and other metabolites showed that none of these substances, when added to partially purified NR at those concentrations occurring in the cytosol of illuminated and darkened spinach leaves, were able to change NR activity to such an extent that the enzyme could be switched off during sudden darkening (25).Recently, Kaiser and Forster ( 11) presented evidence that NR may be altered in its activity by interconversion of the enzyme protein. These authors found that in illuminated spinach leaves the extractable NR activity assayed under standard conditions largely decreased when leaves kept under ambient CO2 were flushed with C02-free air, and the deactivation was reversed when the leaves were brought back to air. Moreover, the extractable NR activity was found to increase when darkened leaves were illuminated (1 1). The deactivation of NR during the exposure of leaves to C02-free air occurred with a half-time of about 20 min (10, 11). To avoid an accumulation of nitrite when a leaf suddenly becomes shaded, a much higher rate of NR deactivation would be required. In the present report, from measurements of nitrite contents and of NR activity in spinach le...

Posttranslational Regulation of Nitrate Reductase in Higher Plants

“…In illuminated spinach leaves from plants fertilized with nitrate, extractable NRA was about 10 to 20 pmol NO3-g-' fresh weight h-', compared to a COZ-assimilation rate of 100 to 200 pmol g-' fresh weight h-'. Upon sudden darkening, NRA decreased by 50 to 85%, with a half-time of only 2 to 15 min (Huber et al, 1992a;Huber et al, 1992c;Kaiser et al, 1992;MacKintosh, 1992;Riens and Heldt, 1992;De Cires et al, 1993).…”

Effect of pod removal on absorption and reduction of nitrate in soybean

“…Kaiser and Spill (1991) suggested that the cytosolic ATP/AMP levels are the central link between the nitrate reductase activity in the cytosol and photosynthesis in the chloroplasts. On the other hand, Riens and Heldt (1992) who observed a rapid inactivation of the nitrate reductase activity of spinach leaves under sudden darkness suggested that this phenomenon was related to the mechanism used for avoiding the accumulation of toxic nitrite in cells. Such a light effect on the nitrate reductase activity may not be involved in the present study, because sampled leaf tissues were quickly frozen with ice immediately after detachment from growing plants under full sunlight.…”

Escarola (Cichorium endivia L.): sistemas de manejo y ciclos como factores de la incidencia de fisiopatías y la acumulación de nitratos.

“…Una vez absorbido, para que el nitrato pueda ser asimilado en forma de estructuras orgánicas, tiene que ser reducido a amonio lo cual se produce mediante dos reacciones catalizadas por las enzimas nitrato reductasa y nitrito reductasa (Ingle et al, 1966;Weissman, 1972;Guardiola, García, 1990;Riens, Heldt, 1992).…”

“…Este proceso requiere energía, pudiendo consumir una parte de los electrones originados en la fotosíntesis (Riens, Heldt, 1992;Bungard et al, 1999).…”