Nitrate reductase from winter wheat leaves is activated at low temperature via protein dephosphorylation

The effect of short-term low temperature treatment on nitrate reductase (NR, EC 1.6.6.1) activity, NR protein and NR transcript levels in excised leaves of winter wheat (Triticum aestivum L. cv. Sadovo-1) was investigated. NR activity, measured in the presence of Mg2+ (NRact), doubled within 2 h at 4 degrees C, whereas NR activity, measured in the presence of EDTA (NRmax), did not respond to the cold treatment. Such an activation of NR occurred only if leaves were exposed to low temperature in the light but not in the dark. It was not affected by feeding cytoplasmic protein synthesis inhibitor, cycloheximide, or protein kinase inhibitor, staurosporin, but was completely prevented by okadaic acid, an inhibitor of protein phosphatases of the type 1 and 2 A. This inhibitory effect decreased gradually when okadaic acid-concentration in the nutrient solution was lowered below 1 &mgr;M and tended to disappear when leaves were fed with 10 nM okadaic acid. It was demonstrated that the cold-induced NR activation was dependent neither on cold-triggered calcium influx nor on high endogenous abscisic acid levels. The increased NRact in cold-exposed leaves was found to correlate with a higher level of NR transcript but not with an increased NR protein level. Feeding okadaic acid to these leaves prevented the cold-induced accumulation of NR mRNA. These data point to protein phosphatases of the type 2 A being involved in NR protein dephosphorylation and NR transcript accumulation as targets of activation by low temperature treatment.