Nitrate reductase dephosphorylation is induced by sugars and sugar-phosphates in corn leaf segments

The in situ and in vitro regulation of nitrate reductase (NR; EC 1.6.6.1) activity by glucose (Glc) and glucose-6-phosphate (Glc-6P) was studied in leaf segments of 7-day-old corn plants. In situ, Glc and Glc-6P not only prevented NR inactivation, but also slightly activated the enzyme relative to that in fresh attached leaves in the light. Glc and Glc-6P also reactivated NR that had previously been inactivated by incubating the segments for 30 min in the dark. Sugars were effective, even in the presence of cycloheximide, but not of cantharidin, an inhibitor of type 2A phosphoprotein phosphatase (PP2A). In segments kept in the dark, the inhibition of protein dephosphorylation by cantharidin showed that the phosphorylation of NR was not inhibited by either Glc or Glc-6P, as the enzyme was inactivated to the same extent whether or not sugars (P) were present in the incubation medium. In vitro, as in situ, neither Glc nor Glc-6P could prevent NR phosphorylation. In spite of some reports showing that sugar-phosphates can act on kinases and prevent NR phosphorylation, the results presented here suggest that, in corn leaves, sugars and their phosphorylated derivatives probably activate NR in situ mainly by inducing protein dephosphorylation. The incubation of crude extract in a water bath at 27degreesC for 45 min resulted in the activation of NR that was blocked by cantharidin, but was not increased by either Glc or Glc-6P. This result suggests that the presence of another metabolite(s) and the maintenance of cell functionality may be necessary for the sugar-induced activation of NR. A sugar-triggered signalling pathway independent of protein synthesis may be involved in the process. L-Glc and 6-deoxyglucose were ineffective in reactivating NR in darkened segments, whilst 2-deoxyglucose was as effective as Glc itself. The effect of sugar analogues shows that, although Glc has to enter the cell and be phosphorylated to activate NR, further metabolism is not necessary. As sugar-phosphates, such as Glc-6P and fructose-6-phosphate (Fru-6P), also activate NR, it seems that hexokinases are not involved in the pathway that leads to the in situ dephosphorylation of NR. In vitro, Glc-6P mildly but rapidly activated NR by a mechanism insensitive to cantharidin. The addition of an increasing concentration of Mg2+ to crude extract containing Glc-6P increased the Mg2+ inhibition of NR. This result suggests that the hexose-phosphate does not prevent Mg2+ association with NR. It is possible that Glc-6P activates NR in vitro by inducing the dissociation of 14-3-3 from the phospho-NR (pNR)/Mg/14-3-3 complex.