Mechanism and importance of post-translational regulation of nitrate reductase.

In higher plants, nitrate reductase (NR) is inactivated by the phosphorylation of a conserved Ser residue and binding of 14-3-3 proteins in the presence of divalent cations or polyamines. A transgenic Nicotiana plumbaginifolia line (S521) has been constructed where the regulatory, conserved Ser 521 of tobacco NR (corresponding to Ser 534 in Arabidopsis) was mutated into Asp. This mutation resulted in the complete abolition of activation/inactivation in response to light/dark transitions or other treatments known to regulate the activation state of NR. Analysis of the transgenic plants showed that, under certain conditions, when whole plants or cut tissues are exposed to high nitrate supply, post-translational regulation is necessary to avoid nitrite accumulation. Abolition of the post-translational regulation of NR also results in an increased flux of nitric oxide from the leaves and roots. In view of the results obtained from examining the different transgenic N. plumbaginifolia lines, compartmentation of nitrate into an active metabolic pool and a large storage pool appears to be an important factor for regulating nitrate reduction. The complex regulation of nitrate reduction is likely to have evolved not only to optimize nitrogen assimilation, but also to prevent and control the formation of toxic, and possibly regulatory, products of NR activities. Phos phorylation of NR has previously been found to influence the degradation of NR in spinach leaves and Arabidopsis cell cultures. However, experiments with whole plants of N. plumbaginifolia, Arabidopsis, or squash are in favour of NR degradation being the same in light and darkness and independent of phosphorylation at the regulatory Ser.