Control of nitrate reductase by circadian and diurnal rhythms in tomato

Nitrate reductase (NR, EC 1.6.6.1) is a key regulatory enzyme in the assimilation of nitrate into amino acids in plant leaves. NR activity is intricately controlled by multifarious regulatory mechanisms acting at different levels ranging from transcription to protein degradation. It is among the few enzymes known to have a robust circadian rhythm of enzyme activity in many plant species. Although many aspects of NR regulation have been studied in depth, how these different types of control interact in a plant to deliver integrated control of activity in leaves over the course of the day has not been systematically investigated. This work documents that NR in young tomato (Lycopersicon esculentum Mill.) leaves has an endogenous rhythm in mRNA and protein level, which in nearly all circumstances are in phase with the rhythm in NR enzyme activity. Our data show that the diurnal control of NR activity in tomato leaves rests primarily with circadian regulation of Nia gene expression. The accompanying oscillations in protein level in tomato are made possible by a short half-life of NR protein that is approx. 6 h under normal conditions and approx. 2.5 h when plants are darkened during mid-day. NR post-transcriptional regulation via phosphorylation and subsequent 14-3-3 protein binding has a physiologically vital but secondary regulatory role in tomato of rapidly deactivating NR in response to changes in light intensity that cannot be anticipated by circadian timing. The post-translational reactivation of phosphorylated NR appears to have its primary physiological role in tomato leaves in reversing the down regulation of NR following transient shading events. Although there is a significant steady-state pool of apparently inactive NR throughout the diurnal, our data indicate that tomato leaves are unable to draw on this reserve to compensate for NR protein that is degraded during shading.