Nitrate reductase in higher plants: A case study for transduction of environmental stimuli into control of catalytic activity

In higher plants, cytosolic NAD(P)H-nitrate reductase (NR) is rapidly modulated by environmental conditions such as light, CO₂, or oxygen availability. In leaves, NR is activated by photosynthesis, reaching an activation state of 60-80%. In the dark, or after stomatal closure, leaf NR is inactivated down to 20 or 40% of its maximum activity. In roots, hypoxia or anoxia activate NR, whereas high oxygen supply inactivates NR. Spinach leaf NR is inactivated by phosohorylation of serine 543 and subsequent Mg²⁺-dependent binding of 14-3-3 proteins at, or close to, this phosphorylation site. At least three different protein kinases (NR-PK) have been identified in spinach leaves that are able to phosphorylate NR on serine 543. Two of them show up as calmodulin-like domain protein kinases (CDPKs), and one as a SNF1-like protein kinase. Dephosphorylation of serine 543 is catalyzed by a Mg²⁺-dependent protein phosphatase and by a type 2A protein phosphatase (NR-PP), which is regulated by a trimer/ dimer interconversion. The NR-PKs, NR-PPs, and 14-3-3s are present even in NR-depleted plant tissues. Artificial activation of NR in vivo is achieved by cellular acidification, by respiratory inhibitors, or by mannose feeding. As for anoxia, these treatments seem to act, at least in part, via cytosolic acidification, mediated by low cytosolic ATP levels. Activation is also achieved by ionophore-induced release of divalent cations from the cytosol. In addition, cytosolic AMP and phosphate esters seem to regulate NR-PK and NR-PP activities, thereby adapting NR activity within minutes to the changing environment.