Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation

Summary In wild‐type Nicotiana plumbaginifolia and other higher plants, nitrate reductase (NR) is rapidly inactivated/activated in response to dark/light transitions. Inactivation of NR is believed to be caused by phosphorylation at a special conserved regulatory Ser residue, Ser 521, and interactions with divalent cations and inhibitory 14‐3‐3 proteins. A transgenic N. plumbaginifolia line (S 521 ) was constructed where the Ser 521 had been changed by site‐directed mutagenesis into Asp. This mutation resulted in complete abolishment of inactivation in response to light/dark transitions or other treatments known to inactivate NR. During prolonged darkness, NR in wild‐type plants is in the inactivated form, whereas NR in the S 521 line is always in the active form. Differences in degradation rate between NR from S 521 and lines with non‐mutated NR were not found. Kinetic constants like K m values for NADH and NO 3 − were not changed, but a slightly different pH profile was observed for mutated NR as opposed to non‐mutated NR. Under optimal growth conditions, the phenotype of the S 521 plants was not different from the wild type (WT). However, when plants were irrigated with high nitrate concentration, 150 m m , the transgenic plants accumulated nitrite in darkness, and young leaves showed chlorosis.