Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation.
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 Km values for NADH and NO3(-) 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 mM, the transgenic plants accumulated nitrite in darkness, and young leaves showed chlorosis.
Lillo
Cathrine
C
Stavanger University College, School of Technology and Science, Box 8002 Ullandhaug, 4068 Stavanger, Norway. cathrine.lillo@tn.his.no
Lea
Unni S
US
Leydecker
Marie-Thérèse
MT
Meyer
Christian
C
D003624:Darkness; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology; D009573:Nitrites / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D030821:Plants, Genetically Modified; D017680:Potassium Compounds / Q000494:pharmacology; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D009566:Nitrates; D009573:Nitrites; D017680:Potassium Compounds; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C023844:potassium nitrate