Role of light and CO2 fixation in the control of nitrate-reductase activity in barley leaves

Nitrate reductase (NR, NADH:nitrate oxidoreductase, EC 1.6.6.1) from barley (Hordeum vulgare L. cv. Hassan) leaves was inactivated during a light-dark transition, losing approx. 50% of activity after 30 min of darkness. The dark inactivation was reversed by illumination of the seedlings, the kinetics of reactivation being similar to those of inactivation. High extractable NR activity and significant differences between illuminated and darkened leaves were observed in media containing EDTA and inorganic phosphate (P_i). Addition of Ca²⁺ ions during extraction and assay decreased NR activity from illuminated and darkened leaves, enhancing the light-dark difference. While no clear correlation could be found between irradiance and NR activity, a hyperbolic correlation appeared between extractable NR activity and in-vivo rates of CO₂ fixation, indicating that NR activation follows saturation kinetics with respect to CO₂ fixation. Furthermore, hexoses and hexose-phosphates fed to the leaves via the transpiration stream protected against the dark-inactivation of NR. The results indicate that carbon-assimilation products are regulatory factors of NR activity in barley leaves, mediating both the light-dark modulation of NR and its dependence upon CO₂ fixation.