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All the activities of the nitrate reductase complex from spinach are irreversibly inactivated by irradiation of the enzyme with blue light in the presence of flavin mononucleotide. The photoinactivation requires oxygen and is prevented by ethylenediaminetetraacetic acid and by reduced nicotinamide adenine dinucleotide, but not by superoxide dismutase p h s catalase. On the other hand, the inactivation is markedly enhanced in 77% deuterated water and it is suppressed by the singlet oxygen quenchers azide, histidine and tryptophan. All these results suggest that singlet oxygen generated by light absorption by flavin mononucleotide, rather than excited flavin mononucleotide or other oxygen species, is the primary agent involved in the photooxidative inactivation of the enzyme.

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Summary.We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO3-, NO2-or NH4+), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO2-or NH4 + the synthesis of NiR and chloroplastic GS (GS2) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO3-concentration indicate that NO3--dependent enzyme induction is elicited by NO3-per se and not by a product of its assimilatory reduction, e.g., NO2 or NH4 +. In the presence of NO3-, light remarkably enhanced the appearance of NR, NiR, and GS2, while the activity of the cytosolic GS isoform (GS1) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light-and NO3--dependent increase of GS2 activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO3-on the appearance of NR, NiR, and GS2 isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO3-and/or for the accumulation of newly formed enzymes in the organelle.