Induction of nitrate reductase, nitrite reductase, and glutamine synthetase isoforms in sunflower cotyledons as affected by nitrate, light, and plastid integrity

Abstract

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 (NO 3 − , NO 2 − or NH 4 + ), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO 2 − or NH 4 + 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 NO 3 − concentration indicate that NO 3 − -dependent enzyme induction is elicited by NO 3 − per se and not by a product of its assimilatory reduction, e.g., NO 2 − or NH 4 + . In the presence of NO 3 − , 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 NO 3 − -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 NO 3 − 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 NO 3 − and/or for the accumulation of newly formed enzymes in the organelle.