Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

Abstract

The effect of nitrogen starvation on the NO3-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH 4 + as the only nitrogen source (NH 4 + -cells) were transferred into NO 3 − medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO 3 − , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m−2 s−1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m−2 s−1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO 3 − -induced NR synthesis when the cells, previously grown in NH 4 + medium, were transferred into NO 3 − medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH 4 + to NO 3 − medium (at time 0 h), NO 3 − induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO 3 − to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO 3 − medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO 3 − uptake and NR induction due to the expression of NR and NO 3 − -transporter mRNAs.