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 NH4+as the only nitrogen source (NH4+-cells) were transferred into NO3-medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO3-, 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 NO3--induced NR synthesis when the cells, previously grown in NH4+medium, were transferred into NO3-medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH4+to NO3-medium (at time 0 h), NO3-induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO3-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 NO3-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 NO3-uptake and NR induction due to the expression of NR and NO3--transporter mRNAs.