Constitutive expression of nitrate reductase changes the regulation of nitrate and nitrite transporters in Chlamydomonas reinhardtii

Abstract

The effect of constitutive expression of nitrate reductase (NR) on the regulation pattern of nitrite and nitrate transporters (NT) has been analysed in Chlamydomonas reinhardtii. Strain T×11-8, bearing the chimeric Nia1 gene for NR under the control of the cabll-1 gene promoter, showed a constitutive NR which was inactive in ammonium-grown cells. In contrast to the wild-type, incubation of T×11-8 in ammonium media containing nitrate at micromolar concentrations resulted in reactivation of inactive NR and excretion of nitrite. In addition, negligible amounts of NT gene transcripts were induced by nitrate in this strain. However, NT transcripts from T×11-8 cells were induced in amounts similar to those in the wild-type, by preventing constitutive NR expression from the cabll-1 gene promoter in the dark. Strains lacking high-affinity NT and nitrite reductase activity and having the chimeric Niai gene have been constructed by genetic crosses. All these strains were sensitive to chlorate in ammonium-containing media and excreted nitrite when nitrate was present at millimolar concentrations. Spontaneous chlorate-resistant mutants isolated from these strains lacked constitutive NR activity though they constitutively expressed NR mRNA, The data strongly suggest that nitrate plays an important post-transcriptional regulatory role and that NR is involved in the regulation of the nitrate/ nitrite transport activities in C. reinhardtii.