Characterization of the alternative oxidase of Chlamydomonas reinhardtii in response to oxidative stress and a shift in nitrogen source

To study the transcriptional regulation of Aox1, the major alternative oxidase (AOX) gene, we fused 1072 bp of its promoter to the promoterless arylsulfatase reporter gene. We find that the reporter is strongly activated when cells are shifted from a medium containing ammonium to one containing nitrate but is unresponsive to treatments known to induce Aox1 in higher plants - H2O2, antimycin A and cold stress. However, induction of Aox1 by all these factors was found when changes in gene expression were monitored using RNA blot analysis. Our data suggest that transcriptional upregulation of Aox1 by H2O2, antimycin A and cold-stress requires one or more enhancers which are not found in the proximal promoter region of the gene. Interestingly, while nitrate, H2O2 and cold stress result in increased AOX abundance and increased alternative pathway respiration, these changes are not seen when cells are treated with antimycin A. We find that H2O2, antimycin A and cold-stress result in an increase in intracellular reactive oxygen species (ROS) but that a significant change in ROS does not occur when cells are shifted into a medium-containing nitrate. Overall, our data are consistent with there being two distinct pathways regulating Aox1 transcription and AOX abundance in Chlamydomonas reinhardtii: one in response to oxidative stress and a second due to metabolic changes brought about by a shift in nitrogen source from ammonium to nitrate.