Transcriptional regulation of the Chlamydomonas alternative oxidase (Aox1) gene studied with a reporter gene construct

Beside the cytochrome pathway of respiration, mitochondria of higher plants and many microeukaryotes possess an alternative pathway that bypasses complexes III and IV to directly catalyze the oxidation of the ubiquinol pool by molecular oxygen. No proton motive force is generated during this reaction and free energy is thus lost as heat. The alternative oxidase (AOX), which is the sole enzyme implicated in this wasteful process, is generally subject to both transcriptional and post-translational regulation. — Recently, we have cloned and characterized two cDNAs and the corresponding gene sequences (AOX1 and AOX2) encoding the alternative oxidase in the green alga Chlamydomonas reinhardtii (1). In order to investigate the transcriptional regulation of the AOX1 gene (which is more strongly expressed than AOX2), we fused the AOX1 promoter to the arylsulfatase-encoding ARS reporter gene. Several transformants expressing the ARS protein were selected and characterized at the molecular level. While AOX transcription is induced or at least enhanced by cytochrome pathway inhibitors in fungi and higher plants, our results indicate that the expression of the AOX1:ARS construct remains unchanged following inhibitor addition in Chlamydomonas. Moreover, its expression was not affected by light, acetate as a carbon source and osmotic stress. A five-to-ten-fold stimulation was however achieved by using nitrate instead of ammonium as a nitrogen source. Effects of toxic compounds (oxidative stress agents and heavy metals) are currently investigated.

This work was supported by grants from the Belgian FRFC (2.4527.97) and Fonds Spéciaux pour la Recherche dans les Universités. D. Baurain is a FNRS Research Fellow.

(1) Dinant, M., Baurain, D., Coosemans, N., Joris, B. and Matagne, R. F. (2001) Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii. Curr. Genet. (in press)