Genetic manipulations in Chlamydomonas reinhardtii: Use of the antisense strategy to inhibit the alternative oxidase

Besides the cytochrome pathway (CP), mitochondria of higher plants and many microeukaryotes possess a second electron pathway, the alternative pathway (AP), that bypasses complexes III and IV and catalyzes the oxidation of the ubiquinol pool by molecular oxygen. Electrons transferred by this way are not coupled to ATP production and free energy is lost as heat. The alternative oxidase (AOX) is the sole enzyme implicated in this process. Despite a great research effort during these last years, the exact role of AP is poorly understood, except in Araceae (such as Sauromatum guttatum, the ‘voodoo lily’) in which it is implicated in the thermogenesis of the inflorescence.
In order to investigate the physiological role of AOX in the unicellular green alga Chlamydomonas reinhardtii, two different plasmids derived from pSP105 (Stevens et al., 1996) were constructed by placing in antisense orientation an AOX cDNA from this species under the control of the regulatory regions of the Chlamydomonas RBCS2 gene. The first plasmid possessed the full promoter, while the second one had a truncated promoter, supposed to be more efficient (Purton, personal communication). Cell wall-deficient arginine-requiring strains were co-transformed by the glass beads method with linearized pASL plasmid (complementing the arginine requirement) and linearized first or second construction. More than 1100 transformants able to grow on an arginine-free medium were screened for AOX alteration by growth tests in the presence of the two complex III inhibitors antimycin A and myxothiazol. Out of seventeen clones selected for their sensitivity to these inhibitors, only one (A1 clone) was totally unable to grow in the presence of antimycin A and myxothiazol. A PCR analysis showed that the seventeen selected clones had integrated the antisense construction. The A1 cell line was further investigated for its mitochondrial respiration activity. The cells grown under mixotrophic conditions (light + acetate as a carbon source), showed a 70% drop of the AP capacity. Moreover, the total respiration rate represented only 60% of the wild-type rate. Interestingly, despite these alterations, the A1 transformant displayed a 25% faster growth rate than the wild-type cultivated under the same mixotrophic conditions.

This work was supported by grants from the Belgian FRFC (2.4527.97). Actions de recherches concertées (ARC 93-98/170) and Fonds spéciaux pour la recherche dans les universités.

Reference
STEVENS D.R., ROCHAIX J.D., and PURTON S. (1996), Mol. Gen. Genet. 251, 23-30