Expression of Chlamydomonas reinhardtii CrGPDH2 and CrGPDH3 cDNAs in yeast reveals that they encode functional glycerol-3-phosphate dehydrogenases involved in glycerol production and osmotic stress tolerance

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) and plays a central role in the synthesis of glycerol and triacylglycerides (TAGs). Osmotic stress has been shown to induce the accumulation of glycerol and TAGs in the green microalga Chlamydomonas reinhardtii. In a previous study, we identified three GPDH homologs (CrGPDH1, CrGPDH2, and CrGPDH3) in this microalga. We found that CrGPDH2 and CrGPDH3 were expressed in response to 200 mM NaCl treatment, suggesting that these two genes play roles in glycerol and TAGs synthesis and in osmotic stress tolerance. In this study, we report on the functional characterization of CrGPDH2 and CrGPDH3. A concentration of NaCl as low as 5 mM for 5 min was sufficient to induce the expressions of both genes. We mapped the cDNA ends of CrGPDH2 and CrGPDH3 using RLM-RACE and cloned their full-length cDNAs. The expression of these two cDNAs in the Saccharomyces cerevisiae gpd1Δgpd2Δ double mutant confirmed that both CrGPDH2 and CrGPDH3 have GPDH activity. The genetic complementation analysis revealed that CrGPDH2 and CrGPDH3 were able to restore glycerol production and rescue the salt sensitivity of this mutant. Compared with CrGPDH3, CrGPDH2 conferred higher glycerol production and greater salt tolerance when expressed in the gpd1Δgpd2Δ double mutant. Together, these findings show that CrGPDH2 and CrGPDH3 encode functional homologs of the S. cerevisiae GPD1 gene that is involved in glycerol synthesis and osmotic stress tolerance.