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 Melissa Lessen Casais-Molina 1 & Santy Peraza-Echeverria 1 & Ileana Echevarría-Machado 2 & Virginia Aurora Herrera-Valencia 1 Received: 28 October 2014 /Revised and accepted: 6 April 2015 # Springer Science+Business Media Dordrecht 2015 Abstract Glycerol-3-phosphate dehydrogenase (GPDH) cat- alyzes 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 accu- mulation of glycerol and TAGs in the green microalga Chlamydomonas reinhardtii. In a previous study, we identi- fied three GPDH homologs (CrGPDH1, CrGPDH2, and CrGPDH3) in this microalga. We found that CrGPDH2 and CrGPDH3 were expressed in response to 200 mM NaCl treat- ment, 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 con- firmed that both CrGPDH2 and CrGPDH3 have GPDH activity. The genetic complementation analysis revealed that CrGPDH2 and CrGPDH3 were able to restore glycerol pro- duction and rescue the salt sensitivity of this mutant. Compared with CrGPDH3, CrGPDH2 conferred higher glyc- erol production and greater salt tolerance when expressed in the gpd1Δgpd2Δ double mutant. Together, these findings show that CrGPDH2 and CrGPDH3 encode functional ho- mologs of the S. cerevisiae GPD1 gene that is involved in glycerol synthesis and osmotic stress tolerance. Keywords Chlamydomonas reinhardtii . Glycerol-3-phosphate dehydrogenase (GPDH) . Glycerol synthesis . Osmotic stress Introduction Chlamydomonas reinhardtii is a freshwater unicellular green alga that is capable of synthesizing glycerol as an osmoregu- latory metabolite in response to osmotic stress. This alga can tolerate up to 200 mM NaCl (León and Galván 1994). C. reinhardtii is also able to accumulate triacylglycerides (TAGs) in response to NaCl ranging from 20 to 100 mM (Siaut et al. 2011). Glycerol is an important osmoregulatory solute induced by osmotic up-shocks in some halophilic green algae, including several species of Chlamydomonas and Dunaliella, as well as almost all yeast species studied to date (Ben-Amotz and Avron 1983; Avron 1986; Hohmann 2002). However, the microalgal genes involved in glycerol and lipid biosynthesis have not yet been fully characterized. Glycerol- 3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) using NADH as an electron do- nor. Then, G3P is dephosphorylated to glycerol by the action Electronic supplementary material The online version of this article (doi:10.1007/s10811-015-0588-3) contains supplementary material, which is available to authorized users. * Virginia Aurora Herrera-Valencia vicky@cicy.mx 1 Unidad de Biotecnología, Centro de Investigación Científica de Yucatán (CICY), Calle 43, No. 130, Col. Chuburná de Hidalgo, 97200 Mérida, YUC, México 2 Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán (CICY), Calle 43, No. 130, Col. Chuburná de Hidalgo, 97200 Mérida, YUC, México J Appl Phycol DOI 10.1007/s10811-015-0588-3