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

Abstract

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.

A multidomain enzyme, with glycerol‐3‐phosphate dehydrogenase and phosphatase activities, is involved in a chloroplastic pathway for glycerol synthesis in Chlamydomonas reinhardtii

“…To assess whether genes encoding enzymes of glycerol metabolism may be transcriptionally activated in cells exposed to environmental stress, their transcript abundance, in wild type CC‐125 and in the Ri16 GPD2/GPD3 RNAi strain, was examined by RT‐PCR (Figure ). As previously reported (Goodenough et al ., ; Schmollinger et al ., ; Casais‐Molina et al ., ), GPD2 mRNA levels were substantially enhanced in CC‐125 under nitrogen deprivation or hyperosmotic stress. However, this up‐regulation was largely suppressed in the Ri16 RNAi strain (Figure ).…”

“…As a control, an aliquot of the wheat germ lysate programmed with an empty vector did not affect the NADH concentration. These observations corroborated that Chlamydomonas GPD2 has glycerol‐3‐phosphate dehydrogenase activity, as previously demonstrated by complementation of a Saccharomyces cerevisiae gpd1Δgpd2Δ double mutant via expression of a GPD2 cDNA (Casais‐Molina et al ., ). Moreover, the GPD activity of GPD2 was no affected by mutations in its PSP domain (Figure a).…”

“…Canonical GPDs catalyze the reversible redox conversion of dihydroxyacetone phosphate to glycerol‐3‐phosphate and our observations substantiated that recombinant GPD2 has G3P dehydrogenase activity in vitro , as previously established by complementation of a S . cerevisiae gpd1Δgpd2Δ double mutant (Casais‐Molina et al ., ). This reductase activity of GPD2 was not affected by mutations in its PSP domain.…”

Two Glycerol-3-Phosphate Dehydrogenases from Chlamydomonas Have Distinct Roles in Lipid Metabolism

“…To date, all of the analysis of Dunaliella spp. and C. reinhardtii GPDH enzymes has focused on glycerol biosynthesis and osmotic stress tolerance (Gee et al, 1993;He et al, 2007He et al, , 2009Cai et al, 2013;Casais-Molina et al, 2016). However, previous expression data showing the induction of C. reinhardtii GPD2, GPD3, and GPD4 under nutrient starvation (Goodenough et al, 2014), which in turn induces TAG accumulation (Schmollinger et al, 2014), provide indirect evidence to suggest that these three GPDH isoforms are involved in the production of G3P for entry into the Kennedy pathway for DAG and subsequently TAG biosynthesis (Fig.…”

“…S2). These discrete changes probably explain the higher GPDH activity of GPD2 compared with GPD3 when expressed in yeast (Casais-Molina et al, 2016), although future studies will need to validate whether there are significant enzymatic differences between GPDH isoforms in C. reinhardtii. A further difference between GPD2 and GPD3 was the observation that GPD2 was the only isoform induced by salt treatment (Fig.…”