## 3 Results
### Control of Glycerol Accumulation under Salinity Stress  
To explore the hypothesis that the GPP domain of GPD2 is activated (or de-repressed) under salinity stress, we examined glycerol accumulation in the wild type and OX-12 strains under a variety of treatments, aimed at identifying a plausible regulatory mechanism(s), including light exposure, scavengers of reactive oxygen species, and inhibitors of protein phosphatases or protein kinases. We first tested whether light was required for glycerol accumulation in _C. reinhardtii_ under hypersalinity conditions. Cells, in the middle of the logarithmic phase, were cultured for three days in minimal HS medium under continuous darkness (to deplete them from stored starch), and then incubated for an additional 6 h under continuous light or dark in HS medium or HS medium containing 100 mM NaCl. Salinity stress triggered substantial glycerol accumulation in light-exposed cells, as before, but this response was abolished in the starch-depleted dark-incubated cells (Figure S2A). The same experiment was repeated with cells cultured in TAP medium, containing acetate as a source of carbon. In this case, dark-incubated cells also were capable of accumulating glycerol upon exposure to NaCl (Figure S2B), suggesting that acetate provided a carbon source for glycerol synthesis. Please also note the change in scale when comparing Figures S2A and S2B, indicating that cells cultured in acetate-containing medium accumulate much higher levels of glycerol than those cultured in minimal HS medium. These results are consistent with prior studies in several _Dunaliella_ species indicating that light does not play a regulatory role in the synthesis of glycerol upon salinity/osmotic stress [12; 46]; although photosynthesis does provide carbon skeletons and reducing power for glycerol production. In _Dunaliella_ spp., the products of both photosynthesis and starch breakdown contribute to glycerol synthesis in the light, whereas in the dark glycerol is mostly synthesized at the expense of starch degradation [12, 46, 47]. Besides its osmotic effects, high salinity can also stimulate the production of reactive oxygen species (ROS), leading to oxidative damage [6, 16]. Thus, we also tested whether ROS signaling might be involved in triggering glycerol synthesis upon exposure of cells to NaCl. \(N\)-acetyl-L-cysteine (NAC) is a synthetic precursor of intracellular cysteine and glutathione, and it has antioxidant activity resulting from its free radical scavenging property, either directly via its thiol-disulfide exchange activity or secondarily via increasing glutathione levels in the cells [48]. NAC has been commonly used as a cell-permeable ROS scavenger in diverse organisms, including _C. reinhardtii_[49, 50]. However, incubation of wild type and OX-12 cells with NAC did not affect the glycerol accumulation triggered by NaCl stress in any of the strains (Figure S2C). The response to hyperosmotic stress and accumulation of glycerol in the yeast _Saccharomyces cerevisiae_ involves several conserved signaling pathways, with the high osmolarity glycerol (HOG) pathway being the most prominent [11, 16]. In this protein kinase cascade, the signal is often transmitted by reversible protein phosphorylation, modulating proteins' activity, localization and interactions with other proteins. Mitogen-activated protein kinases (MAPKs), related to yeast HOG1, have also been implicated in glycerol production under salt stress in \(D\). _salina_ and \(D\). _tertiolecta_[51, 52]. Moreover, proteomics analyses in _C. reinhardtii_ subject to osmotic shock have suggested the involvement of stress-related kinases, such as MAPK6, and phosphatases in the initial cellular responses [16]. Thus, we tested whether protein phosphatase or protein kinase inhibitors might affect glycerol accumulation in our strains.