## 3 Results
### Control of Glycerol Accumulation under Salinity Stress
Okadaic acid (OA), a polyether fatty acid, is a strong inhibitor of protein phosphatases, specifically several serine/threonine phosphatases such as protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) homologs [53, 54]. OA was successfully used to inhibit PP1 phosphatases in _C. reinhardtii_[55], but short incubation times with the inhibitor failed to affect PP2A3 [56]. Based on in vivo studies, the amount of OA required to inhibit protein phosphatases varies, possibly because its uptake can be affected by pH, temperature, and exposure time [56]. Given these observations, we tested several OA concentrations and long, 24 h pre-incubation times in our experiments. Treatment of wild type _C. reinhardtii_ with OA led to somewhat increased glycerol accumulation, relative to untreated cells, upon salt stress (Figure 4A). This was substantiated in a second experiment using the lowest effective concentration of OA (i.e., 10 nM) to minimize any non-specific effects (Figure 4B). However, under the same conditions, OX-12 cells incubated with OA did not show significantly higher glycerol accumulation than untreated cells (Figure 4B). In metazoans and fungi, the target of rapamycin (TOR) protein kinase assembles into two complexes with distinct partners and independent functions [57, 58]. In _S. cerevisiae_, the target of rapamycin complex 2 (TORC2) plays a role, independently from the HOG pathway, in a transient signaling response to hyperosmotic stress. High osmolarity prevents TORC2-mediated phosphorylation and activation of another kinase, YPK1, which in turn phosphorylates and inhibits one of the major glycerol-3-phosphate dehydrogenases, GPD1 [11]. Thus, under normal growth conditions, TORC2 has a repressive effect on GPD1 activity. By contrast, microalgae and other photosynthetic organisms only possess the target of rapamycin complex 1 (TORC1) [58], and the TOR kinase appears to have, at least transiently, a positive role in land plant responses to drought and osmotic stress [59, 60, 61]. In several studies in _C. reinhardtii_[57, 62], Torin1 has been used to inhibit the TOR kinase with high specificity. Interestingly, when wild type _C. reinhardtii_ was incubated with Torin1, glycerol accumulation triggered by NaCl stress was nearly entirely abolished (Figure 4C). In contrast, OX-12 cells treated with Torin1 were still able to synthesize glycerol when stressed with NaCl (Figure 4C). The effect of Torin1 on glycerol accumulation in the wild type and OX-12 strains was similar to that caused by the inhibition of cytoplasmic protein synthesis with cycloheximide (Figure 3B). Indeed, in cells exposed to NaCl, preexistent GPD2 protein was still able to synthesize glycerol in the presence of both Torin1 and cycloheximide (Figure 4D, OX-12 HS+NaCl+Tor1+Chx), suggesting that any post-translational regulatory mechanism(s) might be largely independent of TOR kinase activity. On the other hand, the TOR kinase appears to play a positive role in the accumulation of glycerol in response to salinity stress in wild type _C. reinhardtii_. However, further work will be necessary to assess its actual function, possibly affecting transcriptional and/or translational responses. Figure 4: Effect of protein phosphatase and TOR kinase inhibitors on glycerol accumulation by the wild type and OX-12 strains. (**A**) Glycerol content in the wild type cultured in minimal HS medium or HS medium containing 100 mM NaCl (HS+NaCl) in the presence of the indicated concentrations of the protein phosphatase inhibitor okadaic acid. Values shown are the mean \(\pm\) SD of three independent experiments (n = 3). (**B**) Glycerol content in the wild type and OX-12 strains cultured in HS medium or HS medium containing NaCl, in the absence (Ctrl) or presence of 10 nM okadaic acid (OA). Values indicate the mean \(\pm\) SD of three independent experiments (n = 3). Different lowercase letters indicate a significant difference among means (one way ANOVA with post hoc Tukey HSD test, \(p\) < 0.05).