## 4 Discussion
These observations suggested that GPP domain activity might be context dependent and possibly regulated at the post-translational level. Interestingly, the _C. reinhardtii GPD2_ gene was significantly upregulated upon exposure of cells to NaCl ([15; 19; 20], this work) but, in several proteomic experiments examining cells under osmotic/salt stress, the GPD2 protein was not detected [16; 23]. Likewise, genes encoding certain bidomain GPD enzymes were transcriptionally induced in _Dunaliella_ spp. exposed to hypersalinity [18; 24; 25] but, at least in _D. tertiolecta_[26] and _D. salina_[70], no changes were detected in the abundance of the corresponding proteins. In this study, we observed that the stability of the transgenic GPD2 protein was greatly reduced when cells were cultured in HS medium containing NaCl (Figures 3A and S1). Yet, under these conditions, OX-12 cells (unlike the wild type) were able to accumulate glycerol in the absence of de novo cytoplasmic protein synthesis blocked by the addition of cycloheximide (Figure 3B). Thus, pre-existing transgenic GPD2 protein (and likely also endogenous bidomain GPDs) appears to be altered by NaCl stress; and its destabilization may be related to activation (or de-repression) of the GPP domain allowing glycerol synthesis (please see below). We used several approaches, mostly relying on pharmacological treatments, to explore the mechanism(s) involved in the proposed post-translational GPP domain regulation under salinity stress. However, the ROS scavenger N-acetyl-L-cysteine, the protein phosphatase inhibitor okadaic acid, and the TOR kinase inhibitor Torin1 did not affect glycerol accumulation by transgenic GPD2 strains upon salinity stress (Figure 4 and Figure S2C). Only staurosporine, a non-specific protein kinase inhibitor, was able to prevent the NaCl-induced synthesis of glycerol by pre-existing transgenic GPD2 (Figure 5D). Intriguingly, we still observed GPD2 protein destabilization under these conditions (Figure 5C). Proteomic experiments enriching AcV5-tagged GPD2 protein by affinity purification, followed by identification of differentially phosphorylated peptides by LC-MS/MS only detected Ser145 (within the GPD2 GPP domain) as potentially phosphorylated upon NaCl stress (Figure S3A). While this residue is within a conserved motif in bidomain GPDs from diverse microalgae (Figure S3B), it remains uncertain whether its phosphorylation is functionally relevant. Moreover, the corresponding peptide was only identified at low/medium confidence due to its low abundance. However, in cells grown photobeteorotrophically in TAP medium under normal conditions, the same peptide has been reported to be unphosphorylated [71]. Additionally, we cannot rule out that a kinase(s) inhibited by staurosporine might modify another protein which, in turn, affects GPD2 activity. Nevertheless, our results, taken together, strongly indicate that the GPD2 protein is post-translationally regulated upon NaCl stress. Post-translational regulation of enzymes involved in osmotic/salt stress responses is supported by evidence in various organisms [6, 11, 14, 21, 72]. In _D. salina_, de novo protein synthesis was not required for the osmotic response, and the increase in glycerol content triggered by exposure to high salinity [72]. In _Phyomyces blackseleanus_, a G3P phosphatase has been proposed to be activated by phosphorylation mediated by a cyclic-AMP-dependent protein kinase [73]. Moreover, in _C. reinhardtii_, metabolome analysis revealed that the proline-biosynthetic pathway is strongly induced upon salt stress but, in parallel proteomic analyses, enzymes in this pathway did not change in abundance [21]. Other studies also reported very rapid metabolic changes and remodeling of primary metabolism in _C. reinhardtii_ when exposed to osmotic stress [16]. These observations suggest that initial responses to osmotic/salinity stress may involve the post-translational regulation of existing enzymes, allowing cells to adjust rapidly their metabolism to cope with the stress.