## 4 Discussion  
Many chlorophytes accumulate intracellular glycerol, as a compatible solute, under high salinity [1, 2, 5, 6, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3  
ter salinity [13; 67]. Chloroplast-localized bidomain GPD enzymes, consisting of canonical glycerol-3-phosphate dehydrogenase domains fused with an N-terminal phosphoserine phosphatase-like domain, have been implicated in the response to salt stress [5; 15; 17; 18]. In C. _reinhardtii_, transcriptomic and RNA interference analyses also support a role for these enzymes in triacylglycerglycerol accumulation under nitrogen or phosphorus deprivation [15; 20; 27; 28]. However, the regulation of these bidomain GPDs under different environmental stresses is not clearly understood. As proposed before [15], for membrane glycerolipid and TAG synthesis under normal or nutrient-deprived conditions, the production of G3P catalyzed solely by the canonical GPD domains would be energetically favored. On the other hand, under osmotic/salinity stress, the synthesis of glycerol would also require an active G3P phosphatase domain. The canonical GPD domains from several _Dunaliella_ bidomain enzymes have been reported to be active in diverse contexts: in in vitro assays with purified recombinant proteins [17; 25], in the complementation of yeast GPD deletion mutants [25], and in tests with the partly purified, by DEAE cellulose chromatography, endogenous osmoregulated enzyme [68]. The latter was also the case when the enzyme was purified from cells grown at low NaCl concentration [68], which accumulate very little glycerol [12; 46]. Likewise, _C. reinhardtii_ bidomain GPD enzymes have been reported to display glycerol-3-phosphate dehydrogenase activity in the complementation of yeast deletion mutants [19; 20] and in in vitro assays with recombinant proteins [15]. In addition, _C. reinhardtii_ GPD2 overexpression strains, grown in nutrient replete TAP medium, showed a significant increase in diglycerides (but not glycerol) relative to the wild type [20], as expected if transgenic GPD2 mostly synthesizes G3P under normal environmental conditions. Based on these observations, we favor the hypothesis that the canonical GPD domains of the chloroplast-localized bidomain enzymes are active in vivo under (nearly) all environmental conditions. Additionally, in the experiments reported here, the stability of transgenic GPD2 protein was very similar in cells cultured in nutrient replete medium as in those subject to nitrogen deprivation for 48 h (Figure 3C), possibly suggesting a similar enzyme conformation under these conditions, where mainly G3P synthesis is expected. However, in several algal species, transcriptional (and potentially translational) regulation may constrain endogenous bidomain GPD protein expression only to cells subject to certain environmental stresses [5; 18; 19; 20; 24; 25; 27; 28]. In contrast, conflicting results have been published on the G3P phosphatase activity of bidomain GPD enzymes. An \(\sim\)86 kDa protein (likely a bidomain GPD enzyme), partly purified from _D. salina_, showed unstable G3P phosphatase activity [69]. In addition, its Km for G3P, about 2.7 mM, was considered unusually high [68; 69]. Using purified recombinant proteins in in vitro assays, no G3P phosphatase activity was detected for two _D. viridis_ bidomain GPD enzymes [25]. However, recombinant _C. reinhardtii_ GPD2 and _D. salina_ osmoregulated GPDH were able to convert DHAP directly to glycerol in biochemical assays [15; 17]. Mutation of key aspartate residues in the phosphoserine phosphatase-like domain of GPD2 abolished its G3P phosphatase activity, substantiating its function as a GPP [15]. In the present work with overexpression strains, transgenic GPD2 protein was clearly detected and fairly stable in cells grown photoautotrophically in minimal medium (Figure 3A), but no glycerol accumulation was detected under these conditions (Figure 3B).