## 1 Introduction  
Microalgae can live in a variety of environments, due to their ability to withstand changing and, sometimes, extreme conditions. For instance, unicellular green algae from the genus _Chlamydomonas_ have been found in soil, freshwater, oceans, deserts, and even in snow or sea ice, where they can face substantial variations in salinity [1, 2, 3, 4, 5, 6]. The genus _Chlamydomonas_ currently consists of several hundred species, but it is highly polyphyyletic [7, 8]. _Chlamydomonas reinhardtii_ is a well-established model system for examining photosynthesis, physiology, metabolism and the structure and function of flagella [3, 4, 9]. However, most contemporary laboratory strains of _C. reinhardtii_, including those used in the work described here, are derived from a single zygote isolated from a potato field in Massachusetts in 1945 [3, 4]. Salinity is one of the key environmental factors affecting the growth and distribution of microalgae. Organisms exposed to salt stress have to cope with ionic and osmotic imbalances, as well as toxicity effects, such as oxidative damage [5, 6, 10, 11]. Under these conditions, many microorganisms counteract dehydration through the intracellular accumulation of one or more compatible solutes, small organic molecules characterized by a neutral charge and low toxicity at high concentrations [5, 6, 11]. Glycerol is a common osmolyte synthesized by several yeast species and microalgae, including _C. reinhardtii_ and _Dunaliella_ spp. [1, 2, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17]. This compatible solute contributes to the osmotic balance of cells and the maintenance of enzyme activities under conditions of cell shrinkage, molecular crowding and low water activity [10, 11, 12, 14]. Glycerol is generally synthesized in a two-step pathway from the glycolytic intermediate dihydroxyacetone phosphate (DHAP) (Figure 1), although an alternative pathway via dihydroxyacetone has been proposed in some species [6, 10, 11, 14, 15]. In the first step, NAD(P)+-dependent glycerol-3-phosphate dehydrogenase (GPDs) catalyze the reduction of DHAP to glycerol-3-phosphate (GSP). In the second step, G3P is dephosphorylated to glycerol by glycerol-3-phosphate phosphatases (GPPs). Microalgae in the Chlorophyceae case and Trebouiophyceae classes encode GPDs with an unusual bidomain structure, consisting of a phosphoserine phosphatase-like domain fused to canonical glycerol-3-phosphate dehydrogenase domains [5, 15, 17, 18, 19]. Recent work has demonstrated that two of these bidomain enzymes, _C. reinhardtii_ GPD2 [15] and _D. salina_ osmoregulated GPDH [17], can convert DHAP directly to glycerol, indicating that the phosphoserine phosphatase-like domain actually functions as a GPP. _C. reinhardtii_ possesses five NAD(P)+-dependent GPD homologs and the genes encoding two bidomain isoforms (i.e., GPD2 and GPD3), in particular _GPD2_, were substantially upregulated upon exposure of cells to NaCl [15, 19, 20]. By using pulse labeling with \({}^{13}\)C-arginine in an auxotrophic strain, an increase in the rate of synthesis of the GPD2 protein (ID 146945) was also observed in cells exposed to NaCl [21]. Moreover, consistent with enhanced activity of the corresponding enzyme(s), the acclimation of _C. reinhardtii_ to hyperosmotic stress involved the rapid accumulation of glycerol, as well as several other sugars [13, 16, 22]. However, various proteomic experiments examining cells subjected to osmotic/salt stress failed to detect any bidomain GPD protein [16, 23]. Other chlorophytes also showed increased expression of genes encoding bidomain GPDs in response to osmotic/salinity stress. In a psychrophilic species of _Chlamydomonas_ (UWO 241), a bidomain  
Figure 1: Simplified pathway of glycerol metabolism in _C. reinhardtii_ and connections to membrane glycerolipid and triacylglycerenol synthesis. For additional details, see [PERSON] et al. [15]. Metabolites: DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; Gly, glycerol.