## 3 Results
### Transgenic GPD2 Protein Abundance and Stability  
We designed our transgenic construct with an epitope from the GP64 envelope fusion protein of _Autographa californica_ multiple nuclear polyhedrosis virus (i.e., the AcV5 tag) [33]abundance of transgenic GPD2 protein changed in a similar manner in cells cultured under nitrogen deprivation as in those maintained in HS medium for the same period (Figure 3C). Likewise, in cells incubated with cycloheximide to prevent de novo cytoplasmic protein synthesis, the abundance of transgenic GPD2 protein decreased similarly in cells subject to nitrogen deprivation as in those cultured in nutrient replete medium (Figure 3C, +Chx).  
Figure 3: Analysis of GPD2 protein abundance and glycerol accumulation in overexpression strain OX-12 upon inhibition of cytoplasmic protein synthesis by cycloheximide. Cells were pre-incubated with cycloheximide for 1 h before the start of the experimental treatments. (**A**) Immunoblot analysis of transgenic GPD2 protein levels detected with an anti AcV5 antibody. OX-12 cells were cultured, for the indicated times, in minimal HS medium or HS medium containing 100 mM NaCl (HS+NaCl),  
in the presence or absence of cycloheximide (Chx). Immunodetection of histone H3 was used as a control for equivalent loading of the lanes. The zero time (0 h) controls indicate that 1 h pre-incubation with cycloheximide did not alter, by itself, GPD2 protein abundance. (**B**) Glycerol content in the wild type and OX-12 strains grown for 6 h in HS medium or HS medium containing NaCl. Cells were cultured in the media alone (Ctrl) or with the addition of cycloheximide (Chx) or spectinomycin (Spec). 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, \(p<0.05\)). (**C**) Immunoblot analysis of transgenic GPD2 protein levels detected with an anti AcV5 antibody. OX-12 cells were cultured, for the indicated times, in minimal HS medium or HS medium lacking nitrogen (HS-N), in the presence or absence of cycloheximide (Chx). Immunodetection of histone H3 was used as a control for equivalent loading of the lanes.  
The simplest interpretation of these observations is that the GPP domain of transgenic GPD2 is inactive when cells are grown in HS medium, under normal environmental conditions (please see Discussion). However, under salinity stress in the presence of cycloheximide, pre-existent GPD2 protein becomes activated (or de-repressed) to synthesize glycerol (Figure 3B, OX-12 HS+NaCl+Chx) but, at the same time, it is rapidly destabilized (Figure 3A, OX-12 HS+NaCl+Chx). Both effects might be related to conformational changes in the GPP domain ultimately triggered in response to NaCl stress (please see Discussion). In contrast, transgenic GPD2 protein stability was similar in cells cultured under nitrogen deprivation as in those maintained in nutrient replete medium (Figure 3C). Conceivably, only the canonical GPD domains of GPD2 might be active under these conditions (please see Discussion), where no increases in glycerol content were observed in the OX-12 strain. As previously reported for the wild type [15], _C. reinhardtii_ overexpression strains accumulate appreciable amounts of non-polar lipids when subject to nitrogen deprivation (Figure 2D), suggesting that the G3P backbone is being produced. In nutrient replete medium, however, an expected increase in glycerol-3-phosphate dehydrogenase activity brought about by transgenic GPD2 might not alter the carbon flux directed to the synthesis of membrane glycerolipids (rather than storage TAGs) in rapidly growing cells. Indeed, when cells were grown in nutrient replete TAP medium, GPD2 overexpression strains only showed a significant increase in diglycerides relative to the wild type, but no differences in other lipid classes [20].