## 3 Results  
### Chlamydomonas reinhardtii GPD2 Transgenic Strains  
Since we were primarily interested in examining the proposed post-translational regulation of bidomain GPD2, we decided to generate transgenic strains expressing this protein under the control of the _C. reinhardtii PSAD_ regulatory sequences (i.e., promoter, \(5^{\prime}\) and \(3^{\prime}\) untranslated regions, and terminator) (Figure 2A). _PSAD_ is a nuclear gene that encodes an abundant chloroplast-targeted protein, the subunit II of the Photosystem I reaction center [34]. Various transgenes constructed with _PSAD_ regulatory sequences have been demonstrated to be expressed constitutively and at high levels in _C. reinhardtii_ grown under continuous light [34]. Thus, _PSAD:GPD2:PSAD_ transgenic strains would be expected to express the GPD2 protein in the absence of any environmental stress, when cultured in HS minimal medium under continuous light. We generated several overexpression strains that showed substantial increases in _GPD2_ transcript abundance relative to the wild type (CC-124) when grown in HS medium (Figure 2B, OX-9, OX-12, and OX-30). However, when cells were subject to salinity stress, the upregulated _GPD2_ transcript levels were similar in the wild type and overexpression strains (Figure 2C). We next tested whether the overexpression strains could accumulate glycerol or non-polar lipids, such as TAGs, when cultured under normal environmental conditions in HS medium. Because of similar preliminary results with all overexpression strains, we focused our analyses on OX-12, showing intermediate GDP2 transcript levels (Figure 2B), to avoid potential artifacts caused by excessive protein expression. Interestingly, the OX-12 strain, similarly to the wild type, only showed a significant increase in non-polar lipid content, as examined by staining cells with the lipophilic fluorophore Nile Red, when cells were subject to nitrogen deprivation (Figure 2D). Likewise, glycerol accumulation above background levels was only detected in cells cultured for 6 h in HS medium containing 100 mM NaCl (Figure 2E), and no significant differences in glycerol content were observed between OX-12 and the wild type. A previous study also generated _GPD2_ overexpression strains, showing substantial increases in _GPD2_ transcript levels, by using transgenes constructed with the tandem _HSP70A-RBCS2_ promoter [20]. These strains were grown photoheterotrophically in TAP medium (containing acetate) with or without phosphorus limitation. However, none of the overexpression strains showed changes in glycerol accumulation, relative to the wild type, under the conditions tested. They did exhibit increased total lipid content but only under low phosphorus stress, and the accumulated amounts were slightly lower than in the wild type cultured under the same conditions [20]. These combined results indicated that, despite significant increases in _GPD2_ transcript levels in overexpression strains grown under normal environmental conditions, the contents of expected products of GPD2 activity did not differ from the background levels detected in the wild type. Figure 2: Characterization of _PSAD:GPD2:PSAD_ transgenic strains. (**A**) Schematic diagram of the transgene designed to express the GPD2 protein constitutively. cTP, chloroplast transit peptide; GPP, G3P phosphatase domain (related to phosphoserine phosphatase); GPDH, canonical G3P dehydrogenase domains; AcV5, epitope from the GP64 envelope fusion protein of _A. californica_ multiple nuclear polyhedrosis virus; _PSAD_ Pro, _PSAD_ promoter; _PSAD_ ter, 3\({}^{\prime}\)UTR and terminator from _PSAD_. (**B**) _GPD2_ mRNA abundance in overexpression strains grown in minimal HS medium relative to the wild type (CC-124). Transcript expression was determined by qRT-PCR, and values  
shown are the mean \(\pm\) standard deviation (SD) of three independent experiments (n = 3). Samples indicated with an asterisk are significantly different from the wild type (\(p\) < 0.05) in a two tailed Student's \(t\)-test.