## 3 Results
### Transcriptional Analysis
#### 3.2.2 Gene Expression Profile under Stressed Conditions  
A follow-up of the expression of some genes involved in the caroten accumulation process during the first 72 h of stress time was processed by quantitative PCR. The first gene followed was _ENO_ involved in the glycolysis cycle and giving an idea about carbon origin (polysaccharide degradation, photosynthesis, etc.) and destiny (isoprenoids synthesis, carbohydrate synthesis, etc.) in the cell. Its transcript level decreased 0.48 times during the first 24 h of stress. Then, it tended to re-stabilize after 72 h by having a relative expression level close to 1 (Figure 3a) and without a significant difference in its expression with unstressed cells (Figure 3g). On the other hand, the _DXS_ mRNA level doubled after 24 h of stress (Figure 3b), while after 72 h, the comparison with unstressed culture showed a significant decrease in its expression level (Figure 3g).  
Figure 2: Flow cytometry analysis: counters plots control of population gated under unstressed condition (**(a)**. above) and stressed condition (**(a)**. below). Mean fluorescence intensities of _Dunaliella_ sp. Cells were obtained upon excitation with FL1 (FITC, 530/30 nm), FL2 (PE, 575/26 nm), and FL3 lasers (PerCP, 682/13 nm) (**b**).  
Figure 3: Gene expression levels as a function of stress time. The amounts of _ENO_ (**a**), _DXS_ (**b**), _LCYB_ (**c**), _CGP_ (**d**), _HSP70_ (**e**), and _RPE_ (**f**) mRNA were normalized against that of the \(\alpha\)-_Tub_ gene. Agarose gel electrophoresis of the selected gene in _Dunaliella_ sp. under stressed and unstressed conditions upon 72 h of cell culture (**g**). Bands intensity correlates with values found by qPCR quantification. Linear regression between the relative expression of _LCYB_ and _CGP_, showing a linear relationship between the expressions of these two genes (**h**). An asterisk indicates a significant difference between gene expressions at different stressed times, according to the one-way ANOVA test followed by Tukey test. * \(p\) \(<\)0.05; ** \(p\) \(<\) 0.005; *** \(p\) \(<\) 0.001. Data are expressed as means \(\pm\) SD (n = 3). - Model, ------ confidence interval (Mean 95%), ------ confidence interval (obs. 95%).  
The two genes \(LCYB\) and \(CGP\), which are directly implicated in caroten accumulation, showed the most important rise. Data showed that their expression was increased since 4 h of stress time (Figure 3c,d) at an exponential pace. For the \(LCYB\) gene, its expression was found to be about 3-fold higher in stressed culture than the unstressed one during the first 72 h (Figure 3g). However, for \(CGP\), the expression level in the stressed cell was very high; it was improved by almost 11-fold at 24 h (Figure 3d). There was a drastic increase in their expression between 24 h to 72 h of stress conditions: up to 10-fold for \(LCYB\) and to 54-fold for \(CGP\) (Figure 3c,d). We noticed also a positive correlation in their mRNA expression (R\({}^{2}\) = 0.997).  
The less important increase was registered in HSP70B mRNA expression level, where the increase reached 3 and 2.27 times at 72 h in stressed and non-stressed conditions, respectively (Figure 3e,g). Chloroplast ribulose phosphate-3-epimerase (RPE) transcript level showed no significant variation during the first 72 h of stress (Figure 3f,g).