## 4 Discussion  
_Dunaliella_ is a green microcalga, well-known by its high production of carotenoids under specific conditions. In this context, we characterized carotenoids production among an isolated strain of _Dunaliella_ sp. cultured under two conditions: unstressed and stressed. The growth rate was influenced by the stress condition, causing a decrease in the DSS growth rate after a week. This was probably due to the depletion of the nitrogen source initially used in the medium. Similar results were also found by [PERSON] et al. [56], where _D. salina_ grown under nitrogen limitation maintained the same growth rate as those under nitrogen sufficiency during 8 days and then decreased to the half in about 20 days. The decrease in cell number was accompanied rather with an increase in intracellular carotenoids accumulation and a decrease of chlorophyll amount. [PERSON] et al. in 2016 [21] studied the effect of nutrition depletion on carotenoid production that increased from approximately 2 pg/cell in complete medium to 9 pg/cell in nitrogen-depleted medium on day 15. This observation is in agreement with a previous study showing that the limitation of nitrogen availability accompanied by an increase of salinity and light intensity induced carotenoid accumulation [34]. The increase of salinity engenders also an increase in glycerol and polysaccharide synthesis accompanied by starch degradation [57-59]. For a better characterization of microalgae cells, they were subjected to flow cytometry analysis. Among DSC, we noticed the presence of two populations, which could be due to different evolution cells stage. The analysis of cells at the end of the exponential phase did not permit to display the increase in size and volume of DSS cells versus DSC. [PERSON] et al. [32] showed that during the first day of environmental stress, cells accumulated lipid globules, causing an increase of cell granularity. After two days, this granularity declined as the stress level would be reduced due to the increase in  
Figure 7: 3D model of SUMO (small ubiquitin-related modifier) protein from _Dunaliella_ sp. (**a**) 3D model of LCYB, showing docked FAD molecule, SUMO interaction motifs, and SUMO lysine site in yellow color (**b**). \(\beta\)-carotene accumulation, and the cell's granularity would become almost like the unstressed one [60]. Based on autofluorescence proprieties of chlorophylls and carotenoids pigment, we confirmed the deviation of _Dunaliella_ metabolism to carotenoids accumulation. In view of this change observed at the phenotypic level, we decided to analyze its response during the first three days of stress, at the transcriptional level of _ENO_, _DXS_, _LCYB_, _CGP_, _HSP70_, and _RPE genes_. For this purpose, the first step was the determination of the best reference gene, which appeared to be the _\(\alpha\)-Tub_. Two other studies have also reported the stability of the _\(\alpha\)-Tub_ gene under severe conditions, one of which was conducted with _Dunaliella_ sp. grown under high light stress [61]. Similarly, a second study realized with _Syntrichia caninervis_ under abiotic stress; the _\(\alpha\)-Tub_ gene was also found to be stable amongst 15 reference genes, and _185_ was the less unstable one [41]. Enolase is a ubiquitous enzyme that catalyzes the penultimate reversible step of glycolysis, which is the unique dehydration of D-2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) [62]. Its regulation is a key step in the orientation of the organism's metabolism to glycerol or tricarboxylic acid formation [63]. In our study, we found that the transcript level of _ENO_ declined during the first 24 h of stress, which was in accordance with results found by [PERSON] et al., reporting a decrease in mRNA expression and ENO activity during 12 h of hyperosmolarity stress [59]. The expression of _ENO_ tended to increase and restabilize at 72 h of culture with a relative expression level close to 1 (Figure 3a) and similar to that of DSC (Figure 3h).