# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 3 Results and Discussion
### Molecular Investigation
#### 3.5.4 Differential Expression of the _FtsH2_ Gene Under Various Stress Conditions  
To obtain insight into FtsH metalloproteinase functions, we assessed the expression patterns of the _FtsH2_ gene in _Dunaliella_ sp. under different stress conditions by qRT-PCR. Compared to the control, _FtsH2_ expression was differentially regulated regarding the stress treatment. Interestingly, the highest transcription levels were observed in response to light stress and nitrogen starvation (8.5- and 7.4-fold increases, respectively) (Figure 4d). When salt stress was applied (135 g/L NaCl salinity (equivalent to 2.31 M NaCl)), there was a significant increase in the _FtsH2_ expression level. The variation in pH had a marginal effect on _FtsH2_ expression. The response of the _FtsH2_ gene to hormonal additions was also investigated by treating microalgae with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 6-benzylaminopurine (BAP), gibberell acid (GA3), and kinetin. As shown in Figure 4d, _FtsH2_ expression was upregulated in response to GA3 and IBA, while no significant changes were observed with the other phytohormones. These results, which coincided with the prediction analysis of promoter cis-acting elements of _FtsH2_, demonstrate that this gene is mainly involved in the response to abiotic stressors, including nitrogen limitation, light, and salt stresses, and is directly implied in the process of microalgae growth and development mechanisms. In agreement with previous research on _Arabidopsis thaliana_ and _Solanum tuberosum_, _FtsH2_ was highly expressed under various abiotic stress including high light intensity, salt, oxidative, and osmotic stresses [118; 119]. Moreover, _FtsH2_ from _Mediago truncatula_, _Arabidopsis thaliana_, _Triticum aestivum_, and the cyanobacterium _Synechocystis_ has been reported to repair PSII by degrading damaged D1 protein under high light stress conditions [116; 120; 121]. Several studies have demonstrated that _FtsH_ gene expression was significantly influenced under nutrient-limited conditions in the cyanobacteria _Synechocystis_ sp. [17; 19]. In addition, it has been reported that FtsHs are involved in plant stress resistance by regulating and maintaining chloroplast protein homeostasis. In fact, these metalloproteinases degrade misfolded and aggregated proteins to repair chloroplast damage under environmental stress. Recent studies on _Dunaliella salina_ transcriptomic responses to hypersaline stress showed that some genes, including the _FtsH2_ gene, have high expression within 2 h of stress [122]. [PERSON] et al. [123] also demonstrated that proteins with salinity-induced damage in chloroplasts from _D. salina_ were unfolded and degraded via a well-organized proteolytic mechanism through chloroplast biogenesis, maintenance, and stress response guided by the overexpression and post-translational phosphorylation of FtsH, in addition to the abundance of the Clp protease chain. FtsH can efficiently degrade proteins showing weak thermodynamic stability induced by short-term salinity stress in _D. salina_[124].