Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

Abstract

Background: Dunaliella salina can produce glycerol under salt stress, and this production can quickly adapt to changes in external salt concentration. Notably, glycerol is an ideal energy source. In recent years, it has been reported that the mitogen-activated protein kinase cascade pathway plays an important role in regulating salt stress, and in Dunaliella tertiolecta DtMAPK can regulate glycerol synthesis under salt stress. Therefore, it is highly important to study the relationship between the MAPK cascade pathway and salt stress in D. salina and modify it to increase the prodution of glycerol. Results: In our study, we identified and analysed the alternative splicing of DsMEK1 (DsMEK1-X1, DsMEK1-X2) from the unicellular green alga D. salina. DsMEK1-X1 and DsMEK1-X2 were both localized in the cytoplasm. qRT-PCR assays showed that DsMEK1-X2 was induced by salt stress. Overexpression of DsMEK1-X2 revealed a higher increase rate of glycerol production compared to the control and DsMEK1-X1-oe under salt stress. Under salt stress, the expression of DsGPDH2/3/5/6 increased in DsMEK1-X2-oe strains compared to the control. This finding indicated that DsMEK1-X2 was involved in the regulation of DsGPDH expression and glycerol overexpression under salt stress. Overexpression of DsMEK1-X1 increased the proline content and reduced the MDA content under salt stress, and DsMEK1-X1 was able to regulate oxidative stress; thus, we hypothesized that DsMEK1-X1 could reduce oxidative damage under salt stress. Yeast two-hybrid analysis showed that DsMEK1-X2 could interact with DsMAPKKK1/2/3/9/10/17 and DsMAPK1; however, DsMEK1-X1 interacted with neither upstream MAPKKK nor downstream MAPK. DsMEK1-X2-oe transgenic lines increased the expression of DsMAPKKK1/3/10/17 and DsMAPK1, and DsMEK1-X2-RNAi lines decreased the expression of DsMAPKKK2/10/17. DsMEK1-X1-oe transgenic lines did not exhibit increased gene expression, except for DsMAPKKK9. Conclusion: Our findings demonstrate that DsMEK1-X1 and DsMEK1-X2 can respond to salt stress by two different pathways. The DsMEK1-X1 response to salt stress reduces oxidative damage; however, the DsMAPKKK1/2/3/9/10/17- DsMEK1-X2-DsMAPK1 cascade is involved in the regulation of DsGPDH expression and thus glycerol synthesis under salt stress.

DsLCYB Directionally Modulated β-Carotene of the Green Alga Dunaliella salina under Red Light Stress

“…salina (also named FACHB-435) was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (China), and was passaged and preserved by our laboratory [26]. D. salina was cultivated in liquid DeWalne's medium containing 1.5 M NaCl [26,27] and then grown with shaking [28] under different light conditions including black light (BL), 80 μmol (photons)/m 2 /s, red light (RL), 80 μmol/m 2 /s, and white light (WL, 80 μmol/m 2 /s) using SuperFlux LED, at 26 ± 1°C and 16/8 h (light/dark). When the number of algal cells under RL was about 2 × 10 6 cells/ml, this was considered as the initial sampling time point (0 h).…”

“…Because D. salina cells are sensitive to chloramphenicol, the modified pGreen0029-Cmr vector was used as the D. salina transformation vector [26]. To obtain pGreen-0029-Cmr-DsLCYB-overexpressing strains, the fulllength CDS of DsLCYB was amplified by PCR (primers shown in Table S2) and then inserted into the modified pGreen0029-Cmr vector.…”

“…To obtain pGreen-0029-Cmr-DsLCYB-overexpressing strains, the fulllength CDS of DsLCYB was amplified by PCR (primers shown in Table S2) and then inserted into the modified pGreen0029-Cmr vector. D. salina transformation was performed according to the Agrobacterium-mediated transformation method of Claudia et al [32], with slight modifications [26].…”