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 a large amount of glycerol under salt stress, which can quickly adapt to the change of external salt concentration, and glycerol is one of the ideal energy sources. In recent years, it has been reported that 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 urgent to study the relationship between MAPK cascade pathway and salt stress in D. salina, and help it to increase the content of glycerol. Results： In our study, we identified and analyzed the alternative splicing of DsMEK1 (DsMEK1-X1, DsMEK1-X2) from the unicellular green alga D. salina. DsMEK1-X1, DsMEK1-X2 both localized in the cytoplasm. The qRT-PCR assays showed that DsMEK1-X2 induced by salt stress. Overexpression of DsMEK1-X2 revealed a higher increase rate of glycerol compared to the control and DsMEK1-X1-oe under salt stress. The expression of DsGPDH2/3/5/6 increased in DsMEK1-X2-oe strains compared to the control under salt stress. It means that DsMEK1-X2 is involved in the regulation of DsGPDHs expression and glycerol overexpression under salt stress. Overexpression of DsMEK1-X1 increasing the proline content and reducing the MDA content under salt stress, and DsMEK1-X1 can regulate oxidative stress, thus we speculate that DsMEK1-X1 can reduce the damage of oxidative under salt stress. Yeast two-hybrid analysis showed that DsMEK1-X2 can 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 do not increased genes expression, except for DsMAPKKK9. Conclusion： Our findings demonstrate that DsMEK1-X1 and DsMEK1-X2 can response to salt stress in two different pathways, DsMEK1-X1 response to salt stress by reducing oxidative damage, however, DsMAPKKK1/2/3/9/10/17- DsMEK1-X2-DsMAPK1 cascade is involved in the regulated of DsGPDH expression and thus glycerol synthesis under salt stress.

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

10.21203/rs.2.20554/v2 ◽

2020 ◽

Author(s):

Ziyi Tang ◽

Xiyue Cao ◽

Yiping Zhang ◽

Jia Jiang ◽

Dairong Qiao ◽

...

Keyword(s):

Salt Stress ◽

Protein Kinase ◽

Oxidative Damage ◽

Dunaliella Salina ◽

Splice Variants ◽

Mitogen Activated Protein Kinase ◽

Mitogen Activated Protein ◽

Transgenic Lines ◽

Kinase Cascade ◽

Glycerol Synthesis

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.

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