cDNA cloning and sequence analysis of the DsMEK1 gene
One specific band of the expected length was detected with 2% agarose gel electrophoresis, but DsMEK1 had an additional lower band (Fig. 1A). Considering the potential for AS, these bands were purified and transformed into Escherichia coli DH5α, screened by colony PCR, and clones containing fragments of different lengths were then sequenced and a total of two transcript variants of DsMEK1 were obtained.
Two splicing variants, one in which thirteen introns were complete spliced (DsMEK1-X2), the other transcript variant DsMEK1-X1 lacked four exons (Fig. 1B). The DsMEK1-X1 gene’s CDS is 1020 bp that encodes a 340 amino acid protein with an expected molecular weight of 37.07 kDa. The DsMEK1-X2 gene’s CDS is 1464 bp which encodes a 488 amino acid protein with an expected molecular weight of 53.66 kDa. DsMEK1-X2 code for the full-length protein, whereas DsMEK1-X1 codes for a truncated protein, lacking a partial protein kinase domain and NTF2 domain (Fig. 1C).
A dendrogram was built based on the translated amino acid sequences of DsMEK1-X1, DsMEK1-X2, 10 Arabidopsis members (AtMAPKK1-10), 8 O. sativa members (OsMAPKK1-8), and 1 C. reinhardtii member CrMAPKK1 (Fig. 2). The MAPKK proteins used in the analysis were divided into four groups, A–D. DsMEK1-X1 and DsMEK1-X2 were categorized into subfamily B, which contains AtMKK3 and OsMKK3 suggesting that DsMEK1-X1 and DsMEK1-X2 possessed the close evolutionary relationship with AtMKK3 and OsMKK3. Previous studies revealed that AtMKK3 was induced by ABA or salt stress treatments, and treatment with methyl jasmonate (MeJA) or salicylic acid (SA) could induced the expression of OsMKK3. Thus, the phylogenetic tree analysis suggested that DsMEK1-X1 and DsMEK1-X2 may involve in abiotic stress.
Database search (WoLF PSORT) with the DsMEK1 splice variant sequences pointed to the same targeting (cytoplasm). For validating the prediction, DsMEK1-X1::GFP, and DsMEK1-X2::GFP were expressed in Arabidopsis protoplasts (Fig. 3). As expected the DsMEK1-X1::GFP and DsMEK1-X2::GFP localized to cytoplasm. This suggested that the splicing does not alter the localization of DsMEK1s variants in the D. salina.
DsMEK1 is regulated by alternative splicing
For many genes, AS leads to the production of functionally different protein isoforms, which may exhibit alterations in activity, interactive partners,localization, and patterns of expression. To solve the last issue, we firstly analyzed the transcription levels of DsMEK1 isoforms under salt stress. DsMEK1-X2 was significantly up-regulated along with the salt treatment, while DsMEK1-X1 was nearly unaffected under salt stress (Fig. 4).
Given that DsMEK1-X2 was found to be involved in the regulate of salt stress, we constructed DsMEK1-X1 and DsMEK1-X2 overexpression lines named DsMEK1-X1-oe and DsMEK1-X2-oe, respectively, furthermore, we constructed a DsMEK1-X2 knock down mutant, DsMEK1-X2-RNAi (Fig. 5A). In all the transformants, the Cmr gene (573 bp) was found, confirming the correct insertion of DsMEK1s in the genome of the D. salina (Fig. 5B). Furthermore, qRT-PCR assays of DsMEK1-X1 and DsMEK1-X2 gene were performed in those lines, which confirmed that the related genes were overexpressed or knock down (Fig. 5C).
MDA, proline, total sugar and so on are often used as indicators of salt tolerance in plants. To verify the function of DsMEK1-X1 and DsMEK1-X2, we detected some physiological indexes under salt stress in D. salina. Firstly, we analyzed the growth curve under the salt condition of these lines. The cell growth rate of DsMEK1-X2-oe lines increased and DsMEK1-X1-oe lines were not significantly different with that of control types, while DsMEK1-X2-RNAi lines were lower than that of the control lines (Fig. 6A). These results showed that DsMEK1-X2 can regulate salt stress and affect growth. The contents of total sugar content, proline content and MDA content were determined by the methods described previously. Under salt stress, the DsMEK1-X1-oe, DsMEK1-X2-oe, and DsMEK1-X2-RNAi lines were no difference with the control lines in total sugar content (Fig. 6B). On the other hand, both overexpression strains decreased MDA content compared with control, DsMEK1-X2-RNAi strains increased MDA content under salt stress (Fig. 6C). Furthermore, we also detected the proline content of the DsMEK1 transformants (Fig. 6D). The proline content in DsMEK1-X1-oe and DsMEK1-X2-oe increase compared to the control. Interesting, DsMEK1-X1-oe increase more than DsMEK1-X2-oe. The content of proline in DsMEK1-X2-RNAi was less than control (Fig. 6D). Previous reports that MDA and proline can reduce the damage of oxidative, so we speculate that DsMEK1-X1 involved in mitigating oxidative-stress damage under salt induced. We analyzed the expression level of DsMEK1-X1 and DsMEK1-X2 under oxidative stress. As shown in Fig. 7, DsMEK1-X1 can regulate oxidative stress, however, DsMEK1-X2 does not respond to oxidative stress, it means that DsMEK1-X1 is mainly involved in antioxidant defense in response to salt stress.
As we all know, Dunaliella can survive in a wide range of salt concentrations is attributed to its ability to adjust osmotic potential by changing intracellular glycerol concentration. Given that DtMAPK was found to be involved in the regulation of glycerol synthesis in D tertiolecta , we predicted that the DsMEK1 would regulate glycerol production in response to salt stress. To investigate this, we analysis the glycerol content under high salinity conditions (3.5 M NaCl concentration). As expected, glycerol content in DsMEK1-X1-oe, DsMEK1-X2-oe, and control could be enhanced significantly after salt stress. The glycerol content in DsMEK1-X1-oe has a similar increased rate compared with control, which both increased by about 40% after 30 min. Interesting, glycerol of DsMEK1-X2-oe increased almost 100% after 30 min of salt stress, it's 2.5 times than the control and DsMEK1-X1-oe lines. Then, DsMEK1-X1-oe, DsMEK1-X2-oe and the control have similar glycerol content after 2 h. DsMEK1-X2-RNAi strains have fewer changes in glycerol content under salt stress (Fig. 6E). These results indicating that overexpression of DsMEK1-X2 could enhance glycerol accumulation to mediate salt stress in D. salina cells.
The results reveal that DsMEK1 involved in the regulation of glycerol synthesis under salt stress (Fig. 4, Fig. 6). Glycerol-3-phosphate dehydrogenase (GPDH) is a rate-limiting enzyme in the glycerol synthesis pathway and intracellular glycerol concentration functions as the counterbalancing osmolyte in D. salina. So we further analyzed the transcription levels of all DsGPDH (DsGPDH1-7), which were reported to be involved in glycerol synthesis. The expression profile of these genes in the DsMEK1-X1-oe, DsMEK1-X2-oe, DsMEK1-X2-RNAi lines and control were analyzed by qRT-PCR under salt stress (Fig. 8). There was no difference in DsGPDH1/7 genes between the control strains and the DsMEK1-X1-oe, DsMEK1-X2-oe, DsMEK1-X2-RNAi lines under salt stress. The expression of DsGPDH4 was upregulated in all strains under salt stress. Those results mean that DsGPDH1/4/7 was not regulated by DsMEK1 under salt stress. DsGPDH2/3/5 genes were upregulated in the DsMEK1-X2-oe strains compared to control under salt stress. The expression of DsGPDH2 increased almost 4 times, the expression of DsGPDH3 was upregulated approximately 2.5 times and the expression of DsGPDH5 increased approximately 2 times in the DsMEK1-X2-oe strain compared to control. It is evidenced that DsMEK1-X2 can positively regulate DsGPDH2/3/5 expression and thus glycerol synthesis under salt stress. Furthermore, DsGPDH6 upregulated in the DsMEK1-X2-RNAi strains and control lines, it is speculated that DsGPDH6 was negatively regulated by DsMEK1-X2 under salt stress. Based on the data analysis, it was confirmed that DsMEK1-X2 can mediate DsGPDH2/3/5/6 and is essential for the regulation of glycerol synthesis under salt stress.
Interaction of DsMEK1 Splice Variants with their Upstream and Downstream interactors
Alternative splicing could provide selective advantage for choosing upstream and downstream regulators [32, 39]. So we investigated the effect of AS on the regulation of the variants by performing Y2H assay. The Y2H results showed that DsMAPK1 and DsMAPKKK1/2/3/9/10/17 interacted with DsMEK1-X2 but not with DsMEK1-X1 (Fig. 9).
To further investigate the function of DsMEK1, we analyzed the expression of DsMAPKKK1/2/3/9/10/17 and DsMAPK1 in control, DsMEK1-X1-oe, DsMEK1-X2-oe and DsMEK1-X2-RNAi lines under salt stress (Fig. 10). Notably, DsMAPK1 and DsMAPKKK1/3/10/17 were up-regulated in DsMEK1-X2-oe lines under salt stress, DsMAPKKK2/10/17 were down-regulated in DsMEK1-X2-RNAi lines under salt stress. Combine with the results of Y2H, we confirmed that DsMAPKKK1/3/10/17-DsMEK1-X2-DsMAPK1 positively regulate salt stress, and DsMAPKKK2-DsMEK1-X2 negatively regulate it.