cDNA cloning and sequence analysis of the DsMEK1 gene
To determine the sequence of the DsMEK1 cDNA isolated from the transcriptome, we conducted a RACE analysis (Fig S1). According to the analysis of D. salina transcriptome data, there may be two alternative splices forms in DsMEK1, named DsMEK1-X1 and DsMEK1-X2, in which the length of DsMEK1-X1 is approximately 1000 bp and the length of DsMEK1-X2 is approximately 1400 bp. Two bands were separated by 2% agarose gel electrophoresis, and the sizes of the bands were in accordance with the expected sizes (Fig 1A). These bands were puriﬁed and transformed into Escherichia coli DH5α and screened by colony PCR, clones containing fragments of different lengths were then sequenced, and a total of two transcript variants of DsMEK1 were obtained.
The identification of exon/intron structures for the DsMEK1-X1 and DsMEK1-X2 genes was studied by aligning full-length cDNA sequences and corresponding genomic DNA sequences. Two splicing variants were characterzied; for one, thirteen introns were completely spliced (DsMEK1-X2), and the other variant (DsMEK1-X1) lacked four exons (Fig 1B). The CDS of the DsMEK1-X1 gene is 1020 bp, which encodes a 340 amino acid protein with an expected molecular weight of 37.07 kDa. The CDS of the DsMEK1-X2 gene is 1464 bp, which encodes a 488 amino acid protein with an expected molecular weight of 53.66 kDa. DsMEK1-X2 encodes the full-length protein, whereas DsMEK1-X1 encodes 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, DsMEK2, 10 Arabidopsis members (AtMAPKK1-10), 8 O. sativa members (OsMAPKK1-8), 2 Chlamydomonas reinhardtii members CrMAPKK2 and CrMAPKK3, 1 Ostreococcus lucimarinus OlMAPKK6, and 2 Volvox carteri VcMAPKK1 and VcMAPKK2 (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 a 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 induce the expression of OsMKK3. Thus, the phylogenetic tree analysis suggested that DsMEK1-X1 and DsMEK1-X2 may be involved in abiotic stress.
Database search (WoLF PSORT) with the DsMEK1 splice variant sequences pointed to the same targeting (cytoplasm). To validate the prediction, DsMEK1-X1::GFP and DsMEK1-X2::GFP were expressed in Arabidopsis protoplasts (Fig 3). As expected, DsMEK1-X1::GFP and DsMEK1-X2::GFP localized to the cytoplasm. This finding suggested that splicing does not alter the localization of DsMEK1 variants in 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, patterns of expression and localization,. To solve the last issue, we first analysed the transcription levels of DsMEK1 isoforms under salt stress. DsMEK1-X2 was signiﬁcantly upregulated along with salt treatment, while DsMEK1-X1 was nearly unaffected under salt stress (Fig 4).
Given that DsMEK1-X2 was found to be involved in the regulation of salt stress, we constructed DsMEK1-X1 and DsMEK1-X2 overexpression lines named DsMEK1-X1-oe and DsMEK1-X2-oe, respectively (Fig 5A). Furthermore, we constructed a DsMEK1-X2 knockdown 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 D. salina (Fig 5B). qRT-PCR assays of the DsMEK1-X1 and DsMEK1-X2 genes were performed in those lines, which confirmed that the related genes were overexpressed or knockdown (Fig 5C).
MDA, proline, total sugar and other compounds 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. First, we analysed the growth curve under the salt conditions of these lines. The cell growth rate of the DsMEK1-X2-oe lines increased, and that of the DsMEK1-X1-oe lines was not significantly different from that of the control lines, while that of the DsMEK1-X2-RNAi lines was 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 total sugar content of the DsMEK1-X1-oe, DsMEK1-X2-oe, and DsMEK1-X2-RNAi lines was not different from that of control lines (Fig 6B). On the other hand, both overexpression strains decreased MDA content compared with the control, and 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 increased compared to the control. Interestingly, DsMEK1-X1-oe increased more than DsMEK1-X2-oe. The proline content in DsMEK1-X2-RNAi was less than that in the control (Fig 6D). Previous reports have shown that MDA and proline can reduce oxidative damage; therefore, we speculate that DsMEK1-X1 is involved in mitigating oxidative stress damage under salt induction. We analysed the expression levels 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, which means that DsMEK1-X1 is mainly involved in antioxidant defence in response to salt stress.
Dunaliella species can survive in a wide range of salt concentrations because of their ability to adjust osmotic potential by changing intracellular glycerol concentrations. Given that DtMAPK was found to be involved in the regulation of glycerol synthesis in D tertiolecta, we predicted that DsMEK1 would regulate glycerol production in response to salt stress. To investigate this possibility, we analysed the glycerol content under high salinity conditions (3.5 M NaCl concentration). As expected, glycerol content in DsMEK1-X1-oe, DsMEK1-X2-oe, and the control could be enhanced significantly after salt stress. The glycerol content in DsMEK1-X1-oe had a similar increased rate compared with the control, which both increased by approximately 40% after 30 min. Interestingly, glycerol of DsMEK1-X2-oe increased almost 100% after 30 min of salt stress, which was 2.5 times that of the control and DsMEK1-X1-oe lines. Then, DsMEK1-X1-oe, DsMEK1-X2-oe and the control had similar glycerol contents after 2 h. DsMEK1-X2-RNAi strains had fewer changes in glycerol content under salt stress (Fig 6E). These results indicate that overexpression of DsMEK1-X2 could enhance glycerol accumulation to mediate salt stress in D. salina cells.
The results reveal that DsMEK1 is 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. Therefore, we further analysed the transcription levels of all DsGPDHs (DsGPDH1-7), which were reported to be involved in glycerol synthesis. The expression profiles of these genes in the DsMEK1-X1-oe, DsMEK1-X2-oe, and DsMEK1-X2-RNAi lines and the control were analysed 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, and DsMEK1-X2-RNAi lines under salt stress. The expression of DsGPDH4 was upregulated in all strains under salt stress. These 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 the 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 the control. DsMEK1-X2 can positively regulate DsGPDH2/3/5 expression and thus glycerol synthesis under salt stress. Furthermore, DsGPDH6 was upregulated in the DsMEK1-X2-RNAi strains and control lines, and DsGPDH6 was specalated to be 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 a selective advantage for choosing upstream and downstream regulators[17, 32]. Therefore, we investigated the effect of AS on the regulation of the variants by performing a 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 analysed the expression of DsMAPKKK1/2/3/9/10/17 and DsMAPK1 in the 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 upregulated in DsMEK1-X2-oe lines under salt stress, and DsMAPKKK2/10/17 were downregulated in DsMEK1-X2-RNAi lines under salt stress. Combined with the results of Y2H, we confirmed that DsMAPKKK1/3/10/17-DsMEK1-X2-DsMAPK1 positively regulates salt stress and that DsMAPKKK2-DsMEK1-X2 negatively regulates salt stress.