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

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 These results the regulation of glycerol synthesis by activating the expression of DsGPDHs in response to salt stress. Our work provides insight into the alternative splicing of DsMEK1


Background
Saline soil is a severely adverse environmental factor, and more than 800 million hectares of land is affected by excess salt concentrations [1]. Adaptive responses to salt stress can be grouped into three processes: osmotic stress, ionic stress, and detoxification response [2]. Among the various reported regulation mechanisms of salt stress [3][4][5], there are many studies on the regulation of salt stress by the MAPK cascade pathway [6]. MAPK cascade pathway is minimally composed of three kinase modules, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK) [7]. PLDα1derived PA binds to AtMPK6 and the activated AtMPK6 phosphorylates the Na + /H + antiporter, AtSOS1, reduced Na + accumulation in Arabidopsis leaves under salt stress [8].
DtMAPK in Dunaliella tertiolecta accumulation of intracellular glycerol under hyperosmotic shock [9]. In Arabidopsis, MKK4-MPK3 and MKKK20-MPK6 cascades mediate osmotic stress responses [10,11]. MEKK1-MKK5/6 mediates the salt-induced expression of iron superoxide dismutases in Arabidopsis [12]. It is precise that MAPK cascade pathway plays an important role in the three processes caused by salt, so it is necessary to further study of MAPK cascade pathway.
The activation of these MAPK cascade pathway is a continuous process. In the MAPK cascade pathway, MAPKKK is phosphorylated by external stimuli as the first step of signal transduction, while MAPK regulates the downstream target to respond to external stimuli.
So the current research focus is mainly on MAPKKK and MAPK. For example, AtMAPKKK20 4 mediates osmotic stress induced by salt [10]. OsDMS1, a Raf-like MAPKKK gene, responses to oxidative stress caused by salt by regulating the scavenging of ROS [13]. Furthermore, AtMAPK3/6-HSFA4A is involved in the modulation of ROS metabolism and responses to salt stress [14]. OsWRKY30 could interact with and be phosphorylated by OsMPK3/7/14.
Phosphorylation of OsWRKY30 by MAPKs plays a crucial role in the process in which OsWRKY30 performing its biological function under salt stress [15]. However, there are few reports that MAPKK is involved in the regulation of salt stress.
MAPKK plays a pivotal role in MAPK cascade pathway, and perform different functions in signal distribution. It has been reported that OsMEK1 from Oryza sativa, is involved in different signal transduction processes. OsMEK1-OsMPK1 interaction might be involved in defense against pathogens as well as salt and drought stresses [16]. Meanwhile, the OsMEK1-OsMPK5 pair plays a role in the response to cold stress [17]. This phenomena occur in other species. In Arabidopsis, the AtMKK1-AtMPK6 pair was reportedly a critical mediator common to both sugar and ABA signaling during seed germination [ These results imply that the alternative splicing of MAPKK is responsible for distributing a variety of signal transduction, which allows the MAPK cascade pathway to participate in a wider range of processes. However, the AS regulation mechanism of MAPKK was relatively unexplored, and the AS regulation mechanism of MAPKK has not been reported in salt stress.
Dunaliella salina is a kind of unicellular green algae without a rigid cell wall, which can Here, we report that DsMEK1 gene undergoes alternative splicing, producing two protein splice variants, the full-size DsMEK1-X2 form and the truncated DsMEK1-X1 form that contains the disrupted protein kinase domain. DsMEK1-X2 can interact with DsMAPK1 and 6 DsMAPKKK1/2/3/10/17. However, so far, no interacting protein of DsMEK1-X1 has been found. DsMEK1 alternative splicing is induced under salt stress, producing more DsMEK1- Results 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 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[32],localization [33], and patterns of expression [34]. 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 8 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) [9]. 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 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 [2], 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 [36]. Given that DtMAPK was found to be involved in the regulation of glycerol synthesis in D tertiolecta [ 9], 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 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 [37]. So we further analyzed the transcription levels of all DsGPDH (DsGPDH1-7) [38], 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 Alternative splicing can produce proteins with different biological functions to help organisms deal with a variety of stresses [44]. In Arabidopsis, HAB1 encode HAB1.1 and HAB1.2. HAB1.1 switching the ABA signaling off, while, HAB1.2 keeping the ABA signaling, that play opposing roles in ABA-mediated seed germination and ABA-mediated post-germination developmental arrest [45]. Alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In our research, AS of DsMEK1 can produce two proteins with different biological functions in D.
salina to deal with different effects caused by salt stress. We found that DsMEK1-X1 overexpression strains have a similar increase rate of glycerol with the control after salt stress (Fig. 6E). Hence it can be suggested that DsMEK1-X1 response to salt stress independent with glycerol synthesis. In DsMEK1-X1-oe and DsMEK1-X2-oe lines, the content of MDA both less than control lines under salt stress (Fig. 6C). Furthermore, the proline content both increased in DsMEK1-X1-oe lines, and DsMEK1-X2-oe lines compared with control under salt stress. The increase rate of proline in DsMEK1-X1 lines was slightly higher than that in DsMEK1-X2 lines (Fig. 6D). Previous studies of plant have reported that proline can protect the plasma membrane by upregulating activities of various antioxidant systems to minimize membrane lipid and protein oxidation resulted from salinity-induced oxidative stress [46], MDA is the product of cell membrane lipid peroxidation and the content of MDA increased can leads to plant metabolic disorders [47]. Combining with the qRT-PCR assay shown that DsMEK1-X1 was induced under oxidative stress (Fig. 7). Based on our results, we speculated that DsMEK1-X1 can mediate various antioxidant systems to reduce the damage of oxidative and help D. salina adaptive to salt stress (Fig. 11). The glycerol accumulation rate of DsMEK1-X2-oe strains was 2.5 times than that of the control after 30 minutes of salt stress, however, in DsMEK1-X2-RNAi strains, there was less change in glycerol content under salt stress (Fig. 6E). Furthermore, DsGPDH2/3/5 were upregulated in DsMEK1-X2-oe strains and DsGPDH6 was induced in DsMEK1-X2-RNAi strains under salt stress (Fig. 7). These results revealed that DsMEK1-X2 is essential for the regulation of glycerol synthesis by activating the expression of DsGPDHs in response to salt stress. Our work provides insight into the alternative splicing of DsMEK1 13 responding to different process caused by salt stress.
Summarizing the present knowledge on DsMEK1-X2, we put forward the hypothesis of the MAPK cascade pathway in D. salina (Fig. 11). DsMAPKKK1/2/3/10/17-DsMEK1-X2-DsMAPK1 cascade pathways were essential for the regulation of glycerol synthesis by activating the expression of DsGPDHs in response to salt stress. In Y2H and in vivo overexpression experiments, we did not find the interacting proteins of DsMEK1-X1, so we just constructed the overexpression strain of DsMEK1-X1, knock-down strain of DsMEK1-X1 were not constructed for further study. The difference between DsMEK1-X1 and DsMEK1-X2 interaction networks may be caused by protein structure. DsMEK1-X1 lacks a part of the NTF2 domain (Fig. 1C), and it has been reported that the knockout of NTF2 will lead to the weakening of protein interaction ability [48], so we speculate that the difference of NTF2 domain may lead to the change of DsMEK1-X1 and DsMEK1-X2 regulatory networks.
There are two possible reasons for the interaction network of DSMEK1-X1 was not found in this study: 1. In the initial predicted interaction network, because of the high similarity between DsMEK1-X1 and DsMEK1-X2, the results of the interaction network predicted by STRING database were the same. May have not hit the interaction network of DsMEK1-X1; 2. DsMEK1-X1 has evolved a MAPK-independent phosphorylation pathway, and can directly phosphorylate downstream transcription factors, just like OsMAPK5, directly phosphorylation by CPK18, instead of MKK-dependent phosphorylation pathway [49]. In this paper, the MAPK cascade pathway of D. salina was studied systematically for the first time, which is helpful for us to further study the salt tolerance mechanism of D. salina.

Conclusion
In conclusion, DsMEK1-X1 and DsMEK1-X2 in D. Salina were successfully cloned and characterized. We showed that overexpression of DsMEK1-X2 enhanced cell growth under salt stress. Overexpression of DsMEK1-X2 in D. Salina resulted in glycerol accumulation and DsGPDHs expression level change, and DsMAPKKK1/2/3/10/17-DsMEK1-X2-DsMAPK1 cascade involved in glycerol synthesis under salt stress (Fig. 11). These results confirmed that a MAPK signaling pathway, similar to yeast HOG pathway [50], may be involved in the  [51]. For transformation and selection experiments solid and liquid TAP mediums (Tris-Acetate-Phosphate) were used, adjusted to contain a final concentration of 0.5 M NaCl. For solid medium 2.5% agar was added [52].
Ten milliliters (7 × 10 6 cells) unialgal culture was inoculated in triplicate into 100 ml of De Walne's culture media and was grown under controlled laboratory conditions to study the growth pattern. Growth was measured in terms of cell numbers using Neubaur haemocytometer [53].
Gene cloning, DNA sequencing, bioinformatic analysis, and phylogenetic tree analysis The full-length CDS of DsMEK1 was accessed in D. salina transcriptome database. The forward and reverse primers used to analyze the CDS of DsMEK1 genes (Table S1 No. 1-2).
PCR products were analyzed on a 2% agarose gel,then combined with the pMD19-T Vector The O. sativa MAPKK and Chlamydomonas reinhardtii MAPKK1 sequences were obtained from the NCBI database. Multiple protein sequence alignments were performed using ClustalX 2.0, while a phylogenetic tree was constructed from the amino acid sequences using the neighbor-joining method with MEGA6 software. A bootstrap analysis was performed using 1000 replicates [54]. 16

Subcellular localization
For protein localization observation, DsMEK1-X1 and DsMEK1-X2 were inserted into the p1300-GFP expression vector using gene specific primers (primers No. 3-4 in Table S1) [38]. To transiently express the fusion proteins in protoplasts isolated from Arabidopsis leaves, PEG-mediated transformation was used to transfect the protoplasts with each DsMEK1::GFP construct. GFP was excited by 488 nm laser lines and detected with bandpass 498-543 nm filters [55].
Yeast two-hybrid assays Yeast two-hybrid assays were performed with the Y2H Gold-Gal4 system (Clontech).

Transformation of D . s a l i n a
Constructed plasmids were transformed into D. salina cells using the Agrobacteriummediated transformation method as described previously with slight modifications [56].
Total sugar content Ten-milliliter culture was centrifuged at 5,000 rpm for 5 min and the pellet was resuspended in 4 ml distilled water and incubated at 100 ˚ C for 1 h. Homogenized cells were centrifuged for 10 min at 5,000 rpm and 2 ml upper aqueous solution was transferred in a fresh test tube. A measure of 1 ml phenol solution (5%) was added into the test tube, followed by the addition of 5 ml sulphuric acid. The test tube was kept at room temperature for 30 min. Upper color phase was taken out for the absorbance reading at 485 nm [53]. Reading was compared to find out the total sugar content of samples with a standard curve, which was drawn by the same method, using a known amount of glucose as standard.

Proline content
The proline content of each sample subjected to 3. Glycerol content Intracellular glycerol content of D. salina was measured using the method of Prabhakar et al [57] with slight modifications. All samples (5 mL) were centrifuged (6000 g, 5 min at RT) and the pellets were washed with fresh culture medium. Those pellets were suspended in distilled water (1 mL). The suspensions were freeze-thaw and then centrifuged (12000 g, 10 min, RT). The supernatant (200µL) of each sample was made up to 1 mL by adding distilled water. 1.2 ml sodium periodate reagent was added and mixed followed by the addition of 1.2 mL acetylacetone reagent. These samples were gently mixed and placed in a water bath (60 ˚ C, 10 min) and quickly cooled in an ice-bath. The absorbance of each sample was read at 413 nm after cooling at room temperature.

RNA extraction and qRT-PCR
The total RNA of each sample was isolated with the TRIzol™ Reagent (Invitrogen) according to the manufacturer's instructions. The first cDNAs were synthesized using ExTaq™ PCR Kit following the manufacturer's protocol (Takara, China). qRT-PCR was carried out with BIORAD CF96 Real-Time PCR system using SYBR ® Premix Ex Taq™ II (Takara, China). The primers used for qRT-PCR are listed in Table S2, and the β-tubulin gene was used as an internal reference. Each treatment was repeated three times independently. The 2 − ∆∆Ct method was used to analyze the relative expression of genes.

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional files. Additional Files Table S1. Primers and antisense oligonucleotides used in this study.     The expression levels of DsGPDH in DsMEK1-X1-oe, DsMEK1-X2-oe, and DsMEK1-X2-RNAi lines, were detected by qRT-PCR assays. Data are presented as means (±SE, n = 3). The columns with "*" had a statistical difference (p < .05, fold change > 2).
36 Figure 9 Interactions of DsMEK1 splice variants with six MAPKKKs and one MAPK in yeast two-hybrid assay. We confirm that our bait does not autonomously activate the reporter genes in Y2H Gold in the absence of a prey protein.