The Liriodendron chinense MKK2 Gene Enhances Arabidopsis thaliana Salt Resistance

To adapt and sense environmental perturbations, including a variety of biotic and abiotic stress conditions, plants have developed disparate regulatory pathways. Mitogen-activated protein kinase (MAPK or MPK) signaling cascades are found widespread across the eukaryotic kingdoms of life. In plants, they may regulate signaling pathways aimed at resisting the stressful effects of low temperature, salt damage, drought, touch, and mechanical damage. To date, no conclusive studies into Liriodendron chinense (Hemsl.) Sarg MPK-related stress resistance signaling have been performed. In our study, we cloned three homologous L. chinense MAP kinase kinase family genes: LcMKK2, LcMKK4, and LcMKK6. LcMKK2 and LcMKK6 have their highest expression level in the root, while LcMKK4 is highly expressed in the stem. LcMKK2 showed upregulation in response to salt and cold stress conditions in L. chinense. To further analyze its gene function, we overexpressed LcMKK2 in wild-type Arabidopsis thaliana (L.) Heynh. and obtained transgenic plants. Overexpression of LcMKK2 caused a significant reduction in plant mortality (from 96% to 70%) in response to a 7-day 200 mM NaCl treatment. Therefore, we conclude that LcMKK2 is involved in a signaling response to salt stress, and it could thus prove an effective target gene for breeding strategies to improve Liriodendron salt tolerance.


Introduction
Salt stress has a negative role in most plant growth and development; high salt causes a change in the osmotic potential or ion balance between the plant and its exterior environment, causing a water deficit and plant wilting. Subsequent leaf or root damage will eventually lead to plant death [1]. To respond to biotic or abiotic stresses, including salt stress, plants use a set of sophisticated signaling pathways [2]. Currently, the most studied salt stress regulatory pathways mainly include the MAPK (mitogen-activated protein kinase) signal pathway [3], the Ca 2+ signaling transduction pathway [4], and the ABA signal pathway [5]. In recent years, genetic engineering has been widely used as an effective tool for improving the target plant's abilities in dealing with environmental stresses [6]. Mitogen-activated protein kinases (MAPKs) are one of the largest groups of transferases, catalyzing phosphorylation of appropriate protein substrates on serine or threonine residues [7]. Transient activation of MAPK cascades is one of the first conserved defense responses playing an early triggering role in resistance caused by metabolic changes and transcriptional reprogramming [8]. MAPKs cascades are involved in signaling pathways that respond to various stresses, such as temperature stress or salt stress [9].
A typical MAPK cascade consists of at least three sequentially acting serine/threonine kinases; one MAP kinase kinase kinase (MAPKKK), one MAP kinase kinase (MAPKK), and finally, the MAP kinase (MAPK) itself, with each phosphorylating and thereby activating the next kinase in the cascade [10]. In 1986, Sturgll and Ray [11] first discovered a MAPK in animal cells. Over the next 20 years, a large number of studies have gradually revealed MAPK-related signal transduction pathways in eukaryotes. The exploration of plant MAPK signaling began in the 1990s, starting relatively late but developing very rapidly [12]. The first MAPK gene was isolated and identified from plants in 1993 [12]. In Arabidopsis, studies have revealed the existence of 20 MAPKs, 10 MAPK kinases, and 60 MAPK kinase kinases [13]. The rice (Oryza sativa) genome contains 17 MAPK, 8 MAPKK, and 75 MAPKKK genes [14], whereas the poplar (Populus trichocarpa) genome contains 21 MAPK and 10 MAPKK genes [15]. This indicates that a large number of MAPK cascade kinase genes are prevalent in plants.
In plants, MAPKK families have diverged into four major groups (A, B, C, and D) [15]. Taking Arabidopsis as a reference, group A includes MAPKK1 (MKK1) and MKK2 that act upstream of MPK4 [16]. Extensive genetic studies show that the MEKK1-MKK1/2-MPK4 cascade plays a role in negatively regulating temperature-dependent resistance through maintaining endogenous levels of salicylic acid (SA) and reactive oxygen species (ROS) and preventing programmed cell death [17]. MKK2 is also implicated in responses to cold and salinity, and both MKK1 and MKK2 mediate innate immunity responses [18,19]. In banana (Musa spp.), the MKK2 gene acts as a cold resistance gene [20]. MKK group B in Arabidopsis, including the gene MKK3, can be distinguished because of the presence of a nuclear transfer factor (NTF) domain [21]. Group C includes MKK4 and MKK5, while group D consists of the remaining MKKs (MKK7-10); the genes in these two groups contain no intron [19].
Plant MKK phosphorylation sites consist of the consensus sequence S/TxxxxxS/T, whereas the mammalian equivalent can be represented as S/TxxxS/T [13]. Interestingly, this conserved sequence does not occur in Arabidopsis MKK10 due to a three-amino-acid residue deletion in this region [13]. MKK proteins contain conserved sequences that can be phosphorylated by upstream MAPKKKs, which in turn phosphorylate the Thr and Tyr residues of the T-X-Y motif in downstream MAPKs [22]. Once activated, MPKs phosphorylate diverse substrates mainly located in the cytosol and nucleus to regulate protein function and gene expression for the appropriate cellular response [23]. Therefore, as an intermediate step in plants, MAPKKs play an extremely important role in the MAPK signal transduction pathway.
Studies have shown that the signaling pathway of cellular MKK2 phosphorylation contributes to abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism, phosphorylation of Arabidopsis thaliana MEKK1 via Ca 2+ signaling is a part of the cold stress response, and membrane rigidification functions upstream of the MEKK1-MKK2-MPK4 cascade during cold acclimation in A. thaliana [24]. The AtMKK2 gene is involved in many stress responses in plants, including salt stress and low temperature stress responses [25].
Liriodendron chinense (Hemsl.) Sarg, a well-known forest tree, belongs to the Liriodendron L. genus in the Magnoliaceae family. L. chinense has been identified as a precious commercial tree species with a fast growth rate and good wood texture [26]. It is an excellent tree species for barren mountain industrial timber and also a garden ornamental with great market potential [27]. The publication of the genome of L. chinense has greatly promoted the study of this valuable tree, but no comprehensive studies on genes related to stress response in this species have been reported thus far. The current lack of knowledge on how MAPK cascade genes function in Liriodendron limits their use in genetic improvement programs. In this study, we first report the cloning of L. chinense MAPKK genes. Because of the limitation of the forest tree transgenic system, Arabidopsis was used to study the basic functions of the LcMKK2 gene using cross-species transgenesis. We found that the overexpression of the LcMKK2 gene can improve salt tolerance in Arabidopsis, indicating that this gene may play a similar role in Liriodendron. The different tissues used in this study were harvested from an adult plant of L. chinense growing at the Nanjing Forestry University campus and were immediately frozen in liquid nitrogen and stored at −80 • C for RNA extraction. We used this RNA for gene cloning and qPCR experiments of expression in different parts of L. chinense. The tissue cultures of L. chinense grown in the bottle for 1 month were used for cold and salt stress treatments. Two-month old L. chinense plants were grown under room temperature and 4 • C for 48 h. Then, the first or second true leaves were collected and immediately frozen in liquid nitrogen and stored at −80 • C.

A. thaliana
The A. thaliana Columbia ecotype was used for this study. Transgenic Arabidopsis plants were obtained via the floral dip method [28]. Transgenic and wild-type Arabidopsis seeds were sterilized, then plated on 1 2 Murashige and Skoog (MS) medium with Kanamycin (Kan), and grown for 7 days until the first or second true leaves appeared. Transgenic plants overexpressing the LcMKK2 gene in the T2 generation were genotyped by PCR, after which transgenic plants were transferred onto nutrient soil. After growing in the soil for 2 weeks, the samples were collected for qPCR after being treated with 300 mmol/L NaCl for 2 h. In order to better observe the root system and phenotype of transgenic plants, we treated the transgenic plants under salt stress in 1 2 MS medium. For observing LcMKK2-overexpressing plants, three lines were selected. The NaCl concentration gradient used was 0, 100, and 200 mmol/L. Each treatment was repeated three times with 50 seeds per treatment.
2.1.3. LcMKK2, LcMKK4, and LcMKK6 Gene Cloning Total RNA was extracted from L. chinense leaves using the RNA extraction kit (Bioteke RP3301, Beijing, China) following the manual: (1) 100 mg leaves were completely grinded with liquid nitrogen in a mortar treated with 0.1% DEPC; (2) the lysis solution and β-mercaptoethanol were added into the sample; (3) after completely splitting, the nucleosomes were decomposed after incubation at 65 • C for 5 min; (4) after centrifugation under 12,000 rpm for 5 min, the supernatant was transferred into a new centrifuge tube; (5) 70% ethanol was added into the supernatant with equal volume; (6) the RNA was harvested with the adsorption column, which was digested with DNase I (Takara RR4201, Japan) at 37 • C for 20 min; (7) clean RNA was washed out with RNase-free H 2 O. RNA quality was tested using electrophoresis and ultraviolet spectrophotometry. Integral RNA without DNA contamination was applied for double-stranded cDNA synthesis using reverse transcriptase according to the manufacturer's instructions (Vazyme Biotech Co R312-02, Nanjing, China). The first-strand cDNA was synthesized with 1 µg RNA as template at 25 • C for 5 min, 37 • C for 45 min, and 85 • C for 5 s. We used Primer 5.0 [29] software to design primers, which are shown in Supplementary Table  S1. The TOYOBO's KODPLUS high-fidelity enzyme was used for DNA amplification. A 1.2 Kb band was obtained, which was consistent with the predicted size. The fragment-added poly (A) tail was ligated with the cloning vector PMD19-T Vector (Takara, Japan) and transferred to Escherichia coli for sequencing. XbaI and SacI double restriction sites were selected as the sites for ligation of the LcMKK2 gene to expression vector pBI121. The primers used for ligation are listed in Supplementary Table S2.

Quantitative qPCR Analyses
A Revert Aid Premium First Strand cDNA Synthesis Kit was obtained from Vazyme (Vazyme Biotech Co, Nanjing, China); a SYBR Green PCR master mix kit was purchased from Takara. Total RNA isolation and reverse transcription were performed as mentioned above. Quantitative real-time PCR was performed using a SYBR-Green PCR Mastermix on a LightCycler ® 480 real-time PCR detection Forests 2020, 11, 1160 4 of 12 system (Roche, Basel, Switzerland), according to the manufacturer's instructions. Three independent real-time PCR experiments were carried out. Sequence-specific primers were designed using Primer 5.0 [29] and Oligo 7 [30] and are listed in Supplementary Table S3. Each PCR reaction was repeated three times, and the average value was taken. The housekeeping genes 18S rRNA in L. chinense and ubiquitin10 (UBQ10) in Arabidopsis were used as a reference for expression levels. The expression level of 18S rRNA in stamens was used as a reference for expression normalization to one. Sequence-specific primers, which were designed using Oligo 7, are listed in Supplementary Table S3. The primers for salt-related gene detection are also shown in Supplementary Table S3. 2.1.5. Sequence Analysis of LcMKK2, LcMKK4, and LcMKK6 Identification of LcMKK genes was based on known gene sequences from A. thaliana (https: //www.arabidopsis.com). Orthologs of LcMKK2, LcMKK4, and LcMKK6 from other species were searched using National Center for Biotechnology Information (NCBI) blastp (https://blast.ncbi.nlm. nih.gov/Blast.cgi). The conservative domain of NtCIPK9 was predicted using InterProScan online software (http://www.ebi.ac.uk/InterProScan/). The phylogenetic tree was constructed using the neighbor-joining method [31], with a bootstrap value of 1000 replicates [32]. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 547 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [33]. The accession numbers of identified LcMKK sequences are listed in Supplementary  Table S4.

Identification and Sequence Analysis of LcMKK2, LcMKK4, and LcMKK6
Given the importance of genes in the MAPKK family under abiotic stresses, we wondered whether the function of these genes acts usefully in commercial timber tree species L. chinense. Thus, we isolated the MAPKKs from the leaves of L. chinense based on the MAPKK homologs of Arabidopsis from the NCBI database. Three LcMKK family genes were identified from L. chinense. Sequence analysis showed that LcMKK2, LcMKK4, and LcMKK6 contain a complete open reading frame of 1110, 1049, and 1065 bp, respectively, and encode a protein of 369, 347, and 354 amino acids, respectively. According to the BLAST tool analysis in NCBI, the LcMKK2, LcMKK4, and LcMKK6 proteins share more than 70% sequence identity with other MAPKK family genes, such as those from A. thaliana and P. trichocarpa. Multiple alignment analysis showed that the deduced protein sequence of this clone displayed a high identity with MKK2 orthologs from other species ( Figure 1A). Additionally, LcMKK4 and LcMKK6 are highly similar to MKK4 and MKK6 sequences from other species (Supplementary Figure S1).
To determine whether these identified genes belong to the MKK family and to obtain more insights into the evolutionary relationship between members of the different MKK subfamilies, we performed a phylogenetic analysis, in which we compared the isolated LcMKK proteins and their functionally characterized homologues from A. thaliana and P. trichocarpa on a sequence level. The resulting tree showed that the MAPKK family can be divided into four subfamilies (i.e., groups A, B, C, and D). LcMKK2 and LcMKK6 are classified into group A, whereas LcMKK4 is classified into group C ( Figure 1B).  To determine whether these identified genes belong to the MKK family and to obtain more insights into the evolutionary relationship between members of the different MKK subfamilies, we performed a phylogenetic analysis, in which we compared the isolated LcMKK proteins and their functionally characterized homologues from A. thaliana and P. trichocarpa on a sequence level. The resulting tree showed that the MAPKK family can be divided into four subfamilies (i.e., groups A, B, C, and D). LcMKK2 and LcMKK6 are classified into group A, whereas LcMKK4 is classified into group C ( Figure 1B).

LcMKK2 Responds to Cold and Salinity in L. chinense
The MAPK cascade pathway in plants is widespread in eukaryotes [34]. MAPKK family genes have been cloned in many species, and their signal transduction pathways are widespread in plants. We used real-time fluorescence quantitative PCR to analyze the expression levels of LcMKK2, LcMKK4, and LcMKK6 across different L. chinense tissues. We found that these three genes are expressed in all observed L. chinense tissues, including roots, stems, leaves, buds, flowers, petals, stamens, and pistils (Supplementary Figure S2A,B). However, each of the three MAPKK genes has its own unique expression pattern. LcMKK2 expression is highest in roots and lowest in stems and shows a relatively low expression level in the leaves. Flower organs, except petals, showed the highest expression of LcMKK2. Conversely, LcMKK4 shows its highest expression in the stem and the lowest expression in the leaves. Finally, LcMKK6 has its highest expression level in the root and the lowest in the leaves. Thus, the low expression in leaves is a common feature for these three MAPKK genes in L. chinense. In reproductive organs, these three genes have the highest expression in petals and the lowest expression in stamens.
In order to observe the expression of LcMKK2 in L. chinense under salt and cold stress, we isolated total mRNA from whole plants after a 2-h 100 mM NaCl treatment, as well as after a 48-h cold stress treatment. The qPCR results showed that the expression level of LcMKK2 was upregulated after both salt and cold stress (Figure 2A,B). This result indicates that LcMKK2 responds to both salt and cold stress, after which its expression is significantly upregulated.

LcMKK2 Responds to Cold and Salinity in L. chinense
The MAPK cascade pathway in plants is widespread in eukaryotes [34]. MAPKK family genes have been cloned in many species, and their signal transduction pathways are widespread in plants. We used real-time fluorescence quantitative PCR to analyze the expression levels of LcMKK2, LcMKK4, and LcMKK6 across different L. chinense tissues. We found that these three genes are expressed in all observed L. chinense tissues, including roots, stems, leaves, buds, flowers, petals, stamens, and pistils (Supplementary Figure S2A,B). However, each of the three MAPKK genes has its own unique expression pattern. LcMKK2 expression is highest in roots and lowest in stems and shows a relatively low expression level in the leaves. Flower organs, except petals, showed the highest expression of LcMKK2. Conversely, LcMKK4 shows its highest expression in the stem and the lowest expression in the leaves. Finally, LcMKK6 has its highest expression level in the root and the lowest in the leaves. Thus, the low expression in leaves is a common feature for these three MAPKK genes in L. chinense. In reproductive organs, these three genes have the highest expression in petals and the lowest expression in stamens.
In order to observe the expression of LcMKK2 in L. chinense under salt and cold stress, we isolated total mRNA from whole plants after a 2-h 100 mM NaCl treatment, as well as after a 48-h cold stress treatment. The qPCR results showed that the expression level of LcMKK2 was upregulated after both salt and cold stress (Figure 2A,B). This result indicates that LcMKK2 responds to both salt and cold stress, after which its expression is significantly upregulated.

Overexpression of LcMKK2 Causes Salt Resistance in Arabidopsis
To study whether LcMKK2 has a function in response to salt stress, we overexpressed the LcMKK2 gene in Arabidopsis (LcMKK2-OE). LcMKK2-OE plants developed more lateral branches, as well as more leaves on side branches, than wild-type Arabidopsis ( Figure 3C).

Overexpression of LcMKK2 Causes Salt Resistance in Arabidopsis
To study whether LcMKK2 has a function in response to salt stress, we overexpressed the LcMKK2 gene in Arabidopsis (LcMKK2-OE). LcMKK2-OE plants developed more lateral branches, as well as more leaves on side branches, than wild-type Arabidopsis ( Figure 3C).
We treated transgenic and wild-type seeds on medium containing 100 mM NaCl for a duration of 5 days. On 1 /2 MS control medium without salt, LcMKK2-OE and wild-type plants germinated and there was no significant difference in growth. We did observe an apparent lengthening of the root system in transgenic plants, especially in the OE-2 line ( Figure 3A). After the salt stress treatment, the growth of all plants was inhibited. Under these high salt conditions, wild-type plants are unable to develop green leaves ( Figure 3B). In contrast, within all three LcMKK2-OE lines, a certain percentage of plants developed green leaves ( Figure 3D). These results indicate that overexpression of LcMKK2-OE indeed enhances the salt tolerance of Arabidopsis. We speculate that LcMKK2-OE could improve salt tolerance due to the roots of these transgenic plants being longer than those in wild-type plants [35].  We treated transgenic and wild-type seeds on medium containing 100 mM NaCl for a duration of 5 days. On ½ MS control medium without salt, LcMKK2-OE and wild-type plants germinated and there was no significant difference in growth. We did observe an apparent lengthening of the root system in transgenic plants, especially in the OE-2 line ( Figure 3A). After the salt stress treatment, the growth of all plants was inhibited. Under these high salt conditions, wild-type plants are unable to develop green leaves ( Figure 3B). In contrast, within all three LcMKK2-OE lines, a certain percentage of plants developed green leaves ( Figure 3D). These results indicate that overexpression of LcMKK2-OE indeed enhances the salt tolerance of Arabidopsis. We speculate that LcMKK2-OE could improve salt tolerance due to the roots of these transgenic plants being longer than those in wild-type plants [35].
To further study the effects of LcMKK2-OE on the root system of A. thaliana plants under salt stress, we performed a root length assay after treating the plants with salt stress. On the medium without salt, no difference was observed between LcMKK2-OE and wild-type plants. However, after 5 days of the salt stress treatment, the root system of the transgenic plant was significantly longer than that of the wild-type plants (Figure 4). To further study the effects of LcMKK2-OE on the root system of A. thaliana plants under salt stress, we performed a root length assay after treating the plants with salt stress. On the medium without salt, no difference was observed between LcMKK2-OE and wild-type plants. However, after 5 days of the salt stress treatment, the root system of the transgenic plant was significantly longer than that of the wild-type plants (Figure 4).
Under very high concentrations, plants may be stressed to such a degree that they do not survive [36]. To study the lethality rate of A. thaliana under a high salt concentration, we treated plants for 7 days using medium containing 200 mM NaCl. Under this condition, wild-type plants germinated normally; however, after 3 days on high salt medium, these plants started showing lethality. As the stress time prolonged, the number of dead plants increased. After 7 days, Arabidopsis displayed a lethality rate of 96% ( Figure 5). LcMKK2-OE plants showed a much higher resistance to salt stress and were able to develop green leaves. Their survival rate was relatively high, with a maximum lethality rate of only 70%. Together, these results indicate that the LcMKK2 gene increases salt tolerance when overexpressed, possibly by inducing development of a more extensive root system.  Under very high concentrations, plants may be stressed to such a degree that they do not survive [36]. To study the lethality rate of A. thaliana under a high salt concentration, we treated plants for 7 days using medium containing 200 mM NaCl. Under this condition, wild-type plants germinated normally; however, after 3 days on high salt medium, these plants started showing lethality. As the stress time prolonged, the number of dead plants increased. After 7 days, Arabidopsis displayed a lethality rate of 96% ( Figure 5). LcMKK2-OE plants showed a much higher resistance to salt stress and were able to develop green leaves. Their survival rate was relatively high, with a maximum lethality rate of only 70%. Together, these results indicate that the LcMKK2 gene increases salt tolerance when overexpressed, possibly by inducing development of a more extensive root system.

Enhanced Expression of Salt Stress Genes in LcMKK2-OE
Having found that LcMKK2 overexpression leads to increased salt tolerance in Arabidopsis, we sought to determine the mechanism by which this development occurs. To clarify how LcMKK2-OE increases salt resistance, we tested the expression of salt stress response-related genes after a salt stress treatment. Previous studies have shown that the Arabidopsis MEKK1-MKK2-MAPK4/6 cascade is involved in the regulation of salt stress [37].  Under very high concentrations, plants may be stressed to such a degree that they do not survive [36]. To study the lethality rate of A. thaliana under a high salt concentration, we treated plants for 7 days using medium containing 200 mM NaCl. Under this condition, wild-type plants germinated normally; however, after 3 days on high salt medium, these plants started showing lethality. As the stress time prolonged, the number of dead plants increased. After 7 days, Arabidopsis displayed a lethality rate of 96% ( Figure 5). LcMKK2-OE plants showed a much higher resistance to salt stress and were able to develop green leaves. Their survival rate was relatively high, with a maximum lethality rate of only 70%. Together, these results indicate that the LcMKK2 gene increases salt tolerance when overexpressed, possibly by inducing development of a more extensive root system.

Enhanced Expression of Salt Stress Genes in LcMKK2-OE
Having found that LcMKK2 overexpression leads to increased salt tolerance in Arabidopsis, we sought to determine the mechanism by which this development occurs. To clarify how LcMKK2-OE increases salt resistance, we tested the expression of salt stress response-related genes after a salt stress treatment. Previous studies have shown that the Arabidopsis MEKK1-MKK2-MAPK4/6 cascade is involved in the regulation of salt stress [37].

Enhanced Expression of Salt Stress Genes in LcMKK2-OE
Having found that LcMKK2 overexpression leads to increased salt tolerance in Arabidopsis, we sought to determine the mechanism by which this development occurs. To clarify how LcMKK2-OE increases salt resistance, we tested the expression of salt stress response-related genes after a salt stress treatment. Previous studies have shown that the Arabidopsis MEKK1-MKK2-MAPK4/6 cascade is involved in the regulation of salt stress [37]. qPCR analysis showed that the expressions of the genes related to salt stress were increased in LcMKK2-OE plants after a 2-h salt stress treatment in 300 mM NaCl compared with those of the wild-type plants [38]. Proline is the most important osmoregulation substance in plants under high salt and other stresses. P5CS is a key enzyme in proline biosynthesis, which determines the rate of proline accumulation [39]. The P5CS gene was significantly upregulated in WT plants after salt stress, and it was extremely upregulated in transgenic plants.
The PS5C gene responds to salt-stress in both wild-type and transgenic plants. However, other salt stress response-related genes (ACS6 and ACT3) [25] were also upregulated after the salt treatment, whereas wild-type plants were downregulated after the stress treatment ( Figure 6). These results indicate that the LcMKK2 gene can regulate some other genes related to salt stress to help plants resist abiotic stress. was extremely upregulated in transgenic plants.
The PS5C gene responds to salt-stress in both wild-type and transgenic plants. However, other salt stress response-related genes (ACS6 and ACT3) [25] were also upregulated after the salt treatment, whereas wild-type plants were downregulated after the stress treatment ( Figure 6). These results indicate that the LcMKK2 gene can regulate some other genes related to salt stress to help plants resist abiotic stress. h. Data represent the mean ± SD from three technical replicates. An ANOVA test was used for statistical analysis. *** p < 0.001; ** p < 0.01; * p < 0.05.

Discussion
Soil salinity occurs widely throughout the environment, subsequently eliciting a plant response for survival. Generally, elevated levels of salt are encountered by individual roots, but it is likely that the whole plant will respond to salt stress because of the various signal transductions regulated by gene expression [10]. MKK cascades play an important role in transducing developmental and environmental signals into adaptive responses [20,40]. In previous studies, the MKK2 pathway was found to mediate cold and salt stress signaling in Arabidopsis [25]. In our study, we observed that

Discussion
Soil salinity occurs widely throughout the environment, subsequently eliciting a plant response for survival. Generally, elevated levels of salt are encountered by individual roots, but it is likely that the whole plant will respond to salt stress because of the various signal transductions regulated by gene expression [10]. MKK cascades play an important role in transducing developmental and environmental signals into adaptive responses [20,40]. In previous studies, the MKK2 pathway was found to mediate cold and salt stress signaling in Arabidopsis [25]. In our study, we observed that LcMKK2 overexpression mediates improved growth in Arabidopsis under normal conditions in terms of the root system. Moreover, LcMKK2-overexpressing plants hold a higher resistance to salinity than wild-type plants, as LcMKK2 behaves like other homologous genes in different plants.
Our previous hypothesis is that, under salt stress conditions, overexpressing LcMKK2 transgenic plants may phosphorylate downstream MAPK genes to transduce plant stress signals, causing a cellular response to improve salt tolerance. In previous literature, MPK4 and MPK6 were identified as the strongest interactors with MKK2 in Arabidopsis thaliana. In contrast, LcMKK2 does not depend on MPK4 and MPK6 to function in Arabidopsis in our study. Accordingly, the exact molecular mechanisms employed by LcMKK2 to improve salt resistance and regulate plant root growth are still unknown.
Transgenic analysis showed that overexpression of the P. trichocarpa MAPKK4 (PtMKK4) gene remarkably enhanced drought stress tolerance in transgenic poplar plants. Additionally, some drought marker genes, including PtP5CS, PtSUS3, PtLTP3, and PtDREB8, exhibited higher expression levels in the transgenic lines than in the wide-type plants under drought conditions [41]. OsMKK6, an MKK gene from rice (Oryza sativa), was functionally characterized under plant salt stress conditions by transforming its constitutively active form [42]. Furthermore, previous studies have shown that AtMKK6 plays a role in regulating plant immune responses. Gene transformation in trees remains difficult. Our laboratory has established the L. chinense somatic embryogenesis system, which lays the foundation for future transgenesis. All of this will provide references and research directions for us to continue to study the LcMKK4 and LcMKK6 genes in the future.

Conclusions
First, we cloned three MAPKK genes from L. chinense (i.e., LcMKK2, LcMKK4, and LcMKK6). In L. chinense, LcMKK2 responds to cold and salinity. Next, we transfected LcMKK2 genes into Arabidopsis and obtained positive transgenic plants. After a salt stress treatment, we found that the transgenic plants were more salt-resistant compared with the WT plants. Finally, we validated the genes related to salt stress through qPCR experiments and found that the expressions of these genes were all upregulated. Therefore, we conclude that the LcMKK2 gene can enhance salt resistance in A. thaliana.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4907/11/11/1160/s1, Table S1: Primers used for isolation of LhMKK2, LhMKK4 and LhMKK6 fragments, Table S2: Primers used for isolation of the complete LhMKK2 gene coding region, Table S3: Primers used for qPCR analysis, Table S4: Arabidopsis MAPK family genes used for phylogenic analysis and their NCBI accession numbers. Figure