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Genome-wide identification and analysis of abiotic stress responsiveness of the mitogen-activated protein kinase gene family in Medicago sativa L.

BMC Plant Biology volume 24, Article number: 800 (2024) Cite this article

Abstract

Background

The mitogen-activated protein kinase (MAPK) cascade is crucial cell signal transduction mechanism that plays an important role in plant growth and development, metabolism, and stress responses. The MAPK cascade includes three protein kinases, MAPK, MAPKK, and MAPKKK. The three protein kinases mediate signaling to downstream response molecules by sequential phosphorylation. The MAPK gene family has been identified and analyzed in many plants, however it has not been investigated in alfalfa.

Results

In this study, Medicago sativa MAPK genes (referred to as MsMAPKs) were identified in the tetraploid alfalfa genome. Eighty MsMAPKs were divided into four groups, with eight in group A, 21 in group B, 21 in group C and 30 in group D. Analysis of the basic structures of the MsMAPKs revealed presence of a conserved TXY motif. Groups A, B and C contained a TEY motif, while group D contained a TDY motif. RNA-seq analysis revealed tissue-specificity of two MsMAPKs and tissue-wide expression of 35 MsMAPKs. Further analysis identified MsMAPK members responsive to drought, salt, and cold stress conditions. Two MsMAPKs (MsMAPK70 and MsMAPK75) responds to salt and cold stresses; two MsMAPKs (MsMAPK60 and MsMAPK73) responds to cold and drought stresses; four MsMAPKs (MsMAPK1, MsMAPK33, MsMAPK64 and MsMAPK71) responds to salt and drought stresses; and two MsMAPKs (MsMAPK5 and MsMAPK7) responded to all three stresses.

Conclusion

This study comprehensively identified and analysed the alfalfa MAPK gene family. Candidate genes related to abiotic stresses were screened by analysing the RNA-seq data. The results provide key information for further analysis of alfalfa MAPK gene functions and improvement of stress tolerance.

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Background

Plants are exposed to various biotic or abiotic stresses during different phases of growth and development [1]. To cope with these stresses, plants harbour multiple regulatory mechanisms [2,3,4]. Signal transduction plays an important role in stress responsive mechanism [5]. The MAPK cascade, common in eukaryotes and important component of signalling [6, 7], is central to plant growth and development [8], hormone signal transduction [9], and response to biotic or abiotic stresses [10, 11].

The MAPK cascade is composed of three sequential protein kinases: MAP kinase kinase kinases (MAPKKKs), MAP kinase kinases (MAPKKs), and MAP kinases (MAPKs) [12, 13]. MAPKKKs activates downstream MAPKKs by phosphorylating serine/threonine residues in the MAPKK activation loops S/T-xxxx-S/T [14]. MAPKKs present in the centre of the cascade are protein kinases with dual specificity [15] that can not only accept the activation signal of upstream MAPKKKs but also activate downstream MAPKs by phosphorylating tyrosine/threonine residues in the TXY motifs of the MAPK activation loops [16, 17]. Activated MAPKs can be transported to the cytoplasm or nucleus to phosphorylate other proteins (kinases, transcription factors, cytoskeleton-binding proteins) for regulation of various cellular activities [18,19,20], and hence these downstream MAPKs are crucial for signal transduction.

The MAPK cascade was first identified as a microtubule-associated protein kinase in animal cells in 1986 [21, 22]. This enzyme is called “mitogen-activated protein kinase” due to their response to mitogen phosphorylation at tyrosine residues [23, 24], which is associated with growth and development, hormonal responses, and stress [25,26,27]. The MAPK gene family members of many species have been identified; for example, 20, 38, 54, 15, 19, and 54 MAPKs have been identified in Arabidopsis thaliana [28], Glycine max [29], Triticum aestivum [30, 31], Oryza sativa [32], Zea mays [33] and Gossypium hirsutum [34], respectively. The function of the MAPK cascade pathway has been the subject of recent studies in many species. the MEKK1-MKK4/5-MPK3/6 cascade was the first signalling module identified in A. thaliana, which up-regulates the expression of the WRKY22/29 transcription factor, while enhancing resistance to fungal and bacterial pathogens [35]. In Hordeum spontaneum, three enriched MAPK cascades (MEKK1-MKK2-MPK4/6, MEKK17/18-MKK3-MPK1/2/7/14, MKK3-MPK8) can effectively participate in in salt stress adaptation and tolerance as well as homeostasis of reactive oxygen species (ROS). [36]. In A. thaliana, MPK3 and MPK6 play role in growth and development. For example, MPK3 and MPK6 mediate the guidance response in pollen tubes [37], and MPK3 and MPK6 and their upstream MAPKKs (MKK4 and MKK5) serve as regulators of stomatal development [38]. Further, MPK3 and MPK6 control salicylic acid signaling by upregulating NLR receptor expression in pattern and effector-triggered immune processes [39]. 1-amino-cyclopropane-1-carboxylic acid synthase (ACS) catalyzes the committing and rate-limiting steps in the ethylene biosynthesis pathway. MPK3 and MPK6 can phosphorylate and stabilize ACS2 and ACS6, and regulate the expression of ACS2 and ACS6 genes through another MPK3/MPK6 substrate WRKY33, thereby regulating ethylene synthesis [40]. MPK3/MPK6 can also degrade and destroy the stability of ICE1 (CBF expression inducer 1), which regulates C-repeat-binding factor (CBF) transcription factors associated with cold stress, thereby negatively regulating CBF expression and freezing tolerance in plants [41]. AtMPK6 is found to phosphorylate AtMYB15 to reduce the binding affinity of AtCBF3 and freezing tolerance [42]. In G. max, GmMPK4 is a negative regulator of the defense response and a positive regulator of growth and development, like the function of ATMPK4 in A. thaliana, suggesting functional conservation across plant species, during evolution [43]. GMK1 is regulated by both phosphatidic acid and hydrogen peroxide (H2O2) and is translocated to the nucleus during salt stress [44]. In Z. mays, the transcriptional level of ZmMPK3 was significantly increased when maize seedlings were subjected to exogenous signal molecules such as ABA, H2O2, jasmonic acid and salicylic acid, as well as various abiotic stresses such as salinity. At the same time, it was found that ABA and H2O2 induced a significant increase in ZmMPK3 activity [45]. ZmMPK3 and ZmMPK5 were induced by drought and cold stress [46, 47]. In O. sativa, OsMAPK6 can phosphorylate the OsLIC (zinc finger protein), which regulates the transcription of OsWRKY30 gene and enhancing the response to Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) [48]. OsMAPK2 seems to play roles in stress signal transduction pathways and panicle development in O. sativa [49].

Alfalfa (Medicago sativa L.) is a high-quality legume forage (high nutritional value and good palatability), cultivated in China for more than 2,000 years [50]. However, the MAPK gene family has not been investigated in alfalfa. In this study, the MAPK gene family of alfalfa was comprehensively analyzed via genome-wide screening, phylogenetic analysis, gene structure and conserved motif analysis, and chromosome localization and collinearity analysis. The RNA-seq analysis of alfalfa MAPK genes in different tissues and under different stresses was carried out. The results of this study provide key information for further analysis of the function of MAPKs and their utility in molecular breeding of alfalfa.

Results

Diversity of MAPK genes in M. sativa genome

Using a combination of in silico approaches including Hidden Markov model and domain-based search methods a total of 80 MsMAPK genes were identified from the “Xinjiangdaye” reference genome. The important characteristics of gene and protein sequences are shown in Table 1 and Table S1.

Among all the MsMAPK members, the longest and shortest proteins were MsMAPK27 (716 aa. MW: 81.36 kDa) and MsMAPK13 (137 aa, MW: 15.78 kDa), respectively. The highest and lowest isoelectric points (pIs) were found for MsMAPK7 (9.41) and MsMAPK16 (4.97), respectively, and the instability index ranged from 29.98 (MsMAPK21) to 49.48 (MsMAPK34). The MsMAPK members showed divers localization in the cell, with fifty-six members in the cytosol, five in the chloroplast, three in the cytoskeleton, seven in the mitochondria, eight in the nucleus, and only one in the endoplasmic reticulum (Table S1).

Of the total eighty, 76 MsMAPK genes (MsMAPK1-76) were located on 23 chromosomes (none on chr1.1, chr1.2, chr1.3, chr1.4, chr6.2, chr6.4, chr7.1, chr7.3, or chr7.4), whereas four genes (MsMAPK77, MsMAPK78, MsMAPK79, MsMAPK80) were identified on the unanchored 50,223–50,226. Each chromosome contained various genes, ranging from 1 to 9 (Fig. 1). Finally, 80 MsMAPK genes were renamed according to their chromosomal locations (MsMAPK1-MsMAPK80).

Fig. 1
figure 1

Chromosome distribution of the MAPK genes in M. sativa. Each color represents different groups of chromosomes. MsMAPK genes were renamed as MsMAPK1-MsMAPK80 according to the order of chromosomes and the position of MsMAPK gene on chromosomes. Rectangle represents a tandem duplication event

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Table 1 Basic information of MAPK genes family in M. sativa
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Phylogenetic analysis of MAPK genes in M. sativa

To further explore the evolutionary relationships between 80 MsMAPK members in M. sativa, a phylogenetic tree was constructed, including 20 sequences from A. thaliana, 32 from G. max and, 15 from O. sativa (Fig. 2). According to the classification of MAPK gene family in A. thaliana [51], and based on the conserved phosphorylation motifs (TEY, TDY) in the activation loop [52], the MsMAPK family genes were divided into A, B, C and D subgroups. Among them, MsMAPK members in groups A, B and C have TEY motifs, while MsMAPK members in group D have TDY motifs (Fig. S1), which is consistent with the findings of previous studies. Groups A, B, C and D contained 8, 21, 21 and 30 members, respectively, and some of the results were consistent with the information in previous reports [53].

Fig. 2
figure 2

Phylogenetic tree of MAPK genes in M. sativa, A. thaliana, G. max and O. sativa. Red, orange, green and blue represent the A, B, C and D subgroup, respectively. The red, blue, green and black circles represent A. thaliana, G. max, O. sativa, and M. sativa, respectively

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Analysis of the MAPK gene basic structures and conserved domains of MAPK proteins in M. sativa

To study the structural characteristics of the MAPK family members in M. sativa, the presences of conserved motifs were analyzed using the online tool MEME. Ten motifs were predicted, and the basic information is shown in Fig. S2. All MsMAPK proteins contained Motif 1 (Fig. S1), and several other members contained Motif 2, Motif 3, Motif 4, Motif 5, Motif 6, Motif 7, and Motif 10. Except for MsMAPK29, all group D members contained Motif 8. Except for MsMAPK58, all group A and group B members contained Motif 9 (Fig. 3A). Motif 2 contains a TXY structure. As shown in Fig. S1, all MsMAPK members contained a TXY structure. The C-terminus of the MsMAPK proteins in group A and group B contain a –(LH)DxxDE(P)xC- motif, which is defined as the CD domain and is a site for identifying substrate proteins (Fig. S3) [54].

Gene structure analysis revealed that in addition to MsMAPK79, MsMAPK77 (containing 4 introns) and MsMAPK58 (containing 6 introns) in group A and group B, the other members contained 5 introns each. In group C, 7 members contained 2 introns each, and 14 members contained only 1 intron each. The number of introns in the group D genes significantly differed from that in the genes of groups A, B, and C, ranging from 7 to 14 (Fig. 3B).

Fig. 3
figure 3

Basic structures and motifs of the MsMAPK genes. (A) The structure of MsMAPK protein motif. (B) Basic structures of MsMAPK genes

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Gene duplication events and collinearity analysis of MAPK genes in M. sativa

The gene duplication events of MAPK genes in alfalfa were analyzed. As shown in Fig. 1, a total of 12 tandem duplication events were found, involving 32 MsMAPKs, such as the tandem duplication event MsMAPK10/MsMAPK11/MsMAPK12/MsMAPK13 located on chr3.1 and another tandem duplication event MsMAPK41/MsMAPK42/MsMAPK43/MsMAPK44 located on chr4.3 (Table S2). Moreover, a total of 131 segmental duplication events involving 64 MsMAPK genes were detected (Table S3). As can be seen in Fig. 4, the MsMAPK genes in most of the segmental duplication events are located at similar positions on each chromosome in the homologous chromosome. For example, MsMAPK2/MsMAPK4/MsMAPK6/MsMAPK8 are located on chr2.1, chr2.2, chr2.3 and chr2.4, respectively. There were significantly more segmental duplication events than tandem duplication events. In summary, it can be speculated that the development and evolution of the alfalfa MAPK family genes mainly rely on segmental duplication events.

Next, to clarify the potential evolutionary relationships of the MAPK genes family in different crop species, the evolutionary relationships and collinearity between M. sativa and A. thaliana and between G. max and Medicago truncatula were predicted (Fig. 5). The results showed that 46 MsMAPK genes were collinear with those of A. thaliana, 55 MsMAPK genes were collinear with those of G. max, and 56 MsMAPK genes were collinear with those of Medicago truncatula. At the same time, there were 80, 180 and 90 collinear gene pairs in A. thaliana, G. max and Medicago truncatula, respectively.

Fig. 4
figure 4

Schematic diagram of the syntenic relationships of MsMAPK genes in M. sativa. The gray ribbons represent syntenic blocks in the alfalfa genome, and the segmental duplication events are marked in red

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Fig. 5
figure 5

Collinearity analysis of the MsMAPK genes with those of A. thaliana, Medicago truncatula and G. max. The gray ribbons represent syntenic blocks in the alfalfa genome, and the segmental duplication events are marked in red

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Analysis of cis-acting elements in the promoter regions of MAPK genes in M. sativa

PlantCARE database was used to identify the cis-acting elements in the promoter of the MsMAPK genes. The cis-acting elements were divided into three categories: growth and development, hormone response, and stress response (Table S4). Ten cis-acting elements related to the hormone stress response were screened for analysis (Fig. 6). Among these, abscisic acid responsiveness (ABRE) was present in the most MsMAPK members (65), and flavonoid biosynthesis (MBSI) was present in the least members (7). The results showed that MsMAPKs may play corresponding roles in regulating growth and development, hormone response and stress response.

Fig. 6
figure 6

Cis-acting elements of the MAPK gene promoters in M. sativa.A-D: MsMAPK genes were divided into four groups

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Expression of MAPK genes in different tissues of M. sativa

To clarify the expression patterns of the MsMAPK genes in different tissues, the transcriptome data of six different tissues (roots, elongated stems, pre-elongated stems, leaves, flowers, and nodules) of MsMAPKs were obtained from a public database (Table S5). The results showed that 58 MsMAPK genes were expressed in at least one tissue. Among them, two MsMAPKs showed tissue-specific expression (Fig. 7A), and 35 MsMAPKs were expressed in six tissues (Fig. 7D). For example, MsMAPK18 was only expressed in elongated stems, and MsMAPK7 was expressed in six different tissues. In addition, 3, 4, 7 and 7 MsMAPK genes were expressed in 2, 3, 4 and 5 different tissues, respectively. (Fig. 7A-C). Although some MsMAPK genes can be expressed in a variety of tissues, the expression patterns of these genes in different tissues vary greatly. For example, the expression levels of MsMAPK7 in flowers and leaves were significantly greater than those in the other four tissues. It can be speculated that the MsMAPK genes may play roles in different growth and developmental stages.

Fig. 7
figure 7

Expression of 58 MsMAPK genes in different tissues. The expression levels were normalized by row using the Z − scores algorithm. The color scale on the right side of the heatmap indicates the relative expression levels, and the color gradient from blue to red indicates an increase in expression levels

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Expression of MAPK genes in M. sativa under abiotic stress response

To explore the potential regulatory mechanisms of the MsMAPK genes under different stresses, the RNA-seq data of alfalfa plants under thre abiotic stresses (salt, drought, cold) were analyzed (Table S6). As shown in Fig. 8A-C, multiple MsMAPK genes exhibited responses to salt (11 genes), drought (10 genes), cold (11 genes), respectively. As shown in Fig. 8D, there were 10 MsMAPK genes that responded to only one stress, e.g., MsMAPK3, MsMAPK36, and MsMAPK50 responded only to salt, drought, and cold stress, respectively. Eight MsMAPK genes can respond to two stresses, e.g., MsMAPK1, MsMAPK70, MsMAPK60. However, two MsMAPK genes can respond to all three stresses (MsMAPK5, MsMAPK7).

To verify the RNA-seq data, several key genes were selected for RT‒qPCR analysis. The experimental primers used are shown in Table S7. As shown in Fig. 9, under drought stress, the expression of the MsMAPK7/33/36 increase first but never goes down the control. Under cold stress, the expression of MsMAPK7 gradually increased over time while expression of MsMAPK51 gradually decreased over time. The expression of the MsMAPK53 increase first but never goes down the control. Under salt stress, the expression of the MsMAPK7 and MsMAPK33 increase first but never goes down the control. The RT‒PCR results were consistent with the RNA-seq data.

Fig. 8
figure 8

Expression of MAPK genes in M. sativa under stress. (A) Expression of MAPK genes in M. sativa under salt stress. (B) Expression of MAPK genes in M. sativa under drought stress. (C) Expression of MAPK genes in M. sativa under cold stress. (D) Venn diagrams of MsMAPK genes responding to three stresses. Under salt stress: 0 h as CK and 0.5, 1, 3, 6, 12 and 24 h as S1 to S6, respectively. Under drought stress: 0 h as CK and 1, 3, 6, 12 and 24 h as M1, M2, M3, M4 and M5, respectively. Under cold stress: 0 h as CK and 2, 6, 24 and 48 h as C1, C2, C3 and C4, respectively

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Fig. 9
figure 9

MsMAPK expression under drought, salt and cold stress conditions according to RT–qPCR. (A) Expression of MsMAPKs under drought stress. (B) Expression of MsMAPKs under cold stress. (C) Expression of MsMAPKs under salt stress

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Discussion

The mitogen-activated protein kinase (MAPK) cascade exists widely across eukaryotes and has been studied in many plants, such as A. thaliana [28], G. max [29], and O. sativa [32]. However, the MAPK genes family has not been described in alfalfa. In this study, a total of 80 MAPK genes were identified in the “Xinnjiangdaye” genome. Activated MAPKs can be transported to the cytoplasm or nucleus to phosphorylate other kinases, transcription factors and cytoskeleton-binding proteins to regulate various cellular activities [55]. Subcellular localization prediction of 80 MsMAPKs revealed that 64 genes were predicted to be located in the cytoplasm or nucleus, which was consistent with the findings of previous studies [53]. In A. thaliana, MAPK genes are divided into four different groups according to their evolutionary relationships and the presence of TDY and TEY phosphorylation motifs. Among them, groups A, B and C contain TEY motifs, and group D contains TDY motifs, which is consistent with the results of this study. In other studies, MAPK proteins include not only TEY and TDY motifs but also MEY, TEM, TSY, TEC, TVY, etc. [56]. Presence of only TEY and TDY motifs among alfalfa MAPKs indicate conservation of protein motifs.

The amplification of MAPK family members is essential for plant evolution [12]. Compared with the 20 MAPK genes in A. thaliana, there are 17 MAPK genes in Medicago truncatula, and there are far more MAPK genes in M. sativa than in A. thaliana. The reason may be that A. thaliana and Medicago truncatula use the haploid genome [57, 58], while this study used the “Xinjiangdaye” tetraploid genome [59]. Duplications of individual genes, chromosome segments, or entire genomes are common. In some cases, these duplications can facilitate the evolution of new functions that enable plants to better cope with stress [60]. Plant genomes tend to evolve at a higher rate than other eukaryotic genomes, leading to higher genome diversity [61]. Tandem duplication and segmental duplication are the two main forms of plant gene family expansion [62]. The analysis of gene duplication events revealed segmental duplication events were significantly more than tandem duplication events in MsMAPKs, indicating that segmental duplication is the main mechanism of MAPK gene family evolution and expansion in alfalfa. There were significantly more homologous gene pairs between alfalfa and leguminous plants than between alfalfa and A. thaliana, and the most homologous gene pairs were found in G. max. The homologous gene pairs between alfalfa and legumes were significantly more than those between alfalfa and Arabidopsis, and the most homologous gene pairs were found in soybean, indicating that the distribution of MAPK genes in legumes was relatively conservative.

Cis-acting elements on the MsMAPK gene promoters are involved in a variety of cellular functions. The MAPK cascade is a highly conserved signaling pathway in higher plants that is involved not only in cell division, apoptosis and plant growth and development but also in plant responses to abiotic stress [1]. As mentioned above, MPK3 and MPK6 are related to the formation of pollen tubes and stomata in terms of growth and development [30, 31]. The hormone response is related to the synthesis of salicylic acid and ethylene [32, 33]. In terms of stress responses, they are not only related to cold stress [34] but can also interact with AtPFA-DSP5 to negatively regulate the salt responses of plants [63]. To a certain extent, they can also enhance salt tolerance through their negative regulation of Arabidopsis response regulator 1 (ARR1), ARR10 and ARR12 protein stability [64]. Through BLAST and phylogenetic analyses, the MsMAPK genes with the highest similarity between ATMPK3 and ATMPK6 in alfalfa were identified. Among them, MsMAPK33/38/47/53 had the high similarity with ATMPK3 (identity > 82%), and MsMAPK31/36/45/51 had the high similarity with ATMPK6 (identity > 88%). By analysing the RNA-seq data from different tissues, among them, 6 MsMAPKs were expressed in all six tissues, but at different levels. Except for MsMAPK45, the other five genes had relatively high expression levels in roots. According to previous studies, YODA and MPK6 regulate cell division and mitotic microtubules through an auxin-dependent mechanism and are involved in the development of postembryonic roots [65], therefore, it can be speculated that they may have similar functions. The analysis of RNA-seq data for different stresses revealed that 11, 10 and 11 MsMAPK genes exhibit responses to salt, drought, and cold stress, respectively. Among them, MsMAPK70 and MsMAPK75 can exhibit responses to salt and cold stress. MsMAPK60 and MsMAPK73 can exhibit responses to cold and drought stress. MsMAPK1/33/64/71 can exhibit responses to salt and drought stress. Notably, MsMAPK5 and MsMAPK7 can exhibit responses to four stresses. The above MsMAPK genes may be key for improving the abiotic stress resistance of alfalfa.

Alfalfa is an important forage crop. However, because most of the planting areas in China are located in areas with severe salinization, it is inevitable that many abiotic stresses are encountered during the growth of alfalfa, resulting in a decline in quality. Therefore, it is very important to cultivate new varieties of alfalfa with good stress resistance. In this study, several important stress-responsive MsMAPK genes were predicted by analysing RNA-seq data. In the next study, these several MsMAPK genes can be transgenic or gene edited to verify their functions. With the development of transgenic technology and gene editing technology, it has become possible to breed new alfalfa varieties with high stress resistance through molecular breeding.

Conclusions

In this study, 80 MsMAPK genes were identified in the alfalfa genome; these genes were subdivided into groups A, B, C and D. Group D contained the most MsMAPK genes. All MsMAPK genes contained a TXY domain; MsMAPK genes in groups A, B, and C contained a TEY domain, and MsMAPK genes in group D contained a TDY motif. there were significantly more segmental duplication events than tandem duplication events for the MsMAPK genes. Tissue RNA-seq analysis revealed that 2 MsMAPKs exhibited tissue-specific expression, and 35 MsMAPKs exhibited pantissue expression. Abiotic stress RNA-seq analysis revealed that 11, 10 and 11 MsMAPKs exhibited responses to salt, drought and cold stresses, respectively. Among them, two MsMAPKs (MsMAPK70 and MsMAPK75) responds to salt and cold stresses; two MsMAPKs (MsMAPK60 and MsMAPK73) responds to cold and drought stresses; four MsMAPKs (MsMAPK1, MsMAPK33, MsMAPK64 and MsMAPK71) responds to salt and drought stresses; and two MsMAPKs (MsMAPK5 and MsMAPK7) responded to all three stresses. In summary, we comprehensively described the MAPK genes family of M. sativa. The results lay a foundation for future exploration of the function of the MsMAPK genes and provide new ideas for molecular breeding of alfalfa.

Materials and methods

In silico identification and analysis of MAPK genes in M. sativa genome

The alfalfa genome used in this study was from the Alfalfa Genome Project (https://fgshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380) [66]. The MAPK proteins of A. thaliana (TAIR database: https://www.arabidopsis.org/), G. max (Glycine max genome database: https://www.soybase.org/), and O. sativa (RGAP database: http://rice.uga.edu/) were used as BLAST templates. The hidden Markov model (HMM) was used to obtain the configuration file (PF00069) of the MAPK domain from the Pfam database to further remove redundancy [67]. The theoretical molecular weights (MWs), isoelectric points (pIs) and instability indices of the MsMAPKs were estimated using tools available at the ExPASy database (http://www.ExPASy.org/).

Phylogenetic and gene and protein structure analysis

Phylogenetic trees were constructed based on the MAPK protein sequences of M. sativa, A. thaliana, G. max, and O. sativa using MEGA11, the neighbor-joining (NJ) method and 1000 bootstrap repeats. DNAMAN9 software was used to compare the sequences of alfalfa MAPK proteins. The online MEME website (https://meme-suite.org/meme/tools/meme) was used to analyze the MsMAPK protein motifs, and the number of motifs was set to 10. The conserved domains of the MsMAPK protein were predicted by the NCBI conserved domain database (https://www.ncbi.nlm.nih.gov/cdd/). Structural information of alfalfa MAPK gene was obtained from alfalfa genome Gff annotation file. TBtools-II (v2.110) software was used to visualize the results of above-mentioned analysis.

Chromosomal localization, gene duplication events and collinearity analysis

The chromosomal distribution information of the MsMAPK genes was obtained from the gff file of the alfalfa genome, and visualized by TBtools software [68]. The MCScanX tool was used to analyze the collinearity information of MAPK genes within and between species [69]. Tandem duplication occurs when two or more genes are located within 200 Kb of the same chromosome. Tandem duplication events were identified by comparing the location of the chromosome where the MsMAPK gene is located. The results were visualized with TBtools.

Promoter cis-acting element analysis

TBtools was used to extract 2000 bp fragments upstream of the MsMAPK gene promoters, and the cis-acting elements were identified through the online website PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and visualized using TBtools.

Expression profile analysis using RNA-seq data

RNA-seq data from six alfalfa tissues (roots, elongated stems, pre-elongated stems, leaves, flowers, and nodules) (SRP055547) and studies on response to salt, drought and cold stress (SRR7091780-SRR7091794 and SRR7160313-SRR7160357) were downloaded from the NCBI database for analysis [70, 71]. TBtools was used to visualize the data.

Plant materials, growth and stress conditions, and RT‒qPCR analysis

Alfalfa (Zhongmu No.1) seeds were obtained from the Institute of Animal Science of the Chinese Academy of Agricultural Sciences. Seeds were treated at 4 °C for 3 days and then cultured in a greenhouse for 2 weeks under a 16/8 hours light cycle, 70–80% relative humidity and 24 °C/20 ℃ day/night temperature conditions. Mannitol (400 mM) was used to simulate drought stress, and root tip samples were collected at 1, 3, 6, 12, and 24 h. Plants were subjected to cold stress treatment at 4 °C, 0 h was selected as the control group, and 2, 6, 24 and 48 h were selected as the sampling time points to take leaf samples. NaCl (250 mM) treatment was used to simulate salt stress. The root tip samples were collected, 0 h was used as the control group, and 0.5, 1, 3, 6, 12 and 24 h were used as the sampling time points. There were three replicates for each stress treatment, and five seedlings were pooled in each replicate. Untreated control plants were cultured normally.

Total RNA was extracted from all samples using TRIzol reagent according to the manufacturer’s instructions. The corresponding cDNA was obtained using an EasyScript First-Strand cDNA Synthesis Kit (random primer (N9)). The primers used in the study were designed using Primer 5.0 software. SYBR Premix Ex Taq (Takara, Japan) and a 7500 real-time fluorescent quantitative PCR system (Applied Biosystems, Foster City, CA, USA) were used for the RT‒qPCR experiments. Three replicates were analyzed for each sample, and the data were standardized relative to alfalfa actin gene expression. Relative expression levels were calculated by using the 2 − ΔΔCT method with Actin2 used as the internal reference.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

MAPK/MPK:

Mitogen-activated protein kinase

NLR:

Nucleotide-binding domain leucine-rich repeat receptor

ACS:

1-amino-cyclopropane-1-carboxylic acid synthase

CBF:

C-repeat-binding factor

ICE1:

Inducer of CBF expression 1

ABA:

Abscisic acid

LIC:

Leaf and tiller angle increased controller

MW:

Molecular weight

pI:

Isoelectric points

aa:

Amino acid

PFA-DSP:

Plant and fungi atypical dual-specificity phosphatases

ARR:

Arabidopsis response regulator

HMM:

Hidden Markov model

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Funding

This work was supported by the National Natural Science Foundation of China (32371757), the Ordos Science and Technology Plan (2022EEDSKJZDZX011), the major demonstration project “The Open Competition” for Seed Industry Science and Technology Innovation in Inner Mongolia (No. 2022JBGS0016), the Central Public-interest Scientific Institution Basal Research Fund (No. 2022-YWF-ZYSQ-04), and the Agricultural Science and Technology Innovation Program (ASTIP No. CAAS-ZDRW202201).

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  1. Hao Liu, Xianyang Li and Fei He contributed equally to this work.

Authors and Affiliations

  1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

    Hao Liu, Xianyang Li, Fei He, Mingna Li, Ruicai Long, Junmei Kang, Qingchuan Yang & Lin Chen

  2. College of Grassland Science, Qingdao Agricultural University, Qingdao, 266109, China

    Hao Liu

  3. Institute of Forage Crop Science, Ordos Academy of Agricultural and Animal Husbandry Sciences, Ordos, 017000, China

    Yunfei Zi, Guoqing Zhao, Lihua Zhu, Ling Hong & Shiqing Wang

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Contributions

Experimental design and planning and first draft writing, H.L., L.C.; preparation and modification of the images, F.H., X.L.; data processing, manuscript modification, Y.Z., G.Z.; data analysis and test data accuracy, L.Z., L.H. and M.L.; application and analysis of the software used in the experiment, S.W., R.L.; data and manuscript review, J.K., Q.Y.; funding acquisition, L.C. All the authors contributed to the article. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Lin Chen.

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Liu, H., Li, X., He, F. et al. Genome-wide identification and analysis of abiotic stress responsiveness of the mitogen-activated protein kinase gene family in Medicago sativa L.. BMC Plant Biol 24, 800 (2024). https://doi.org/10.1186/s12870-024-05524-4

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