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Article

In Silico Analysis of the MAPK Gene Family in Cabbage and Its Expression during Development and Stress Response

by
Min Wang
,
Jinxiu Chen
,
Xiaowei Zhu
,
Xiang Tai
* and
Tianyue Bo
*
Horticultural Research Institute, Shanghai Key Laboratory of Protected Horticultural Technology, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2023, 9(10), 1119; https://doi.org/10.3390/horticulturae9101119
Submission received: 24 August 2023 / Revised: 22 September 2023 / Accepted: 2 October 2023 / Published: 10 October 2023
(This article belongs to the Special Issue Recent Scientific Developments in Genetic Improvement of Vegetables for Resistance to Biotic and Abiotic Stresses)
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Abstract

:
Cabbage is often affected by an adverse environment and pathogens during its growth, resulting in a decline in yield and quality. Mitogen-activated protein kinases (MAPKs) play crucial roles in the development process, hormones, abiotic and biotic stresses, and cell division. So far, MAPKs have been characterized in various species. However, no systematic analysis of the MAPK family in cabbage has been performed. A total of 24 BoMAPK genes were identified and renamed according to Arabidopsis homologs; they were unevenly distributed on nine chromosomes. According to the conserved domain and phylogenetic relationship, BoMAPKs were divided into four subgroups, of which three belonged to subgroup A, four to subgroup B, three to subgroup C, and fourteen to subgroup D, respectively. There were 13 segmental duplication events in the cabbage genome. The Ka/Ks ratios for duplicated gene pairs of the MAPK family were less than 0.2, indicating that these BoMAPK genes have undergone purification selection in evolution. There were various cis-acting elements associated with stress, hormones, and development found in the promoter regions of most BoMAPK genes. Transcriptome data showed a high expression of BoMAPK3, BoMAPK4.1, and BoMAPK6.1 genes in various tissues. BoMAPK17.2, BoMAPK20.1 and BoMAPK20.2 were significantly induced by Plasmodiophora brassicae, as evidenced in the quantitative PCR (RT-qPCR) analysis, revealing their enormous potential to regulate stress response. RT-qPCR results showed how the transcriptional levels of BoMAPK genes varied substantially under abiotic stress at different time points. These analysis results provide a reference to further explore the function and evolution of BoMAPK genes in cabbage.

1. Introduction

Mitogen-activated protein kinase cascades (MAPK cascades), which are an evolutionarily conserved and functionally important signal transduction system in eukaryotes, are activated by a series of extracellular stimulation signals and mediate the transmission of signals from the cell membrane to the nucleus [1]. The MAPK cascade regulates many physiological activities, such as plant growth and development, immune reactions, and environmental stress responses [2]. It is composed of three types of kinases and transmits stress signals through sequential phosphorylation: MAPKKKs-MAPKKs-MAPKs. MAPKKK usually acts on the S/T-xxxxx-S/T site of MAPKK to phosphorylate and activate it, and then the activated MAPKK phosphorylates threonine and tyrosine residues of MAPK to activate MAPK [3]. MAPKs, the bottom kinase in MAPK cascades, phosphorylates downstream substrates, such as protein kinases, transcription factors, structural proteins, and other enzymes, to activate cellular responses [4]. The MAPK protein has conserved regions (motifs) with a signature TXY activation motif between the subdomains VII and VIII. The TXY motif is the ATP phosphorylation site or active fragment, which is the key structure to determine the activity of various MAPK kinases and plays a key role in the MAPK cascade pathway [1,5]. MAPK genes in plants can be divided into TEY and TDY subtypes according to the different types of amino acids in the middle of the TXY motif. The TEY type is further classified into three subgroups: A, B, and C. The terminal extension region of subgroup A and B MAPK proteins contains an evolutionarily conserved C-terminal common docking (CD) domain, which serves as a docking site for MAPKKs, MAPK protein substrates, and phosphatase. The TDY type is separately classified as subgroup D, which lacks the CD domain [6].
To date, multiple MAPK genes have been identified in various species, such as 20 in Arabidopsis [7], 15 in rice [8], 19 in maize [9], 54 in barley [10], and 12 in strawberry [11], and the functions of only some MAPKs have been characterized. At the junction of environmental and developmental signals, MPK3/MPK6 regulates inflorescence architecture development, root growth, and anther development in Arabidopsis [12,13,14]. The mpk4 mutant (which harbors transposon-inactive AtMAPK4) exhibits increased resistance to virulent pathogens [15]. Moreover, the functions of MAPK genes have also been investigated in other species. FaMAPK5/FaMAPK10 participated in ABA-mediated H2O2 signal transduction to regulate strawberry fruit ripening quality and disease [16]. FvMAPK3 negatively regulated strawberry fruit ripening, anthocyanin content, and the biosynthesis gene [17]. The transient expression of FtMAPK1 enhanced resistance to salt stress by increasing the expression of downstream stress-related genes [18]. OsMAPK3 phosphorylated the OsbHLH002 protein and slowed down its degradation rate, which promoted the expression of OsTPP1, leading to an enhanced chilling tolerance [19]. OsMPK4 phosphorylates IPA1, thereby promoting the ubiquitination-mediated degradation of IPA1, reducing its protein levels, and ultimately enhancing salt tolerance [20]. So far, several MAPK signaling cascades have been established in Arabidopsis. The MEKK1-MKK2-MPK4/MPK6 module (first identified MAPK cascade) participates in response to salt and cold stress [21,22,23]. In Arabidopsis, the MEKK1-MKK1/MKK2-MPK4 cascade negatively regulates immunity [24], and the MEKK1-MKK4/5-MPK3/6 module confers resistance to bacterial and fungal pathogens in Arabidopsis [25,26]. A cascade reaction YODY(MAPKKK)-MKK4/MKK5-MPK3/MPK6 was found in Arabidopsis to regulate stomatal development and patterning [27]. The reported MAPK cascade pathways in rice included OsMKKK10-OsMKK4-OsMAPK6 and OsMKKKε-OsMKK4/5-OsMAPK3/6, which are involved in rice grain type and disease resistance response, respectively [28,29].
Cabbage (Brassica oleracea var. capitata L.) is an economically important vegetable crop. In recent years, the disease caused by the pathogen Plasmodiophora brassicae has led to a significant decline in cabbage yield and quality [30]. MAPK family members play a key role in plant growth, development, and response to various stresses; however, the MAPK gene family has not been characterized thoroughly in the cabbage genome. In this study, a total of 24 BoMAPK genes were identified and comprehensively analyzed for their phylogeny, gene structure, chromosome location, collinearity, promoters, and protein interaction prediction. In addition, the expression characteristics of BoMAPKs in different tissues, in response to pathogen infection and abiotic stresses were analyzed by data mining using transcriptome data and RT-qPCR. Overall, these studies might provide insight into the molecular mechanisms of these genes and their application in cabbage disease resistance breeding.

2. Materials and Methods

2.1. Identification and In Silico Analysis of MAPK Family Members in Cabbage

The genome, protein, and CDS sequences of the cabbage genome (JZS v2) were downloaded from the BRAD database (http://brassicadb.cn (accessed on 9 June 2022)) [31,32]. Arabidopsis MAPK protein sequences were retrieved from TAIR (http://www.arabidopsis.org/ (accessed on 14 June 2022)), and rice MAPK sequences were obtained from the Rice Genome Annotation Project (http://rice.uga.edu/ (accessed on 14 June 2022)). HMMER 3.0 software [33] was used to screen out MAPK proteins using the kinase protein profile (PF00069) downloaded from the Pfam database with the e-value ≤ 1 × 10−5. Arabidopsis and rice MAPK protein sequences were used as queries to search for the cabbage proteins using Local Blastp in BioEdit software [34]. Then, the two candidate gene sets were merged, and the redundant protein sequences were manually removed. The online software Pfam (http://pfam.xfam.org/ (accessed on 9 June 2022)) and NCBI with a conserved structure (http//www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 9 June 2022)) were used to ascertain that the MAPK domain existed. The Expasy software (ExPASy-ProtParam tool) was used to predict the physical and chemical properties of BoMAPK proteins, including molecular weight, isoelectric point, and hydrophilicity. Through WoLF PSORT (https://wolfpsort.hgc.jp (accessed on 9 June 2022)), subcellular localization prediction was performed. The MAPK protein sequences of Brassica napus and Brassica rapa were also extracted from the BRAD (Brassica Database: http://brassicadb.cn (accessed on 14 June 2022)) [31].

2.2. Multiple Sequence Alignment and Construction of Phylogenetic Tree

The multiple sequence alignment of BoMAPK protein sequences was carried out using DNAMAN6 software (Lynnon Biosoft, Vaudreuil-Dorion, QC, Canada). MAPK protein sequences of cabbage were compared with that of Arabidopsis thaliana (At), Oryza sativa (Os), Brassica oleracea (Bol), Brassica napus (Bna), and Brassica rapa (Bra) by Clustal W; the clustering results were then used to generate the phylogenetic tree with MEGA5.0 using the neighbor-joining method (Bootstrap: 1000 with Poisson model, complete deletion). The evolutionary tree containing only BoMAPK protein sequences was also constructed using the same methodology. The secondary structures of BoMAPK proteins were predicted using the SOPMA method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html (accessed on 8 November 2022)). The Swiss-Model online tool (https://swissmodel.expasy.org/ (accessed on 14 July 2022)) was used to predict the tertiary structure of BoMAPK proteins with homology modeling.

2.3. Chromosomal Localization and Synteny Analysis of BoMAPK Genes

TBtools v1.108 software was used to extract the position annotation of MAPK gene family members in the cabbage genome and draw the chromosome distribution map [35]. The whole genome collinearity analysis of cabbage and collinearity analysis with Arabidopsis were analyzed [36]. The values of Ka, Ks, and Ka/Ks (the ratio between the nonsynonymous substitution rate (Ka) and synonymous substitution rate (Ks) of two protein-coding genes) were also calculated using the TBtools v1.108 software [35].

2.4. Gene Structure and Motif Analysis of BoMAPK Genes

The conserved motifs of BoMAPK proteins were predicted using MEME (https://meme-suite.org/meme/ (accessed on 18 August 2023)) with the following search parameters: the maximum number of motifs at 15 and optimal motif width at 6–50. Furthermore, TBtools were used to visualize the gene structure and motif distribution [35].

2.5. Cis-Acting Elements Analysis in BoMAPK Genes Promoter

We extracted the 2000-bp sequence upstream of the translation initiation codon (ATG) of each BoMAPK gene as the promoter fragment. Then, the online program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 17 June 2022)) was used to identify the number and types of cis-acting elements.

2.6. Expression Profiles of BoMAPK Genes in Different Tissues

The available transcriptome data set (GSE42891) from the NCBI database was downloaded and used to analyze the expression of BoMAPK genes in seven different organs (leaf, stem, bud, callus, silique, flower, and root) in the cabbage homozygous line “02–12” [37,38]. Then, transcriptome data were used to analyze the expression levels (log2FPKM) of all BoMAPK genes in seven tissues. The heatmap was visualized using TBtools software [35].

2.7. Plant Materials and Treatment Methods

High-generation inbred line “Zhengfu” seeds were sown in a nutritive cube and placed in an artificial climate chamber with a 16 h light/8 h dark photoperiod at 25 °C with 60% relative humidity and 400 μmol/m2/s light intensity at Shanghai Academy of Agricultural Sciences in Shanghai, China. The pathogen Plasmodiophora brassica was obtained from the Xiannv mountain vegetable base in Wulong Chongqing. According to Williams’ classification, the pathogen was identified as the race 4 strain. When cabbage seedlings were in the three-leaf stage, 4 mL of spore suspension with a concentration of 1 × 108 spores/mL were injected into the roots of cabbage seedlings. No treatment was given to the control group. For each biological replicate, the roots from ten cabbage seedlings were analyzed. The root samples at 7 and 21 days (F7I and F21I) after inoculation with pathogen and control samples (F7CK and F21CK) were taken for RT-qPCR. For salt stress treatment, five-week-old cabbage seedlings were subjected to a 200 mmol/L NaCl solution [39]. To investigate the expression profiles of BoMAPK genes under cold stress, plants were incubated in a light incubator (16/8 h light/dark) and set at 4 °C. Leaves were collected at 0 h (CK), 3 h, 6 h, and 24 h after the treatments for RT-qPCR analysis.

2.8. RNA Extraction and RT-qPCR

Total RNA was isolated using a universal plant RNA extraction Kit (Bioteke Beijing, Beijing, China). The PrimeScript™ FAST RT reagent Kit with gDNA Eraser (TaKaRa Code No. RR092A) was used to transcribe the total RNA into cDNA. The RT-qPCR-specific primers were designed using Primer 5.0 software (Table S1), and assays were performed in a Quantum Studio Q5 instrument. The volume of each reaction was 20 μL, which included 1μL of the diluted cDNA template, 0.4 μL of each primer (10 μmol/L), 10 μL of TB Green® Premix Ex TaqTM II (Takara Code No. RR820A), and 8.2 μL of ddH2O. The PCR reaction procedure: 95 °C 30 s, 95 °C 5 s, 60 °C 30 s 40 cycles, followed by the melting curve analysis. The Actin gene of cabbage was used as the internal reference gene [40]. The relative expression level of BoMAPKs was calculated using the 2−ΔΔCt method [41].

2.9. Protein–protein Interaction Network Prediction

A protein–protein interaction network was constructed in the STRING program (https://string-db.org/cgi/input.pl (accessed on 22 September 2023)) according to the Arabidopsis association model with the interaction score of high confidence (0.700).

3. Results

3.1. Identification of MAPK Gene Family Members in Cabbage

In order to identify MAPK gene family genes in cabbage, the latest protein and gene sequences of cabbage (JZS_V2.0) were obtained from the Brassicaceae Database (BRAD). A total of 24 BoMAPK genes were identified by screening the MAPK protein database using the Blastp software and HMMER3.0 program and were renamed according to this homologous sequence’s similarity with Arabidopsis (Table S2). The physical and chemical properties of MAPK gene family members were analyzed using the ExPASY tool (Table 1). The BoMAPK proteins’ length ranged from 369 (BoMAPK1) to 840 (BoMAPK20.1) amino acids (AA) with the molecular weight (MW) from 42,485.25 (BoMAPK1) to 95,445.32 (BoMAPK20.1), and the isoelectric point (pI) from 5.10 (BoMAPK4.2) to 9.48 (BoMAPK19.2). The aliphatic index (AI) varied from 76.42 (BoMAPK20.1) to 98.78 (BoMAPK2). This variation range meant that different BoMAPK proteins may operate in different microenvironments. The grand average of hydropathicity (GRAVY) for all 24 BoMAPK proteins was negative, indicating that these proteins were all hydrophilic proteins. Subcellular localization prediction showed that half of the BoMAPK genes were located in the cytoplasm. Another four BoMAPK genes were located in the nucleus; five were located in the chloroplast, one was located in the mitochondrion, and one in the cytoskeleton. This indicates that BoMAPK genes may play different roles in diverse cellular environments.

3.2. Phylogenetic Analysis of BoMAPK Gene Family

The MAPKs are defined by the presence of a signature TEY or TDY activation motif. The multiple sequence alignment showed that all 24 BoMAPK proteins contained the highly conserved core amino acid sequence “T(E/D)YV(V/A)TRWYRAPEL” (Figure 1). In order to further explore these evolutionary relationships, 24, 29, 14, 20, and 17 MAPK protein sequences from B. oleracea, B. napus, B. rapa, A. thaliana, and O. sativa were used to construct the phylogenetic tree (Figure 2). The BoMAPK gene family could be divided into two categories, “TEY” and “TDY”, based on different phosphorylation sites. The BoMAPKs with “TEY” phosphorylation sites were further divided into subgroups A, B, and C, in accordance with their previous classification in Arabidopsis, whereas only BoMAPK proteins belonging to subgroup D contained the TDY motif. BoMAPK3, BoMAPK6.1, and BoMAPK6.2 were clustered into subgroup A. There were four and three BoMAPKs in subgroups B and C, respectively. The BoMAPKs in subgroup D expanded significantly, with a total of 14 members in the cabbage, as also observed for the other four species (Table S3). The amplification of subgroup D BoMAPK genes provided an important reference for the evolution of cabbage. BoMAPK genes in subgroup D also contained the TEY/SDY motif at the N-terminal in Figure 1, which could be the target phosphorylation sites of other kinases. Besides these conserved motifs, the N-terminal of all BoMAPKs contained I-G-X-G-X-Y-G-X-V. Each subgroup included at least one BoMAPK gene from three species, indicating that the MAPK protein domain was relatively conserved. The secondary structure prediction indicated that cabbage BoMAPK proteins were mainly consisted of Alpha Helix (34.05–47.03%), and random coil (32.07–46.84%), followed by extension chains (10.75–16.79%), and β-turns (3.92%–7.07%) (Table S4). The tertiary structures of BoMAPK proteins from the same group were similar to each other (Figure S1).

3.3. Chromosome Location and Synteny Analysis of BoMAPK Genes

The genomic distribution of BoMAPKs was carried out using TBtools software [35]. The 24 candidate BoMAPKs were mapped to 9 chromosomes of cabbage (Figure 3). The largest number of BoMAPKs was found on chromosome 3 with five genes. In contrast, chromosomes 2 and 7 had the least number of genes, with only BoMAPK17.2 and BoMAPK17.1, respectively. Interestingly, chromosome 6 contained three members, which all belonged to subgroup D. Therefore, the BoMAPKs presented an uneven distribution in all regions of these chromosomes.
Among the cabbage MAPK gene family, thirteen gene pairs could be assigned to segmental duplication events (Table S5, Figure S2) [36]. However, no tandem duplication events were found. Most gene duplication events occurred on chromosomes 5 and 6. To further explore the origin and evolution of the BoMAPK gene family, the collinearity map between cabbage and Arabidopsis at the whole genome level was constructed. There were 38 (orthologous) collinear gene pairs identified between cabbage and Arabidopsis, which suggested that most BoMAPKs had ortholog genes in Arabidopsis (Table S6). To detect the selection pressure on segmentally duplicated gene pairs, the nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and Ka/Ks ratios were calculated. As a result, the Ka/Ks rations of all segmental duplication gene pairs ranged from 0.05 to 0.17, with a clear peak at 0.1–0.2 (Table S5). The average Ka/Ks ratios of collinear gene pairs between cabbage and Arabidopsis were 0.08, demonstrating that most MAPK genes were under purifying selection (Table S6).

3.4. Conserved Motifs and Gene Structure Analysis of BoMAPK Gene Family

By comparing the cDNA sequence and genomic DNA of BoMAPKs, the number and location of exons-introns for each BoMAPK gene were revealed (Figure 4). In subgroup A, BoMAPK6.1, BoMAPK3, and BoMAPK6.2 contained 4, 5, and 6 introns, respectively. BoMAPK1, BoMAPK2, and BoMAPK7 in subgroup C were only composed of one intron. All BoMAPKs in subgroup B had five introns, with each exon being conservative. The intron and exon distribution of BoMAPKs in subgroup D was more complicated. BoMAPK8.2, BoMAPK8.1, and BoMAPK15 had ten introns. BoMAPK15 contained a relatively longer intron. However, BoMAPK16.2 and BoMAPK20.1 contained more introns, 12 and 13, respectively. The intron number of other BoMAPKs ranged from 6 to 9. Most BoMAPKs within the same subgroups displayed similar gene structures. Meanwhile, the motifs of BoMAPK protein sequences were analyzed using the MEME program (Figure 4). A total of 15 motifs were identified, and the number of motifs in BoMAPK proteins ranged from 7 to 13. Motif 9 represented the conserved T-E-Y motif in the activation loop region, which was the most conserved and appeared in all subgroup A-C BoMAPK proteins. By contrast, motif 8 represented the conserved T-D-Y motif, which appeared in all subgroup D BoMAPK proteins. Several motifs only existed in specific subgroups, such as motif 12, which only existed in BoMAPK proteins of subgroups A and B, while motif 6 and 15 only existed in all members of subgroup D. Motif distribution showed that members in the same branch had a similar gene structure. The disparity of motif distribution for the BoMAPK protein in different subgroups was the structural basis of functional gene diversity.

3.5. Number and Types of Cis-Acting Elements of BoMAPK Genes Promoter

We predicted and analyzed the number and types of cis-acting elements in the 2000 bp promoter sequence of each BoMAPK so as to understand the potential regulation mechanism of these genes. After screening, six stress-related elements, nine hormone-related elements, and four development-related elements were discovered (Figure 5 and Figure S3, Table S7). The stress-related cis-acting elements included ARE (abscisic acid responsiveness), LTR (low temperature), W-box (a binding site for WRKY TFs), MBS (drought stresses), TC-rich repeats (cis-acting elements involved in defense and stress responsiveness), and the GC-motif (anoxic specific inducibility). In the analysis, MBS (drought stresses), and LTR (low temperature) were found in the promoter regions of 11 and 12 BoMAPK genes, respectively. Only ten gene promoters contained varying numbers of TC-rich repeat elements (cis-acting element involved in defense and stress responsiveness). Cis-acting elements involved in plant development were present in 15 BoMAPK genes. Among them, eleven CAT-boxes (meristem expression regulatory elements) existed in ten BoMAPKs. The GCN4_motif (involved in endosperm expression) was located in the promoter regions of six BoMAPKs. The number of hormone-response elements in BoMAPKs varied greatly. The ABRE (abscisic acid responsiveness element) was the most common element and existed in the upstream sequences of 21 BoMAPKs. The second one was salicylic acid responsiveness (TCA-element), which appeared on the 15 BoMAPK promoters. BoMAPK17.2 had the largest number of hormone response elements (15). Therefore, the cis-element analysis revealed that the expression levels of BoMAPK genes in cabbage might be associated with different environmental factors.

3.6. Prediction and Analysis of Protein–protein Interaction Network

To understand the interaction and relationships between BoMAPK proteins and other proteins in cabbage, the interaction network was analyzed based on orthology analysis with AtMAPK proteins (Figure 6). In general, 17 MAPK family proteins in Arabidopsis and 15 BoMAPK protein orthologs were identified in the correlation network. The results indicated that BoMAPK6.1 and BoMAPK6.2 (the Arabidopsis MAPK6 homologs) showed an interaction with BoMAPK3 (a homolog of Arabidopsis MAPK3) and BoMAPK4 (a homolog of Arabidopsis MAPK4). Furthermore, MAPK6, MAPK3, and MAPK4 also showed a complex interaction with MKK1, MKK2, MKK6, MKS1 (VQ motif-containing protein), and DSPTP1B (Dual specificity protein phosphatase 1B). MAPK8 (BoMAPK8.1 and BoMAPK8.2), MAPK9 (BoMAPK9), and MAPK18 (BoMAPK18.1 and BoMAPK19.1) only interacted with DSPTP1B, while the other MAPK proteins were predicted to have a correlation with different MAPKK proteins. These protein interaction predictions could help investigate the function of cabbage BoMAPK proteins.

3.7. Expression Analysis of BoMAPKs in Different Tissues and Development

In order to explore the role of MAPK genes during the development of cabbage, the expression level of BoMAPKs in seven different tissues and organs (root, callus, leaf, bud, siliques, stem, and flower) were investigated based on transcriptome data (Figure 7, Table S8). Ten BoMAPKs showed different expressions across tissues. BoMAPK3 had a high expression level in all tissues, especially in the callus, root, and stem. The expression level of BoMAPK6.1 in flower is higher than that of other genes, while the expression of BoMAPK17.2 and BoMAPK8.1 showed a higher expression in root and flower, respectively. BoMAPK4.1 was expressed similarly in the seven tissues. BoMAPK9 was slightly expressed in all tissues.

3.8. Expression Pattern of BoMAPKs under Clubroot Disease Infection

More evidence shows that BoMAPKs are involved in plant responses to external environmental stimuli, including pathogens. As shown in Figure 8 and Table S9, most BoMAPKs were induced by Plasmodiophora brassicae through RT-qPCR, while nine BoMAPKs were rarely expressed. Eight BoMAPKs (BoMAPK2, BoMAPK4.1, BoMAPK9, BoMAPK18.1, BoMAPK19.1, BoMAPK19.2, BoMAPK20.1, and BoMAPK20.2) were highly expressed in the F21I condition. Among them, the transcription level of BoMAPK20.2 was the highest; the expression value was three times more than that of the control. The expression levels of BoMAPK17.1 and BoMAPK18.2 were up-regulated at 7d after infection by Plasmodiophora brassicae. Whie BoMAPK17.2 exhibited significantly increased expression levels both in F7I and F21I conditions.

3.9. Expression Profiles of BoMAPKs under Cold and Salt Stress Treatment

In response to salt stress (Figure 9A and Figure S4 and Table S10), three BoMAPKs, including BoMAPK1, BoMAPK7, and BoMAPK15, were significantly induced throughout the treatment process. The expression levels of three BoMAPKs (BoMAPK8.2, BoMAPK18.2, and BoMAPK20.2) were down-regulated. The expression of BoMAPK4.1, BoMAPK6.1, and BoMAPK9 were increased first and then returned to normal levels. The majority of BoMAPK genes were up-regulated after cold stress (Figure 9B and Figure S5). Half of the BoMAPKs were found to be up-regulated only at 6 h. BoMAPK6.2, BoMAPK15, BoMAPK18.1, and BoMAPK18.2 were up-regulated at both 6 h and 24 h. Two BoMAPK genes (BoMAPK5 and BoMAPK17.1) exhibited suppressed expression.

4. Discussion

MAPK cascades are significant signaling pathways involved in hormone signal transduction, growth and development, and plant disease resistance [42]. MAPK is a class of hybrid serine/threonine kinases, which are located downstream of MAPK cascades and can phosphorylate a variety of substrates [3]. In our study, 24 BoMAPK genes were identified in cabbage, and this number was larger than that reported previously [43]. This is largely due to the different versions of the genome used. Based on the Arabidopsis MAPK proteins classification and the phylogenetic tree, all 24 cabbage MAPKs were phylogenetically divided into four subgroups. Each subgroup contained MAPK genes from five species (Table S3). The number of MAPK genes in each subgroup was similar between B. napus, B. oleracea and Arabidopsis.
Gene duplication has played an important role in the evolution of gene families and the generation of new functions [44,45]. Thirteen segmental duplication gene pairs, including 17 BoMAPK genes, were identified in cabbage without tandem duplication events. In previous reports, there have been 3, 9, and 19 segmental duplication gene pairs in the genomes of Fagopyrum tataricum, A. thaliana, and Gossypium raimondii, respectively [18]. In our study, most BoMAPK genes in cabbage were collinear with homologous genes in the Arabidopsis genome. Therefore, we inferred that gene segmental duplication events could contribute to the expansion of MAPK gene family members in cabbage. In order to further explore the macroevolutionary model of cabbage, the Ka/Ks value of duplicated gene pairs was estimated. Interestingly, the average Ka/Ks ratio for Bo-Bo and Bo-At gene pairs were 0.11 and 0.08, respectively, implying a strong selection constraint and purifying selection in BoMAPK genes.
In Arabidopsis, AtMAPK3 and AtMAPK6 participated in stomatal development [27], anther development [14], pollen development [46,47], lateral root development [48], and shoot apical meristem [49]. Similarly, the orthologs for AtMAPK3 and AtBoMAPK6, BoMAPK3, and BoMAPK6.1 were found to be highly expressed in all tissues. The results suggest the BoMAPKs in subgroup A play a key role during plant growth and development, similar to that in Arabidopsis. Meantime, AtMPK3 and AtMPK6 play key roles in resistance to disease by regulating various defense responses [50]. In the present study, BoMAPK6.1 and BoMAPK6.2 were also induced by pathogen infection. The overexpression of B. napus BnMPK4 enhanced defense response and resistance to Sclerotinia sclerotiorum in oilseed rape [51]. BoMAPK4, a homologous gene of BnMAPK4, was up-regulated in clubroot infection, suggesting that this gene was positively regulating response to disease resistance. Four subgroup D members (BoMAPK19.1, BoMAPK19.2, BoMAPK20.1, and BoMAPK20.2) were up-regulated at 21 d after clubroot disease infection. Gene expression regulation was mainly carried out at the transcriptional level, which is coordinated by a variety of cis-acting elements and trans-acting factors. TC-rich elements were found in the promoter regions of 10 BoMAPK genes. Among them, six BoMAPK genes were significantly expressed after clubroot infection. A large number of studies demonstrated that MAPK cascades also play critical roles in plant abiotic stress. In Arabidopsis, MPK3, MPK4, and MPK6 were rapidly activated after cold treatment [22]. MPK3 and MPK6 negatively regulated ICE1’s stability and attenuated freezing tolerance in Arabidopsis [52]. However, the MEKK1-MKK2-MPK4 pathway positively regulated cold response [22]. MKK5-MPK3/6 cascades enhanced salt tolerance in Arabidopsis through phosphorylation and the degradation of ARR1/10/12 [53]. BoMAPK3, BoMAPK4.1, BoMAPK4.2, BoMAPK6.1, and BoMAPK6.2, which were orthologous to the AtMAPK3, AtMAPK4, and AtMAPK6 genes, exhibited high expression levels at 6 h after cold treatment, implying that these genes may play a similar role in cold stress. The RT-qPCR results show that BoMAPK19.1 exhibited relatively higher expression under three types of stress involved in this study; this is regarded as a candidate gene for further research.

5. Conclusions

In this study, 24 BoMAPK genes were identified from the cabbage genome, and all of the BoMAPKs contained TEY or TDY characteristic motifs. Members in the same subgroup had a highly similar gene structure and motif composition. Furthermore, segmental duplication events contributed to the expansion of the MAPK gene family in cabbage. The BoMAPKs showed various transcription levels in different tissues. RT-qPCR showed that BoMAPK17.2, BoMAPK20.1 and BoMAPK20.2 were significantly induced by Plasmodiophora brassicae infection. The transcriptional levels of BoMAPK genes varied greatly under abiotic stress at different time points. These findings can help us to better understand the role of BoMAPK genes in response to biotic and abiotic stresses.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9101119/s1, Figure S1. Predicted three-dimensional domains of MAPK proteins from cabbage. 3D models of BoMAPKs according to SWISS-MODEL. In BoMAPK3 (group A), the yellow mark represents the TEY motif in the activation loop region. Similarly, in BoMAPK8.1 (group D), the conserved activation loop TDY motif is indicated by a yellow mark; Figure S2: Collinearity analysis of MAPK gene family in B. oleracea. Grey lines represent the collinear blocks within the genome, while the red lines highlight the duplicated MAPK gene pairs; Figure S3. Cis-element analysis of BoMAPKs promoter. (A). Proportion of cis-elements in the BoMAPKs promoter; (B). The number of BoMAPKs in each class of cis-element; Figure S4: Gene expression of BoMAPK genes under NaCl treatment was analyzed using RT-qPCR. The different lowercase letters indicate significant differences at the p < 0.05 level; Figure S5: Gene expression of BoMAPK genes under cold treatment was analyzed using RT-qPCR. The different lowercase letters indicate significant differences at the p < 0.05 level. Table S1: The specific primers of all BoMAPK genes used in RT-qPCR analysis; Table S2. List of MAPK genes identified in B. oleracea and A. thaliana. Table S3: Size of the MAPK subgroups in different plant species; Table S4: Secondary structural elements of BoMAPK proteins; Table S5. The Ka/Ks value of segmental duplication gene pairs in cabbage; Table S6: The Ka/Ks value of collinearity gene pairs between cabbage and Arabidopsis; Table S7: The number of cis-acting elements in BoMAPKs promoter; Table S8: Expression profiles of BoMAPK genes in different tissues; Table S9: Expression analysis of BoMAPK genes under culbroot infection by RT-qPCR; Table S10: Expression analysis of BoMAPK genes under different salt and cold stress, as determined by RT-qPCR.

Author Contributions

Conceptualization, M.W. and T.B.; software, M.W. and X.Z.; investigation, M.W. and J.C.; resources, X.T.; data curation, M.W. and X.T.; writing—original draft preparation, M.W.; writing—review and editing, M.W. and T.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the “14th Five-Year Plan” National key Research and Development program (Grant No. 2023YFD1200005-1) and the Excellent Team Project (Nongkezhuo 2022(005)).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Protein sequence comparison of cabbage MAPK proteins using DNAMAN software. The T(E/D)Y, IGxGxYGxV and (LH)DXXDE(P)XC domains are marked by red boxes.
Figure 1. Protein sequence comparison of cabbage MAPK proteins using DNAMAN software. The T(E/D)Y, IGxGxYGxV and (LH)DXXDE(P)XC domains are marked by red boxes.
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Figure 2. Phylogenetic tree of MAPK gene family in B. oleracea (Bo), B. rapa (Bra), B. napus (Bna), A. thaliana (At), and O.sativa (Os). The tree was constructed based on the full length of MAPK protein sequences with MEGA5.0 and the neighbor-joining method. MAPK proteins were divided into four subgroups (A, B, C, and D). The red circle represents B. oleracea MAPK (BoMAPK); pink square represents B. rapa MAPK; green diamond represents B. napus MAPK (BnaMAPK); blue triangle represents A. thaliana MAPK (AtMAPK); and the green hollow circle represents O. sativa MAPK (OsMAPK).
Figure 2. Phylogenetic tree of MAPK gene family in B. oleracea (Bo), B. rapa (Bra), B. napus (Bna), A. thaliana (At), and O.sativa (Os). The tree was constructed based on the full length of MAPK protein sequences with MEGA5.0 and the neighbor-joining method. MAPK proteins were divided into four subgroups (A, B, C, and D). The red circle represents B. oleracea MAPK (BoMAPK); pink square represents B. rapa MAPK; green diamond represents B. napus MAPK (BnaMAPK); blue triangle represents A. thaliana MAPK (AtMAPK); and the green hollow circle represents O. sativa MAPK (OsMAPK).
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Figure 3. Chromosomal distributions of BoMAPK genes. The vertical bars mark the chromosomes of cabbage. Chromosome numbers are on the left of each chromosome. The left most scale indicates the length of the chromosome.
Figure 3. Chromosomal distributions of BoMAPK genes. The vertical bars mark the chromosomes of cabbage. Chromosome numbers are on the left of each chromosome. The left most scale indicates the length of the chromosome.
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Figure 4. Structural analysis of cabbage MAPK gene family. The phylogenetic tree of cabbage MAPK proteins on the left; the motif composition in the middle; the exon-intron structure of cabbage MAPK genes on the right.
Figure 4. Structural analysis of cabbage MAPK gene family. The phylogenetic tree of cabbage MAPK proteins on the left; the motif composition in the middle; the exon-intron structure of cabbage MAPK genes on the right.
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Figure 5. Cis-element analysis of BoMAPKs promoter. Analysis of cis-regulatory elements in promoter regions of 24 BoMAPKs. The black line indicates the promoter length of each BoMAPK gene. The boxes with different colors represent different types of cis-acting elements.
Figure 5. Cis-element analysis of BoMAPKs promoter. Analysis of cis-regulatory elements in promoter regions of 24 BoMAPKs. The black line indicates the promoter length of each BoMAPK gene. The boxes with different colors represent different types of cis-acting elements.
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Figure 6. An interaction network analysis of BoMAPK proteins according to STRING. The nodes represent proteins, the lines between nodes indicate the interaction between proteins, and different colors correspond to different interaction types.
Figure 6. An interaction network analysis of BoMAPK proteins according to STRING. The nodes represent proteins, the lines between nodes indicate the interaction between proteins, and different colors correspond to different interaction types.
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Figure 7. Heatmap showing the expression profile of BoMAPK genes in different tissues.
Figure 7. Heatmap showing the expression profile of BoMAPK genes in different tissues.
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Figure 8. Expression analysis of BoMAPK genes under culbroot infection using RT-qPCR. F7I: roots of cabbage seedlings at 7 days after inoculation, F7CK: untreated roots of cabbage seedlings after 7 days, F21I: roots of cabbage seedlings at day 21 after inoculation, F21CK: untreated roots of cabbage seedlings at day 21. Lowercase letters represent significant differences (p < 0.05).
Figure 8. Expression analysis of BoMAPK genes under culbroot infection using RT-qPCR. F7I: roots of cabbage seedlings at 7 days after inoculation, F7CK: untreated roots of cabbage seedlings after 7 days, F21I: roots of cabbage seedlings at day 21 after inoculation, F21CK: untreated roots of cabbage seedlings at day 21. Lowercase letters represent significant differences (p < 0.05).
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Figure 9. Expression analysis of BoMAPK genes under two abiotic stresses using RT-qPCR. (A) The relative expression level of BoMAPK genes under Nacl stress. (B) The relative expression level of BoMAPK genes under cold stress.
Figure 9. Expression analysis of BoMAPK genes under two abiotic stresses using RT-qPCR. (A) The relative expression level of BoMAPK genes under Nacl stress. (B) The relative expression level of BoMAPK genes under cold stress.
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Table 1. Identification and characteristic of MAPK gene family in cabbage.
Table 1. Identification and characteristic of MAPK gene family in cabbage.
Gene
Name		Gene IDNumber of AAMW (Da)pIAIGRAVYLocalization
Prediction
BoMAPK1BolC05g008000.2J36942,485.256.6798.27−0.248Cytoplasm
BoMAPK2BolC01g035020.2J37042,488.036.2398.78−0.223Cytoplasm
BoMAPK3BolC03g065400.2J37042,592.775.7092.00−0.312Cytoskeleton
BoMAPK4.1BolC03g035260.2J37342,513.485.8589.14−0.332Cytoplasm
BoMAPK4.2BolC08g057380.2J37143,151.415.1089.60−0.371Cytoplasm
BoMAPK5BolC09g032170.2J37342,809.885.5791.98−0.302Cytoplasm
BoMAPK6.1BolC04g005090.2J39244,850.295.2791.35−0.299Nucleus
BoMAPK6.2BolC03g026650.2J41447,076.995.3593.12−0.220Chloroplast
BoMAPK7BolC09g012900.2J36842,234.857.1894.29−0.238Cytoplasm
BoMAPK8.1BolC08g051030.2J58265,637.186.5777.42−0.554Cytoplasm
BoMAPK8.2BolC05g015790.2J58465,863.306.0878.51−0.527Chloroplast
BoMAPK9BolC05g047830.2J50157,381.468.1280.06−0.479Cytoplasm
BoMAPK13BolC04g067890.2J37242,589.858.0388.79−0.305Cytoplasm
BoMAPK15BolC06g032120.2J57765,327.037.2980.12−0.571Cytoplasm
BoMAPK16.1BolC09g053590.2J56764,784.078.8177.23−0.517Mitochondrion
BoMAPK16.2BolC03g009910.2J69879,579.799.2776.98−0.549Chloroplast
BoMAPK17.1BolC07g031210.2J48755,627.596.5181.25−0.419Cytoplasm
BoMAPK17.2BolC02g049250.2J48855,755.706.6480.68−0.434Cytoplasm
BoMAPK18.1BolC06g008540.2J60267,838.369.2076.64−0.446Nucleus
BoMAPK18.2BolC05g051570.2J59667,131.799.2678.05−0.476Nucleus
BoMAPK19.1BolC06g014820.2J56163,486.849.2783.28−0.377Chloroplast
BoMAPK19.2BolC01g048030.2J64872,685.319.4878.12−0.465Chloroplast
BoMAPK20.1BolC04g004430.2J84095,445.329.0076.42−0.460Nucleus
BoMAPK20.2BolC03g026050.2J62470,463.188.5881.97−0.450Cytoplasm
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Wang, M.; Chen, J.; Zhu, X.; Tai, X.; Bo, T. In Silico Analysis of the MAPK Gene Family in Cabbage and Its Expression during Development and Stress Response. Horticulturae 2023, 9, 1119. https://doi.org/10.3390/horticulturae9101119

AMA Style

Wang M, Chen J, Zhu X, Tai X, Bo T. In Silico Analysis of the MAPK Gene Family in Cabbage and Its Expression during Development and Stress Response. Horticulturae. 2023; 9(10):1119. https://doi.org/10.3390/horticulturae9101119

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Wang, Min, Jinxiu Chen, Xiaowei Zhu, Xiang Tai, and Tianyue Bo. 2023. "In Silico Analysis of the MAPK Gene Family in Cabbage and Its Expression during Development and Stress Response" Horticulturae 9, no. 10: 1119. https://doi.org/10.3390/horticulturae9101119

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