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Article

Genome-Wide Characterization of CaM/CML Gene Family in Cabbage (Brassica oleracea var. capitata): Expression Profiling and Functional Implications During Hyaloperonospora parasitica Infection

by
Yuankang Wu
1,2,†,
Bin Zhang
3,†,
Xuehui Yao
1,
Limei Yang
1,
Mu Zhuang
1,
Honghao Lv
1,
Yong Wang
1,
Jialei Ji
1,
Xilin Hou
2,* and
Yangyong Zhang
1,*
1
State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2
National Key Laboratory of Crop Genetics & Germplasm Innovation and Utilization, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (East China), Ministry of Agriculture and Rural Affairs of China, Engineering Research Center of Germplasm Enhancement and Utilization of Horticultural Crops, Ministry of Education of China, Nanjing Agricultural University, Nanjing 210095, China
3
State Key Laboratory of Vegetable Biobreeding, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Science, Beijing 100097, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(7), 3208; https://doi.org/10.3390/ijms26073208
Submission received: 18 November 2024 / Revised: 15 March 2025 / Accepted: 28 March 2025 / Published: 30 March 2025
(This article belongs to the Special Issue New Horizons in Vegetable Genetics and Genetic Breeding (Second Edition))
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Abstract

:
Calmodulin (CaM) and calmodulin-like proteins (CMLs) are crucial for calcium signal transduction in plants. Although CaM/CML genes have been extensively studied in various plant species, research on these genes in Brassica oleracea is still limited. In this study, 14 BoCaM and 75 BoCML genes were identified in the B. oleracea genome through a genome-wide search. Phylogenetic analysis categorized these genes, along with their homologs in Arabidopsis and rice, into six distinct groups. All BoCaM/BoCML genes were unevenly distributed across the nine chromosomes of B. oleracea, with 52 of them lacking introns. Collinearity analysis revealed that CaM/CML genes in Arabidopsis are present in multiple copies in the B. oleracea genome. Moreover, the majority of BoCaM/BoCML genes exhibited distinct expression patterns across the different tissues, indicating their role in the growth and development of B. oleracea. A clustering heatmap of BoCaM/BoCML gene expression showed distinct patterns before and four days after Hyaloperonospora parasitica infection, dividing the genes into five groups based on their expression patterns. Notably, BoCML46-2 is significantly downregulated in both susceptible and resistant materials, suggesting that it plays an important role in responding to H. parasitica infection. This study conducted a comprehensive survey of the BoCaM/BoCML gene family in B. oleracea. It could serve as a theoretical foundation for further functional identification and utilization of family members and their role in the interaction between B. oleracea and H. parasitica.

1. Introduction

Plants encounter numerous biotic and abiotic stresses during their growth and development, which can lead to significant reductions in yield. They respond to these pressures through intricate internal signaling mechanisms [1]. Serving as a ubiquitous intracellular coordinator, calcium (Ca2+) mediates decoding of environmental stimuli into stress-adaptive signaling cascades. Almost all types of environmental stresses lead to changes in the cytosolic free Ca2+ levels within plant cells and affect the movement of Ca2+ between cellular organelles [2,3]. When faced with environmental stresses, plants translate Ca2+ signals into specific downstream responses through a complex array of calcium sensor proteins, thereby precisely regulating internal homeostasis [4]. In Arabidopsis, over 250 calcium sensor proteins have been identified, including calcineurin B-like proteins (CBLs), calmodulin (CaM), calmodulin-like proteins (CMLs), calcium-dependent protein kinases (CPKs), and calcium and calmodulin-dependent protein kinase (CCaMK), and all of them contain different numbers of EF-hand motifs [5,6,7,8,9,10].
CaMs and CMLs are essential types of Ca2+ sensors and are crucial components in Ca2+ signal transduction [11]. CaMs, which contain four EF-hand motifs, are conserved Ca2+ sensors found in both plants and animals [12]. On the other hand, CMLs, which typically contain 1–6 EF-hand motifs, show some sequence similarity to CaM and display structural variations in plants [13]. Genome-wide identification and analysis of CaM/CML genes have been conducted for numerous plant species, including Arabidopsis (7 CaMs and 50 CMLs), rice (Oryza sativa, 5 CaMs and 32 CMLs), and Brassica napus (25 CaMs and 168 CMLs) [7,14,15]. Although CMLs and CaMs are homologous, the number of CMLs in plants is significantly higher than that of CaMs. The roles of CaMs and CMLs in responding to stress have been well documented. In Arabidopsis, the AtCaM3 knockout mutant exhibits reduced heat tolerance, whereas transgenic lines overexpressing AtCaM3 demonstrate enhanced heat tolerance [16]. AtCML8 and AtCML9 enhance Arabidopsis resistance to Pseudomonas syringae via the ABA and SA pathways, respectively [17]. The AtCML24 gene plays a role in inhibiting pathogen-induced nitric oxide (NO) generation [18]. In cotton, GhCML11 interacts with GhMYB108, acting as a positive regulator in defense against Verticillium dahliae infection [19]. In pepper, CaCML13 acts as a positive regulator of immunity against Ralstonia solanacearum inoculation by forming a positive feedback loop with CabZIP63 [20]. Furthermore, OsCaML2 can serve as a target gene for osa-miR1432, and its overexpression enhances resistance to Xanthomonas oryzae pv. Oryzae [21]. Recently, SlCML55 has been identified as a novel calmodulin-like protein in tomato that negatively regulates plant immunity by inhibiting Phytophthora capsici infection and interacting with the SA signaling pathway [22]. Although the roles of CaMs/CMLs in response to various stimuli have been extensively studied in several plant species, a comprehensive genome-wide analysis of the CaM/CML gene families in cabbage (Brassica oleracea var. capitata L.) is lacking, and the potential functions of cabbage CaM/CML genes remain unclear.
Cabbage is one of the most widely cultivated vegetables globally, with a significant quantity of this crop consumed annually. The production of cabbage is significantly impacted by four prominent diseases: wilt, black rot, root knot disease, and downy mildew. Downy mildew, caused by the oomycete H. parasitica, has emerged as a significant threat to cabbage production in recent years [23]. However, research efforts directed towards identifying resistance genes against downy mildew in cabbage remain scarce. Investigating cabbage resistance genes through various approaches is essential for elucidating the molecular mechanisms of downy mildew resistance and accelerating molecular breeding for resistance.
In this study, 14 BoCaM and 75 BoCML genes were identified in B. oleracea based on their homology to those in Arabidopsis thaliana. The characteristics of the B. oleracea CaM/CML protein including molecular weight, theoretical pI, grand average of hydropathicity, and subcellular localization. Additionally, intron-exon structures, chromosomal localizations, EF-hand motifs, and phylogenetic relationships of the BoCaM and BoCML genes were analyzed. Additionally, the tissue-specific expression of BoCaM and BoCML genes in various cabbage tissues was analyzed, and their differential expression profiles in response to Hyaloperonospora parasitica infection were compared between resistant and susceptible cabbage varieties. These findings lay a theoretical foundation for future studies on the functional identification and utilization of BoCaM/BoCML genes, particularly in understanding their role in the interaction between B. oleracea and downy mildew.

2. Results

2.1. Identification and Characterization of BoCaM/BoCML Genes in B. oleracea

To identify the putative CaM and CML genes in B. oleracea, 7 CaM and 50 CML genes of Arabidopsis were used to blastp against B. oleracea genome protein sequence in the BRAD database (Supplementary Table S1). The results of this blastp search were then analyzed using Pfam, InterProScan, and the NCBI CD-search tool to identify the CaM/CML genes in B. oleracea. A total of 14 BoCaM and 75 BoCML genes have been identified and were named as BoCaM1-1 to BoCML50-2 based on their homologous relationship to Arabidopsis CaM/CML genes. The lengths of the BoCaM/BoCML genes ranged from 78 amino acids (BoCML28-2) to 436 amino acids (BoCML21-2), with varying numbers of EF-hand motifs. Their isoelectric points (pI) and theoretical molecular weights (MW) ranged from 4.00 to 6.89 and 8.55 to 49.74 kDa, respectively. Notably, the Grand Average of Hydropathicity (GRAVY) results indicate that most BoCaM/BoCML genes exhibit relatively high hydrophilicity, while BoCML15-1 and BoCML47 display relatively high hydrophobicity. Subcellular localization predictions revealed that the majority (62) of BoCaM/BoCML genes are located in the nucleus, while only BoCML21-2 is located in the mitochondrion. Furthermore, the instability index and aliphatic index of BoCaMs/BoCMLs were also provided (Supplementary Table S2).

2.2. Phylogenetic Analysis, Protein Motifs, and Gene Structure of BoCaM/BoCML Genes

A total of 14 BoCaM and 75 BoCML genes were classified into six groups (Groups I–VI) based on their evolutionary relationships (Figure 1). Notably, Group IV has the largest number of members, including all BoCaM genes, whereas Group V has only four members (BoCML21-1, BoCML21-2, BoCML22-1, BoCML22-2). BoCML genes originating from the same Arabidopsis gene (e.g., BoCML25-1 to BoCML25-6, BoCML46-1 to BoCML46-4) are clustered together in the same group. Additionally, there are 63 members with four EF-hand motifs, 14 members with three EF-hand motifs, and 12 members with two EF-hand motifs. All BoCaM genes, except for BoCaM4-2, which possesses three EF-hand motifs, exhibit four EF-hand motifs, which corresponds to their relatively distant placement from other BoCaM genes in the phylogenetic tree. Furthermore, the majority (52) of CaM/CML members have only a single exon without intron regions, and their genes are relatively short. However, BoCML2-1, BoCML11-3, and BoCML8-2 have large introns, resulting in gene lengths of approximately 8000 bp. Overall, genes within the same subcategories exhibit close evolutionary relationships, considerable sequence similarity, and comparable genetic architectures.

2.3. Phylogenetic Relationship of CaM/CML Genes Among B. oleracea, Arabidopsis, and Rice

Protein sequences for 14 BoCaM and 75 BoCML genes in B. oleracea, 7 AtCaM and 50 AtCML genes in Arabidopsis, and 5 OsCaM and 32 OsCML genes in Oryza sativa were obtained. Phylogenetic analysis was performed using the full protein sequences of these genes from B. oleracea, Arabidopsis, and rice (Figure 2). All CaM/CML genes were clustered into six groups (Groups I–VI). The results suggest that most CaM/CML genes originate from a common ancestor. Group III contains the largest number of members (75), whereas Group IV has the fewest members (BoCML36, AtCML36, AtCML35, BoCML35-1, BoCML35-2), with no OsCaM/OsCML genes present. In the clustering analysis, all CaM genes, whether from dicotyledonous (B. oleracea, Arabidopsis) or monocotyledonous (rice) plants, were grouped into Cluster VI. Additionally, most of the BoCaM/BoCML genes are homologous to those in Arabidopsis and rice. These findings suggest that CaM/CML genes are conserved across diverse plant species.

2.4. Chromosome Distribution and Collinearity Analysis of BoCaM/BoCML Genes

To analyze the chromosomal localization of BoCaM and BoCML genes, the genes were mapped onto the chromosomes of the B. oleracea genome (B. oleracea JZS V2.0) (Figure 3). All 89 BoCaM/BoCML genes were located on the nine chromosomes of B. oleracea. The largest number (16) of BoCaM/BoCML genes were located on chromosome C03, while only 3 BoCaM/BoCML genes (BoCML29, BoCaM7-4, and BoCML43-3) were mapped to chromosome C02. The remaining BoCaM/BoCML genes were unevenly distributed across the other chromosomes.
To gain deeper insights into the evolutionary lineage of CaM and CML genes in B. oleracea and Arabidopsis thaliana, the duplication events of candidate CaM/CML genes within their genomes were investigated (Figure 4). Among the CML genes in Arabidopsis, only 2 (AtCML21 and AtCML49) lacked homologs in B. oleracea, whereas the remaining 55 CaM/CML genes in Arabidopsis had at least one homolog in B. oleracea. In the genomes of B. oleracea and Arabidopsis, a total of 959 collinear blocks were identified, encompassing 50,429 genes. This set included 89 BoCaM/BoCML genes from B. oleracea and 57 AtCaM/AtCML genes from A. thaliana. For instance, AtCaM7 had six homologous genes within the genome of B. oleracea, whereas AtCML9 had only a single homolog.

2.5. Expression Profiling of BoCaM/BoCML Genes in Different Tissues

To determine the expression profiles of BoCaM/BoCML genes in leaves, stems, flowers, siliques, buds, calli, and roots, the RNA-Seq dataset (GSM1052958-964) was used for a comprehensive analysis across these tissues. The expression of 64 BoCaM/BoCML genes across different tissues was detected. The heatmap of the expression profiles for BoCaM/BoCML genes was generated using log2 FPKM values (Figure 5). The results show that most BoCaM genes (except BoCaM4-2) exhibited higher expression levels in all tissue compared to BoCML genes. BoCaM4-2 was not detected in any tissues, suggesting that its expression may be specific to certain developmental stages. Specifically, BoCML27-1, BoCML27-2, BoCML35-1, BoCML35-2, and BoCML49 exhibited relatively higher expression in all tissues compared to other BoCML genes, suggesting that their functions may be more extensive. BoCML2-2 and BoCML15-2 were expressed only in flowers and buds, suggesting that they may be involved in the development of floral organs or other related biological processes. BoCML3-1, BoCML15-1, and BoCML28-2 were expressed in flowers, buds, and roots, implying that they play a vital role in the morphogenesis of B. oleracea. BoCML3-3 and BoCML25-1 were specifically expressed in flowers, buds, and siliques, indicating their essential roles in the reproductive regulation of B. oleracea. BoCML34-1 and BoCML34-2 were specifically expressed in siliques, suggesting that they play a unique role in silique development. Additionally, the expression of BoCML39 was detected only in leaves and roots, highlighting its pivotal role in nutrient supply in B. oleracea.

2.6. The Relative Expression of BoCaM/BoCML Genes Under H. parasitica Infection

To investigate the differential responses of BoCaM/BoCML genes in B. oleracea under H. parasitica infection, we conducted inoculation experiments using the resistant inbred line 20-2221 and the susceptible inbred line 20-2229. The symptoms of downy mildew infection in lines 20-2221 and 20-2229 were observed both before inoculation and four days post-inoculation. Four days after inoculation, no symptoms were observed on the abaxial leaf surface of 20-2221, whereas the abaxial leaf surface of 20-2229 exhibited not only lesions but also spore germination (Figure 6A). To investigate the role of BoCaM and BoCML genes in cabbage’s response to H. parasitica infection, RNA-seq was performed on resistant and susceptible leaves collected both before and four days after inoculation. Based on the RNA-seq data (BioProject ID: PRJNA1146208), the differential expression of BoCaM and BoCML genes in response to H. parasitica infection was analyzed. Using the differential expression data of BoCaM and BoCML genes in B. oleracea collected before and four days after H. parasitica infection, a heatmap of gene expression was generated (Figure 6B). The heatmap revealed that all BoCaM/BoCML genes clustered into five groups based on their responses to H. parasitica infection in both resistant and susceptible varieties. Genes in Cluster I (26) exhibited high expression levels in both resistant and susceptible materials but did not show changes in response to H. parasitica infection, suggesting that these genes are not involved in the defense response to H. parasitica infection. Genes in Cluster II (11) also displayed high expression in the leaves, but their expression significantly decreased in both resistant and susceptible materials four days post-infection, indicating that H. parasitica infection suppresses their expression. Genes in Clusters III (13) and IV (15) exhibited low expression levels in the leaves and similarly showed reduced expression in response to H. parasitica infection. Genes in Cluster V (24) were almost undetectable in the leaves, consistent with previously observed tissue-specific expression patterns. Six BoCaM/BoCML genes were randomly selected for qRT-PCR validation, and the results confirmed the reliability of the RNA-seq data (Figure 6C). Additionally, to identify the genes with the most significant response to downy mildew infection, RNA-seq and qRT-PCR results were combined, revealing that BoCML46-2 exhibited the most significant reduction in expression following infection. Following infection by H. parasitica, BoCML46-2 expression decreased nearly fivefold in both 20-2221 and 20-2229. Thus, it is hypothesized that BoCML46-2 is a key BoCaM/BoCML gene that responds to H. parasitica infection.

3. Discussion

In this study, a genome-wide identification of BoCaM and BoCML genes in cabbage was conducted, resulting in the identification of 14 BoCaM and 75 BoCML genes, along with their collinearity, structures, chromosomal locations, and expression patterns across different tissues. Additionally, the differential expression profiles of BoCaM and BoCML genes in response to H. parasitica infection were analyzed. This study provides comprehensive insights into the BoCaM and BoCML gene families in cabbage.
The EF-hand motif is a conserved feature in both CaM and CML genes. Whereas CaM genes consistently contain four EF-hand motifs, the number of EF-hand motifs in CML genes varies from one to six [12,13]. The number of EF-hand motifs in CML genes varies across plant species. For instance, CmCML13 and AtCML43 contain three EF-hand motifs, whereas MsCML10 and CpCML15 possess four, which are involved in biotic and abiotic stress responses, respectively [24,25,26,27]. These imply that the variation in EF-hand motif numbers may play pivotal roles in differential stress response mechanisms. Interestingly, BoCML genes contain between two and four EF-hand motifs. Most paralogous genes contain the same number of EF-hand motifs (Figure 1); however, within the BoCML46 family, BoCML46-2 has three EF-hand motifs, whereas BoCML46-1, BoCML46-3, and BoCML46-4 each have only two. This variation suggests that BoCML46-2 may be involved in a broader range of pathways compared to the other three paralogous genes.
Genes that lack introns or have fewer introns are generally considered to be expressed more rapidly in plants, enabling a quicker response to biotic and abiotic stressors [28]. Most BoCML genes (52) have only one exon and lack introns, whereas BoCaM genes typically contain one to three introns. Additionally, most paralogous genes exhibit similar gene structures (e.g., BoCML13-1 to BoCML13-3 and BoCML46-1 to BoCML46-4), although exceptions do exist. For example, BoCML25-2 and BoCML25-3 each contain one intron, whereas the other BoCML25 paralogs lack introns. The majority of the BoCML genes identified in this study contain only one exon, and these genes may play a key role in the rapid response to various abiotic and biotic stresses.
Previous studies have demonstrated that CaM/CML genes play a crucial role in plant responses to pathogen infection [29]. Knockout of OsCML31 has been shown to increase the susceptibility of rice to Meloidogyne graminicola [30]. As a positive regulatory factor, the overexpression of AhCML69 in tobacco leaves enhances resistance to Ralstonia solanacearum infection and induces the expression of defense-related genes [31]. CML43 serves as a key mediator of Ca2+-dependent signaling in the plant immune response to bacterial pathogens [24]. Overexpression of the rice gene OsCaML2 confers resistance to Xanthomonas oryzae pv. Oryzae [21]. AtCML46, together with AtCML47, negatively regulates salicylic acid accumulation and immunity in Arabidopsis. The cml46 and cml47 double mutant displays increased resistance to Pseudomonas syringae and exhibits altered expression patterns of key immune regulators [32]. In this study, all BoCaM/BoCML genes responsive to H. parasitica infection were down-regulated, with BoCML46-2 exhibiting a significant fivefold reduction in expression. Thus, it is hypothesized that BoCML46-2 functions as a negative regulator in response to H. parasitica infection. The findings of this study will facilitate further investigation into the functions of BoCaM/BoCML genes in cabbage, particularly those related to responses to biotic and abiotic stressors.

4. Materials and Methods

4.1. Genome-Wide Identification of BoCaM/BoCML Genes in B. oleracea

The protein sequences of AtCaM/AtCML and OsCaM/OsCML genes were obtained from their respective databases based on previous research [15,33,34,35]. The BLASTP program (https://blast.ncbi.nlm.nih.gov/, accessed on 6 July 2024) was used to identify BoCaM/BoCML genes in B. oleracea genome by comparing them with the protein sequences of AtCaM/AtCML and OsCaM/OsCML genes [36,37]. The sequences of the 89 candidate BoCaM/BoCML genes were subjected to domain analysis using InterPro, the Conserved Domain Database, Pfam, and SMART, with the EF-hand domain IDs being cd00051, PF00036, IPR002048, and SM00054, respectively [38,39,40,41]. False BoCaM/BoCML gene candidates were filtered out based on conserved domains and the number and characteristics of EF-hand motifs, leading to the identification of 14 BoCaM genes and 75 BoCML genes. Based on the homologous sequences of these genes in Arabidopsis, the genes were designated sequentially as BoCaM1-1 to BoCML50-2.

4.2. Prediction of Basic Infomation of BoCaM/BoCML Genes

To obtain information on the amino acid count, molecular weight (MW), theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY) for the BoCaM and BoCML proteins, their sequences were submitted to ProtParam [42]. Each predicted BoCaM/BoCML genes was analyzed for EF-hand motifs using the InterPro tool (https://www.ebi.ac.uk/interpro/, accessed on 6 July 2024), and the GSDS 2.0 (Gene Structure Display Server; https://gsds.gao-lab.org/Gsds_help.php, accessed on 10 October 2024.) tool was used to analyze the exons and introns of the genes [39]. The PlantCARE analysis (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 6 July 2024) was performed with default parameters, focusing on core promoter elements and stress-responsive motifs (Supplementary Table S4).The BUSCA (Bologna Unified subcellular Component Annotator) tool was used to predict the subcellular localization of each BoCaM/BoCML gene, with the taxonomic origin set to eukaryotic plants [43].

4.3. Phylogenetic Analysis

Phylogenetic analysis of BoCaM/BoCML genes was conducted using MEGA 7.0 software. Multiple sequence alignment was performed with ClustalW using default parameters, and a neighbor-joining phylogenetic tree was constructed based on p-distance with bootstrap values of 1000 replicates [44]. The protein sequences of AtCaM/AtCML genes in Arabidopsis and OsCaM/OsCML genes in Oryza sativa L. were obtained from the Arabidopsis Genome Database (The Arabidopsis Information Resource, TAIR; https://www.arabidopsis.org/, accessed on 6 July 2024) and the rice genome database (The Institute for Genomic Research, TIGR; http://rice.plantbiology.msu.edu/, accessed on 6 July 2024) according to precious research [15,34]. Phylogenetic trees for BoCaM/BoCML genes, AtCaM/AtCML genes, and OsCaM/OsCML genes were constructed using the same methods as described above.

4.4. Chromosome Location and Collinearity Analysis

The genome GFF3 file and the BoCaM/BoCML gene ID file, both downloaded from the Brassica oleracea genome JZS 2.0 (BRAD: http://brassicadb.cn/#/, accessed on 6 July 2024), were submitted to the functional module “Gene Location Visualize from GTF/GFF” in TBtools (v2.210) to map the chromosome localization of BoCaM/BoCML genes. The genome FASTA files for B. oleracea and Arabidopsis were submitted to the functional module “Fasta Stats” of TBtools to obtain the respective genome annotation files. Subsequently, the genome annotation files and FASTA files of B. oleracea and Arabidopsis were submitted to the functional module “One Step MCScanX-Super Fast” for collinearity analysis [45].

4.5. Expression Analysis of BoCaM/BoCML Genes Using RNA-Seq Data

To analyze the tissue-specific expression patterns of BoCaM/BoCML genes, RNA-seq data from various tissues, including leaves, stems, flowers, siliques, buds, calli, and roots, were downloaded from the NCBI database (GSM1052958-964). To analyze the expression patterns of BoCaM/BoCML genes in response to H. parasitica infection, leaves were collected from both resistant line 20-2221 and susceptible line 20-2229 at two time points: before inoculation and four days post-inoculation. Transcriptome sequencing was performed on these samples, and the RNA-seq data have been uploaded to NCBI (BioProject number: PRJNA1146208). Gene expression levels were calculated using FPKM values, and the FPKM algorithm was employed to normalize the gene expression data. Heat maps of hierarchical clustering were constructed using TBtools software [45].

4.6. Plant Materials and Treatments

H.parasitica used in this research was propagated and preserved by our laboratory for many years. Before inoculation, spores were harvested from the susceptible plant variety 20-2229, and an inoculum of 1 × 105 spores/mL was prepared. The seeds of resistant variety 20-2221 and susceptible variety 20-2229 were sown in seedling trays, each measuring 10 cm by 10 cm, and filled with a sterilized substrate conducive to germination. H. parasitica inoculation was carried out using a sprayer when the seedlings had developed two true leaves. At this stage, the prepared spore suspension was evenly applied to the underside of the leaves. Each treatment included 30 seedlings, with 10 seedlings constituting one biological replicate. After inoculation, the plants were subjected to a 24 h dark incubation period. Subsequently, the plants were transferred to a greenhouse for routine cultivation under conditions of 16 h of light and 8 h of darkness at a temperature of 23–25 °C. Six days later, the plants underwent another 24 h dark incubation, with the relative humidity maintained at 95%.

4.7. Total RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis

Total RNA was extracted from cabbage samples using the TIANGEN RNAprep Pure Plant Kit according to the supplier’s instructions (Transgen, Beijing, China). Then, the RNA purity and quality were assessed using a spectrophotometer (BioDrop, Cambridge, UK) and 1% formaldehyde gel electrophoresis. First-strand cDNA was synthesized using the FastKing RT Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. Specific primers for BoCaM/BoCML genes were designed using Premier 3.0 (Supplementary Table S3). qRT–PCR was performed using a TransStart Top Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China) on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). Three technical and biological replicates were conducted for each reaction.

5. Conclusions

In this study, 14 BoCaM and 75 BoCML genes were identified in the B. oleracea genome and classified into six subgroups (Groups I–VI). Through bioinformatics and qRT-PCR analyses, the gene structures, phylogenetic relationships, chromosomal locations, gene duplications, and expression patterns of BoCaM/BoCML genes were examined. Eleven BoCaM/BoCML genes were down-regulated in response to H. parasitica infection, with BoCML46-2 exhibiting the most significant decrease, nearly fivefold, in both resistant and susceptible varieties, suggesting that BoCML46-2 may play a crucial role in the response to H. parasitica. This study provides comprehensive genome-wide information on BoCaM/BoCML genes in cabbage, which will facilitate the identification of their roles in cabbage growth, development, and stress responses.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26073208/s1.

Author Contributions

Y.Z. conceived and designed the experiments; Y.W. (Yuankang Wu) and B.Z. performed the experiments and analyzed the data; Y.W. (Yuankang Wu), X.Y. and Y.Z. wrote and revised the paper; and L.Y., M.Z., H.L., Y.W. (Yong Wang), J.J. and X.H. coordinated and designed the study. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from Beijing Natural Science Foundation (6232037); the earmarked fund for the Modern Agro-Industry Technology Research System, China (CARS23); Chongqing-Sichuan Science and Technology Innovation Cooperation Plan (CSTB2024TIAD-CYKJCXX0022); the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS); and Chongqing-Sichuan Science and Technology Innovation Cooperation Plan (CSTB2024TIAD-CYKJCXX0022). The work was performed in the State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China. The funder was not involved in the design, data analysis, or writing associated with the study.

Institutional Review Board Statement

All the plant materials are from the Institute of Vegetables and Flowers, Chinese Academy of Agriculture Sciences (IVFCAAS, Beijing, China). The utilization of these plant materials in this study complies with the guidelines and legislation of China.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supplementary Information Files. The raw sequencing data used during this study are available in the NCBI SRA database (BioProject number: PRJNA1146208). The B. oleracea reference genome ‘Braol JZS V2.0’ used in this study can be found at the link http://brassicadb.cn/#/, accessed on 10 July 2024. The A. thaliana genome can be found at the link https://www.arabidopsis.org/index.jsp, accessed on 6 July 2024. The protein database of National Center for Biotechnology Information (NCBI) can be found at the link https://www.ncbi.nlm.nih.gov/, accessed on 6 July 2024. All these databases are open to public access.

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. Characterization of the identified BoCaM/BoCML genes in B. oleracea. (A) Phylogenetic relationships and classification of BoCaM/BoCML proteins. (B) Distribution of conserved EF-hand motifs among the BoCaM/BoCML proteins. (C) Exon–intron structure of BoCaM/BoCML genes.
Figure 1. Characterization of the identified BoCaM/BoCML genes in B. oleracea. (A) Phylogenetic relationships and classification of BoCaM/BoCML proteins. (B) Distribution of conserved EF-hand motifs among the BoCaM/BoCML proteins. (C) Exon–intron structure of BoCaM/BoCML genes.
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Figure 2. Phylogenetic tree of CaM/CML proteins in B. oleracea, Arabidopsis, and rice constructed using the neighbor-joining method.
Figure 2. Phylogenetic tree of CaM/CML proteins in B. oleracea, Arabidopsis, and rice constructed using the neighbor-joining method.
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Figure 3. Chromosome distribution of BoCaM/BoCML genes in B. oleracea.
Figure 3. Chromosome distribution of BoCaM/BoCML genes in B. oleracea.
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Figure 4. Collinearity analysis of CaM/CML genes in Arabidopsis and B. oleracea.
Figure 4. Collinearity analysis of CaM/CML genes in Arabidopsis and B. oleracea.
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Figure 5. Differential expression patterns of BoCaM/BoCML genes in leaf, stem, flower, silique, bud, callus, and root of B. oleracea.
Figure 5. Differential expression patterns of BoCaM/BoCML genes in leaf, stem, flower, silique, bud, callus, and root of B. oleracea.
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Figure 6. Phenotypes, heatmap, and RT-qPCR analysis related to the response of BoCaM/BoCML genes to H. parasitica infection. (A) Phenotypes of two cabbage cultivars before and four days after H. parasitica infection. The red arrows indicate the downy mildew-infected lesions (caused by Peronosporaceae) in the 20-2229 experimental material. (B) Heatmap of BoCaM/BoCML gene expression before and four days after H. parasitica infection. (C) qRT-PCR expression patterns of BoCaM/BoCML genes in B. oleracea before and four days after H. parasitica infection. Stars above the bars indicate significant differences among treatments. “ns” denotes no significant difference. Two stars (**) indicate a significant level (p < 0.01), and three stars (***) denote a highly significant level (p < 0.001).
Figure 6. Phenotypes, heatmap, and RT-qPCR analysis related to the response of BoCaM/BoCML genes to H. parasitica infection. (A) Phenotypes of two cabbage cultivars before and four days after H. parasitica infection. The red arrows indicate the downy mildew-infected lesions (caused by Peronosporaceae) in the 20-2229 experimental material. (B) Heatmap of BoCaM/BoCML gene expression before and four days after H. parasitica infection. (C) qRT-PCR expression patterns of BoCaM/BoCML genes in B. oleracea before and four days after H. parasitica infection. Stars above the bars indicate significant differences among treatments. “ns” denotes no significant difference. Two stars (**) indicate a significant level (p < 0.01), and three stars (***) denote a highly significant level (p < 0.001).
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Wu, Y.; Zhang, B.; Yao, X.; Yang, L.; Zhuang, M.; Lv, H.; Wang, Y.; Ji, J.; Hou, X.; Zhang, Y. Genome-Wide Characterization of CaM/CML Gene Family in Cabbage (Brassica oleracea var. capitata): Expression Profiling and Functional Implications During Hyaloperonospora parasitica Infection. Int. J. Mol. Sci. 2025, 26, 3208. https://doi.org/10.3390/ijms26073208

AMA Style

Wu Y, Zhang B, Yao X, Yang L, Zhuang M, Lv H, Wang Y, Ji J, Hou X, Zhang Y. Genome-Wide Characterization of CaM/CML Gene Family in Cabbage (Brassica oleracea var. capitata): Expression Profiling and Functional Implications During Hyaloperonospora parasitica Infection. International Journal of Molecular Sciences. 2025; 26(7):3208. https://doi.org/10.3390/ijms26073208

Chicago/Turabian Style

Wu, Yuankang, Bin Zhang, Xuehui Yao, Limei Yang, Mu Zhuang, Honghao Lv, Yong Wang, Jialei Ji, Xilin Hou, and Yangyong Zhang. 2025. "Genome-Wide Characterization of CaM/CML Gene Family in Cabbage (Brassica oleracea var. capitata): Expression Profiling and Functional Implications During Hyaloperonospora parasitica Infection" International Journal of Molecular Sciences 26, no. 7: 3208. https://doi.org/10.3390/ijms26073208

APA Style

Wu, Y., Zhang, B., Yao, X., Yang, L., Zhuang, M., Lv, H., Wang, Y., Ji, J., Hou, X., & Zhang, Y. (2025). Genome-Wide Characterization of CaM/CML Gene Family in Cabbage (Brassica oleracea var. capitata): Expression Profiling and Functional Implications During Hyaloperonospora parasitica Infection. International Journal of Molecular Sciences, 26(7), 3208. https://doi.org/10.3390/ijms26073208

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