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Article

A Genome-Wide Analysis of the VuR2R3-MYB Gene Family in Cowpea and Its Expression in Anthocyanin Accumulation

by
Yi Yang
1,2,†,
Canye Yu
3,†,
Xuan Zhou
1,2,
Zengxiang Wu
1,2,
Zhuo Shen
1,2,
Tinyao Li
1,2,* and
Yan Zhang
1,2,*
1
Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
2
Guangdong Key Laboratory for New Technology Research of Vegetables, Guangzhou 510640, China
3
Jieyang Institute of Agricultural Sciences, Jieyang 515500, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(5), 1075; https://doi.org/10.3390/agronomy15051075
Submission received: 25 March 2025 / Revised: 23 April 2025 / Accepted: 25 April 2025 / Published: 28 April 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

:
Purple cowpea accumulates abundant anthocyanins in its epidermis, with R2R3-MYB transcription factors serving as potential regulators of anthocyanin accumulation. This study systematically deciphered the genome-wide characteristics of cowpea R2R3-MYB transcription factors, elucidating their critical roles in plant anthocyanin accumulation. Employing a combined strategy of HMMER Hidden Markov Model searches and BLASTP homology alignment, we successfully identified 127 non-redundant VuR2R3-MYB transcription factors. The encoded proteins exhibited remarkable physicochemical diversity: the average length reached 338.8 amino acid residues, with theoretical isoelectric points distributed between 4.79 and 10.91 residues. When performing a phylogenetic analysis with Arabidopsis homologs, 27 distinct subgroups were identified. Among them, the S4–S7 clades showed conserved protein architectures, which might play a role in regulating the phenylpropanoid pathway. An analysis of the gene architecture revealed patterns of intron/exon organization. Specifically, 85 out of 127 loci (66.9%) presented the typical two-intron configuration, whereas 18 genes had no introns. An investigation of the promoters found that, on average, each gene had 52 cis-regulatory elements. These elements were mainly light-responsive motifs and phytohormone-related elements. Chromosomal mapping indicated an uneven distribution of these genes across 11 chromosomes. Duplication analysis further showed 13 tandem repeats and 54 segmentally duplicated pairs. An analysis of evolutionary constraints demonstrated that purifying selection was predominant (Ka/Ks < 0.5) among paralogous pairs. Through comparative transcriptomics of pod color variants, 19 differentially expressed MYB regulators were identified. These included VuR2R3-MYB23 (MYB3 homolog), VuR2R3-MYB95 (MYB4 homolog), VuR2R3-MYB53 (MYB114 homolog), and VuR2R3-MYB92 (MYB5 homolog), which showed a strong correlation with the patterns of anthocyanin accumulation. Our findings are expected to contribute to elucidating the potential regulatory mechanisms through which R2R3-MYB transcription factors mediate anthocyanin biosynthesis and accumulation.

1. Introduction

Cowpea (Vigna unguiculata L. Walp., 2n = 2x = 22), which is endemic to sub-Saharan Africa, is cultivated as a grain, vegetable, and livestock feed worldwide. Cowpea has evolved into two main sub-species. The vegetable type (V. unguiculata L. Walp. ssp. sesquipedalis) is heat-tolerant and drought-resistant, leading to its widespread cultivation in East and Southeast Asia due to its adaptability to warm climates [1,2,3]. This sub-species, commonly known as asparagus bean, yardlong bean, or snake bean, is characterized by exceptionally long pods (reaching 50–100 cm in length) [4]. Grain cowpea (V. unguiculata L. Walp. ssp. unguiculata) is nutrient-rich, offering high-quality protein, vitamins, minerals, and fiber. This makes it valuable for combating hidden hunger and supporting food security [5,6,7].
Seed and pod coloration constitute critical quality traits in cowpea, directly influencing consumer sensory evaluation and purchasing decisions [2,8]. Anthocyanin-mediated pigmentation patterns are frequently associated with predetermined nutritional value and culinary appeal in market selection [9]. Anthocyanins act as antioxidants, mitigating oxidative stress by scavenging ROS and modulating inflammation, thereby reducing chronic disease risks [10,11]. In plants, they enhance drought and UV tolerance through ROS neutralization and photoprotection, preserving cellular integrity under stress [10,12]. This dual role highlights their evolutionary significance in stress-resilient crops.
The R2R3-MYB transcription factors (TFs) constitute one of the largest and most functionally diverse gene families in plants, regulating secondary metabolism [13], developmental processes [14], and abiotic stress responses [15,16,17]. The N-terminal region has two conserved MYB domains (R2 and R3) that bind specific DNA motifs (e.g., MRE, AC elements) in gene promoters. In contrast, the C-terminal domain varies and controls transcriptional activation or repression [18].
The anthocyanin pathway in plants is regulated by the MBW complex (MYB-bHLH-WD40), with R2R3-MYB transcription factors (TFs) as central components that activate key structural genes, such as Chalcone Synthase (CHS), Dihydroflavonol 4-Reductase (DFR), and Anthocyanidin Synthase (ANS) [19,20]. The MBW complex comprises MYB (DNA-binding promoter), bHLH (co-activator), and WD40 (scaffold protein), with its structure being highly conserved [21,22]. The functional preeminence of R2R3-MYB TFs is vividly illustrated by their intricate molecular mechanisms that govern the dynamic buildup of anthocyanins in a tissue-specific and stress-responsive manner.
In legume plants, R2R3-MYB TFs serve a central function in legume plants by interacting with bHLH and WD40 proteins to form MBW complexes, which dynamically control anthocyanin buildup and tissue-specific pigmentation. For instance, in soybean (Glycine max), tandem duplication causes the subfunctionalization of MYB genes. GmMYBA5 drives the delphinidin pathway, while GmMYBA2 and GmMYBA1 boost anthocyanin accumulation by collaborating with bHLH and WD40 proteins, creating detailed pigmentation in seed coats and flowers [23]. Similarly, in alfalfa (Medicago sativa), the MsMYB206-MsMYB450-MsHY5 complex regulates flavonoid biosynthesis through circadian rhythms, enabling responses to abiotic stresses and ROS balance [24]. Moreover, Wu et al. [25] found a MYB113 homolog cluster on chromosome Pv08 in the common bean (Phaseolus vulgaris) through genomic analysis. This cluster regulates the C locus, affecting seed coat patterns, and underscores the importance of MYB TFs in legume evolution and adaptation. Recent studies on related cereals provide further insights; for example, in qingke barley (Hordeum vulgare L. var. nudum Hook. f.), the HvnAnt2 gene, an R2R3-MYB TF, has been shown to regulate anthocyanin accumulation in grains of different colors, highlighting its role in pigment diversity [26]. In sorghum (Sorghum bicolor), transcriptome profiling revealed cultivar-specific MYB signatures associated with phenolic compound biosynthesis, emphasizing the transcriptional regulation of anthocyanin-related pathways [27]. These findings illustrate the conserved yet diversified roles of R2R3-MYB TFs in anthocyanin metabolism across cereals and legumes.
In cowpea, previous studies have identified VuMYB114 (VuR2R3-MYB54) as a key regulator of anthocyanin accumulation in pods, activating genes like VuDFR (Dihydroflavonol Reductase gene) and VuANS (Anthocyanin Synthase gene) [28]. The VuMYB90-1/2/3 gene cluster identified in the purple-podded cultivar ‘China Red Dragon’ can synergistically form the MBW complex with bHLH and WD40 proteins. This complex significantly enhances secondary metabolite diversity and promotes pigment deposition through targeted binding to promoters of anthocyanin/flavonoid biosynthetic genes [29]. Although R2R3-MYB transcription factors have been functionally validated as key regulators of anthocyanin biosynthesis in model crops [30,31,32], cowpea-specific research remains fragmented. Current studies lack genome-wide characterization of the MYB gene family (e.g., subfunctionalization analysis) and multi-omics integration (transcriptome–metabolome networks) [33,34,35,36,37], which are critical for developing molecular strategies to enhance anthocyanin-mediated traits.
Therefore, the objectives of this study are as follows: (1) to systematically identify the R2R3-MYB transcription factor family in cowpea using whole-genome data; (2) to integrate transcriptome and metabolome analyses to reveal the potential regulatory network in anthocyanin accumulation; (3) to identify potential regulatory candidate genes, providing a foundation for subsequent functional validation and breeding applications. This not only fills the gaps in molecular biology research on cowpea but also contributes new insights to enhancing crop adaptability and nutritional value.
Through integrated transcriptomic and metabolomic profiling of three cowpea accessions exhibiting deep green (DGH), light purple (LPH), and purple (PRS) pod phenotypes, we detected 19 differentially expressed VuR2R3-MYB TFs. Among these, VuR2R3-MYB23, VuR2R3-MYB95, VuR2R3-MYB92, and VuR2R3-MYB53 emerged as potential regulatory candidates for anthocyanin biosynthesis. These findings advance the understanding of molecular regulatory networks governed by R2R3-MYB TFs and provide a framework for subsequent functional studies on anthocyanin metabolism in cowpea.

2. Material and Methods

2.1. Genome-Wide Identification of VuR2R3-MYB TFs in Cowpea

In this study, the assembled potential R2R3-MYB family members. Using Arabidopsis R2R3-MYB family as reference, blast alignment analysis (E-value < 1 × 10−10) was performed for further candidate screening [38]. Domain annotation of candidate sequences was conducted via Pfamscan software (v1.6) with the Pfam database [39], retaining sequences containing the PF00249 domain as complete MYB proteins. Finally, the physicochemical properties (amino acid length, molecular weight, isoelectric point) of cowpea R2R3-MYB family members were analyzed using the ProtParam online tool (https://web.expasy.org/protparam/, accessed on 15 June 2024).

2.2. Phylogenetic Analysis and Conservation Analysis of VuR2R3-MYB Proteins

Phylogenetic analysis was conducted using MAFFT v7.427 software (default parameters) to perform multiple sequence alignment of R2R3-MYB protein sequences from cowpea and Arabidopsis. An evolutionary tree was constructed via the Neighbor-Joining method (default parameters) in MEGA 10.0 software [40], with branch reliability assessed through 1000 bootstrap replicates. Final tree visualization and annotation were implemented using the iTOL v6 online tool (https://itol.embl.de/, accessed on 18 June 2024). The sequence logos of the R2 and R3 repeats were generated by submitting the multiple sequences to https://weblogo.berkeley.edu/logo.cgi (accessed on 20 June 2024).

2.3. Analysis of Gene Structure and Promoter Characteristics of VuR2R2-MYBs

Members sharing similar conserved motifs often exhibit analogous functions. To investigate motif conservation and diversity in cowpea R2R3-MYB proteins, conserved motifs were analyzed using MEME v5.0.5 (http://meme-suite.org/, accessed on 18 June 2024) [41], with the maximum motif search set to 15. Gene structure features were resolved via TB-tools software to delineate intron–exon distribution patterns, revealing structural characteristics and evolutionary insights. Additionally, 2 kb upstream regions were extracted as promoter sequences. Transcription factor (TF) binding sites were predicted using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 18 June 2024), with positional annotations of binding sites displayed on promoter physical maps, showing only the top 12 transcription factor families.

2.4. VuR2R3-MYB Physical Localization, Collinearity Analysis, and Ka/Ks Calculation of Duplicated VuR2R3-MYB TFs

Based on cowpea genome annotation data, chromosomal physical mapping of the VuR2R3-MYB TF family was performed using the MG2C tool (http://mg2c.iask.in/mg2c_v2.1/, accessed on 20 June 2024). Intra- and inter-species synteny analyses were conducted with MCScanX v2 software (default parameters), and visualizations were generated via Circos [42] and TBtools-II [43]. To assess evolutionary pressures on gene duplication events, nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and their ratio (Ka/Ks) were calculated for VuR2R3-MYB duplicated gene pairs using KaKs_Calculator2.0 (https://sourceforge.net/projects/kakscalculator2/files/?source=navbar, accessed on 25 June 2024), elucidating evolutionary selection patterns.

2.5. Transcriptome Date Analysis

To elucidate the molecular mechanisms underlying pod coloration in cowpea, this investigation employed three cultivars exhibiting distinct color gradients: dark green (DGH), light purple (LPH), and purple (PRS). Epidermal tissue samples were collected from market-stage pods, with three biological replicates per cultivar. Each replicate comprised three independent pods, which were pooled for analysis following extraction. Total RNA was extracted from the samples using the RNAprep Pure Plant Plus Kit (TIANGEN, Beijing, China). RNA concentration was quantified by a NanoDrop 2000 spectrophotometer, and RNA integrity was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) with LabChip GX microfluidic chips. The obtained RNA met the quality criteria for library construction, with OD₂₆₀/₂₈₀ ratios ranging from 1.7 to 2.5 and RNA Integrity Numbers (RINs) ≥ 6.5. Library preparation and sequencing were conducted by Biomarker Technologies Corporation (Beijing, China). Following the bioinformatics pipeline provided by the BMK Cloud platform (www.biocloud.net), the raw sequencing data underwent quality filtering (removing low-quality reads) to obtain high-quality data, followed by alignment to the cowpea reference genome [22] using HISAT2 [44]. We used StringTie [45] to assemble the aligned reads, and applied the maximum flow algorithm to normalize the data using FPKM (Fragments Per Kilobase of transcript per Million mapped reads) [46] as a metric for measuring transcript or gene expression levels. A threshold of fold change ≥ 2 and FDR < 0.01 was set as the selection criteria, and differential expression analysis was performed using the DESeq2 software (1.30.1) [47].

2.6. Verification of RNA-Seq Data by qRT-PCR

To confirm the reliability of the transcriptome data, quantitative real-time PCR (qRT-PCR) was conducted to validate several candidate genes potentially involved in anthocyanin biosynthesis pathways. Total RNA was isolated from the tissue samples described earlier, and first-strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit with a gDNA Eraser (TRAN®, Beijing, China) to ensure efficient reverse transcription. For the qRT-PCR reactions, a total mixture volume of 10 µL was prepared, including 5 µL of 2x Green qPCR SuperMix (which contains optimized concentrations of Taq polymerase, dNTPs, and reaction buffer, as per the manufacturer’s specifications), 1 µL of cDNA template (derived from the reverse transcription of approximately 1 µg total RNA per reaction), 0.4 µL of forward primer and 0.4 µL of reverse primer (both at a working concentration of 10 µM), and 3.2 µL of nuclease-free water to adjust the volume. The primer sequences were designed using Primer3Plus (https://www.primer3plus.com/, accessed on 16 January 2025) to ensure specificity and efficiency, with VuActin selected as the internal reference gene for normalization (Supplementary Table S7). All the qRT-PCR assays were performed with at least three biological replicates to account for biological variability, and the gene expression data were analyzed using the 2−ΔΔCt method [48].

2.7. Metabolome Detection and Analysis

For metabolomic analysis, the plant cultivars, developmental time points, and sampling protocols were identical to the parameters used in transcriptomic studies. Freeze-dried samples were pulverized using a mixer mill (MM 400, Retsch, Hann, Germany) with zirconium beads at 30 Hz for 1.5 min. A 100 mg aliquot of the powder was weighed and extracted with 0.6 mL of 70% aqueous methanol (v/v) at 4 °C overnight (approximately 16 h). The extract was centrifuged at 10,000× g for 10 min, filtered through a 0.22 μm membrane (SCAA-104), and subjected to UPLC-MS/MS analysis. The sample extract was analyzed using a UPLC-ESI-MS/MS system (UPLC: Shim-pack UFLC SHIMADZU CBM30A; MS: Applied Biosystems 4500 QTRAP) equipped with a Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm). The mobile phase consisted of solvent A (pure water with 0.04% acetic acid) and solvent B (acetonitrile with 0.04% acetic acid). The gradient elution was set as follows, based on optimizations from prior work, to achieve effective metabolite separation: 0–2 min at 5% B, a linear increase to 95% B from 2 to 10 min, a hold at 95% B from 10 to 12 min, a rapid return to 5% B between 12 and 12.1 min, and equilibration at 5% B until 15 min. The column oven was maintained at 40 °C, and the injection volume was 4 μL. The effluent was connected to an ESI-triple quadrupole-linear ion trap (QTRAP) mass spectrometer, operating in both positive and negative ion modes to capture diverse metabolite ions. The mass spectrometry parameters included an ion spray voltage of 5500 V, electrospray ionization temperature of 55 °C, and curtain gas pressure of 25.0 psi [49]. For targeted quantification, MRM (multiple reaction monitoring) mode was used, with ion pairs selected based on metabolite-specific m/z values and fragment ions derived from databases such as HMDB and previous studies, ensuring high sensitivity and specificity through pre-acquisition calibration. Data processing was performed using Analyst 1.6.3 software (AB Sciex). Significantly regulated metabolites between groups were identified based on VIP ≥ 1 and absolute Log2FC (fold change) ≥ 1. VIP values were extracted from the OPLS-DA results, which included score plots and permutation plots generated using the R package MetaboAnalystR v2.0 [50].

3. Results

3.1. Identification and Characteristics of R2R3-MYB TF Family Members in Cowpea

To identify members of the R2R3-MYB TFs family in cowpea, we constructed a Hidden Markov Model (HMM) using protein sequences of the Arabidopsis thaliana R2R3-MYB family. This model was then employed to screen the coding protein sequences of cowpea. Candidate TFs were further refined by aligning them with Arabidopsis R2R3-MYB reference sequences via BLAST (v2.10.1+), followed by the validation of structural domains using the Pfam database to remove any incomplete sequences. A total of 127 cowpea R2R3-MYB TFs were identified and designated as VuR2R3-MYB1 to VuR2R3-MYB127 based on their chromosomal locations (Supplementary Table S1).
To further characterize the R2R3-MYB proteins, we analyzed their physicochemical properties including protein length, molecular weight, and isoelectric point (pI) (Supplementary Table S1). Our analysis revealed that these proteins had an average length of 338.8 amino acids. For instance, VuR2R3-MYB49 was the shortest, with 166 amino acids and a molecular weight of 18.78 kDa, while VuR2R3-MYB50 represented the longest protein (969 amino acids) with the largest molecular weight (109.16 kDa). The predicted pI values ranged from 4.79 to 10.91, with 59.8% of the proteins showing acidic isoelectric points (pH < 7) and 40.2% displaying basic isoelectric points (pH > 7).

3.2. Phylogenetic Analysis of VuR2R3-MYB TFs in Cowpea

To examine the phylogenetic relationships among the VuR2R3-MYB transcription factors (TFs), we constructed a Neighbor-Joining tree using MEGA software(v10), which included 127 VuR2R3-MYB sequences from cowpea and 125 AtR2R3-MYB sequences from Arabidopsis. Based on established classification criteria for plant R2R3-MYB subfamilies [18], the 127 VuR2R3-MYB TFs were categorized into 27 distinct subfamilies, as illustrated in Figure 1. The tree revealed clear clustering patterns, with many cowpea TFs grouping closely with their Arabidopsis counterparts, indicating potential evolutionary conservation. For example, the S4 subfamily in cowpea showed high sequence similarity to Arabidopsis S4 members, which have been associated with phenylpropanoid metabolism in previous studies. Similarly, the S5, S6, and S7 subfamilies displayed comparable clustering, aligning with Arabidopsis genes linked to anthocyanin and proanthocyanidin pathways [51]. Consequently, we hypothesize that the 16 members of the cowpea S4, S5, S6, and S7 subfamilies are candidate regulators involved in anthocyanin synthesis and accumulation. Notably, a new cluster was identified in the phylogenetic tree, which we designated as N1. It appears to represent a group of unique transcription factors. This clustering suggests that subfamily members may share structural similarities, potentially reflecting conserved functional roles in biological processes.

3.3. Analysis of Gene Structure, Conserved Motifs, and Protein Sequence Conservation in VuR2R3-MYB TFs

The VuR2R3-MYB TFs in cowpea exhibited continuous intron number variation, ranging from 0 to 15 (mean of 2.56), with 87 TFs (66.9%) demonstrating a conserved two-intron/three-exon structural configuration. Notably, VuR2R3-MYB88/89 (S22 subfamily) displayed intron loss characteristics, while the N1 subfamily contained the maximum intron–exon numbers (Supplementary Table S2). Using the MEME suite, we predicted 15 conserved motifs among the 127 VuR2R3-MYB proteins. These motifs varied greatly in length, with Motif 9 being the shortest (6 amino acid residues) and Motifs 1/5 the longest (50 residues each). Most TFs (87, ~68.5%) demonstrated the core motif combination (Motifs 1–3), except those in the N1 and S27 subfamilies. Motifs 5/7 were exclusively present in the N1 subfamily and Motif 11 was specifically distributed in S27, while Motif 10 served as a signature element for the S9 subfamily. Phylogenetic clustering analysis revealed members with analogous gene structures and motif compositions grouped into distinct evolutionary clades (Figure 2), suggesting functional conservation within clusters. For instance, Motif 12 in the S9 subfamily may regulate epidermal cell growth, whereas the S1-specific Motif 15 is potentially associated with abiotic stress responses.
Proteins within the R2R3-MYB subfamily have high sequence similarity in the DNA-binding domain (DBD), in contrast to the substantial variability observed in amino acid length and composition outside this region [51]. To assess the conservation at each residue position within the R2 and R3 repeat sequences of the R2R3-MYB proteins, we conducted a detailed sequence analysis in cowpea (Supplementary Figure S1A). Our findings reveal that these two MYB repeats encompass approximately 106 amino acid residues, inclusive of linker regions, with only minor instances of deletions or insertions. The R2 and R3 repeats of the MYB domain exhibit distinct patterns of characteristic amino acids, yet they remain highly conserved across species such as pea and Arabidopsis. Specifically, the R2 repeat contains three regularly spaced tryptophan (W) residues (depicted in Supplementary Figure S1A), which serve to interconnect the three helices. In the R3 repeat, two highly conserved W residues correspond to the second and third positions in R2, while the first W residue is often substituted by phenylalanine (F), isoleucine (I), tryptophan (W), or leucine (L) (Supplementary Figure S1B). Overall, conserved residues are less prevalent in helix1 and helix2 of the repeat sequences, whereas helix3 shows greater conservation, owing to its critical function in DNA recognition, underscoring the pronounced evolutionary stability of the R2R3-MYB DBD in cowpea.

3.4. Cis-Acting Element Analysis of VuR2R3-MYB TF Promoters in Cowpea

Cis-regulatory elements play pivotal roles in transcriptional regulation and initiation. Within the 2000 bp promoter region upstream of the VuR2R3-MYB TF initiation codons, we identified 35 cis-acting elements categorized into four functional groups: phytohormone-responsive elements, environmental/stress-responsive elements, light-responsive elements, and growth/development-associated elements. The light-responsive group comprised 16 types, including Box 4, G-box, and GT1-motif. The hormone-responsive elements (nine types) encompassed signaling pathways for abscisic acid (ABRE), methyl jasmonate (CGTCA-motif), salicylic acid (TCA-element), gibberellin (GARE-motif, P-box), and auxin (AuxRR-core, TATC-box). Additionally, five growth-related elements and five biotic/abiotic stress-responsive elements were detected (Figure 3). These findings suggest that VuR2R3-MYB TF expression may be modulated by a range of mechanisms involving hormonal crosstalk during cowpea growth, the activation of light-responsive elements, and the mediation of defense signaling pathways. This sophisticated regulatory network enables dynamic adaptation to environmental fluctuations and internal physiological changes, ensuring proper growth and development (Supplementary Table S3).

3.5. Chromosomal Distribution, Tandem and Segmental Duplications Analysis, and Ka/Ks Analysis of VuR2R3-MYB TFs

Chromosomal localization analysis showed an uneven distribution of the 127 VuR2R3-MYB TFs across 11 chromosomes. This distribution pattern likely reflects functional diversification within the TF family and evolutionary trajectory diversity. For instance, chromosomes 6 and 2 had the lowest gene densities, indicating constrained expansion potential, whereas chromosome 3 showed high-density clustering (23 TFs), suggesting its central role in maintaining TF family diversity and functional complexity (Figure 4).
Tandem duplication and segmental duplication were identified as primary drivers of TF family expansion. We detected 13 tandemly duplicated VuR2R3-MYB TF pairs, demonstrating localized genomic replication as a foundation for family expansion. Furthermore, the in-depth analysis of cowpea genome data uncovered 54 syntenic TF pairs (Figure 5). These syntenic relationships reflect conserved evolutionary features and provide critical insights into the functional diversification and complexity of the VuR2R3-MYB family.
The Ka/Ks analysis of 62 syntenic VuR2R3-MYB TF pairs showed them all to have Ka/Ks ratios below 0.5, indicating predominant purifying selection during evolution (Supplementary Table S4).

3.6. Evolutionary Analysis of R2R3-MYB TFs Across Multiple Species

To elucidate the evolutionary characteristics of the cowpea VuR2R3-MYB TFs, we conducted cross-species comparative genomic analyses encompassing cowpea, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), peanut (Arachis hypogaea), and soybean (Glycine max). The analysis revealed significant orthologous relationships between cowpea and legume species: ortholog numbers with soybean (112 pairs) and peanut (95 pairs) substantially exceeded those with the non-legume species Arabidopsis (68 pairs) and rice (33 pairs) (Figure 6). This distribution pattern demonstrates the remarkable evolutionary conservation of R2R3-MYB TF families in legumes, particularly within the cowpea–soybean–peanut system, suggesting that legume-specific TF retention mechanisms critically shaped family evolution.

3.7. Integrated Transcriptomic and Metabolomic Analysis with Anthocyanin Biosynthesis Candidate TF Screening

The targeted metabolomic profiling of three cowpea accessions with different pod phenotypes—deep green (DGH), light purple (LPH), and purple (PRS)—(Figure 7A) identified 465 metabolites (Supplementary Table S5). Among these, differential metabolites were primarily enriched in the flavonoid and anthocyanin biosynthesis pathways (Figure 7B). Six anthocyanins and their precursors were detected, including anthocyanins such as pelargonidin, cyanidin 3-O-arabinose, pelargonidin 3-glucoside, pelargonidin 3-O-galactoside, delphinidin 3-O-glucoside (mirtillin), and cyanidin O-syringic acid, as well as precursors like phenylalanine, coumaroyl-CoA, and naringenin. These compounds showed notable accumulation differences across the pod phenotypes (Supplementary Table S5).
The transcriptomic sequencing of the three pod phenotypes yielded 64.77 Gb of clean data post-quality control, with individual samples containing ≥6.26 Gb and >93.83% Q30 bases. The clean reads from the biological replicates achieved 95.25–96.81% alignment efficiencies to the reference genome. Under stringent thresholds (|FC| ≥ 2 and FDR < 0.01), 19 differentially expressed VuR2R3-MYB TFs were identified (Figure 7C, Supplementary Table S6), suggesting their potential regulatory roles in anthocyanin biosynthesis.
Based on functional studies in Arabidopsis, R2R3-MYB TFs in the S4–S7 subfamilies are known to influence anthocyanin biosynthesis. Using homology comparisons and expression data, we identified the following VuR2R3-MYB TFs as potential candidates for regulating anthocyanin biosynthesis in cowpea: VuR2R3-MYB23 (MYB3 homolog), VuR2R3-MYB95 (MYB4 homolog), VuR2R3-MYB53 (MYB114 homolog), and VuR2R3-MYB92 (MYB5 homolog) (Figure 7D).

3.8. Expression Analysis of VuR2R3-MYBs Through qRT-PCR

To validate the transcriptomic findings and further explore the potential regulatory roles of candidate R2R3-MYB transcription factors in anthocyanin biosynthesis, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) to assess the expression of four selected genes—VuR2R3-MYB53, VuR2R3-MYB92, VuR2R3-MYB23, and VuR2R3-MYB95—across three pod phenotypes (deep green DGH, light purple LPH, and purple PRS). The qRT-PCR results (Figure 8, Supplementary Table S7) revealed clear expression patterns that were consistent with the transcriptomic data (Supplementary Table S6). For example, the expression of VuR2R3-MYB53 was verified to be significantly higher in PRS compared to LPH and DGH, as confirmed by Duncan post hoc tests. This observed pattern correlated with the higher accumulation of anthocyanins, such as cyanidin-O-syringic acid, in PRS, as detected through metabolomic analysis. Similarly, qRT-PCR confirmed that VuR2R3-MYB23 was upregulated in PRS and LPH but was nearly absent in DGH, with significant differences between groups. In contrast, VuR2R3-MYB95 and VuR2R3-MYB92 exhibited moderate expression in LPH and PRS but lower levels in DGH, which may suggest their involvement in precursor allocation rather than direct anthocyanin synthesis, though this remains a hypothesis requiring further investigation. By integrating these qRT-PCR data with metabolomic profiles and transcriptomic FPKM values, we strengthened the evidence for gene–metabolite correlations; for instance, the elevated expression of VuR2R3-MYB53 in PRS was associated with increased anthocyanin concentrations, potentially indicating its role as a positive regulator. Overall, the qRT-PCR validation confirmed the reliability of the RNA sequencing data and provided greater resolution for identifying potential regulatory factors in the anthocyanin biosynthesis pathway.

4. Discussion

This genome-wide identification and expression analysis of the R2R3-MYB TF family in cowpea provides critical insights into the molecular mechanisms potentially associated with anthocyanin biosynthesis and pod pigmentation. By integrating gene family evolution, structural diversity, and transcriptomic dynamics, this study elucidates the regulatory landscape of R2R3-MYBs in cowpea and their possible connections to secondary metabolism and abiotic stress adaptation.
The identification of the VuR2R3-MYB TF family in cowpea, encompassing 127 members, highlights the expansion patterns typical of legume plants, mirroring those seen in soybean (Glycine max, 244 R2R3-MYBs) [53] and peanut (Arachis hypogaea, 196 R2R3-MYBs) [54]. Our analysis of chromosomal distribution in cowpea showed that the dense clustering of 23 transcription factors on chromosome 3 might reflect adaptive evolutionary processes specific to this species. For comparison, studies in other herbaceous crops, such as wheat (Triticum aestivum), have demonstrated that tandem duplication events can improve abiotic stress adaptability [55]. In soybean (Glycine max), similar events have led to the subfunctionalization of genes like GmMYBA5, GmMYBA2, and GmMYBA1, where GmMYBA5 mainly influences the delphinidin pathway, while GmMYBA2 and GmMYBA1 promote anthocyanin accumulation. Working with bHLH and WD40 co-factors, these genes help create complex pigment patterns in seed coats and floral tissues. This evidence suggests that comparable evolutionary mechanisms could be operating in cowpea’s MYB gene cluster, as shown by the 13 pairs of tandem duplications and 54 pairs of segmental duplications in Figure 5. Such processes may have supported the development of abiotic stress tolerance and the specialization of MYB genes. The Ka/Ks analysis of collinear gene pairs revealed strong purifying selection (Ka/Ks < 0.5), indicating evolutionary constraints on functionally critical residues. This aligns with findings in Arabidopsis, where anthocyanin-related MYBs (e.g., PAP1/MYB75) are under intense selective pressure to maintain regulatory specificity [56].
Our phylogenetic clustering into 27 subfamilies (Figure 1) mirrors the classification in Arabidopsis [3,51], with the S4, S5, S6, and S7 subfamilies implicated in phenylpropanoid metabolism. These subfamilies include homologs of AtMYB3/4/7/32 [57,58,59], which are known as repressors or activators of lignin and flavonoid biosynthesis, and this similarity points to their potentially conserved role in cowpea. Gene structure analysis revealed striking diversity through the presence of intronless TFs (S22 subfamily) versus intron-rich members (N1 subfamily). Intron loss in MYBs, as seen in the S22 subfamily, is often associated with transcriptional efficiency and stress-responsive regulation [60,61], while intron retention (e.g., N1 subfamily) may facilitate alternative splicing for functional plasticity [62]. MEME-based motif prediction identified 15 conserved motifs, with Motifs 1–3 being ubiquitous across subfamilies, while Motif 11 (S27-specific) and Motif 10 (S9-specific) suggest subfunctionalization. Similar motif distributions have been observed in tomato (Solanum lycopersicum) MYBs, where lineage-specific motifs regulate fruit ripening [63].
Promoter analysis revealed 35 cis-elements, including light-responsive (G-box, GT1-motif) and hormone-related motifs (ABRE, CGTCA-motif), suggesting the integration of environmental and hormonal signals. For instance, strigolactones, as emerging phytohormones, have been shown to enhance abiotic stress tolerance by modulating antioxidant defenses and interacting with pathways like anthocyanin biosynthesis [64]. This regulatory architecture likely enables cowpea to fine-tune anthocyanin synthesis under stresses like drought or UV radiation.
Through comparative transcriptomic analysis of three cowpea pod color phenotypes (green, DGH; light purple, LPH; and deep purple, PRS), we found 19 R2R3-MYB transcription factors that were significantly differentially expressed (|log2FC| ≥ 1, FDR < 0.01). When combined with metabolomic data, this helped us to identify four candidate R2R3-MYB genes that might be linked to anthocyanin biosynthesis, based on their expression patterns at a single time point. We also used qRT-PCR to confirm the transcriptome results for these candidates. In contrast, VuR2R3-MYB23 (S4 subfamily), despite belonging to the repressive MYB3 clade, showed significant upregulation in PRS, potentially indicating negative feedback regulation under high anthocyanin flux. A similar mechanism has been validated in Arabidopsis MYBL2 studies, where MYBL2 inhibits anthocyanin biosynthesis gene expression by interacting with bHLH to interfere with MBW complex assembly [65]. Notably, VuR2R3-MYB95 (S4 subfamily) was entirely absent in DGH and displayed moderate expression in LPH/PRS. As an ortholog of AtMYB4—a known suppressor of phenylpropanoid pathway genes such as C4H—its expression pattern suggests evolutionary adaptation to balance precursor allocation between anthocyanin synthesis and competing metabolic pathways [66]. VuR2R3-MYB92 (S7 subfamily) exhibited low expression in DGH and moderate expression in PRS and LPH. Its Arabidopsis homolog, AtMYB5, has not been reported to regulate anthocyanin biosynthesis. Three other S7 subfamily members (MYB11, MYB12, and MYB111) were identified as co-activators, initiating diverse branches of flavonoid biosynthesis [67]. These proteins form MYB-bHLH-WD40 (MBW) complexes with TT8 and TTG1, directly activating the flavonoid biosynthetic pathway [66]. In strawberry (Fragaria × ananassa), FaMYB5 has been reported to positively regulate anthocyanin and proanthocyanidin accumulation through the trans-activation of Flavanone 3′-hydroxylase (F3′H) and Leucoanthocyanidin Reductase (LAR) [68]. Similarly, NnMYB5 in lotus (Nelumbo nucifera) participates in petal anthocyanin accumulation [69]. The S6 subfamily member VuR2R3-MYB53 exhibited PRS-specific high expression, demonstrating a significant positive correlation (r = 0.92, p < 0.05) with cyanidin O-syringic acid accumulation. The ortholog gene AtMYB114 regulates anthocyanin biosynthesis by forming an MBW complex with the bHLH cofactor TT8 to directly activate structural genes such as DFR and ANS [51]. In pear, PyMYB114 has also been reported to participate in anthocyanin biosynthesis metabolism [70,71].
In our earlier investigations, we successfully introduced the coding sequence (CDS) of VuMYB114(VuR2R3-MYB54) from cowpea into the model plant Arabidopsis thaliana, generating transgenic lines that displayed a range of purple phenotypes [28]. This approach conclusively established VuMYB114 as a central regulatory transcription factor driving anthocyanin accumulation in pod tissues, while also illuminating the conserved mechanisms of VuR2R3-MYB transcription factors in anthocyanin biosynthesis across cowpea, but this validation is limited to heterologous systems. These findings provide robust empirical support for unraveling the molecular underpinnings of pigment regulation in legume crops more broadly. However, a key limitation of the current study is the absence of temporal expression data, which is essential for establishing whether these genes act before anthocyanin accumulation. Without time-course analysis, we cannot determine the sequence of events, and, thus, interpretations should be cautious and focused on associations rather than causation. Given the underdeveloped transgenic systems in cowpea itself, the functional validation of candidate genes identified in our screening efforts continues to face significant constraints. Looking ahead, it will be imperative to pursue more targeted functional assays directly in cowpea, such as CRISPR/Cas9-based gene editing or overexpression experiments, to gain a deeper understanding of the intricate details within these potential regulatory networks and ultimately close the gaps present in current research.

5. Conclusions

This study presents the first genome-wide identification of the R2R3-MYB TF family in cowpea, revealing 127 members with marked structural heterogeneity, evolutionary diversity, and regulatory complexity. Phylogenetic analysis classified them into 27 subfamilies, among which the S4–S7 clades exhibited high conservation and associations with phenylpropanoid and anthocyanin metabolic pathways. Gene structure variations (0–15 introns) and lineage-specific motifs (e.g., Motif 10 in the S9 subfamily) collectively elucidate functional divergence mechanisms, while purifying selection (Ka/Ks < 0.5) underscores the functional constraints during family evolution. Promoter cis-element analysis identified synergistic interactions among light-responsive, hormone-regulatory, and stress-responsive modules, indicating a multidimensional integrated regulatory network. Chromosomal expansion analysis demonstrated that tandem (18 pairs) and segmental duplications (6 pairs) drove family diversification, with chromosome 3 emerging as an evolutionary hotspot, containing 23 high-density TFs. Transcriptomic profiling revealed 19 differentially expressed VuR2R3-MYB TFs significantly correlated with pod pigmentation phenotypes. By combining metabolomic profiling and qRT-PCR validation, we pinpointed four candidate transcription factors—VuR2R3-MYB53, VuR2R3-MYB23, VuR2R3-MYB92, and VuR2R3-MYB95—as potentially linked to anthocyanin biosynthesis processes, based primarily on observed expression correlations at a single time point. This integrated classification and transcriptional–metabolic approach establishes a solid foundation for future research to explore the molecular mechanisms governing anthocyanin synthesis in cowpea.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15051075/s1, Figure S1. The sequence logo diagram of the conserved R2 and R3 repeat sequences in VuR2R3-MYB. Table S1. Information of 127 VuR2R3MYB family proteins in cowpea. Table S2. Genomic architecture of the VuR2R3-MYB transcription factors. Table S3. Promoter cis-acting elements of the VuR2R3-MYB gene. Table S4. Ka, Ks, and Ka/Ks values for the deplication gene pairs from cowpea. Table S5. Anthocyanin and proanthocyanidin content, fold changes, and significant differences. Table S6. Transcriptomic data of anthocyanin biosynthetic pathway structural genes. Table S7. Primers utilized for qRT-PCR.

Author Contributions

Conceptualization, Data Curation, Formal Analysis: Y.Y. and C.Y.; Formal Analysis and Validation: X.Z.; Formal Analysis and Validation: Z.W.; Planting Management and Investigation: Z.S.; Funding Acquisition and Project Administration: Y.Y., T.L., and Y.Z.; Resources: Y.Y. and T.L.; Writing—Original Draft: C.Y.; Writing—Review and Editing: Y.Y., C.Y., X.Z., Z.W., Z.S., T.L., and Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Key Realm R&D Program of Guangdong Province (Grant No. 2020B020220002), the 2023 Special Topic on Basic and Applied Basic Research of Guangzhou Science and Technology Bureau (2023A04J0820) and Innovation Fund Project of Guangdong Academy of Agricultural Sciences (202207).

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic analysis of R2R3-MYB proteins. Arabidopsis proteins (125) marked with red circles and cowpea proteins (127) with green squares. Total of 252 R2R3-MYB members classified into 32 evolutionary subfamilies (indicated by distinct color blocks).
Figure 1. Phylogenetic analysis of R2R3-MYB proteins. Arabidopsis proteins (125) marked with red circles and cowpea proteins (127) with green squares. Total of 252 R2R3-MYB members classified into 32 evolutionary subfamilies (indicated by distinct color blocks).
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Figure 2. The phylogenetic relationships, exon–intron gene structures, and conserved motifs of the VuR2R3-MYB TFs in cowpea are presented. (A) The phylogenetic tree of the VuR2R3-MYB proteins was constructed using Mega software (v10), illustrating their evolutionary relationships. (B) The structure of the VuR2R3-MYB TFs is shown, with the orange boxes representing exons, the black lines indicating introns, and the blue boxes denoting UTRs (untranslated regions). (C) The compositions of the conserved motifs within the VuR2R3-MYB family in cowpea are detailed. (D) Sequence information for the conserved motifs is provided.
Figure 2. The phylogenetic relationships, exon–intron gene structures, and conserved motifs of the VuR2R3-MYB TFs in cowpea are presented. (A) The phylogenetic tree of the VuR2R3-MYB proteins was constructed using Mega software (v10), illustrating their evolutionary relationships. (B) The structure of the VuR2R3-MYB TFs is shown, with the orange boxes representing exons, the black lines indicating introns, and the blue boxes denoting UTRs (untranslated regions). (C) The compositions of the conserved motifs within the VuR2R3-MYB family in cowpea are detailed. (D) Sequence information for the conserved motifs is provided.
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Figure 3. Prediction of cis-acting elements in cowpea VuR2R3-MYB TF promoters. (A) Phylogenetic tree of VuR2R3-MYB proteins. (B) Distribution of cis-regulatory elements in promoter regions (color-coded by element type). (C) Heatmap of element quantities per TF (showing top 16 most abundant elements). (D) Stacked bar plot illustrating distribution of four regulatory element categories across TFs.
Figure 3. Prediction of cis-acting elements in cowpea VuR2R3-MYB TF promoters. (A) Phylogenetic tree of VuR2R3-MYB proteins. (B) Distribution of cis-regulatory elements in promoter regions (color-coded by element type). (C) Heatmap of element quantities per TF (showing top 16 most abundant elements). (D) Stacked bar plot illustrating distribution of four regulatory element categories across TFs.
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Figure 4. Chromosomal localization of VuR2R3-MYB TFs.
Figure 4. Chromosomal localization of VuR2R3-MYB TFs.
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Figure 5. Intra-species synteny relationship diagram. Outermost text represents TF names; colored rings indicate different chromosomes; gray lines represent whole-genome synteny within species; colored lines illustrate syntenic relationships of VuR2R3-MYB TF family within species.
Figure 5. Intra-species synteny relationship diagram. Outermost text represents TF names; colored rings indicate different chromosomes; gray lines represent whole-genome synteny within species; colored lines illustrate syntenic relationships of VuR2R3-MYB TF family within species.
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Figure 6. A homology analysis of the VuR2R3-MYB TFs in cowpea with four representative plants was conducted. The synthesis relationships were analyzed using TB-tools software (v2.210). The gray lines in the background represent the syntenic blocks between cowpea and other species, while the blue lines indicate the duplicated R2R3-MYB TF pairs.
Figure 6. A homology analysis of the VuR2R3-MYB TFs in cowpea with four representative plants was conducted. The synthesis relationships were analyzed using TB-tools software (v2.210). The gray lines in the background represent the syntenic blocks between cowpea and other species, while the blue lines indicate the duplicated R2R3-MYB TF pairs.
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Figure 7. (A) Three distinct pod color phenotypes in cowpea: deep green (DGH), light purple (LPH), and purple (PRS). (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of differentially accumulated metabolites in three cowpea varieties. (C) The heatmap displays the expression profiles of 19 differentially expressed genes (DEGs) within the VuR2R3-MYB gene family across three cowpea varieties. FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values subjected to log2 transformation, followed by row-wise Z-score normalization. (D) Regulatory network of anthocyanin biosynthesis [51,52] and VuR2R3-MYB candidate TFs. Red squares denote transcriptome FPKM values following log2 transformation and Z-score standardization, whereas purple blocks denote metabolite abundance after log2 transformation and Z-score standardization.
Figure 7. (A) Three distinct pod color phenotypes in cowpea: deep green (DGH), light purple (LPH), and purple (PRS). (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of differentially accumulated metabolites in three cowpea varieties. (C) The heatmap displays the expression profiles of 19 differentially expressed genes (DEGs) within the VuR2R3-MYB gene family across three cowpea varieties. FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values subjected to log2 transformation, followed by row-wise Z-score normalization. (D) Regulatory network of anthocyanin biosynthesis [51,52] and VuR2R3-MYB candidate TFs. Red squares denote transcriptome FPKM values following log2 transformation and Z-score standardization, whereas purple blocks denote metabolite abundance after log2 transformation and Z-score standardization.
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Figure 8. qRT-PCR and RNA-seq were employed to investigate the potential transcription factors associated with anthocyanin biosynthesis. The findings are presented as means ± standard deviation, derived from three biological replicates. The statistical evaluations were conducted using one-way analysis of variance (ANOVA) coupled with Duncan’s test, with different lowercase letters denoting significant differences at the p < 0.05 level.
Figure 8. qRT-PCR and RNA-seq were employed to investigate the potential transcription factors associated with anthocyanin biosynthesis. The findings are presented as means ± standard deviation, derived from three biological replicates. The statistical evaluations were conducted using one-way analysis of variance (ANOVA) coupled with Duncan’s test, with different lowercase letters denoting significant differences at the p < 0.05 level.
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MDPI and ACS Style

Yang, Y.; Yu, C.; Zhou, X.; Wu, Z.; Shen, Z.; Li, T.; Zhang, Y. A Genome-Wide Analysis of the VuR2R3-MYB Gene Family in Cowpea and Its Expression in Anthocyanin Accumulation. Agronomy 2025, 15, 1075. https://doi.org/10.3390/agronomy15051075

AMA Style

Yang Y, Yu C, Zhou X, Wu Z, Shen Z, Li T, Zhang Y. A Genome-Wide Analysis of the VuR2R3-MYB Gene Family in Cowpea and Its Expression in Anthocyanin Accumulation. Agronomy. 2025; 15(5):1075. https://doi.org/10.3390/agronomy15051075

Chicago/Turabian Style

Yang, Yi, Canye Yu, Xuan Zhou, Zengxiang Wu, Zhuo Shen, Tinyao Li, and Yan Zhang. 2025. "A Genome-Wide Analysis of the VuR2R3-MYB Gene Family in Cowpea and Its Expression in Anthocyanin Accumulation" Agronomy 15, no. 5: 1075. https://doi.org/10.3390/agronomy15051075

APA Style

Yang, Y., Yu, C., Zhou, X., Wu, Z., Shen, Z., Li, T., & Zhang, Y. (2025). A Genome-Wide Analysis of the VuR2R3-MYB Gene Family in Cowpea and Its Expression in Anthocyanin Accumulation. Agronomy, 15(5), 1075. https://doi.org/10.3390/agronomy15051075

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