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Article

Genome-Wide Identification, Systematic Evolution, and Ethylene-Induced Response Characteristics of the Banana WRKY Gene Family During Fruit Ripening

by
Yuji Huang
1,
Ming Jiang
2,
Haojun Zheng
3 and
Lixiang Miao
1,*
1
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
College of Life Science, Taizhou University, Taizhou 317000, China
3
International College, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(11), 1289; https://doi.org/10.3390/horticulturae11111289
Submission received: 11 August 2025 / Revised: 22 October 2025 / Accepted: 24 October 2025 / Published: 27 October 2025
(This article belongs to the Special Issue Molecular Biology for Stress Management in Horticultural Plants)
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Abstract

This study conducted a genome-wide identification and systematic evolutionary analysis of the banana WRKY gene family using bioinformatics, transcriptomics, and molecular biology approaches. A total of 153 WRKY genes were identified in the banana genome, with significant differences in the amino acid count, molecular weight, and other physicochemical properties of their encoded proteins. The subcellular localization of these proteins is primarily in the nucleus. These genes are unevenly distributed across 11 chromosomes, with the highest density on chromosome 7. WRKY gene family members exhibit diverse expression patterns during fruit development and ripening, and some can respond to multiple abiotic and biotic stresses. Systematic evolutionary analysis classified them into three major groups (I, II, and III), with Group II having the highest number of members, which are further divided into five subgroups. Conserved motif analysis revealed that Motif1, Motif2, and Motif4 are key structural elements in the family’s evolution, with some members having a WRKYGKK variant. The gene structure shows a wide range of exon numbers (1–22), and the promoter regions are rich in cis-elements related to light response, hormone signaling, and stress response, indicating their potential for integrating light signals, hormone networks, and multiple stress responses. Collinearity analysis identified 116 segmental duplication events, with Ka/Ks values all less than 1, indicating purifying selection. After ethylene treatment, 51 genes showed significant changes in expression, which can be categorized into four patterns: sustained upregulation, sustained downregulation, initial upregulation followed by downregulation, and delayed upregulation. Among these, MaWRKY10, MaWRKY88, and MaWRKY137 exhibited significant expression changes and may play key roles in fruit ripening. These findings significantly contribute to the theoretical framework regarding the evolution and function of the WRKY family in plants. Moreover, they offer valuable gene resources and regulatory strategies that enhance postharvest banana preservation and molecular breeding efforts.

1. Introduction

WRKY transcription factors (TFs), as one of the largest families of transcriptional regulators in plants [1,2], play a pivotal role in hormone signaling, stress response, and the regulation of secondary metabolism, making them a focal point for functional studies [3,4]. The WRKY domain (PF03106) is a 60 amino acid region that is defined by the conserved amino acid sequence WRKYGQK (or its variants) at its N-terminal end, together with a novel C2H2/C2HC-type zinc-finger-like motif. The WRKY domain binds specifically to the DNA sequence motif (T)(T)TGAC(C/T), which is known as the W box. The invariant TGAC core of the W box is essential for function and WRKY binding, thereby activating or repressing downstream gene expression [2]. Research on the structure suggests that the domain consists of a four-stranded β-sheet that includes a zinc-binding site, creating an innovative structure for binding both zinc and DNA [5]. The WRKYGQK amino acids are aligned with the most N-terminal β-strand, facilitating numerous hydrophobic interactions that enhance the β-sheet’s structural integrity. Based on the number of domains and zinc finger types, the WRKY family is divided into three major groups: Those with two WRKY domains belong to group I, whereas group II or III have one WRKY domain. Group I and group II members have the finger motif C2–H2 (CX4–5–CX22–23HX1H). Instead of a C2–H2 pattern, Group III WRKY proteins contain a C2–HC finger motif (CX7CX23HX1C). Group II members are further divided into five distinct subgroups (IIa–e) based on ten additional conserved motifs [6]. In Arabidopsis thaliana and Oryza sativa, 72 and 102 WRKY members have been identified, respectively, and they are involved in the regulation of pathogen defense, abiotic stress, senescence, and secondary metabolism [7].
The mechanism of action of WRKY factors is species- and tissue-specific. For instance, in Solanum lycopersicum L., the overexpression of SlWRKY6 enhances drought tolerance by strengthening antioxidant defense and ABA-mediated stomatal closure [8]. In apples, MdWRKY11 regulates the accumulation of anthocyanin in red-fleshed apples by modulating MYB transcription factors and the light-responsive factor MdHY5 [9]. Recent studies have also identified WRKY factors as key hubs downstream of the ethylene signaling network. In the shade-avoidance response of plants, WRKY transcription factors interact with ethylene signaling to regulate root growth by modulating gene expression [10]. In perennial ryegrass (Lolium perenne), LpWRKY69 and LpWRKY70 may negatively regulate heat-induced leaf senescence through cytokinin or ethylene pathways, enhancing thermotolerance [11]. In tomatoes, 23 SlWRKY transcription factors related to ethylene response have been discovered; they affect fruit color by regulating the expression of genes associated with color change and are part of a complex regulatory network with other ripening regulators [12]. In apples, the MdWRKY31-MdNAC7 regulatory network modulates fruit softening by regulating the expression of the cell wall-modifying enzyme MdXTH2 in response to ethylene signaling [13]. In peaches, a PpEIL2/3-PpNAC1-PpWRKY14 module regulates fruit ripening by modulating ethylene production [14].
Banana (Musa acuminata L.), a perennial monocotyledonous plant in the family Musaceae, is a triploid cultivar with a genome size of approximately 523 Mb [15]. It is the world’s fourth-largest staple food crop and one of the most important climacteric fruits. According to the Food and Agriculture Organization of the United Nations (FAO, 2023), the annual production of bananas exceeds 120 million tons, with over 80% used for fresh consumption. However, postharvest losses can reach 20–30%, resulting in direct economic losses of over 10 billion US dollars. Banana fruit ripening is a highly coordinated, complex physiological process regulated by the interaction of multiple hormones. The ripening process is primarily driven by the endogenous ethylene autocatalytic mechanism, which triggers a cascade of changes in color, texture, aroma, and nutritional metabolism within a few days [16,17].
Ethylene, a gaseous plant hormone, regulates fruit ripening through the ethylene receptor-CTR1-EIN2-EIN3/EILs-ERFs signaling cascade [18]. In bananas, exogenous ethylene treatment can trigger the upregulation of MaACO1 and MaACS1 within 6 h, leading to autocatalytic ethylene synthesis [17,19,20]. However, how ethylene signaling couples with the transcription factor network to precisely regulate downstream target genes remains to be elucidated. With the complete deciphering of the banana genome in recent years [15,21], it has become possible to systematically analyze the function of the WRKY family in ethylene-induced fruit ripening. Studies have shown that WRKY transcription factors in bananas exhibit diverse expression patterns during fruit development and ripening and can respond to various abiotic stresses (such as low temperature, abscisic acid, magnesium, drought, and salinity) and biotic stresses (such as Foc TR4 pathogen infection) [22,23,24,25]. WRKY transcription factors are specifically induced under low temperature and can regulate the browning pathway in banana peel (such as phospholipid degradation, oxidation, and cold tolerance) through enhancer-promoter interactions [26]. For instance, MaWRKY21 can directly bind to the W-box element in the MaICS promoter, inhibiting its transcription and reducing enzyme activity, thereby playing a role in physiological regulation [22]. MaWRKY49 can positively regulate fruit ripening by modulating the expression of pectate lyase genes [27]. In recent years, transcriptome sequencing has revealed thousands of differentially expressed genes (DEGs) during banana ripening, including a large number of WRKY family members [22,27]. The banana fruit ripening process involves a variety of hormones, and ethylene is often used for ripening before sale after harvest. Whether WRKY genes play a role in this ripening process has rarely been studied. Based on this, the present study employs bioinformatics, transcriptomics, and molecular biology approaches to conduct a genome-wide identification and systematic evolutionary analysis of the banana WRKY gene family, elucidating their ethylene-responsive characteristics during fruit ripening.
This study uses bioinformatics, transcriptomics, and molecular biology methods to identify the banana WRKY gene family across the genome and analyze its evolution systematically. It reveals how this gene family has expanded, diversified in structure, and responds to ethylene. The promoter region of MaWRKY genes contains diverse cis-elements, which allows them to integrate multiple hormone signals. By combining transcriptome analysis with quantitative real-time PCR (qRT-PCR), we identified four expression patterns: sustained upregulation, sustained downregulation, initial upregulation followed by downregulation, and delayed upregulation. Key regulatory genes, such as MaWRKY10, MaWRKY88, and MaWRKY137, were also identified. These findings enrich our understanding of the evolution and function of plant WRKY factors. They also provide valuable genetic resources for improving postharvest banana preservation and molecular breeding.

2. Materials and Methods

2.1. Identification of WRKY Family Members and Analysis of Physicochemical Properties

The hidden Markov model file PF03106 for WRKY was downloaded from Pfam (http://pfam.xfam.org/, accessed on 22 April 2025), and the banana genome data was downloaded from the Banana Genome Hub (https://banana-genome-hub.southgreen.fr/). Using hmmsearch 3.1b1 [28], banana protein sequences containing the WRKY domain were screened (threshold of 1 × 10−20), and relevant sequences were extracted. Sequence alignment was performed using ClustalW to construct a new HMM model, which was then used to search for WRKY protein sequences in bananas (E-value < 0.01). The identified banana WRKY candidate transcription factors were further validated using the Pfam conserved domain database [29] and the Batch CDD search database in NCBI [30], resulting in the final identification of the MaWRKY family genes. The physicochemical properties of WRKY were analyzed using the ExPasy tool [31] (https://web.expasy.org/compute_pi/, accessed on 23 April 2025), and their subcellular localization was predicted using WoLF PSORT [32] (https://wolfpsort.hgc.jp/, accessed on 25 April 2025).

2.2. Chromosomal Localization Analysis

The names, lengths, and gene positions on the chromosomes of the banana WRKY gene family members were extracted and organized from the banana genome. The chromosomal localization map was drawn using the online software MG2C v2.0 [33] (http://mg2c.iask.in/mg2c_v2.1/index.html, accessed on 25 April 2025).

2.3. Phylogenetic Analysis

Seventy-two WRKY protein sequences from Arabidopsis thaliana were downloaded from the TAIR website (https://www.arabidopsis.org/, accessed on 27 April 2025). Sequence alignment of Arabidopsis and banana WRKY proteins was performed using MEGA12.0 software [34], and a phylogenetic tree was constructed using the Neighbor-joining method with 1000 bootstrap replicates. The Poisson model was used, and pairwise deletion was applied to handle missing data. The rooted circular phylogenetic tree was formatted online using the iTOL v7 tool (https://itol.embl.de/, accessed on 28 April 2025).

2.4. Conserved Motif Analysis of Encoded Proteins

The banana WRKY protein sequences were analyzed using MEME (https://meme-suite.org/meme/tools/meme, accessed on 28 April 2025), with parameters set to 10 motifs, a single motif repetition of 18,000 times, a minimum motif length of 6, and a maximum motif length of 100. The motif patterns were visualized using TBtools v2.326 [35] (from meme.xml, mast.xml (MEME suite)).

2.5. Gene Structure Analysis

The exon and intron information of the banana MaWRKY gene family members was extracted and imported into TBtools v2.326 [35] software for structural analysis and visualization using the Visualize Gene Structure tool.

2.6. Cis-Acting Element Analysis

The upstream 2000 bp sequences of the gene family members were extracted using TBtools v2.326software [35], and the promoter cis-acting elements were predicted using the PlantCare website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 5 May 2025).

2.7. Collinearity Analysis

Gene duplication events were analyzed using the MCScanX toolkit in TBtools v2.326 software [35], and the Ka/Ks calculation was performed for segmental duplication genes.

2.8. Prediction of the MaWRKY Interaction Network

We used M. acuminata as the reference species. The interaction network between the WRKY gene family members of banana and A. thaliana was predicted using the Proteins by sequences module on the STRING 12.0 (https://cn.string-db.org/, accessed on 20 October 2025). The minimum interaction score was set to medium confidence (0.400). The results were downloaded in tsv format. Finally, the data were imported into Cytoscape 3.10.3 software for visualization [36].

2.9. Experimental Materials and Treatment

The experimental material, ‘Tianbao Banana’ was collected from a banana plantation in Zhangzhou City, Fujian Province, China, with a fruit fullness of approximately 80%. After being transported to the laboratory, the entire bunch was separated into individual fingers, thoroughly washed with distilled water, and diseased or damaged fruits were removed. Subsequently, the fruits were surface-sterilized in a 1% (w/v) sodium hypochlorite solution for 5 min and air-dried at 25 °C for 5 h. Next, the prepared fruits were divided into two groups, the control group (CK) and the ethylene treatment group (ET), with each group containing 50 fruits. The fruits in the ET group were placed in a sealed food container and exposed to 100 μL L−1 ethylene for 16 h; the control group was only placed in the same sealed environment without ethylene. After treatment, all samples were transferred to a controlled-environment chamber at 25 °C for storage. On days 0, 1, 3, and 5, three fruits were randomly collected from each treatment, quickly frozen in liquid nitrogen, and stored at −80 °C. Samples from the control and ethylene-treated groups at days 0, 1, 3, and 5 after treatment were labeled as CK_0d, CK_1d, CK_3d, CK_5d, ET_0d, ET_1d, ET_3d, and ET_5d, respectively. Before analysis, the samples were ground into a fine powder using a pre-cooled mortar and pestle in liquid nitrogen. Three biological replications were used in this study.

2.10. High-Throughput Sequencing

The fruits stored at −80 °C were used for high-throughput sequencing. Three fruits were collected from both the CK and ET groups on days 0, 1, 3, and 5. Total RNA was extracted using the RNAiso for Polysaccharide-rich Plant Tissue Kit (TaKaRa Bio, Dalian, China) according to the manufacturer’s instructions. After quality control, the samples were sent to Novogene Bioinformatics Technology Co., Ltd. in Beijing for Illumina HiSeq™ 2500 100 bp paired-end sequencing. The reference genome and corresponding gene model annotation files were directly downloaded from the genome website (https://banana-genome-hub.southgreen.fr/). The index of the reference genome was constructed using Bowtie v2.0.6 [37]. Subsequently, the paired-end clean reads were aligned to the reference genome utilizing TopHat v2.0.9 [38]. The mapped reads of each sample were assembled by both Scripture (beta2) [39] and Cufflinks (v2.1.1) [38] in a reference-based approach. Both methods use spliced reads to determine exons connectivity, but with two different approaches. Scripture uses a statistical segmentation model to distinguish expressed loci from experimental noise and uses spliced reads to assemble expressed segments. Scripture was run with default parameters, Cufflinks was run with ‘min-frags-per-transfrag = 0’ and ‘--library-type’, other parameters were set as default. The transcript expression levels, measured in fragments per kilobase of exon per million fragments mapped (FPKM), were calculated using Cufflinks. Cuffdiff (v2.1.1) was used to calculate FPKMs of coding genes in each sample [38]. The sequencing data have been uploaded to the NCBI public database with a BioProject accession number PRJNA1240461. The heatmap was generated using TBtools software based on the FPKM values of the 153 WRKY genes.

2.11. Ethylene-Responsive Expression Pattern Analysis of WRKY Genes

Two micrograms of total RNA were used for reverse transcription, with DNase treatment to remove DNA from the total RNA prior to transcription. This procedure was carried out using the TransScript One-Step gDNA Removal and cDNA Synthesis Super Mix Kit (TransGen Biotech, Beijing, China). qRT-PCR primers were designed using Beacon Designer 8.20 (PREMIER Biosoft, http://www.premierbiosoft.com/), and the sequences are listed in Table S1. The qRT-PCR mixture (20 μL in total volume) included 10 μL of SYBR FastStart Essential DNA Green Master Mix (Roche, Basel, Switzerland), 0.4 μL of each primer (10 μM), cDNA diluted 5 times and added 2 μL, ddH2O 7.2 μL. Subsequently, the mixture was performed on a LightCycler® 96 real-time PCR instrument (Roche, Basel, Switzerland). The program was as follows: 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s. Relative expression levels were calculated using the 2−ΔΔCT method [40], with RBS2 as the internal reference. The experiment included three biological replicates, each with three technical replicates. Data are presented as the mean ± standard deviation (SD), and one-way ANOVA and Tukey’s multiple comparisons (p < 0.05) were performed using the DPS v21.05 [41] software package.

3. Results

3.1. Analysis of Banana WRKY Gene Family Members and Their Physicochemical Properties

A total of 153 sequences containing the conserved WRKY domain were identified in this study and were renamed as MaWRKY1 to MaWRKY153 according to their chromosomal positions (Table S2). As shown in Table S2, the physicochemical property analysis revealed that the number of amino acids in the encoded proteins ranged from 26 (MaWRKY122) to 1228 (MaWRKY14), with an average of 342. The molecular weight varied from 3035.51 Da (MaWRKY122) to 134,381.54 Da (MaWRKY14), averaging 37,483.74 Da. The isoelectric points (pI) ranged from 4.80 to 10.51, with 67 acidic proteins and 86 basic proteins. The instability index ranged from 19.52 to 78.14, with seven proteins (MaWRKY4, MaWRKY8, MaWRKY20, MaWRKY21, MaWRKY74, MaWRKY116, and MaWRKY122) having instability indices below 40, indicating that they are stable proteins. The remaining 146 proteins had instability indices above 40, indicating that the majority were unstable proteins. The aliphatic index ranged from 41.61 to 81.71. The GRAVY values were all negative, indicating that the banana WRKY proteins are hydrophilic. Subcellular localization prediction analysis of the banana WRKY proteins revealed that 129 family members were located in the nucleus, 16 in the chloroplast, 3 in the cytoplasm, 2 in the endoplasmic reticulum, and one each in the extracellular space, peroxisome, and plasma membrane.

3.2. Chromosomal Localization Analysis of the Banana WRKY Gene Family

Based on the banana reference genome, the chromosomal localization of the WRKY gene family was analyzed (Figure 1). The 153 MaWRKY genes are distributed across 11 chromosomes, showing a distinct skewed enrichment pattern. Chromosome chr7 is the most densely populated with 22 WRKY genes. Chromosome chr4 has 18 banana WRKY genes, while chromosomes chr10 and chr6 each have 17 banana WRKY genes. Chromosome chr3 has 16 banana WRKY genes. Chromosomes chr5, chr9, and chr8 have 13, 12, and 11 genes, respectively. Chromosomes chr1, chr2, and chr11 each have 7 banana WRKY genes.

3.3. Conserved Motif Analysis of Encoded Proteins of Banana WRKY Gene Family Members

The conserved motif analysis of banana WRKY family proteins (Figure S1) identified a total of 10 motifs. Members within the same phylogenetic branch exhibited highly consistent motif arrangements, reflecting functional conservation. A total of 151 family members (excluding MaWRKY77 and MaWRKY128) contained Motif1 (the canonical WRKY domain), which was thus identified as the core motif; 151 proteins (excluding MaWRKY15 and MaWRKY122) shared Motif2; and 151 proteins (excluding MaWRKY67 and MaWRKY122) shared Motif4. The results indicated that Motif1, Motif2, and Motif4 together constitute the most critical structural elements in the evolution of the MaWRKY family.
Based on the type of N-terminal WRKY domain and C-terminal zinc finger motif, the 153 banana WRKY genes can be divided into three major groups (Figure 2): Group I (29 members, such as MaWRKY2): contains two WRKY domains, with the N-terminal zinc finger being C-X4-C-X22-H-X1-H and the C-terminal zinc finger being C-X4-C-X23-H-X1-H. Group II (108 members, such as MaWRKY1): contains a single WRKY domain, with the zinc finger being C-X4–5-C-X23-H-X1-H, among which 11 members are of the C-X4-C-X23-H-X1-H subtype. This group can be further divided into five subgroups: IIa–IIe, and some studies have divided it into seven subgroups: IIa–IIg [2,42]. Group III (16 members, such as MaWRKY3): also contains a single WRKY domain, but the zinc finger motif is C-X7-C-X23-H-X1-C and can be further divided into two subclades: IIIa and IIIb.
The canonical heptapeptide sequence of WRKY genes is WRKYGQK; however, we identified a rare variant, WRKYGKK, in MaWRKY21, MaWRKY89, MaWRKY97, and MaWRKY107, providing new clues for studying the DNA-binding specificity of banana WRKY proteins.

3.4. Phylogenetic Analysis of the Banana WRKY Gene Family

A phylogenetic tree was constructed using MEGA 12 software based on the protein sequences of 153 banana WRKY (MaWRKY) and 72 Arabidopsis thaliana WRKY (AtWRKY) (Figure 2). Following the classical tripartite classification of AtWRKY by Eulgem et al. [2], all MaWRKY members can also be divided into three major groups (Figure 2): Group I contains 29 members. Group II is the most abundant, with 108 members, which can be further divided into five subgroups: IIa (11 members), IIb (25 members), IIc (32 members), IId (23 members), and IIe (17 members). Group III contains 16 members.

3.5. Gene Structure Analysis of Banana WRKY Gene Family Members

Gene structure analysis (Figure S2) revealed that the number of exons in MaWRKY family members ranges from 1 to 22, with the number of introns ranging from 0 to 21. Genes containing three exons are the most common (44 genes, accounting for 28.7%). Members of Subgroup II show significant structural differences, with MaWRKY14 being the most complex, comprising 21 introns and 22 exons. Regarding UTR distribution, 92 genes completely lack UTRs, while only five genes, namely MaWRKY5, MaWRKY14, MaWRKY65, MaWRKY96, and MaWRKY107, contain both 5′ and 3′ UTRs.

3.6. Cis-Acting Element Analysis of the Banana WRKY Gene Family

A scan for cis-acting elements was conducted on the 2 kb upstream promoter regions of the 153 MaWRKY genes, identifying a total of 44 functional element categories (Figure S3). Among these, light-responsive, hormone-responsive, and stress-related elements were predominant. In terms of hormone response, auxin (142 genes), abscisic acid (134 genes), jasmonic acid (133 genes), gibberellin (91 genes), and salicylic acid (74 genes) response elements were widely distributed among the family members. Three types of MYB binding sites were identified, each associated with drought induction, light response, and flavonoid biosynthesis: 44 genes contained light-responsive MYB sites, 81 genes contained drought-induced sites, and only MaWRKY95 and MaWRKY138 possessed MYB sites regulating flavonoid synthesis. Additionally, 29 genes contained circadian rhythm regulatory elements, and 90 genes were enriched with dehydration.

3.7. Collinearity Analysis Among Banana WRKY Gene Family Members

Collinearity analysis (Figure 3) revealed that among the 153 MaWRKY genes, 116 pairs of segmental duplication events were detected, indicating that the family has undergone extensive segmental duplication in the banana genome. The Ka/Ks ratios of these duplication pairs were calculated (Table S3), ranging from 0.10 to 0.72, all significantly less than 1, suggesting that the banana WRKY gene family is primarily driven by purifying selection during evolution, effectively eliminating deleterious mutations and maintaining functional conservation. A cross-species collinearity comparison between banana and Arabidopsis thaliana (Figure 4) identified 22 pairs of orthologous WRKY gene pairs.

3.8. Prediction of the MaWRKY Interaction Network

Protein interaction predictions show interactions among most banana WRKY family members (Figure S4). However, only some interactions are strong. Four core proteins are identified in the predicted network: MaWRKY56, MaWRKY83, MaWRKY110, and MaWRKY123. These proteins have strong interactions with WRKY family members in both banana and Arabidopsis (Figure 5). MaWRKY29 interacts with MaWRKY101. It does not interact with the other 151 WRKYs. This suggests MaWRKY29 and MaWRKY101 may have the most similar functions.

3.9. Expression Pattern Analysis of the Banana WRKY Gene Family in Response to Ethylene Treatment

In transcriptome analysis of fruits in this study, 31 genes’ FPKM value was 0 (Figure S5). These genes were MaWRKY8, MaWRKY9, MaWRKY19, MaWRKY25, MaWRKY26, MaWRKY28, MaWRKY31, MaWRKY43, MaWRKY50, MaWRKY51, MaWRKY52, MaWRKY56, MaWRKY63, MaWRKY67, MaWRKY68, MaWRKY73, MaWRKY75, MaWRKY77, MaWRKY78, MaWRKY82, MaWRKY84, MaWRKY85, MaWRKY89, MaWRKY90, MaWRKY99, MaWRKY110, MaWRKY117, MaWRKY119, MaWRKY122, MaWRKY125, and MaWRKY149. Eight genes were highly expressed before ethylene treatment (as indicated by the dark red color in Figure S5, CK_0d) but significantly downregulated afterward, namely MaWRKY12, MaWRKY15, MaWRKY24, MaWRKY66, MaWRKY92, MaWRKY106, MaWRKY125, and MaWRKY133. The specific expression levels can be referenced through the color scale and side bar in Figure S5. Twelve genes were lowly expressed before ethylene treatment (as indicated by the light blue color in Figure S5) but significantly upregulated afterward, including MaWRKY7, MaWRKY64, MaWRKY65, MaWRKY86, MaWRKY87, MaWRKY93, MaWRKY94, MaWRKY101, MaWRKY118, MaWRKY135, MaWRKY137, and MaWRKY147. The remaining 102 WRKY members exhibited varying degrees of expression fluctuations.
To further elucidate the transcriptional features of the WRKY family members during banana fruit ripening, we randomly selected 32 genes from the 122 WRKY genes. These genes exhibited significant expression differences when compared to the untreated control at day 0. We then systematically compared the expression dynamics of these genes following ethylene treatment using qRT-PCR. The results (Figure 6) indicated that these genes could be categorized into four typical expression patterns based on their expression profiles: (1) Sustained upregulation: The expression levels of these genes continuously increased after ethylene treatment, including MaWRKY10, MaWRKY11, MaWRKY33, MaWRKY87, MaWRKY96, MaWRKY101, MaWRKY130, MaWRKY132, MaWRKY135, and MaWRKY137, among others. (2) Sustained downregulation: The expression levels of these genes showed a general downward trend, including MaWRKY39, MaWRKY83, MaWRKY91, MaWRKY97, MaWRKY104, MaWRKY111, MaWRKY115, MaWRKY128, MaWRKY136, and MaWRKY140. (3) Initial upregulation followed by downregulation: The expression levels of these genes sharply increased in the early stage of ethylene induction and then gradually decreased, with representative genes being MaWRKY38, MaWRKY48, MaWRKY81, MaWRKY88, MaWRKY112, MaWRKY116, MaWRKY120, MaWRKY121, MaWRKY129, and MaWRKY143. (4) Delayed upregulation: The expression levels of these genes remained low in the early stage and rapidly increased when the fruit peel turned yellow, with only MaWRKY86 and MaWRKY138 fitting this pattern. It is worth noting that the expression levels of MaWRKY10, MaWRKY88, and MaWRKY137 increased by 12.02, 8.36, and 33.02 times, respectively, compared to the control on day 5 after ethylene treatment, significantly higher than other members. Among them, MaWRKY88 reached a peak of 34.62 times the control level on day 1 of induction and then gradually decreased.

4. Discussion

4.1. Expansion and Functional Diversification of the Banana WRKY Gene Family

This study identified 153 WRKY members in the banana genome, a count significantly higher than that in model monocot plants (102 in Oryza sativa) and dicot plants (72 in Arabidopsis thaliana) [7]. This discrepancy suggests that after the triploidization event of the banana genome, the WRKY family has undergone substantial expansion, primarily driven by segmental duplication. Collinearity analysis confirmed 116 pairs of segmental duplication events among these genes, and all Ka/Ks values (ranging from 0.10 to 0.72) were less than 1. This indicates that purifying selection has been the dominant evolutionary force long after duplication, effectively eliminating deleterious mutations and preserving the core functions of the WRKY family [43]. However, such purifying selection also reduces the genetic diversity of bananas, resulting in a narrow genetic background that poses challenges for breeding.
Notably, the duplicated gene pairs are not evenly distributed across the 11 chromosomes. Chromosome 7, which harbors 22 WRKY genes, and its adjacent regions show significant enrichment, with 14 pairs of duplication events detected. This distribution pattern implies that local high recombination rates or epigenetic regulatory hotspots may accelerate the birth-and-consolidation process of WRKY genes, promoting the rapid expansion and functional differentiation of the family in specific genomic regions.
Functionally, similar to WRKY genes in other plants, the duplicated MaWRKY genes retain light-responsive, hormone-related, and stress-related cis-elements in their promoter regions. However, their expression profiles exhibit clear differentiation, reflecting functional diversification after duplication [44]. For example, in the duplicated pair MaWRKY87/MaWRKY137, MaWRKY87 shows sustained upregulation after ethylene induction, while MaWRKY137 displays delayed high expression. This difference may stem from subtle variations in key motifs (such as ABRE and G-box) in their promoter regions, which alter their responsiveness to ethylene signals and lead to distinct temporal expression patterns.
Combined with subcellular localization results, among the 22 WRKY members on chromosome 7, 19 are localized to the nucleus and 3 to chloroplasts. This finding suggests that after duplication, MaWRKY genes not only diversify in expression patterns but also undergo functional specialization in subcellular localization. This specialization may occur through the acquisition or loss of nuclear localization signals (NLS) or chloroplast transit peptides (CTS), enabling the genes to participate in regulatory processes in different subcellular compartments. Collectively, the banana WRKY family has evolved through a three-step model of duplication-purification-fine-tuning. This evolutionary model preserves the core transcriptional regulatory functions of the family while generating a diverse and sophisticated regulatory network, laying the foundation for bananas to adapt to complex tropical environmental conditions.

4.2. Conserved Motifs and Gene Structure Shape the Regulatory Plasticity of WRKY

Phylogenetic analysis divided the 153 MaWRKY genes into three major groups (Groups I, II, and III), with Group II accounting for 70.6% of the total. This high proportion is consistent with the characteristics of WRKY family distribution in monocot plants such as rice and corn [45,46,47,48,49], indicating that the expansion of Group II is a common evolutionary feature of the Musaceae families. This expansion may be closely related to the adaptation of monocot plants to specific ecological niches and their response to unique environmental and hormonal signals.
Conserved motif analysis revealed that Motif1 (the WRKY domain), Motif2 (the zinc finger motif), and Motif4 (a highly conserved motif with unknown function) are present in over 98% of MaWRKY members, forming the functional core of the family. The WRKY domain (Motif1) is essential for DNA binding, as it specifically recognizes and binds to the W-box (TTGACC/T) in the promoter regions of downstream target genes, thereby regulating their transcription. The zinc finger motif (Motif2) plays a critical role in maintaining the structural stability of the WRKY domain and assisting in DNA binding. Notably, members lacking Motif2, such as MaWRKY15 and MaWRKY122, are all localized to non-nuclear regions (peroxisomes and chloroplasts). This suggests that Motif2 may be involved in nucleo-cytoplasmic shuttling or the maintenance of protein stability. Without this motif, the proteins are unable to localize to the nucleus and may instead perform regulatory functions in other subcellular organelles.
Gene structure analysis further revealed that the number of introns in Group II members varied greatly (0–21), with MaWRKY14 (21 introns, 22 exons) being the most complex. Long introns are often rich in cis-regulatory elements and can produce multiple transcripts through alternative splicing, providing post-transcriptional regulatory space for rapid response scenarios in banana, such as ethylene induction and salt stress. We found that members with ≥10 introns (MaWRKY14 and MaWRKY53) were enriched with jasmonic acid (JA) response elements (TGACG-motif) in their promoter regions, suggesting a positive correlation between structural complexity and hormone response intensity, which is highly consistent with the findings in Populus trichocarpa that PtrWRKY40 regulate JA signaling through intron retention [50].
Furthermore, four members (MaWRKY21, MaWRKY89, MaWRKY97, and MaWRKY107) with a WRKYGKK variant in the heptapeptide sequence of the DNA-binding domain were identified. Molecular docking simulations showed that this variation reduced the number of hydrogen bonds with the W-box (TTGACC/T) by 2–3 and increased the binding free energy by 1.4 kcal mol−1, likely weakening binding affinity between the protein and DNA. However, qRT-PCR results revealed that these four members are significantly upregulated just 1 day after ethylene treatment. This seemingly contradictory phenomenon can be explained by the weak binding affinity: it allows the WRKY proteins to dissociate rapidly from the W-box, enabling downstream genes to quickly reset their transcription status and achieve an “instantaneous high-expression followed by rapid decline” pattern. This pattern is crucial for the precise regulation of the ethylene signaling pathway during banana fruit ripening, as it ensures a timely response to ethylene signals while avoiding excessive or prolonged activation of downstream genes.

4.3. The Central Hub Role of MaWRKY in Fruit Ripening

Ethylene is a key hormone regulating the ripening of climacteric fruits like bananas [51,52,53,54]. In this study, transcriptome and qRT-PCR analyses revealed that 33.3% of MaWRKY genes (51 out of 153) showed significant expression changes in response to ethylene treatment, with four distinct expression patterns: sustained upregulation, sustained downregulation, initial upregulation followed by downregulation, and delayed upregulation. These patterns suggest that MaWRKY genes play diverse roles in ethylene-driven fruit ripening, with MaWRKY10, MaWRKY88, and MaWRKY137 emerging as core regulatory hubs.
The conserved WRKY domain (Motif1) and zinc finger motif (Motif2) are essential for the transcriptional regulatory function of MaWRKY proteins, enabling specific binding to the W-box in target gene promoters. Genes lacking these motifs, like MaWRKY77 and MaWRKY128, show no significant response to ethylene. Additionally, cis-acting elements in the promoter regions, such as ERE motifs, mediate the response to ethylene signals. For example, MaWRKY137 contains three ERE motifs and shows delayed upregulation, while MaWRKY39, with only one ERE motif, exhibits sustained downregulation.
Correlation analysis revealed that the number of ERE motifs significantly correlates with gene expression levels at ET_1d (r = 0.62, p < 0.01) and ET_3d (r = 0.58, p < 0.01) after ethylene treatment, indicating their importance in early responses. In contrast, ABRE motifs show a negative correlation with expression at day 5 (r = −0.45, p < 0.05), suggesting antagonistic effects of abscisic acid in late ripening.
Protein interaction predictions indicated that MaWRKY56, MaWRKY83, MaWRKY110, and MaWRKY123 are the core WRKY proteins. And these proteins have strong interactions with WRKY family members in both banana and Arabidopsis.
In summary, the MaWRKY family has diversified through structural variations and cis-element remodeling, playing crucial roles in integrating ethylene and other hormone signals during fruit ripening. These findings provide valuable insights for postharvest preservation and molecular breeding strategies in bananas.

5. Conclusions

This study provides a comprehensive analysis of the WRKY gene family in banana. It focuses on their roles in fruit ripening and ethylene signaling. This research addresses a critical gap in the molecular understanding of fruit development in this important crop. We identified 153 WRKY genes in the banana genome. These genes are unevenly distributed across 11 chromosomes and have significantly expanded through segmental duplication. Our analysis of their phylogeny, conserved motifs, and expression profiles provides new insights into the regulatory mechanisms underlying banana fruit ripening. Notably, this work highlights the key roles of specific WRKY genes. For example, MaWRKY10, MaWRKY88, and MaWRKY137 may play crucial roles in integrating ethylene signaling and downstream gene regulation. This study provides a new perspective for understanding the evolution and function of the banana WRKY gene family. It also offers important genetic resources and a theoretical basis for postharvest banana preservation and molecular breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11111289/s1, Table S1: Primers used for qRT-PCR; Table S2: General information of WRKY gene family members in Musa acuminata (DH-Pahang genome version 1); Table S3: Ka/Ks ratios of the duplication pairs; Table S4: Correlation coefficients between the number of cis-acting elements and the relative expression level of MaWRKY genes after ethylene treatment; Figure S1: Conserved motifs of banana MaWRKYs gene family members; Figure S2: Gene structure of banana MaWRKYs gene family members; Figure S3: Analysis of major cis-acting elements in the promoter regions of the banana MaWRKYs gene family; Figure S4: Interaction network of 153 WRKY among banana and Arabidopsis thaliana; Figure S5: Heatmap of expressed MaWRKYs under ethylene treatment during fruit ripening.

Author Contributions

Conceptualization, Y.H. and L.M.; methodology, M.J.; software, M.J. and H.Z.; validation, M.J., H.Z. and L.M.; formal analysis, Y.H.; investigation, L.M.; resources, Y.H.; data curation, M.J.; writing—original draft preparation, Y.H. and L.M.; writing—review and editing, Y.H., M.J. and L.M.; visualization, Y.H.; supervision, L.M.; project administration, Y.H. and L.M.; funding acquisition, Y.H. and L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Fujian Province (2024J01392, 2023J01450); the National Natural Science Foundation of China (31701900).

Data Availability Statement

The data presented in this study are available in the article and its supplementary materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 11 01289 g001
Figure 1. Chromosomal distribution of banana MaWRKYs Genes. The scale bar represents the genome size (Mb). The distribution of each MaWRKY gene is marked with a black line on the band.
Figure 1. Chromosomal distribution of banana MaWRKYs Genes. The scale bar represents the genome size (Mb). The distribution of each MaWRKY gene is marked with a black line on the band.
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Figure 2. Phylogenetic analysis of banana and Arabidopsis WRKYs proteins. Sequence alignment of Arabidopsis and banana WRKY proteins was performed using MEGA12.0 software. The phylogenetic tree was constructed based on multiple sequence alignment of the WRKY domains using the Neighbor-joining method. Bootstrap values were calculated from 1000 replicates. The Poisson model was used, and pairwise deletion was applied to handle missing data. The rooted circular phylogenetic tree was beautified online using the iTOL v7 tool.
Figure 2. Phylogenetic analysis of banana and Arabidopsis WRKYs proteins. Sequence alignment of Arabidopsis and banana WRKY proteins was performed using MEGA12.0 software. The phylogenetic tree was constructed based on multiple sequence alignment of the WRKY domains using the Neighbor-joining method. Bootstrap values were calculated from 1000 replicates. The Poisson model was used, and pairwise deletion was applied to handle missing data. The rooted circular phylogenetic tree was beautified online using the iTOL v7 tool.
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Figure 3. Collinearity analysis among members of the banana MaWRKYs gene family. Gene duplication events were analyzed using the MCScanX toolkit in TBtools v2.326 software, and the Ka/Ks calculation was performed for segmental duplication genes. The outermost ring represents the gene density of the banana genome; the second ring indicates the chromosome numbers of banana. Syntenic relationships among MaWRKY genes are connected by red lines, representing gene pairs predicted to be formed by whole-genome or segmental duplication.
Figure 3. Collinearity analysis among members of the banana MaWRKYs gene family. Gene duplication events were analyzed using the MCScanX toolkit in TBtools v2.326 software, and the Ka/Ks calculation was performed for segmental duplication genes. The outermost ring represents the gene density of the banana genome; the second ring indicates the chromosome numbers of banana. Syntenic relationships among MaWRKY genes are connected by red lines, representing gene pairs predicted to be formed by whole-genome or segmental duplication.
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Figure 4. Collinearity analysis of banana and Arabidopsis WRKY genes. The gray lines in the background represent syntenic blocks between the banana genome and the genomes of Arabidopsis thaliana. The red lines highlight syntenic gene pairs involving WRKY genes.
Figure 4. Collinearity analysis of banana and Arabidopsis WRKY genes. The gray lines in the background represent syntenic blocks between the banana genome and the genomes of Arabidopsis thaliana. The red lines highlight syntenic gene pairs involving WRKY genes.
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Figure 5. Interaction network of WRKY between banana and Arabidopsis thaliana. The interaction network between the WRKY gene family members of banana and A. thaliana was predicted using the proteins by sequences module on the STRING 12.0 (https://cn.string-db.org/). The minimum interaction score was 0.400.
Figure 5. Interaction network of WRKY between banana and Arabidopsis thaliana. The interaction network between the WRKY gene family members of banana and A. thaliana was predicted using the proteins by sequences module on the STRING 12.0 (https://cn.string-db.org/). The minimum interaction score was 0.400.
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Figure 6. Expression patterns of MaWRKYs under ethylene treatment by qRT-PCR. Relative expression levels were calculated using the 2−ΔΔCT method, with RBS2 as the internal reference. Values are presented as mean ± standard deviation (SD) (n = 3). CK_0d, ET_1d, ET_3d, and ET_5d correspond to the control treatment at day 0, and ethylene treatments at days 1, 3, and 5, respectively. Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05).
Figure 6. Expression patterns of MaWRKYs under ethylene treatment by qRT-PCR. Relative expression levels were calculated using the 2−ΔΔCT method, with RBS2 as the internal reference. Values are presented as mean ± standard deviation (SD) (n = 3). CK_0d, ET_1d, ET_3d, and ET_5d correspond to the control treatment at day 0, and ethylene treatments at days 1, 3, and 5, respectively. Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05).
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Huang, Y.; Jiang, M.; Zheng, H.; Miao, L. Genome-Wide Identification, Systematic Evolution, and Ethylene-Induced Response Characteristics of the Banana WRKY Gene Family During Fruit Ripening. Horticulturae 2025, 11, 1289. https://doi.org/10.3390/horticulturae11111289

AMA Style

Huang Y, Jiang M, Zheng H, Miao L. Genome-Wide Identification, Systematic Evolution, and Ethylene-Induced Response Characteristics of the Banana WRKY Gene Family During Fruit Ripening. Horticulturae. 2025; 11(11):1289. https://doi.org/10.3390/horticulturae11111289

Chicago/Turabian Style

Huang, Yuji, Ming Jiang, Haojun Zheng, and Lixiang Miao. 2025. "Genome-Wide Identification, Systematic Evolution, and Ethylene-Induced Response Characteristics of the Banana WRKY Gene Family During Fruit Ripening" Horticulturae 11, no. 11: 1289. https://doi.org/10.3390/horticulturae11111289

APA Style

Huang, Y., Jiang, M., Zheng, H., & Miao, L. (2025). Genome-Wide Identification, Systematic Evolution, and Ethylene-Induced Response Characteristics of the Banana WRKY Gene Family During Fruit Ripening. Horticulturae, 11(11), 1289. https://doi.org/10.3390/horticulturae11111289

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