Progress in Understanding WRKY Transcription Factor-Mediated Stress Responses in Strawberries

Abstract

Strawberry is an economically important horticultural crop cultivated worldwide. However, its growth, yield, and fruit quality are severely constrained by abiotic stresses, such as salinity, drought, and low temperature, as well as biotic stresses including pathogen attack and pest infestation. WRKY transcription factors (TFs) have been extensively characterized in model plants such as Arabidopsis and rice, and increasing evidence highlights their functional diversification and regulatory importance in horticultural crops, including tomato and grapevine. In this review, we summarize recent advances in understanding the roles of WRKY TFs in strawberry responses to both biotic and abiotic stresses, based on studies in both the diploid woodland strawberry (Fragaria vesca L.) and the octoploid cultivated strawberry (Fragaria × ananassa Duchesne). We discuss their involvement in hormone crosstalk, redox regulation, and transcriptional control within complex stress-response networks, while distinguishing expression-based associations from experimentally validated regulatory functions. To provide a clear framework for evaluating the current evidence, we categorize the findings according to a hierarchy of experimental validation, ranging from direct functional characterization in strawberry, to transient assays, heterologous systems (e.g., Arabidopsis or tobacco), transcriptomic inferences, and predictions based on sequence homology. Finally, we outline potential future directions for exploiting strawberry WRKY TFs as candidate regulators in molecular breeding, thereby providing a theoretical basis for future functional studies and breeding applications.

1. Introduction

1.1. Current Challenges in Strawberry Production and Disease Control

In the natural ecological environment, biotic and abiotic stresses are the main factors restricting agricultural production and affecting global food security [1]. Strawberry (Fragaria spp.), a perennial horticultural plant belonging to the family Rosaceae, is widely appreciated for its distinctive flavor and rich nutritional value [2]. It was first cultivated in Europe in the 18th century and is a hybrid of the Chilean strawberry (Fragaria chiloensis (L.) Mill.) and the Virginia strawberry (Fragaria virginiana, native to the eastern United States), collectively known as Fragaria × ananassa [3]. Strawberry fruits are rich in bioactive components and valued for their nutritional quality and reported health-related properties [4,5]. Due to their attractive appearance and desirable taste, strawberries are widely cultivated and constitute economically important crops [6].

However, strawberries are highly vulnerable to multiple stresses during both pre-harvest growth and post-harvest storage and transportation. This vulnerability is largely attributable to the fact that the edible part is primarily the receptacle, which lacks a protective fruit peel, as well as to relatively shallow root systems. These risks include biotic stresses, such as the frequent infection by Botrytis cinerea Pers. and Colletotrichum spp. and other pathogenic fungi that cause diseases, fruit rot, quality deterioration, and significant post-harvest losses. In addition, intensive pesticide application during disease control increases the risk of residue accumulation, thereby posing potential food safety concerns. Moreover, these characteristics make strawberries more sensitive to abiotic stresses such as drought and salinity during the growth stage, leading to significant reductions in yield and quality stability [7,8]. These complex stresses and their impact on fruit quality are ultimately governed by transcriptional regulatory networks. WRKY transcription factors function as central hubs within these networks, integrating defense signaling, hormone crosstalk, and redox homeostasis—processes intimately linked to both stress tolerance and fruit development in strawberry [9,10]. Elucidating WRKY-mediated regulation is therefore essential for improving strawberry resilience without compromising fruit quality.

1.2. Plant Defense Responses and Transcriptional Regulation

1.2.1. Core Components of Plant Defense Responses

During growth and development, plants are constantly exposed to various biotic stresses, including fungi, bacteria, viruses, and nematodes, as well as abiotic stresses, such as drought, salinity, extreme temperatures, and heavy metals. These stress factors collectively disrupt key physiological processes, including photosynthesis, osmotic regulation, nutrient uptake, hormone homeostasis, reactive oxygen species (ROS) balance, and membrane integrity [11,12]. However, the activation of plant defense responses is not cost-free. Excessive defense activation often results in growth inhibition, reduced fruit size, delayed flowering, changes in acid-base balance, and shortened post-harvest longevity, ultimately constraining normal growth and development. Therefore, plant immunity has evolved as a regulatory mechanism for balancing growth and defense, allowing plants to optimize fitness under fluctuating environments. This phenomenon is referred to as the growth-defense trade-off [13,14].

In strawberries, this trade-off has particularly pronounced consequences due to the economic importance of the fruit itself. Unlike model plants such as Arabidopsis, where defense-related growth inhibition primarily affects vegetative biomass, strawberry defense activation directly impacts the harvested organ—the receptacle-derived fruit. Enhanced disease resistance often correlates with reduced fruit size, altered sugar-acid balance, decreased firmness, and shortened post-harvest shelf life—all of which compromise commercial value [15,16]. Moreover, because strawberry fruits are actively growing and ripening tissues with ongoing metabolic and hormonal dynamics, the allocation of resources toward defense can interfere with ripening programs, affecting color development, flavor formation, and softening [17]. This intimate link between stress responses and fruit quality traits makes the growth-defense trade-off a central concern in strawberry production and a key target for genetic improvement.

The plant immune system is generally described as a two-tiered defense architecture consisting of pattern-triggered immunity (PTI), initiated by the recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), and effector-triggered immunity (ETI), activated upon effector recognition by intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) [18]. Although mechanistically distinct, PTI and ETI share overlapping downstream responses, including reactive oxygen species (ROS) bursts, hypersensitive cell death, stomatal closure, and the accumulation of defense-related phytohormones. Major defense signaling pathways include ROS signaling and phytohormone-mediated pathways involving salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) [19,20,21].

Importantly, these multilayered signaling cascades ultimately converge on large-scale transcriptional reprogramming, which determines the specificity, amplitude, and duration of stress adaptation. The integration of immune signaling with hormonal and redox pathways relies on tightly controlled transcriptional networks. Among the diverse transcription factor families involved, WRKY proteins function as central regulatory nodes linking upstream stress perception to downstream defense gene activation, thereby coordinating growth–defense balance at the transcriptional level.

1.2.2. Transcriptional Regulation in Plant Defense

Transcriptional regulation acts as a central hub for integrating diverse signaling inputs and initiating specific defense programs. Signals from multiple pathways converge on the activation and modulation of transcription factors (TFs). Accumulating evidence indicates that the “growth-defense trade-off” [22] is tightly controlled by post-translational modifications, such as phosphorylation and ubiquitination, mediated by kinases, E3 ubiquitin ligases, and other regulatory enzymes. These modifications directly influence the activity, stability, and subcellular localization of TFs, thereby reprogramming gene expression networks and contributing to the coordinated regulation of plant defense and growth [23,24,25].

Based on their molecular structural features, TFs can be classified into several major families, including WRKY, MYB (Myeloblastosis), NAC (NAM/ATAF/CUC), ERF, bZIP (Basic leucine zipper), and bHLH (Basic helix-loop-helix). Acting as molecular regulators, TFs bind to cis-acting elements of target gene promoters to control gene transcription. Among these families, WRKY TFs are widely recognized as a prominent transcription factor family in plants due to their broad distribution and functional diversity. WRKY proteins play a central role in sensing environmental cues and developmental signals, integrating multiple defense pathways, and coordinating downstream gene expression networks [26,27]. Therefore, a comprehensive understanding of how WRKY and other TFs integrate the growth-defense trade-off signals and precisely regulate associated gene networks is essential for elucidating the molecular mechanisms underlying plant stress resistance [28] (Figure 1).

1.3. The Purpose and Framework of This Review

The regulatory mechanisms of WRKY TFs have been extensively characterized in well-studied model plants such as Arabidopsis thaliana (L.) Heynh. and rice (Oryza sativa L.), providing a solid theoretical foundation for stress biology research. In contrast, horticultural crops exhibit remarkable diversity in growth and developmental patterns, reproductive strategies, secondary metabolism, and quality formation. During stress responses, WRKY genes in horticultural species often form distinct regulatory networks that are closely associated with species-specific agronomic traits, including fruit and postharvest quality traits. Therefore, building upon knowledge obtained from model plants, a systematic investigation of WRKY-mediated regulatory mechanisms in horticultural crops represents an important and emerging research area in plant stress resistance.

This review aims to systematically summarize the regulatory role of WRKY TFs in plant defense, with a particular focus on strawberries as a representative horticultural crop. We review the identification and classification of the WRKY gene family in strawberries and summarize recent progress in functional studies under biotic and abiotic stress conditions. In addition, we discuss how strawberry WRKY genes are involved in hormone signaling crosstalk, regulation of downstream defense-related metabolic networks, and specific biological processes such as fruit development and ripening. Finally, we highlight the potential application of WRKY genes in the molecular breeding of strawberry, with the goal of providing a theoretical framework for understanding stress resistance mechanisms in horticultural crops and the development of high-quality, stress-resistant varieties.

2. Overview of WRKY Transcription Factors

WRKY transcription factors (TFs) were first identified in sweet potato (Ipomoea batatas L.) in 1994 [29], and later formally characterized as a distinct TF family in 2000 [30]. Since then, WRKY genes have been identified in a wide range of species, revealing considerable variation in family size. For example, the Arabidopsis thaliana genome contains 74 WRKY genes, while soybean (Glycine max (L.) Merr.) harbors 197, maize (Zea mays L.) 120, rice (Oryza sativa L.) 103, cucumber (Cucumis sativus L.) 61, and grape (Vitis vinifera L.) 62 [31,32].

2.1. WRKY Domain and Taxonomic Classification

The WRKY transcription factor family is named after its highly conserved DNA-binding domain, the WRKY domain, which is approximately 60 amino acids in length and contains the core motif WRKYGQK. WRKY proteins typically harbor one or two WRKY domains, most of which are located in the N-terminal region. The conserved W, K, and Y residues enable specific recognition and binding to W-box cis-acting elements (TTGAC[T/C]) in the promoter of target genes, thereby activating or repressing downstream gene expression [30,33]. In addition, the C-terminal region of WRKY proteins contains a zinc finger motif, generally classified as either the C₂H₂ (C-X4-5-C-X22-23H-X1-H) or C₂HC (C-X7-C-X23-H-X1-C) [34]. This zinc finger structure plays a critical role in stabilizing protein-DNA interactions and enhancing binding affinity, both of which are essential for the regulatory function of WRKY TFs [31,35].

Based on the number of WRKY domains and the type of zinc finger motif, the WRKY gene family is classically divided into three major groups (I, II, and III). Group I proteins contain two WRKY domains and a C₂H₂-type zinc finger. Group II proteins harbor a single WRKY domain with a C₂H₂ zinc finger and can be further subdivided into five subgroups (IIa–IIe) based on phylogenetic analysis. Group III proteins possess one WRKY domain and a distinctive C₂HC-type zinc finger. In addition, a more detailed classification system has been proposed based on phylogenetic relationships, conserved domain features, and intron positions, categorizing WRKY genes into Groups I (Ia and Ib), IIa–IIb, IIc, IId–IIe, and Group III [36] (Figure 2). Two conserved intron splicing patterns, referred to as PR and VQR introns, are commonly observed within the WRKY domain. In Groups I and IIc, IId, IIe, and III, introns are predominantly inserted after the codons encoding the conserved PR motif, thereby separating the WRKY domain from the zinc finger region. In contrast, introns in Groups IIa and IIb are typically located before the conserved VQR motif [37].

As key regulatory hubs that enable plants to adapt to environmental challenges [26,38], WRKY TFs play essential roles in plant growth and development, metabolic regulation, and responses to biotic and abiotic stresses, owing to their precise DNA-binding properties and transcriptional regulatory functions [39].

2.2. Research Progress on WRKY-Mediated Defense in Model Plants

Studies in model plants such as Arabidopsis and rice have revealed that WRKY transcription factors are established as important hubs within a complex regulatory matrix, integrating hormonal, developmental, and metabolic signals to orchestrate the “growth-defense trade-off” [22]. Several core modules appear to be conserved across angiosperms: in coordinating plant defense responses downstream of major phytohormone signaling pathways, including those involving SA, JA, and ET. In these systems, the “growth-defense trade-off” [22] is regulated through complex signaling networks, in which multiple signaling components modulate the transcriptional activity of WRKYs via post-translational modifications. The activated WRKY TFs subsequently bind to specific cis-regulatory DNA elements in the promoters of stress-response and growth-related genes, thereby fine-tuning gene expression in response to environmental challenges [26]. For example, AtWRKY40 in Arabidopsis thaliana is involved in ABA-related stress responses within the Gβ/ABA signaling context [40]. In rice, infection by Magnaporthe oryzae B.C. Couch induces strong expression of OsWRKY31, which activates resistance-related genes associated with JA and SA signaling. This response enhances disease resistance while simultaneously suppressing plant growth through inhibition of auxin signaling [41]. AtWRKY33 has been proposed as a key regulatory node linking JA and SA signaling pathway, it positively regulates JA-responsive genes during Botrytis cinerea infection while negatively regulating SA signaling, thereby contributing to a balanced defense output [42]. In addition, AtWRKY54 and AtWRKY70 have been identified as negative regulators of SA accumulation [43]. Together with AtWRKY46, they participate in diverse biological processes, including brassinosteroid (BR) signaling, leaf senescence, and drought tolerance, highlighting the involvement of WRKY TFs in hormone crosstalk and stress adaptation pathways [44]. Similarly, in rice, OsWRKY45 positively regulates resistance to rice blast disease through coordinated activation of SA- and JA-dependent defense pathways [45]. Beyond hormone signaling, WRKY TFs also connect defense responses with secondary metabolism networks. OsWRKY62 and OsWRKY76 have been shown to influence metabolic reprogramming by modulating SA- and JA- mediated signaling pathways. In double knockout mutants (dsOW62/76), the accumulation of secondary metabolites such as terpenoids and serotonin is increased, whereas flavonoid biosynthesis is reduced [46,47]. These paradigms provide a powerful framework for investigating WRKY function in any plant species. However, direct extrapolation to the genus Fragaria (strawberry) warrants considerable caution. Firstly, much of our current understanding is derived from annual model plants with relatively simple genomes, whereas the cultivated strawberry (Fragaria × ananassa) is an octoploid perennial with a complex genetic architecture. Polyploidy can lead to the subfunctionalization or neofunctionalization of duplicated gene copies, suggesting that orthologs of AtWRKY40 or OsWRKY31 may have acquired novel or partitioned roles in strawberry. Second, the specific selective pressures inherent to a horticultural context—particularly the demand for simultaneously optimizing high yield, fruit quality, and disease resistance—have likely uniquely shaped the WRKY regulatory network. The balance of the growth-defense trade-off is probably calibrated differently in a perennial fruit crop than in an annual model like Arabidopsis or a grain crop like rice. Finally, while the involvement of WRKYs in processes such as secondary metabolism (e.g., the biosynthesis of terpenoids and flavonoids) is conserved, the specific metabolic outputs are often lineage-specific. Therefore, although the overarching logic of the regulatory network is transferable, the precise identity of the relevant WRKY family members, their target genes, and their ultimate phenotypic impact must be empirically determined within the Fragaria context.

2.3. Research Progress on WRKY-Mediated Defense in Horticultural Plants

Building upon mechanistic insights gained from model plants, research into the important roles of WRKY transcription factors in stress resistance has expanded rapidly in horticultural crops in recent years (Table 1). A comparative analysis across these species reveals that WRKYs function through several conserved regulatory modules. These findings can be broadly categorized into three interconnected modules: direct defense against fungal pathogens, mediation of abiotic stress through phytohormone signaling, and the regulation of protective secondary metabolite biosynthesis.

The first major functional module involves direct defense against fungal pathogens, where WRKY TFs often integrate salicylic acid (SA) and jasmonic acid (JA) signaling pathways. In the context of disease and pest resistance, studies in wild grape (Vitis quinquangularis Rehder) have shown that proanthocyanidins (PAs) play an important role in defense against fungal pathogens, with VqWRKY56 contributing to SA accumulation and PA biosynthesis, thereby enhancing resistance to powdery mildew caused by Erysiphe necator Schwein [48]. This mechanism contrasts with the strategy observed in cultivated grapevine against downy mildew, VvWRKY1 has been reported to enhance resistance to downy mildew through activation of JA-related signaling pathways [49]. These examples from different grape species highlight how WRKY TFs can deploy distinct hormonal strategies—SA-dependent versus JA-dependent—to achieve resistance against different fungal pathogens, even within the same plant family.

Another module centers on ABA-mediated abiotic stress signaling, which frequently intersects with biotic stress pathways. Regarding abscisic acid (ABA) and abiotic stress adaptation, exogenous ABA treatment has been shown to induce the expression of CsWRKY2 in tea (Camellia sinensis (L.) O. Kuntze) plant, linking this TF to cold and drought stresses responses [50]. However, the downstream effects can be species-specific. For example, in tomato, SlWRKY8 can interact with SlGSTU43, which can enhance ability to clear ROS. participating in the regulation of lignin biosynthesis to enhance tomato salt stress tolerance [51].

The third module highlights the role of WRKY TFs in orchestrating secondary metabolism, particularly the biosynthesis of flavonoids and related compounds that serve dual roles in stress adaptation. Furthermore, the regulation of flavonoid metabolism emerges as a key downstream function; VqWRKY56 directly influences PA accumulation [48], a mechanism also observed in other species where WRKYs activate secondary metabolite biosynthesis to reinforce cellular barriers against pathogens. Similarly, in tomato, stress adaptation is closely tied to metabolic modulation. SlWRKY50 promotes cold tolerance by controlling JA biosynthesis which not only govern defense but also influence the accumulation of secondary metabolites [52]. Collectively, these studies demonstrate that WRKY TFs are broadly involved in regulating stress responses across diverse horticultural species, While the core logic of these modules is conserved, functioning in both biotic and abiotic stress adaptation through species-specific regulatory mechanisms.

Table 1. Examples of the Functions of WRKY Genes in Representative Horticultural Crops.

Species (Scientific Name)	WRKY Gene	Main Function	Regulatory Pathway/Target	Experimental System	Evidence Level 1
Vitis quinquangularis (Wild Grape)	VqWRKY56	Enhances resistance to Erysiphe necator	Promotes the accumulation of SA and proanthocyanidins	Transient overexpression in grape leaves	Gain-of-function phenotype + biochemical target validation
Vitis vinifera (Grapevine)	VvWRKY1	Enhances resistance to downy mildew	Associated with JA-related gene expression	Transient overexpression in grape leaves	Gain-of-function phenotype + gene expression analysis
Vitis vinifera (Grapevine)	VvWRKY2 [53]	Confers broad-spectrum fungal resistance	Not fully elucidated (verified via heterologous expression in tobacco)	Transient overexpression in grape leaves	Gain-of-function phenotype + gene expression analysis
Solanum lycopersicum (Tomato)	SlWRKY8	Promotes resistance to pathogen infection and mediates drought/salt tolerance	Enhances antioxidant defense/stress-response pathways	Stable transgenic tomato lines	Loss-of-function phenotype + gene expression analysis
Fragaria vesca (Woodland Strawberry)	FvWRKY50	Delays flowering and leaf senescence; promotes anthocyanin accumulation in fruit	Regulates vegetative and reproductive growth balance	Stable transgenic F. vesca lines	Gain-/loss-of-function phenotype + gene expression analysis
Fragaria × ananassa (Cultivated Strawberry)	FaWRKY71 [54]	Promotes anthocyanin synthesis and regulates fruit softening	Activates structural genes in the flavonoid pathway and related transporters	Stable transgenic F. × ananassa lines (RNAi)	Loss-of-function phenotype + gene expression analysis
Fragaria vesca (Woodland Strawberry)	FvWRKY48 [55]	Regulates fruit softening	Binds to the promoter of the FvPLA gene	Stable transgenic F. vesca lines + Y1H/EMSA	Gain-of-function phenotype + direct target validation

¹ Evidence level notes: “Gene expression correlation analysis” indicates that gene expression patterns correlate with specific physiological processes; “Gain-/loss-of-function phenotype” indicates that reproducible biological phenotypes were obtained through overexpression or knockdown/knockout; “Direct target validation” indicates that direct binding of the WRKY to the promoter of a downstream target gene was confirmed through biochemical experiments (e.g., EMSA, ChIP, Y1H).

Table 1. Examples of the Functions of WRKY Genes in Representative Horticultural Crops.

Species (Scientific Name)	WRKY Gene	Main Function	Regulatory Pathway/Target	Experimental System	Evidence Level 1
Vitis quinquangularis (Wild Grape)	VqWRKY56	Enhances resistance to Erysiphe necator	Promotes the accumulation of SA and proanthocyanidins	Transient overexpression in grape leaves	Gain-of-function phenotype + biochemical target validation
Vitis vinifera (Grapevine)	VvWRKY1	Enhances resistance to downy mildew	Associated with JA-related gene expression	Transient overexpression in grape leaves	Gain-of-function phenotype + gene expression analysis
Vitis vinifera (Grapevine)	VvWRKY2 [53]	Confers broad-spectrum fungal resistance	Not fully elucidated (verified via heterologous expression in tobacco)	Transient overexpression in grape leaves	Gain-of-function phenotype + gene expression analysis
Solanum lycopersicum (Tomato)	SlWRKY8	Promotes resistance to pathogen infection and mediates drought/salt tolerance	Enhances antioxidant defense/stress-response pathways	Stable transgenic tomato lines	Loss-of-function phenotype + gene expression analysis
Fragaria vesca (Woodland Strawberry)	FvWRKY50	Delays flowering and leaf senescence; promotes anthocyanin accumulation in fruit	Regulates vegetative and reproductive growth balance	Stable transgenic F. vesca lines	Gain-/loss-of-function phenotype + gene expression analysis
Fragaria × ananassa (Cultivated Strawberry)	FaWRKY71 [54]	Promotes anthocyanin synthesis and regulates fruit softening	Activates structural genes in the flavonoid pathway and related transporters	Stable transgenic F. × ananassa lines (RNAi)	Loss-of-function phenotype + gene expression analysis
Fragaria vesca (Woodland Strawberry)	FvWRKY48 [55]	Regulates fruit softening	Binds to the promoter of the FvPLA gene	Stable transgenic F. vesca lines + Y1H/EMSA	Gain-of-function phenotype + direct target validation

¹ Evidence level notes: “Gene expression correlation analysis” indicates that gene expression patterns correlate with specific physiological processes; “Gain-/loss-of-function phenotype” indicates that reproducible biological phenotypes were obtained through overexpression or knockdown/knockout; “Direct target validation” indicates that direct binding of the WRKY to the promoter of a downstream target gene was confirmed through biochemical experiments (e.g., EMSA, ChIP, Y1H).

A key advantage of the strawberry system lies in the comparison between diploid Fragaria vesca and octoploid Fragaria × ananassa. This system provides an opportunity to examine how WRKY regulatory networks are reshaped by polyploidy and linked to fruit development and stress responses. While Arabidopsis has provided foundational insights into WRKY function, direct extrapolation to strawberry requires caution due to polyploid-related complexity and species-specific traits.

Comparative functional genomics reveals a mixed landscape of conservation and divergence. Several strawberry WRKY orthologs, such as FaWRKY1, have demonstrated functional conservation in core defense signaling when heterologously expressed in Arabidopsis mutants, suggesting that certain downstream regulatory modules are deeply conserved. However, the octoploid strawberry genome, with its four subgenomes, exhibits a more complex WRKY gene family, with evidence of both subfunctionalization and neo-functionalization following polyploidization. For instance, homoeologs of a single Arabidopsis WRKY gene may have evolved to partition roles between biotic stress resistance and the regulation of fruit softening or anthocyanin accumulation in strawberry. This divergence implies that while Arabidopsis remains an invaluable tool for dissecting fundamental molecular mechanisms, direct functional extrapolation to strawberry must be undertaken with caution. The polyploid context introduces an additional layer of regulatory complexity where gene dosage, promoter divergence, and homoeolog expression partitioning can lead to phenotypic outcomes not predictable from diploid models.

A more nuanced perspective emerges when comparing the two strawberry systems directly. In the diploid F. vesca, which retains a simpler genome architecture, functional studies have identified WRKY genes involved in fundamental processes such as basal defense against fungal pathogens and responses to oxidative stress. These investigations in the diploid progenitor provide a baseline understanding of ancestral WRKY functions. By contrast, studies in the octoploid F. × ananassa have revealed a more elaborate regulatory landscape shaped by polyploidy. Genome-wide analyses indicate that many WRKY homoeologs are retained across subgenomes, but their expression patterns often exhibit subgenome dominance or tissue-specific partitioning. For example, homoeologous copies of a single ancestral WRKY gene may display divergent expression during fruit ripening, with one copy maintaining pathogen-responsive expression while the other acquires a novel role in regulating anthocyanin biosynthesis. This expression bias, coupled with evidence of promoter sequence divergence among homoeologs, points to subfunctionalization as a major force shaping the WRKY gene family in cultivated strawberry. Such partitioning allows the octoploid genome to uncouple stress responses from developmental programs, potentially enabling simultaneous optimization of fruit quality and stress resilience—a regulatory flexibility not possible in the diploid progenitor.

Therefore, the “safety” of transferring functional knowledge between these species is context-dependent. Core defense pathways show high translatability, but integrating this knowledge into strawberry improvement programs requires empirical validation within the polyploid genome to account for subgenome dominance and functional redundancy. By systematically comparing what has been functionally validated in F. vesca with findings from F. × ananassa, this review highlights not only the conserved core of WRKY-mediated regulation but also species-specific complexities.

3. Systematic Identification and Characterization of the WRKY Family in Strawberries

WRKY family are regulatory proteins that play important roles in plant defense as well as in growth and development. Originating in primitive eukaryotes, the WRKY family has undergone extensive expansion in the plant kingdom through whole-genome, segmental, and tandem duplication events, and is now recognized as one of the largest transcription factor families in higher plants [56,57]. The genus Fragaria comprises approximately 24 species, with ploidy levels ranging from diploid to decaploid [58]. Among them, the diploid woodland strawberry possesses a relatively small genome and was sequenced in 2011, making it an excellent model for functional genomics research in the Rosaceae family. In contrast, the cultivated strawberry (Fragaria × ananassa), an allo-octoploid species, has also been sequenced and progressively annotated, providing a solid foundation for systematic analysis of gene family evolution and function [59]. Co-linearity analysis indicates that strawberry WRKY genes exhibit a high degree of conservation with their homologs in Arabidopsis and grape, suggesting functional conservation of this family during evolution [60,61]. Compared with model plants such as Arabidopsis and rice, the WRKY family in strawberry has undergone pronounced expansion during the octoploidization process. This expansion is mainly attributed to whole-genome duplication followed by segmental duplication events, particularly in specific subgroups such as Group IIc, in which the number of members has increased substantially. Such expansion may be associated with the adaptation of the strawberry to complex growth environments and diverse biotic and abiotic stress conditions [62,63,64].

3.1. Systematic Identification and Classification

Based on published genome data of the cultivated strawberry, 257 FaWRKY genes were identified. According to the number of WRKY domains and the characteristics of their zinc finger motifs, these genes were classified into three major phylogenetic groups: Group I (containing two WRKY domains), Group II (further subdivided into IIa, IIb, IIc, IId, IIe), and Group III. The nomenclature of strawberry WRKY genes was established by integrating multiple criteria, including their orthologous relationships with F. vesca WRKY genes, subgenome source (A, B, C, D), and the presence of duplication-derived gene copies. This naming strategy enables consistent identification and comprehensive analysis of WRKY family members across diploid and octoploid strawberry genomes [10].

Based on the comprehensive genomic data from Garrido-Gala [10], we have constructed

Table 2. Equivalence table for strawberry WRKY gene nomenclature. Current names follow the format FaWRKY [number] [subgenome] [duplicate number]. Subgenome designations: A = F. nipponica, B = F. iinumae, C = F. viridis, D = F. vesca. Duplicate numbers (.1, .2, .3) indicate paralogous copies arising from recent gene duplication events. Historical names are provided where genes have been previously characterized in the literature; genes without prior functional characterization are noted as “Not previously named”. Complete sequence information for all genes is available in Tables S2 and S3 of Garrido-Gala [10].

Historical Name	Current Name	F. vesca Ortholog	Homoeologs	Subgenome (A = F. nipponica, B = F. iinumae, C = F. viridis, D = F. vesca)
FaWRKY1	FaWRKY24A	FvWRKY24	FaWRKY24A, FaWRKY24B, FaWRKY24D	A, B, D
FaWRKY1	FaWRKY24D	FvWRKY24	FaWRKY24A, FaWRKY24B, FaWRKY24D	D
Not previously named	FaWRKY20A	FvWRKY20	FaWRKY20A	A
Not previously named	FaWRKY21C	FvWRKY21	FaWRKY21B, FaWRKY21C	B, C
Not previously named	FaWRKY21B	FvWRKY21	FaWRKY21B, FaWRKY21C	B
Not previously named	FaWRKY51A.2	FvWRKY51	FaWRKY51A.1, FaWRKY51A.2, FaWRKY51A.3	A
Not previously named	FaWRKY38A	FvWRKY38	FaWRKY38A, FaWRKY38B	A, B
Not previously named	FaWRKY39A	FvWRKY39	FaWRKY39A	A
Not previously named	FaWRKY40D	FvWRKY40	FaWRKY40D, FaWRKY41D, FaWRKY42D	D
Not previously named	FaWRKY41D	FvWRKY41	FaWRKY40D, FaWRKY41D, FaWRKY42D	D
Not previously named	FaWRKY42D	FvWRKY42	FaWRKY40D, FaWRKY41D, FaWRKY42D	D
Not previously named	FaWRKY11A	FvWRKY11	FaWRKY11A, FaWRKY11C, FaWRKY12A, FaWRKY12C	A, C
Not previously named	FaWRKY12A	FvWRKY12	FaWRKY11A, FaWRKY11C, FaWRKY12A, FaWRKY12C	A
Not previously named	FaWRKY55B.1	FvWRKY55	FaWRKY55B.1, FaWRKY55B.2	B
Not previously named	FaWRKY55B.2	FvWRKY55	FaWRKY55B.1, FaWRKY55B.2	B
Not previously named	FaWRKY43B.2	FvWRKY43	FaWRKY43B.1, FaWRKY43B.2	B
Not previously named	FaWRKY29A	FvWRKY29	FaWRKY29A, FaWRKY29B, FaWRKY29D.1, FaWRKY29D.2	A, B, D
Not previously named	FaWRKY29B	FvWRKY29	FaWRKY29A, FaWRKY29B, FaWRKY29D.1, FaWRKY29D.2	B
Not previously named	FaWRKY29D.1	FvWRKY29	FaWRKY29A, FaWRKY29B, FaWRKY29D.1, FaWRKY29D.2	D
Not previously named	FaWRKY29D.2	FvWRKY29	FaWRKY29A, FaWRKY29B, FaWRKY29D.1, FaWRKY29D.2	D
Not previously named	FaWRKY48A	FvWRKY48	FaWRKY48A, FaWRKY48B, FaWRKY48C, FaWRKY48D	A, B, C, D
Not previously named	FaWRKY48B	FvWRKY48	FaWRKY48A, FaWRKY48B, FaWRKY48C, FaWRKY48D	B
Not previously named	FaWRKY48C	FvWRKY48	FaWRKY48A, FaWRKY48B, FaWRKY48C, FaWRKY48D	C
Not previously named	FaWRKY48D	FvWRKY48	FaWRKY48A, FaWRKY48B, FaWRKY48C, FaWRKY48D	D
Not previously named	FaWRKY53A	FvWRKY53	FaWRKY53A, FaWRKY53B, FaWRKY53C, FaWRKY53D	A, B, C, D
Not previously named	FaWRKY53B	FvWRKY53	FaWRKY53A, FaWRKY53B, FaWRKY53C, FaWRKY53D	B
Not previously named	FaWRKY53C	FvWRKY53	FaWRKY53A, FaWRKY53B, FaWRKY53C, FaWRKY53D	C
Not previously named	FaWRKY53D	FvWRKY53	FaWRKY53A, FaWRKY53B, FaWRKY53C, FaWRKY53D	D
Not previously named	FaWRKY9D	FvWRKY9	FaWRKY9B, FaWRKY9C, FaWRKY9D	B, C, D
Not previously named	FaWRKY17A	FvWRKY17	FaWRKY17A, FaWRKY17B, FaWRKY17C.1, FaWRKY17C.2, FaWRKY17D	A, B, C,
Not previously named	FaWRKY57A	FvWRKY57	FaWRKY57A.1, FaWRKY57A.2	A

, which provides a systematic correspondence between historical gene names, current standardized names, their Fragaria vesca orthologs, octoploid homoeologs, and subgenome assignments. This comprehensive equivalence table enables straightforward cross-referencing between historical literature and the standardized nomenclature adopted in this study, facilitating accurate interpretation of functional data across different strawberry WRKY genes and their homoeologous copies.

3.2. Gene Structure and Conserved Motifs

Bioinformatics analysis indicates that most FaWRKY genes encode nuclear- localized proteins containing predicted nuclear localization signals, consistent with their proposed roles as transcription factors. Compared with their diploid counterparts (FvWRKY homologs), the majority of FaWRKY proteins exhibit high sequence conservation, particularly within the WRKY DNA-binding domain. Nevertheless, domain diversification has also occurred during evolution. For example, FaWRKY26B.2 was found to harbor an additional Myb-like DNA-binding domain, suggesting potential functional diversification or regulatory specialization, thus necessitating experimental validation [63,64]. Gene structure analysis further revealed that WRKY members within the same phylogenetic group generally share conserved exon-intron architectures, whereas notable differences exist among different groups. In particular, Group III WRKY genes tend to possess fewer introns and simpler gene structures, a feature that may reflect their rapid evolutionary diversification and stress-responsive functions [65,66,67].

The octoploid nature of cultivated strawberry (Fragaria × ananassa) has profound implications for WRKY gene family evolution and function. Polyploidization events have generated a large repertoire of homoeologous and paralogous FaWRKY genes, with differential retention patterns observed across subgenomes following whole-genome duplication. This expanded genetic toolkit provides raw material for functional diversification, wherein homoeolog pairs may undergo subfunctionalization through partitioning of ancestral functions, or neofunctionalization, enabling the acquisition of novel regulatory roles. Subgenome dominance, with the Fragaria vesca-derived subgenome showing preferential gene retention and expression, likely contributes to expression biases among FaWRKY homoeologs across tissues and stress conditions. Such subgenome partitioning may facilitate regulatory specialization and enhance phenotypic plasticity in this economically important crop. Future studies integrating homoeolog-specific expression profiling with functional characterization will be essential to dissect how polyploidy has shaped WRKY-mediated regulatory networks in strawberry.

3.3. Tissue Specificity and Induction Expression Patterns

Analysis of the expression profile of woodland strawberry revealed that FvWRKY10, FvWRKY2, FvWRKY7, and FvWRKY33 are constitutively expressed across most tissues, with the exception of pollen. This broad expression pattern suggests that these WRKY genes may be involved in fundamental physiological processes—a hypothesis requiring functional testing. In contrast, many other FvWRKY members exhibit pronounced tissue-specific expression or are differentially induced during fruit ripening and pathogen infection, indicating potential roles in developmentally regulated and stress-responsive pathways. Phylogenetic analyses have identified multiple FvWRKY genes as putative orthologs of defense-related WRKY TFs in Arabidopsis, including FvWRKY3/8 corresponding to AtWRKY50/51, and FvWRKY24/30/53 corresponding to AtWRKY45/75. Promoter analysis further revealed that these genes are enriched in cis-acting elements responsive to JA and SA. Notably, these regulatory elements are highly conserved among their octoploid strawberry homologs. This conservation supports the hypothesis that WRKY-mediated hormone-responsive transcriptional regulation may be evolutionarily conserved within strawberry defense signaling networks. However, direct experimental evidence linking these cis-elements to hormone-dependent transcriptional regulation in strawberry remains to be established. In cultivated strawberry, evidence for WRKY involvement in defense responses can be categorized into three distinct tiers based on available data. Currently, no FaWRKY genes have been functionally validated through transgenic approaches in octoploid strawberry. This represents a critical gap in the field. However, based on phylogenetic conservation, expression patterns, and promoter analyses, several FaWRKY genes emerge as strong candidates for defense-related functions. Transcriptome data analysis revealed that several defense-related WRKY homologs—such as the AtWRKY54/70 homologs FaWRKY38B, FaWRKY39A, and FaWRKY60B, as well as AtWRKY75 homologs FaWRKY24A and FaWRKY24D were significantly upregulated upon anthracnose infection. In contrast, the expression of the AtWRKY53 homologs FaWRKY29A-D.2 was markedly downregulated. These expression patterns are broadly consistent with previously reported roles of their homologous WRKY genes in SA and JA-associated defense networks, while the direction of regulation (downregulation) and absence of mechanistic data preclude robust functional inferences. Similarly, although the expression patterns of anthracnose-responsive FaWRKY genes are broadly consistent with reported roles of their homologs in SA- and JA-associated defense networks, current evidence is primarily correlational. Direct demonstration of hormone pathway engagement—through hormone measurements, pathway-specific marker gene expression, or genetic interaction studies—is required to confirm their proposed roles in SA/JA/ABA signaling networks.

This tiered framework highlights substantial opportunities for future functional characterization, particularly for strong candidate genes identified through integrated phylogenetic, promoter, and expression analyses [43,68].

4. Function of Strawberry WRKY Protein in Biological Stress

As strawberry cultivation has shifted from traditional seasonal open-field systems to year-round and controlled-environment production systems, the cultivated area has continued to expand. Concomitantly, problems associated with pest and disease pressures have intensified, rendering strawberry plants more vulnerable to biotic stresses, including pathogen infection, herbivore feeding, and competition with weeds [69]. Under these conditions, WRKY TFs are key regulatory nodes downstream of plant defense signaling pathways. They respond to multiple hormonal and stress-related signals and modulate the transcription of defense-related genes, thereby contributing to the orchestration of immune responses [70]. Based on the preceding overview of the strawberry WRKY gene family, this section focuses on summarizing current knowledge regarding their regulatory role in response to major biotic stress factors.

4.1. Defense Function Against Pathogens

Pathogen infection is a major biotic stress limiting strawberry production. The primary pathogens affecting strawberries include fungi, bacteria, and viruses, among which fungal pathogens are considered the most destructive. They can infect multiple organs, including roots, leaves, flowers, and fruits, often leading to growth inhibition, fruit decay, and, in severe cases, overall plant decline [35,71]. To maintain yield and fruit quality, fungicides are currently widely applied in strawberry production. However, excessive and repeated use of fungicides can promote the emergence of resistant pathogens and result in pesticide residues, thereby raising concerns regarding food safety and environmental sustainability. Consequently, the development of alternative and sustainable disease management strategies has become increasingly important. In recent years, considerable efforts have focused on molecular breeding and disease-control strategies targeting key regulatory factors, including WRKY TFs, which aim to improve disease resistance in strawberries [72].

An important conceptual framework for understanding WRKY-mediated defense lies in the distinct hormonal signatures associated with different pathogen lifestyles. Necrotrophs, hemibiotrophs, and biotrophs differentially engage SA and JA/ET signaling pathways [73]. WRKY transcription factors occupy a central position in this regulatory network, integrating and fine-tuning these hormone signals to establish appropriate defense outputs [74,75]. Their ability to modulate the balance between SA and JA/ET pathways makes them key determinants of defense prioritization against pathogens with different infection strategies [75,76]. In the following sections, we summarize current knowledge on how strawberry WRKY TFs contribute to resistance against representative fungal pathogens with contrasting lifestyles (

Table 3. Representative fungal diseases of strawberry are discussed in this review.

Pathogen	Typical Symptoms	Environmental Preference	Trophic Type
Botrytis cinerea	Gray mycelial growth and soft rot	Cool and humid conditions	Necrotroph
Colletotrichum spp.	Sunken brown lesions with pink conidial masses	Warm and humid conditions	Hemibiotroph
Podosphaera aphanis (Wallr.) U. Braun & S. Takam	White, powdery mycelial layer on plant surface	Germination at moderate temperature, dry but high humidity	Biotroph

), highlighting their roles in shaping hormone-mediated defense responses.

4.1.1. Defense Mechanism of the Strawberry WRKY Family Against Botrytis cinerea

Gray mold disease caused by Botrytis cinerea is one of the most destructive fungal diseases affecting strawberry production worldwide. As a typical necrotrophic pathogen, B. cinerea kills host cells and feeds on dead tissues, often activating JA/ET-mediated defense responses in plants [77,78]. This pathogen frequently causes severe fruit rot during post-harvest storage and transportation, leading to substantial economic losses. Under high humidity conditions, Botrytis cinerea can damage more than 80% of strawberry flower buds and fruits if fungicides are not applied [77].

In recent years, significant progress has been made in studies of the WRKY gene family in strawberry defense. Multiple WRKY genes, including FaWRKY11, FaWRKY25, FaWRKY29, FaWRKY33-2, FaWRKY64, and FvWRKY50, have been associated with defense response against Botrytis cinerea (Figure 3A).

Among these, FaWRKY11 and FvWRKY50 have been shown, through overexpression and loss-of-function analyses, to act as positive regulators of resistance to Botrytis cinerea. Previous studies indicate that these WRKY TFs are involved in defense responses that are associated with JA-related signaling and the expression of defense-related genes. For example, overexpression of FaWRKY11 enhances resistance to Botrytis cinerea and is accompanied by altered expression of MAPK- and pathogenesis-related genes, whereas silencing of this gene results in increased disease susceptibility. Notably, this functional analysis was conducted using strawberry fruits at the white stage approaching ripening, with Agrobacterium-mediated transient transformation and Botrytis cinerea inoculation performed 3 days post-injection on GFP-positive fruits, and FaWRKY11 shows higher expression levels in mature fruits compared with immature fruits, suggesting a potential link between fruit development and disease resistance [79].

Similarly, overexpression of FvWRKY50 has been reported to enhance resistance to gray mold, possibly through coordinated regulation of JA biosynthesis, JA signaling components, and other defense-related pathways, including antimicrobial protein production. This functional analysis was conducted using octoploid strawberry fruits (Fragaria × ananassa) through Agrobacterium-mediated transient transformation, with Botrytis cinerea inoculation performed 3 days post-injection, and disease resistance was assessed by measuring lesion areas on fruit surfaces [80].

In the regulatory network underlying strawberry responses to Botrytis cinerea infection, different WRKY TFs have been reported to play distinct regulatory roles. FaWRKY25 has been reported to act as a negative regulator associated with JA-related defense signaling, and changes in its expression are correlated with altered expression of defense-related genes, such as PR, PGIP, and chitinase genes, as well as with changes in JA levels, thereby affecting disease resistance. These findings were obtained from experiments using strawberry fruits at different developmental stages, with post-harvest Botrytis cinerea inoculation and JA content measurements across stages [71]. These findings, however, are inferred from expression profiling and hormone measurements and lack direct functional validation.

In addition, FaWRKY29 and FaWRKY64 have been reported as negative regulators contributing to increased susceptibility to gray mold. Their effects have been linked to suppression of ABA- and JA-related signaling, impaired cell wall-associated defenses, and disturbance of reactive oxygen species (ROS) homeostasis, collectively weakening strawberry resistance to Botrytis cinerea. Loss-of-function mutations of these genes alleviate their negative regulatory effects and result in enhanced resistance. These functional analyses were carried out in octoploid cultivated strawberry (Fragaria × ananassa) using fruits at the white stage for Agrobacterium-mediated RNAi transient assay, with Botrytis cinerea inoculation performed five days post-injection on red-stage fruits, and disease resistance assessed post-harvest [9]. These findings are based on RNAi-mediated functional assays.

Moreover, FaSnRK1α has been shown to interact with FaWRKY33-2 in the SA-associated signaling context, contributing to enhanced resistance of strawberry fruit to Botrytis cinerea [81]. This regulatory interaction was identified through protein–protein interaction studies. In contrast, FaWRKY47, a member of the IIc subfamily, has been reported to be involved in multiple defense-related processes, including JA synthesis, activation of phenylpropanoid and anthocyanin metabolic pathways, and maintenance of ascorbic acid-glutathione (AsA-GSH) redox homeostasis, which together are associated with improved control of gray mold and delayed fruit senescence. This study was conducted using strawberry fruits at the white stage (Fragaria × ananassa cv. ‘Hongyan’) for Agrobacterium-mediated transient overexpression assays, with Botrytis cinerea inoculation performed at 96 h post-injection, followed by assessment of disease resistance and defense-related metabolic and antioxidant pathways [82]. These functions were elucidated using transient overexpression assays.

In summary, among these WRKY TFs, FaWRKY11 and FvWRKY50 act as positive regulators of resistance via JA signaling, whereas FaWRKY25, FaWRKY29, and FaWRKY64 function as negative regulators (susceptibility factors) associated with suppression of ABA/JA signaling, impaired cell wall defenses, and disturbed ROS homeostasis. FaWRKY33-2 contributes to resistance through SA-associated pathways, and FaWRKY47 operates via JA synthesis, phenylpropanoid/anthocyanin metabolism, and redox homeostasis.

The persistence of negative regulators such as FaWRKY25, FaWRKY29, and FaWRKY64 in the strawberry genome can be explained by their role in balancing the growth-defense trade-off (Section 1.2.1). By restraining excessive defense activation under pathogen-free conditions, they help prioritize resources for growth and fruit development. Although such regulators may reduce defense responses during pathogen attack, their context-dependent functionality is evolutionarily conserved to prevent autoimmunity and fitness penalties.

4.1.2. Defense Mechanism of the Strawberry WRKY Family Against Anthracnose

Strawberry anthracnose is another major fungal disease that severely affects strawberry production worldwide. This disease is caused by Colletotrichum species, which exhibit a hemibiotrophic lifestyle: an initial biotrophic phase followed by a destructive necrotrophic phase. This dual infection strategy often involves complex crosstalk between SA and JA signaling pathways [83]. Anthracnose outbreaks mainly occur during the nursery and early planting stages, leading to characteristic symptoms such as dark lesions on leaves and sunken necrotic spots on petioles and stolons. In severe cases, systemic infection can result in plant collapse and death. Under conditions of high temperature and frequent rainfall, anthracnose can spread rapidly and may destroy entire nurseries within one to two weeks [83].

In addition to their roles in resistance against Botrytis cinerea, WRKY transcription factors have been increasingly implicated in strawberry defense responses to anthracnose. Transcriptomic and expression analyses have shown that infection by Colletotrichum spp. induces the differential expression of multiple FaWRKY genes, including FaWRKY19, FaWRKY1, FaWRKY33-1, FaWRKY33-2, FaWRKY179, FaWRKY205, among others (Figure 3B).

Early studies demonstrated that FaWRKY1 (also referred to as FaWRKY24 in some reports) is inducible by multiple phytohormones, including ABA, SA, JA, and ET, and heterologous or transient expression assays suggested its potential involvement in immune-related regulatory networks [84]. However, functional analyses conducted within strawberry–Colletotrichum interaction systems revealed that FaWRKY1 acts as a negative regulator of anthracnose resistance, as gene silencing enhanced resistance, whereas overexpression led to increased susceptibility. This functional analysis was conducted using strawberry fruits (F. × ananassa cv. Primoris) at the pink/turning stage through Agrobacterium-mediated transient transformation with a same-fruit half-design, inoculating with Colletotrichum acutatum J.H. Simmonds (10⁵ conidia/mL) 2 days post-injection and assessing disease resistance 5 days post-inoculation [68]. Recent studies further reveal that the expressions of FaWRKY1 and FaWRKY19 are positively regulated by FaNPR3. This regulatory relationship was identified using strawberry fruits (F. × ananassa cv. Primoris) at the early red stage (approximately 25% pigmentation) through Agrobacterium-mediated transient transformation with a same-fruit half-design, followed by Colletotrichum acutatum inoculation and gene expression analysis [85], which has refined the upstream regulatory mechanism of FaWRKY1. This dual feature—broad hormone responsiveness yet negative defense outcome—suggests that FaWRKY1 may act as a hormonal integrator that fine-tunes defense responses rather than simply activating immunity. Its negative regulatory role could reflect an adaptive mechanism to restrain excessive defense activation under specific conditions, thereby prioritizing growth or fruit development in line with the growth-defense trade-off discussed in Section 1.2.1. In contrast, FaWRKY33-1 and FaWRKY33-2 are strongly induced upon Colletotrichum infection, suggesting their potential involvement in SA- and JA-associated defense pathways, although their precise regulatory roles remain to be functionally validated. These expression analyses were based on RNA-seq data from strawberry fruits at different developmental stages and leaves infected with Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde, as well as SA/MeJA-treated plants [10,86]. Comparative analyses using the strawberry cultivars ‘Benihoppe’ and ‘Sweet Charlie’ showed that the expression levels of FaWRKY179 and FaWRKY205 were significantly reduced following anthracnose infection, implying a possible role in ABA-related defense regulation. These findings were obtained through qRT-PCR analysis of leaves inoculated with Colletotrichum fructicola under controlled conditions, with samples collected at multiple time points post-inoculation, alongside samples from different fruit developmental stages. Notably, both genes are upregulated during fruit ripening, indicating potential dual functions in coordinating pathogen defense and fruit developmental processes. In addition, FaWRKY46, FaWRKY155, FaWRKY156, and FaWRKY115 have been proposed to negatively regulate anthracnose responses by modulating ABA signaling pathways [87]. Together with previous findings on the role of ABA in strawberry fruit development [88], these studies highlight the complex interplay between hormone-mediated defense signaling and developmental regulation during anthracnose infection.

Furthermore, transcriptomic analyses have expanded the landscape of WRKY involvement in strawberry anthracnose, identifying additional members such as FaWRKY24A/D, FaWRKY30A/B/D, FaWRKY38B, FaWRKY39A, FaWRKY43B.2/D, FaWRKY55D, FaWRKY60B, FaWRKY181, and FaWRKY207 as anthracnose-responsive genes [10,87]. Among these, FaWRKY1 stands out as the only functionally validated regulator, whereas FaWRKY46, FaWRKY155, FaWRKY156, and FaWRKY115 are supported by expression-hormone correlations but lack direct functional evidence. The remaining transcriptome-identified genes require initial expression validation before they can be prioritized for mechanistic studies. These findings collectively demonstrate that WRKY transcription factors are extensively involved in the regulatory networks underlying strawberry defense against anthracnose.

4.1.3. Defense Mechanism of the Strawberry WRKY Family Against Powdery Mildew

The Powdery mildew caused by Podosphaera aphanis is a major fungal disease affecting strawberry leaves, stems, flowers and fruits. As an obligate biotroph, P. aphanis derives nutrients from living host cells and typically triggers SA-mediated defense responses [89,90]. Infection inhibits photosynthesis, leading to leaf withering and premature senescence, while infected fruit rot does not typically occur; the commercial value of infected fruits is severely reduced [89].

WRKY TFs have also been implicated in the regulation of strawberry responses to powdery mildew (Figure 3C). Expression and functional analysis suggest that FaWRKY70 is associated with SA-related defense responses and may participate in SA-induced resistance to powdery mildew [90]. In addition, heterologous expression studies have shown that FvWRKY42 enhances powdery mildew resistance and ABA sensitivity in Arabidopsis thaliana, accompanied by activation of the antioxidant defense system and altered root growth, suggesting a potential conserved function for its strawberry homologs, although direct functional evidence in strawberry is still lacking [91]. Comparative evolutionary and expression analyses further indicate that the strawberry homologs of FvWRKY42 and FvWRKY20 are significantly upregulated upon powdery mildew infection, supporting their possible involvement in defense responses in strawberry, but this remains to be functionally validated [92]. Moreover, FvWRKY56 and FvWRKY27 exhibit transcriptional responses to powdery mildew inoculation and hormone treatment, implying potential functional similarities in strawberry to immune-related homologs such as AtWRKY46, AtWRKY70 and AtWRKY53 based on expression patterns, although direct confirmation is needed. Notably, FvWRKY50 and FvWRKY62 are rapidly induced during the early stage of infection and respond to multiple hormone treatments and abiotic stresses, suggesting that these TFs may be involved in coordinating defense-related signaling in strawberry under diverse conditions [93]. Recent studies have further shown that FvPR10.14 positively contributes to powdery mildew resistance via SA- and ROS-associated pathways. FaMYB63 and FvWRKY75 directly bind to the promoter of FvPR10.14 and activate its transcription. In addition, FvWRKY75 negatively regulates FaMYB63 expression [94].

4.2. The Defense Mechanisms of the Strawberry WRKY Family Against Other Biotic Stresses

4.2.1. Major Harmful Organisms Affecting Strawberry Growth

During growth and cultivation, plants are frequently challenged by various arthropod pests, among which Aphids (Aphidoidea), spider mites (Tetranychus spp.) and thrips are the most prevalent. Aphids are typical piercing-sucking insects that preferentially colonize young leaves and growing points, where they impair plant development. In addition, aphids serve as important vectors for the transmission of strawberry viruses, thereby increasing the incidence and complexity of viral diseases [95,96].

Spider mites primarily feed on the abaxial side of leaves, causing chlorotic spotting, reduced photosynthetic capacity, and premature leaf senescence. Due to their rapid population growth and high potential for pesticide resistance, spider mites are considered one of the most difficult pest groups to manage in protected cultivation systems [95,97]. Collectively, those biotic stresses impose substantial pressure on strawberry growth and productivity, highlighting the importance of understanding plant defense regulatory mechanisms, including those mediated by WRKY TFs in strawberry.

4.2.2. Candidate WRKYs Associated with Mite Response in Strawberry

In recent years, transcriptomic and expression analyses have identified several WRKY genes as potential candidates involved in strawberry response to mite infestation. Expression profiling of group III FaWRKY genes following mite infestation revealed that FaWRKY25, FaWRKY32, FaWRKY43, FaWRKY44, and FaWRKY45 were significantly upregulated, while FaWRKY31 showed sustained downregulation. Notably, this expression pattern was similar to that observed under ABA treatment, suggesting a possible association between mite-induced response and ABA-related signaling [16]. It should be noted that the focus on group III WRKY genes in this section reflects the scope of the available study; whether other WRKY groups contribute to mite defense in strawberry remains to be investigated. Based on expression pattern and comparative analyses, FaWRKY25 has been proposed to be associated with increased susceptibility to mite infection. This hypothesis is supported by functional analogies to CmWRKY53 in chrysanthemum [98] and AtWRKY22 in Arabidopsis, which have been reported to modulate aphid sensitivity through regulation of hormone-related signaling pathway or secondary metabolism [99]. In addition, exogenous SA treatment induces the expression of FaWRKY31, FaWRKY32, FaWRKY44, and FaWRKY45, indicating that group III FaWRKY genes are broadly responsive to immune-related hormone cues. Other upregulated genes, including FaWRKY32, FaWRKY43, FaWRKY44, and FaWRKY45, have therefore been suggested as putative contributors to mite-related defense responses, with a functional pattern partially resembling those of tomato SlWRKY41 and SlWRKY54, which are implicated in antiviral defense (Figure 3D).

Collectively, these findings provide a systematic overview of the transcriptional response of group III FaWRKY genes under mite stress and offer insights into their potential involvement in strawberry- mites interaction. Rather than indicating uniform regulatory functions, the data suggest diverse and context-dependent roles of group III WRKY TFs in biotic stress responses. Among them, FaWRKY25 represents a potential candidate for further functional characterization and breeding-oriented studies aimed at improving pest resistance [16]. Future investigations combining a genome-editing approach will be required to clarify the regulatory pathway and physiological functions.

4.3. WRKY Transcription Factors in Response to Complex Field Stresses: The Case of Continuous Cropping

In addition to pest infestations, continuous cropping (CC) represents a major constraint on strawberry yield and quality, posing a serious challenge to sustainable strawberry production and resulting in substantial agricultural losses worldwide [100]. Long-term CC leads to complex alterations in soil structure and microenvironment, exposing strawberry plants to multiple biotic stresses, such as accumulation of soil-borne pathogens and plant parasitic nematodes, as well as abiotic stresses, including nutrient imbalance, deterioration of soil physicochemical properties, and the accumulation of autotoxic compounds [101].

Transcriptomic analyses investigating the strawberry response to CC stress have suggested that several group III FaWRKY members, including FaWRKY25, FaWRKY32, and FaWRKY45, may be associated with CC-related stress responses. These genes show altered expression patterns and enrichment in plant hormone-related signaling pathways and plant-pathogen interaction pathways, indicating their potential involvement in complex defense responses triggered by CC [102]. These expression patterns suggest a possible association between these genes and CC responses, although their functional roles remain to be experimentally validated. Sequence alignment analysis further reveals that these FaWRKY genes share high sequence similarity with defense-related WRKY TFs in Arabidopsis, such as AtWRKY53, AtWRKY70, and AtWRKY41 [103,104]. While these Arabidopsis homologs are known to participate in defense regulation, the functional significance of their strawberry counterparts under CC stress has not yet been determined. In Arabidopsis, WRKY TFs are generally activated downstream of the MAPK cascade and regulate the expression of defense-related genes, including pathogenesis-related (PR) proteins, peroxidases, and hormone-responsive genes [35,105]. For example, AtWRKY70 is known to participate in SA and JA crosstalk pathways, while AtWRKY41, AtWRKY53, and AtWRKY70 are induced by SA. These well-characterized regulatory features in Arabidopsis provide a comparative framework for understanding the potential role of homologous FaWRKY genes in strawberry responses to CC stress.

Under CC conditions, the expression levels of FaWRKY25, FaWRKY32, FaWRKY33, and FaWRKY45 are significantly altered [69]. These WRKY TFs possess conserved W-box binding domains, consistent with their role in transcriptional regulation networks. Based on sequence similarity to AtWRKY70, it has been speculated that FaWRKY32 might participate in SA-related regulation under CC stress, but this hypothesis requires direct functional testing [106]. Consistent with this hypothesis, the expression of several SA-associated genes, including peroxidase-and amine oxidase-encoding genes, is elevated under CC conditions.

Continuous cropping stress is known to alter soil microbial community composition and often leads to the accumulation of soil-borne pathogens, which can suppress plant growth and increase disease pressure. In response to pathogen challenge and other stress signals, plants can activate ROS-associated defense pathways as signaling components of immune responses, although ROS involvement varies with context and cannot be assumed in all cases. Transcriptional regulators such as FaWRKY32 linked to SA signaling may modulate downstream defense genes and effector components, enhancing antimicrobial capacity under continuous cropping stress through hormone signaling and plant–pathogen interaction pathways [69,106,107].

Overall, the current evidence for WRKY involvement in strawberry CC responses is primarily based on transcriptomic and comparative analyses. These findings provide a basis for prioritizing candidate genes for future functional studies aimed at improving continuous cropping tolerance (Figure 3D).

5. The Function of WRKY Transcription Factors in Strawberry Abiotic Stress

In addition to biotic stress, WRKY transcription factors are also deeply involved in responses to abiotic stresses in strawberry, including salinity, drought, and low temperature. Elucidating the regulatory mechanisms underlying WRKY-mediated abiotic stress responses and identifying key regulatory genes are essential for advancing molecular breeding strategies and enhancing strawberry tolerance to adverse environmental conditions.

5.1. Involvement of WRKY Genes in Salt and Drought Stress Responses in Strawberries

Excessive salinity disrupts ionic homeostasis and cellular osmotic balance in plant cells, severely affecting photosynthesis and cellular energy metabolism. Plant salt tolerance is a complex trait regulated by multiple genes. As sessile organisms, plants respond to drought stress through coordinated mechanisms, including osmotic adjustment, hormone signal reprogramming, and metabolic reconfiguration.

Both salinity and drought induce osmotic stress in plants, particularly during early stress perception, leading to partially overlapping response mechanisms [108,109]. Prolonged salt stress further results in ionic toxicity and nutrient imbalance.

Plant salt tolerance is a complex trait regulated by multiple genes. WRKY TFs have been widely implicated in plant responses to both drought and salinity stresses [39]. In woodland strawberry, 62 FvWRKY genes have been identified, and approximately 70% of them show differential expression under drought and NaCl treatments. During drought stress, FvWRKY23 is strongly induced, whereas FvWRKY27, FvWRKY41, and FvWRKY42 are mainly upregulated from the middle to late stages. Notably, FvWRKY42 exhibits a rapid and robust response to drought, while FvWRKY27 responds most rapidly under NaCl treatment [93].

In octoploid strawberries, FaWRKY40 has been reported as a positive regulator associated with salt tolerance. Functional analyses suggest that FaWRKY40 binds to the promoters of salt-responsive genes, including FaRbohD, FaSOS1, and FaNHX1, and is associated with enhanced ROS-related signaling and Na⁺ homeostasis in roots under salt stress. In contrast, FaWRKY70 is proposed to act as a negative regulator of salt tolerance. Its expression is induced by salt stress but suppressed by exogenous ALA treatment. Under such conditions, increased expression of FaWRKY40, FaNR1, and FaHKT1 has been observed, which correlates with improved salt tolerance [110] (Figure 4B). In addition, a recent study found that when exogenous γ-aminobutyric acid (GABA) is applied, FaWRKY46, FaWRKY51, and FaWRKY70 may positively participate in GABA-induced strawberry adaptation to salt stress by regulating chlorophyll metabolism, thereby enhancing salt tolerance in strawberry seedlings [111,112] (Figure 4A). It should be noted that the “FaWRKY70” described in these studies may refer to different genes under distinct naming systems, which could explain the contrasting regulatory roles reported. Functional characterization of drought-responsive WRKY genes in octoploid strawberry remains limited, representing an important avenue for future investigation. The antagonistic relationship between FaWRKY40 and FaWRKY70 raises important questions regarding the evolutionary and functional organization of WRKY-mediated stress responses. This regulatory configuration—wherein closely related WRKY family members exert opposing effects on a common stress pathway—may represent a conserved mechanism for fine-tuning stress outputs. In Arabidopsis, WRKY40 and WRKY70 have been implicated in both abiotic and biotic stress responses, with WRKY70 functioning as a node of convergence between SA- and JA-mediated defense signaling [113,114]. WRKY40, together with WRKY18 and WRKY60, forms a regulatory network modulating ABA-responsive gene expression [113]. In Pak-choi, WRKY40 and WRKY70 act as key regulators within co-regulatory networks mediating responses to multiple abiotic stresses [115]. However, whether the specific antagonistic module observed in strawberry represents a salinity-specific adaptation or reflects a more widely conserved regulatory paradigm remains to be determined. Comparative functional studies across diverse species and stress contexts will be essential to establish whether WRKY40/WRKY70 antagonism constitutes a general regulatory module for stress homeostasis or a strawberry-specific innovation in salt tolerance mechanisms.

Stomatal closure represents a conserved physiological response to both salt and drought stress, reducing transpirational water loss, thereby maintaining water balance, but it also limits gas exchange and reduces photosynthetic efficiency [116]. This process is primarily mediated by ABA signaling in guard cells and involves multiple signaling components such as ROS, reactive carbonyl compounds (RCS), nitric oxide (NO), and Ca²⁺ (Figure 4). ABA plays a central role in coordinating physiological responses to salt and drought stress, with WRKY transcription factors emerging as potential integrators of ABA-mediated signaling networks. In diploid strawberries, FvWRKY75 has been reported to be associated with enhanced salt tolerance through modulation of antioxidant capacity, ROS homeostasis, and the expression of salt stress-responsive genes [117], suggesting this WRKY may function within ABA-regulated ROS signaling cascades that mediate stress adaptation. ABA also plays a central role in regulating root architecture under salt and drought conditions. Elevated endogenous ABA levels under moderate to high salinity (75–150 mM NaCl), suppress lateral root formation and promote the development of the Casparian strip in the endodermis, thereby restricting Na⁺ diffusion into the stele [118,119]. Under drought stress, ABA-mediated primary root elongation is associated with activation of H⁺-ATPase-dependent auxin transport, sustaining water and nutrient uptake from deeper soil layers [120]. Transcriptome analysis identified 11 FvWRKY genes (FvWRKY23, 24, 27, 34, 35, 41, 42, 44, 50, 56, and 62) that respond to both salt and drought stress. Among them, FvWRKY42 shows strong induction and has been implicated in ABA-associated signaling, antioxidant responses, and root growth regulation, suggesting a potential role in coordinating drought and salinity tolerance. Notably, FvWRKY42 is highly homologous to AtWRKY33, while FvWRKY46 shares homology with AtWRKY40, indicating functional conservation in WRKY-mediated stress regulatory networks [93] (Figure 4A,C,D). In Arabidopsis, WRKY33 and WRKY40 integrate ABA, ROS, and pathogen signaling pathways, raising the possibility that their strawberry orthologs may similarly coordinate multiple stress response outputs. However, direct experimental evidence linking these FvWRKYs to specific ABA-mediated physiological processes—such as guard cell regulation, Casparian strip deposition, or root elongation—remains to be established. This integrated framework provides testable hypotheses for future functional characterization of WRKY-mediated stress tolerance mechanisms in strawberry (Figure 4).

These findings indicate that WRKY TFs are closely associated with ABA-related signaling, ROS homeostasis, and broader stress-related signaling networks involved in salt and drought responses [121,122,123,124,125]. However, the depth of functional characterization varies considerably between stress types and ploidy levels, with salinity response mechanisms in octoploid strawberry being substantially better understood than drought response mechanisms in this economically important crop.

5.2. WRKY Gene Family in Strawberries: Regulation of Cold Stress

To cope with temperature fluctuations, plants have evolved complex signal transduction mechanisms that regulate metabolic states and cellular functions, thereby enhancing tolerance to cold stress. Cold stress, including both chilling (>0 °C) and freezing (<0 °C) conditions, is a major abiotic factor limiting strawberry production. Previous studies have shown that WRKY TFs participate in plant cold stress responses by modulating ABA-associated signaling pathways and the expression of cold-responsive (COR) genes, suggesting important regulatory roles in cold tolerance [121]. However, functionally validated examples in strawberry remain limited.

Accumulation of ROS is a common feature of plants exposed to prolonged biotic or abiotic stress. Among ROS, hydrogen peroxide (H₂O₂) functions as a key signaling molecule that initiates stress-responsive signal cascades, whereas excessive ROS accumulation causes oxidative damage and lipid peroxidation, ultimately inhibiting plant growth [126,127,128,129]. In strawberries, current evidence for WRKY involvement in cold stress is predominantly derived from transcriptome analyses. Transcriptome analyses revealed that low temperature treatment (4 °C) induces the expression of several FvWRKY genes, including FvWRKY24, FvWRKY27, FvWRKY41, FvWRKY50, FvWRKY56, and FvWRKY62, indicating their potential involvement in cold stress responses [93] (Figure 4C). These genes can therefore be considered cold-responsive candidate WRKYs meriting further investigation.

Whether the cold-responsive FvWRKY candidates participate in ROS homeostasis or antioxidant gene regulation under low temperature stress remains unexplored in strawberry.

5.3. Emerging Abiotic Stress Responses with Limited Evidence

Beyond salinity, drought, and cold stress, strawberry is also exposed to additional abiotic factors including heat, nutrient imbalance, ultraviolet radiation, and prolonged darkness. Current evidence for WRKY involvement in these responses remains preliminary and uneven across stress types. Heat stress at 42 °C significantly induces FvWRKY34 expression, suggesting a potential role in thermotolerance. Additionally, FvWRKY27, FvWRKY35, and FvWRKY50 respond to multiple hormone treatments, indicating their potential roles in the hormone-associated stress signaling networks.

Notably, FvWRKY50 and FvWRKY62 are responsive to both hormone treatments and diverse abiotic stresses, suggesting that these WRKY TFs may act as integrators of multi-stress and hormone signaling networks rather than regulators of single stress pathways [93] (Figure 4C). However, functional validation for their roles in responses to ultraviolet radiation, nutrient deficiency, or darkness stress is currently lacking. These emerging environmental factors represent important frontiers for future research, particularly given their increasing relevance under changing climatic conditions and controlled environment agriculture systems (

Table 4. Key Information of Strawberry WRKY Transcription Factors in Plant Defense.

Homologous Genes in Arabidopsis	Gene	Defense Type	Mechanism	Pathway	References
AtWRKY31	FaWRKY11	Botrytis cinerea	Positive regulator; activates defense genes via JA pathway.	JA	[79]
AtWRKY25/33/26	FaWRKY19	Colletotrichum spp.	Negative regulator; expression positively regulated by FaNPR3.	SA	[85]
AtWRKY75	FaWRKY1 (FaWRKY24)	Colletotrichum spp.	Negative regulator of anthracnose (context-dependent).	ABA, SA, JA, ET	[68,84,85]
AtWRKY25	FaWRKY25	Botrytis cinerea, Mite, Continuous cropping	Negative regulator via JA pathway (B. cinerea); May enhance susceptibility; expression upregulated by ABA(Mite); Upregulated; mediates stress response via hormone signaling [59].	SA, ABA, JA	[69,71,98,101,102]
AtWRKY53	FaWRKY29	Botrytis cinerea	Negative regulator/susceptibility factor; inhibits ABA/JA signaling, ROS homeostasis.	ABA, JA, ROS	[9]
AtWRKY54	FaWRKY31	Mite	Expression is continuously downregulated under stress, possibly due to negative regulatory factors.	ABA	[16]
AtWRKY54	FaWRKY32	Mite, Continuous cropping	Resistance to CC, putative positive regulator (induced by mite infestation; ABA-associated expression pattern)	ABA, SA	[16,69,102]
AtWRKY33	FaWRKY33	Continuous cropping	May be resistant to CC	-	[69]
AtWRKY33	FaWRKY33-1	Colletotrichum spp.	Expression is significantly induced upon infection, suggesting potential involvement in SA/JA-mediated defense regulation. Its specific regulatory role requires further functional validation.	SA, JA	[10,86]
AtWRKY25/33/26	FaWRKY33-2	Botrytis cinerea, Colletotrichum spp.	Interacts with FaSnRK1α via SA pathway to enhance resistance to B. cinerea. Induced upon Colletotrichum infection.	SA, JA	[81,86]
AtWRKY55	FaWRKY43	Mite	ABA may contribute to the expression of FaWRKY Group III genes; ABA content may be enhanced due to mite infestation.	ABA, SA	[16]
AtWRKY54	FaWRKY44	Mite	ABA may contribute to the expression of FaWRKY Group III genes; ABA content may be enhanced due to mite infestation.	ABA, SA	[16]
AtWRKY41	FaWRKY45	Continuous cropping, Mite	May be resistant to CC, negative regulator of mite resistance.	SA, JA, ABA	[16,69,102]
AtWRKY57	FaWRKY47	Botrytis cinerea	Promote JA synthesis and upregulate phenylpropanoid/flavonoid defense pathways	JA	[82]
AtWRKY53	FaWRKY64	Botrytis cinerea	Negative regulator of Botrytis cinerea resistance.	ABA, JA, ROS	[9]
AtWRKY72	FaWRKY179	Colletotrichum spp.	Expression suppressed upon infection; may coordinate defense and fruit ripening via ABA.	ABA	[87]
AtWRKY68	FaWRKY181	Colletotrichum spp.	May be a key switch gene regulating the disease resistance/susceptibility response.	ABA	[87]
AtWRKY68	FaWRKY205	Colletotrichum spp.	Expression suppressed upon infection; may coordinate defense and fruit ripening via ABA.	ABA	[87]
AtWRKY66	FaWRKY207	Colletotrichum spp.	May be of great importance in maintaining resistance.	ABA	[87]
AtWRKY33	FvWRKY20	Podosphaera aphanis	Upregulated after infection; likely functions similarly to AtWRKY33.	-	[92]
AtWRKY53	FvWRKY27	Podosphaera aphanis	Responds to powdery mildew and hormones; similar to AtWRKY53.	-	[93]
AtWRKY33	FvWRKY42	Podosphaera aphanis	Positive regulation of ABA signaling pathway, antioxidant defense system and root growth, significantly enhance the resistance of Arabidopsis to powdery mildew; abiotic (salt/drought) tolerance and ABA sensitivity.	SA, JA, ABA, ET	[92]
AtWRKY50/75	FvWRKY50	Botrytis cinerea, Podosphaera aphanis	Positive regulatory factors of gray mold resistance.	JA (and possibly integrates multiple signals)	[80,93]
AtWRKY54/70	FvWRKY56	Podosphaera aphanis	Responds to infection and hormones; similar to AtWRKY46/70/53.	-	[90,93]
AtWRKY75	FvWRKY62	Podosphaera aphanis	Rapidly activated early in infection; integrates multiple signals.	-	[93]
AtWRKY70	FaWRKY70	Podosphaera aphanis	May act in SA-induced defense network	SA	[90]
AtWRKY40	FaWRKY40	Salinity stress	Positive regulation of plant salt tolerance	NO-H2O2	[110]
AtWRK40, 46	FaWRKY46, 51	Salinity stress	Activate antioxidant genes	GABA	[112]
AtWRKY70	FaWRKY70	Salinity stress	inhibit the expression of FaWRKY40 to increase salt sensitivity.	NO-H2O2	[110]
AtWRK75	FvWRKY75	Salinity stress	Salt stress positive regulatory factors enhance the activity of the antioxidant system, regulate ROS clearance, and upregulate stress-related genes to improve salt tolerance.	ROS	[117]
AtWRK71, 3, 53, 33, 75, 22, 54/70, 41/53, 75	FvWRKY15, 24, 27, 41, 50, 53, 56, 59, 62	Cold stress	Potential regulation of plant response to low temperatures	ROS, ABA	[93]
AtWRKY57, 3, 53, 25, 54, 33, 61, 44, 75, 54/70, 75	FvWRKY23, 24, 27, 34, 35, 41, 42, 44, 50, 56, 62	Salinity stress, Heat stress	FvWRKY42 positively regulates the ABA signaling pathway, antioxidant defense system, root growth, tolerance to abiotic stress (salt/drought), and ABA sensitivity. Other genes may also potentially positively regulate responses to drought and salt stress.	SA, JA, ABA, ET	[93]
AtWRKY53, 54, 75	FvWRKY27, 35, 50	Hormone response	Genes are significantly upregulated, potentially regulating plant sensitivity to hormones.	ABA, SA, JA	[93]
AtWRKY25	FvWRKY34	Heat stress	Genes are significantly upregulated, potentially playing a role in regulating the response to heat stress.	ROS, ABA	[93]

).

6. WRKY Transcription Factors in Strawberry Growth, Fruit Development and Quality Formation

Beyond their roles in stress responses, WRKY transcription factors also participate in the regulation of fruit development and quality in strawberry—processes intimately linked to stress adaptation in this horticultural crop.

6.1. WRKY TFs in Fruit Development and Ripening

Fruit development in strawberry involves coordinated genetic programs from fertilization through maturation. Transcriptomic analyses have shown that multiple WRKY genes are differentially expressed during the transition from green to white to red stages. Functional studies have begun to elucidate their roles: FvWRKY48 has been identified as a regulator of fruit softening by directly repressing pectin degradation genes [55]. Similarly, FaWRKY50 and FaWRKY61 exhibit ripening-associated expression patterns and may integrate hormonal signals during fruit maturation [2,10]. These findings indicate that WRKY TFs act as key transcriptional regulators of the ripening program.

6.2. WRKY TFs Regulating Fruit Quality Traits and the Interplay with Stress Responses

Fruit quality in strawberry encompasses color, flavor, texture, and post-harvest longevity. WRKY transcription factors contribute to these traits through diverse mechanisms. For coloration, WRKY genes regulate anthocyanin biosynthesis through multiple mechanisms. FvWRKY50 directly upregulates the expression of anthocyanin structural genes by binding their promoters, while FaWRKY71 promotes anthocyanin accumulation by activating structural genes in the flavonoid pathway [2,54]. For sugar-acid metabolism, expression correlations have been observed between certain WRKYs and genes involved in sucrose and organic acid pathways [93]. Post-harvest shelf life is influenced by WRKY-mediated control of cell wall modification during storage [9,55].

The dual involvement of WRKY TFs in stress defense and fruit quality highlights their role as integrators of the growth-defense trade-off discussed in Section 1.2.1. Activation of stress responses often compromises fruit quality [9,82], and WRKY proteins positioned at the intersection of these pathways may serve as molecular switches balancing resource allocation between defense and reproduction. Understanding how specific WRKY genes coordinate these competing demands is essential for breeding strawberry varieties with robust stress tolerance and superior fruit quality.

7. Conclusions and Prospects

WRKY transcription factors are pivotal regulators in plant defense regulation. Foundational studies in model species such as Arabidopsis and rice have established a well-defined theoretical framework for WRKY-mediated immunity. These works have highlighted conserved mechanisms by which WRKYs balance plant growth and defense, largely through the integration of phyto-hormone signals, particularly SA and JA. This conceptual foundation provides a critical basis for exploring WRKY function in horticultural crops.

In this review, we summarized current understanding of strawberry WRKY transcription factors, elucidating how they coordinate stress responses, hormone signaling, and fruit trait development. In horticultural crops like strawberries, WRKY genes exhibit pronounced species specificity. They not only contribute to disease resistance and stress tolerance but also broadly regulate the formation of quality traits including fruit color and flavor, and their expression is often tightly controlled in tissue- and developmental stage-specific manner.

Despite these advances, the functions of many WRKY TFs still remain unresolved due to functional redundancy and the complexity of polyploid genomes. In particular, the mechanisms by which individual WRKY members coordinate disease resistance with fruit quality formation are poorly understood. Future research should therefore focus on: (i) systematic functional dissection of strawberry WRKY genes using gene editing and related technologies; (ii) construction of WRKY-centered co-regulation networks integrating multi-omics data; (iii) cross-species comparative analyses to distinguish evolutionarily conserved functions from species-specific regulatory innovations.

In summary, situating strawberry WRKY research within the conceptual framework of “from model systems to horticultural crops” not only deepens our understanding of biological functions of WRKY but also reveals a broader TF-mediated regulatory paradigm coordinating growth, defense, and quality traits. A deeper understanding of the bidirectional regulation between stress-response signaling and growth-related pathways, together with elucidation of the hub role of WRKY transcription factors within this complex network, may provide new opportunities to recalibrate the trade-off [22] between stress tolerance and growth, ultimately facilitating the development of stress-tolerant, high-yielding crop varieties.