Multifaceted roles of WRKY transcription factors in abiotic stress and flavonoid biosynthesis

Increasing biotic and abiotic stresses are seriously impeding the growth and yield of staple crops and threatening global food security. As one of the largest classes of regulators in vascular plants, WRKY transcription factors play critical roles governing flavonoid biosynthesis during stress responses. By binding major W-box cis-elements (TGACCA/T) in target promoters, WRKYs modulate diverse signaling pathways. In this review, we optimized existing WRKY phylogenetic trees by incorporating additional plant species with WRKY proteins implicated in stress tolerance and flavonoid regulation. Based on the improved frameworks and documented results, we aim to deduce unifying themes of distinct WRKY subfamilies governing specific stress responses and flavonoid metabolism. These analyses will generate experimentally testable hypotheses regarding the putative functions of uncharacterized WRKY homologs in tuning flavonoid accumulation to enhance stress resilience.

In sweet potato (Ipomoea batatas), after the inaugural gene was found (Ishiguro and Nakamura, 1994), WRKYs have been discovered extensively in terrestrial plants, primitive protozoans and slime molds, affirming the ancient evolutionary origin (Zhang and Wang, 2005;Pan et al., 2009).Highly conserved WRKY structural domains paralleling the zinc finger (ZF) motif is an essential feature of this protein family.The WRKY domain directly bind to the major cis-element, W-box (TGACCA/T) (Rushton et al., 2010;Chen et al., 2019).Although slight variations in some WRKYs, the conserved motif (WRKYGQK) forms the ZF to confer structural stability (Yamasaki et al., 2005;Cheng et al., 2021).
Phylogenetically, WRKYs are classified into three distinct groups depended on the number of WRKY domains together with the type of ZFs.Two WRKY domains and a C2H2 (CX4-5CX22-23HXH) ZF are present in Group I, whereas only one WRKY domain combined with C2H2 or altered C2HC (CX7CX23HXC) ZF appears in Groups II and III (Eulgem et al., 2000;Li et al., 2010;Chen et al., 2020).According to the variation of ZF, Group II can be subclassified into subgroups IIa ~IIe (Zhang and Wang, 2005).The presence of Group I WRKYs in primitive organisms suggests that they are the ancestors of other groups (Wei et al., 2018).WRKY domains are conserved, but different WRKY groups have evolved specialized functions (He et al., 2016;Wang et al., 2022).
Although the functional roles of plant WRKY transcription factors in stress responses are well-documented, the relationship between sequence divergences across distinct WRKY domains and their varying biological activities is still unclear.In order to fully understand the functional specificity encoded within different WRKY phylogenetic clades, further investigation is necessary Furthermore, as critical homeostatic regulators of ROS, numerous flavonoid biosynthetic genes harbor abundant W-box cis-elements within their promoters (Liu et al., 2019a;Su et al., 2022a).Growing evidence suggests that specific WRKY proteins play pivotal roles governing flavonoid metabolism to enhance plant stress adaptation (An et al., 2019;Wang et al., 2022).However, the specific contributions of individual members of the WRKY subfamily in directing flavonoid accumulation are not well understood.In this review, we will refine WRKY phylogenetic frameworks by incorporating additional plant species with WRKY genes that have been implicated in stress tolerance and flavonoid regulation.Through these analyses, we aim to elucidate the roles of specific WRKY clades in modulating flavonoid biosynthesis under defined abiotic stresses.
The phylogenetic analysis showed that the 816 WRKYs were segregated into three canonical groups, designated as Roman numerals (I, II, III), with seven distinct groups and represented by colored outer circles (Figure 1), consistent with the phylogenetic system defined by Zhang and Wang (2005).Further examination reveals that Group II can be categorized into five unique subdivisions: IIa, IIb, IIc, IId, and IIe (Figure 1).Subgroup I, with 172 members, is the most numerous of the 7 subgroups.Whereas groups IIa contained only 49 members.The further division of Group II into 5 subgroups is due to specific sequence variations in the ZF motifs (Xie et al., 2005).
When compared to Zhang and Wang's system (Zhang and Wang (2005)), three WRKY proteins of the present phylogenetic analysis had distinct classes.For example, AtWRKY10, AtWRKY19, AtWRKY45 from subgroup 1 are reassigned to II c, II c, and III, respectively.
3 WRKYs bind to specific cis-acting regions of target genes A platform for systematic investigations of WRKY family is provided by the expanding genome sequencing data from many plant species.The identification of genes crucial for specific processes has been made possible by genome sequences, which have enabled transcriptome profiling of some families (such as WRKY) during particular situations (Arndt et al., 2022;Shende et al., 2022;Zhang et al., 2022e).Furthermore, the genome-wide mapping of WRKY binding sites and target genes has been made possible by high-throughput sequencing after chromatin immunoprecipitation (ChIPseq) (Zhou et al., 2021;Xu et al., 2022).These cutting-edge approaches have profoundly expanded the understanding of WRKY gene functions and regulatory networks.
The C-terminus contains the ZF structure C2H2 or C2HC, while the WRKYGQK motif, which forms the core region, is present at the N-terminus (Yamasaki et al., 2005;Chen et al., 2010;Song et al., 2018;Gu et al., 2019;Wang et al., 2021a).Some WRKY proteins also contain WRRY, WSKY, WKRY, WVKY, or WKKY motifs in place of the WRKY domain (Xie et al., 2005;Duan et al., 2007;Li et al., 2020b).The binding of DNA by WRKY proteins necessitates the ZF motif (Phukan et al., 2017).Replacing the zinc ion in WRKY domains with the metal chelator 1,10-phenanthroline abolishes DNA binding, indicating that WRKY proteins possess ZFs structures.The WRKY domain is composed of four b-sheets, a zincbinding pocket, and coordination with cysteine or histidine residues make up the WRKY domain.The C-terminal of the WRKY domain stabilizes the sequence-specific interaction between the projecting N-terminal WRKYGQK sequence and the 6 bp DNA groove.Therefore, WRKY proteins may attach to the W-box clusters, primarily the TTGACC/T motif, in promoters of target genes.This enables them to regulate the dynamic network of signals and responses (Yamasaki et al., 2005;Li et al., 2020b).
The core TGAC sequence is highly conserved and principally responsible for binding by WRKY proteins (Mare et al., 2004;Ciolkowski et al., 2008;Rahman et al., 2021).In contrast, variations in the number, sequence, and nucleotide composition of the flanking bases in W-boxes of target genes contribute to the binding specificities of different WRKYs (Cheng et al., 2019).
Indeed, most target genes of WRKY were discovered to contain Wbox cis-elements in the promoters, as identified through various approaches.Chromatin immunoprecipitation analyses revealed that Parsley (Petroselinum crispum) WRKY1 binds to W-boxes in the promoters of PcWRKY3 and Pathogenesis-Related1-1 (PcPR1-1) (Turck et al., 2004), and pepper (Capsicum annuum) WRKY6 was able to bind to the W-boxes of the CaWRKY40, as well as defense genes Capsicum annuum defensin 1 (CaDEF1), EXTRACELLULAR PEROXIDASE 2 (CaPO2), and small heat shock protein 24 (CaHSP24) (Hussain et al., 2018).Furthermore, pull-down assays was used to identify candidate W-box containing genes (AtWRKY58, Phylogenetic analysis of identified WRKY proteins in Arabidopsis thaliana, Glycine max, Oryza sativa, Populus trichocarpa and Zea mays.A phylogenetic tree was established using the MEGA 7.0 program and the neighborhood joining method, drawing on WRKY domain sequences.Different colors have been utilized to distinguish between various groups or subgroups.WRKY protein sequences were obtained from NCBI (https://www.ncbi.nlm.nih.gov)database.The 72 proteins highlighted in the inner circle originate from Arabidopsis thaliana (Supplementary Table S1), while WRKY genes from Groups I to III are color-coded in Supplementary Table S2.The functional categories of WRKY, annotated in this review, are identified by black asterisks in Supplementary Table S3.Supplementary Table S4 provides a comprehensive list of the protein IDs and full sequences for all analyzed WRKYs (Jeyasri et al., 2021;He et al., 2012;Zhang et al., 2017;Yu et al., 2016).
In essence, WRKY takes part in a variety of regulatory processes by regulating the expression of various cis-elements of target genes (Table 1).Current studies on the cis-elements of WRKY has primarily focused on six species, including Oryza sativa, Arabidopsis thaliana, Petroselinum crispum, Hordeum vulgare, Nicotiana tabacum and Capsicum annuum.It appears that members of WRKY subgroup 2e have a preference for the PRE4 element.WRKYs in subgroup 1 typically bind to the sulfurresponsive element (SURE), while the those in subgroup 3 interact with both the WT-box and the traditional W-box.

Regulatory mechanism of WRKYs involved in their transactivation capabilities
Extensive researches unveiled that plant WRKYs were crucial orchestrators involved in two branches of plant innate immunity (PII) (Jones and Dangl, 2006).
Upon perception of PAMPs, the mitogen-activated protein kinase (MAPK) cascade is activated, leading to upregulation of AtWRKY22 and AtWRKY29, which are two positive regulators from 2e subgroup (Asai et al., 2002;Hsu et al., 2013).Certain WRKYs also fulfill indispensable regulatory roles in ETI (Adachi FIGURE 2 Various mechanisms of WRKY transcriptional activation regulation.WRKY transcription factors are regulated via a plethora of mechanisms.These range from binding to an assortment of cis-regulatory elements such as SURE, P1BS, and PRE4, to post-translational modifications including ubiquitination, methylation, and phosphorylation, which either activate them or mark them for degradation.Regulation may also involve altered chromatin conformations mediated by changes in histone acetylation or methylation, as well as modulation via interactions with other nuclear proteins like WRKY and ERF.Zhang et al. 10.3389/fpls.2023.1303667Frontiers in Plant Science frontiersin.orget al., 2015).A plethora of WRKY genes are auto-regulated or crossregulated through W-box elements embedded in the promoters (Robatzek and Somssich, 2002;Turck et al., 2004;Besseau et al., 2012;Yang et al., 2012;Adachi et al., 2015).For instance, CaWRKY6 activates CaWRKY40 to elevate pepper tolerance to heat and high humidity (Cai et al., 2015).Post-translational modifications, especially phosphorylation, constitute key mechanisms orchestrating the transactivation capability of WRKYs.MAPKs phosphorylate WRKY proteins, either the DNA binding affinity is directly or indirectly altered, or the transcriptional activity is altered (Miao et al., 2007;Adachi et al., 2015).Prolonged MAPK activation increases the proportion of phosphorylated WRKYs (Figure 2), amplifying downstream signaling (Ishihama et al., 2011).For example, a MAPK kinase kinase 1 (MEKK1) phosphorylates AtWRKY53 to increase its DNA binding activity (Miao et al., 2007).In addition, MAPK 4 (MPK4) phosphorylates MKS1 (MAP kinase substrate 1) to liberate AtWRKY33 from the MKS1-WRKY33 complex, so as to targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) for the synthesis of antimicrobial camalexin (Qiu et al., 2008).
In conclusion, WRKYs emerged as pivotal regulators in regulating plant responses to the environment.The diverse functions are fine-tuned through intricate interaction networks and post-translational modifications.Further elucidating these regulatory mechanisms will provide valuable insights into optimally modulating WRKY functions.

WRKYs regulate plant responses to abiotic stresses
Plants are routinely subjected to various stresses, which impair the normal growth and plant yield (Thomashow, 1999;Khoso et al., 2022).To surmount the stresses, sophisticated defense mechanisms have formed (Rushton et al., 2010;Schluttenhofer and Yuan, 2015;Sood et al., 2021).Recent research has shown that WRKYs have important functions in plant defenses against various stresses, including pathogens, cold, salinity, drought, and nutrition.The majority of WRKYs with known functions can be classified into three subfamilies, as shown in Figure 1; Table 2. Interestingly, WRKY members that regulate drought stress response are mostly found in subgroups 2b, 2c, 2d, and 2e, while those regulate salt resistance mainly belong to the 2a, 2c, and 3 subgroups.Additionally, WRKYs that regulate cold tolerance are mainly found in subgroups 1 and 2c, and those involved in plant responses to heavy metal poisoning are primarily found in subgroup 2c.

Involvement in drought stress
Drought stress results in cell dehydration, which threatens plant growth and yield worldwide.Long-term adaptation and evolution have led plants to have a variety of mechanisms to combat droughtinduced water deficits, including closing stomata to curb water loss, a response mediated mostly by the abscisic acid (ABA) signaling (Lee et al., 1999;Finkelstein et al., 2002;Oztur et al., 2002;Shinozaki et al., 2003;Tang et al., 2016;Ahammed et al., 2020a).Under drought stress, the abscisic acid (ABA) signaling pathway often leads to stomatal closure (Ng et al., 2001;Qiu and Yu, 2009).
In summary, these compelling findings highlight the significant roles of WRKYs, particularly in governing ABA signaling, osmolyte metabolism and ROS homeostasis, in calibrating plant adaptation to drought stress.

Regulation of plant resistance to salinity
The growing issue of soil salinization is having a negative impact on plant growth and reducing crop yields on a global scale.Salt stress can significantly hinder plant growth, leading to harmful consequences for agricultural production worldwide (Hasegawa et al., 2000;Yan et al., 2022).
Several WRKYs were revealed as positive regulators in plant tolerance to salinity.Employing CRISPR-Cas9 to knockout OsWRKY54, Huang et al. (2022a) revealed its beneficial effect on conferring rice tolerance to salinity.Similarly, by upregulating GarWRKY17 and GarWRKY104, the salt tolerance of Arabidopsis was enhanced at different developmental stages (Fan et al., 2015).Wu et al. (2017) found that overexpressing PeWRKY83 in Arabidopsis substantiated salt tolerance, resulting in increased proline accumulation, higher germination rates, less electrolyte leakage, and lower membrane damage under salt stress.
In contrast, WRKYs could also act as negative regulators on salt resistance.Jiang et al. (2021) demonstrated that overexpressing of PalWRKY77 in poplar compromised salt tolerance through inhibition of ABA-responsive genes.Bo et al. (2022) indicated that the maize (Zea mays) ZmWRKY20-ZmWRKY115 complex in nucleus bound to promoters of basic leucine zipper (ZmbZIP111) to inhibit the expression of ZmbZIP111, which elevated the salt sensitivity of maize seedlings.

Orchestrating plant responses to cold stress
Cold stress is unfavorable to normal plant development and poses a major constraint on agricultural productivity (Andaya and Mackill, 2003).Plants have evolved various physiological, biochemical and molecular adaptation mechanisms to improve cold tolerance (Ishitani et al., 1997;Ding et al., 2019;Ding and Yang, 2022;Khoso et al., 2022).Analyzing the regulatory mechanisms and elucidating the transcriptional networks has uncovered many cold stress-related genes (Ritonga et al., 2021).
In particular, WRKYs are instrumental to cold tolerance across plant species.WRKY members in 1 and 2c, were found to regulate the chilling stress.Overexpressing CsWRKY46 from cucumber (Cucumis sativus L.) caused higher tolerance to freezing by upregulating the expression of Responsive to Desiccation gene (RD29A) and cold regulated 47 gene (COR47), and by positively regulating expression of some ABA-regulated genes under low temperature stress (Zhang et al., 2016).By contrast, rice OsWRKY63 downregulated various genes related to chilling response and ROS-scavenging, and negatively regulated chilling tolerance via the WRKY63-WRKY76-DREB1B regulatory cascade (Zhang et al., 2022d).CdWRKY2 positively regulated cold responses by binding to promoters of sucrose phosphate synthase1 (CdSPS1) and C-repeat binding factor 1 (CdCBF1) in bermudagrass (Cynodon dactylon), thereby coordinating sucrose biosynthesis and the CBF pathway (Huang et al., 2022b).

Fine-tuning plant responses to nutrient deficiency and other stresses
Insufficient or excessive levels of soil nutrients impede plant growth.Nutrient deficiency symptoms in plants vary from element to element.As one of the important plant nutrients, phosphorus deficiency affects the photosynthetic rate of plant leaves, the growth of plant stems and the formation of reproductive organs (Barry, 1988;Lauer et al., 1989).Besides, deficiencies of boron or nitrogen (NH 4 + ) also seriously affect plant growth (Tanada, 1978;Krueger et al., 1987;Zhang et al., 2021b).
WRKY also has multiple roles in regulating these plant nutrients.For instance, suppression of OsWRKY28 resulted in decreased phosphate (Pi) accumulation in rice (Wang et al., 2018).In addition, OsWRKY21 and OsWRKY108 can positively regulate the expression level of phosphate transporter (OsPHT1;1) for Pi accumulation (Zhang et al., 2021a).In apple, overexpression of MdWRKY39 led to phosphorus deficiency through up-regulating MdPHT1;7 (Zhou et al., 2023).In poplar, a phosphate starvation response 1 (PHR1) homolog can interact with PtoWRKY40 to inhibit the transcription of PtoPHT1, thereby mediating an increase in Pi content (Chen et al., 2022).Besides, PdeWRKY6 and PdeWRKY65 modulated tissue Pi concentration by coordinating the expression of PdePHT1;9 (Yang et al., 2022).CaWRKY58 activated PHT1 and coordinated with Ca14-3-3 to improve Pi concentration under the Piinsufficient conditions (Cai et al., 2021).In addition, BnaA9.WRKY47 enhanced the tolerance of rapeseed to boron deficiency by upregulating the boric acid channel gene (BnaA3.NIP5;1), thus increasing boron uptake (Feng et al., 2020).AtWRKY46 suppressed the expression of GDP-D-mannose pyrophosphohydrolase (NUDX9) and IAA-conjugating genes, resulting in root tolerance to NH 4 + (Di et al., 2021).WRKY also play important roles in regulating plant responses to other stresses, including heat and cadmium toxicity.Qin et al. (2022) hypothesized that Arabidopsis ABT1/WRKY14 plays a key negative regulatory role in plant thermomorphogenesis.Zhang et al. (2020) demonstrated that Cd induces WRKY13 to activate D-CYSTEINE DESULFHYDRASE (DCD) expression to elevate H 2 S level and enhance Cd tolerance in Arabidopsis.

Regulation of flavonoid biosynthesis in plants
Flavonoids encompass several subclasses, including anthocyanins, proanthocyanidins, flavones, flavanols, flavonols, flavanones and isoflavones (Panche et al., 2016;Durazzo et al., 2019;Luo et al., 2019).More than 10, 000 diverse variants are generated by glycosylation and other modifications (Weston and Mathesius, 2013).Anthocyanins in particular confer bright colors to plant tissues and also act as antioxidants.Flavonoid biosynthesis proceeds through a branched pathway catalyzed by sequential enzymatic reactions.The biosynthesis of flavonoid is regulated by an intricate transcriptional network.Distinct WRKYs can integrate in various ways and exert different regulatory effects on flavonoid synthesis (Table 3; Figure 3).
Furthermore, WRKY forms transcriptional complexes with other transcription factors.MYB, bHLH and WD40 compose the MBW complex to modulate PA synthesis and anthocyanin (Nesi et al., 2001;Ramsay and Glover, 2005;Gou et al., 2011).WRKYs participate in regulating the MBW complex.For example, VvWRKY26 is absorbed into the MBWW complex by VvMYB5a to regulate flavonoid hydroxylation in grape (Amato et al., 2017;Amato et al., 2019).
Thus, the biosynthesis of flavonoids is regulated by a complex transcriptional network.WRKYs utilize various strategies, such as directly binding to target promoters, interacting with other regulators, or forming transcriptional complexes, to regulate flavonoid biosynthesis.The combined efforts of multiple WRKYs and the interactions with other transcription factors allow for precise control over this metabolic pathway.

Elevation of plant stress tolerances through flavonoid synthesis
WRKYs play pivotal roles in modulating the synthesis of various flavonoid for regulating plant responses to various abiotic stresses caused by UV-B, O 3 , and wounding, etc. UV-B-induced apple MdWRKY71-L promotes in apple anthocyanin accumulation by directly activating MdUFGT and MdMYB1 (Su et al., 2022a).When exposed to UV-B radiation in the transgenic calli, MdWRKY72 directly control anthocyanin synthesis via promoting MdMYB1, or indirectly regulates by interacting with MdMYB16 (Hu et al., 2020;Mao et al., 2021).In addition, McWRKY71 directly binds to the McANR, thus regulate the PA biosynthesis in regard to O 3 stress in crabapple (Zhang et al., 2022b).Moreover, MdWRKY40, interacting with MdMYB1, enhance the activation of target genes in reaction to injuries (An et al., 2019).
Apart from abiotic stresses, WRKY also partakes in biotic stresses by regulating the biosynthesis of flavonoids.For example, expression of VqWRKY56 activates PA biosynthesis genes (VvCHS3, VvLAR1 and VvANR) (Wang et al., 2023d).The upregulation of GhMYC2 by group IIc WRKYs induced GhMKK2-GhNTF6 signaling and increased cotton resistance to Fusarium oxysporum via enhancing flavonoid biosynthesis.This demonstrates a novel defense mechanism mediated by WRKY-MAPK-regulated flavonoid biosynthesis against pathogen infection in cotton (Wang et al., 2022).
In short, WRKYs participate in almost all stages of flavonoid synthesis and regulate flavonoid synthesis genes through diverse mechanisms.However, the contribution of WRKY-mediated flavonoid synthesis to plant tolerance has been poorly characterized.

Conclusion and perspective
In this review, we optimized existing WRKY phylogenetic trees and tried to deduce unifying themes of distinct WRKY subfamilies governing specific stress responses and flavonoid metabolism.
Analysis of documented data reveals WRKY members across all subgroups participate in flavonoid synthesis.However, WRKYs regulating salt tolerance mainly belong to subgroups 2a, 2c and 3. Given their shared protein motifs (Tables 2, 3; Figure 1), it is reasonable to hypothesize these WRKY subgroups promote flavonoid accumulation to enhance plant salinity tolerance.Similar hypotheses could be proposed for WRKY homologs tuning flavonoids to elevate plant resistance to cold, drought and nutrient deficiency.However, only a handful of studies have demonstrated direct relationships between WRKY-modulated flavonoid synthesis and stress tolerance.Perhaps the significant contribution of flavonoids has been overlooked in analyzing stress resistance, and roles of the WRKY-flavonoid interplay in plant stress tolerance deserve greater attention in future work.Nonetheless, laboratory validation remains necessary to verify these hypotheses.
On the other hand, research on WRKY-mediated flavonoid regulation has focused on few species like apple, Arabidopsis, grapevine and crabapple.Could WRKY regulate flavonoid synthesis in other species via distinct pathways?Molecular mechanisms underlying flavonoid-enhanced plant stress resilience remain largely unclear.Particularly, flavonoid regulation by WRKY transcription factors and subsequent impacts on plant stress response warrant deeper exploration.

FIGURE 3
FIGURE 3 Depiction of the role of WRKY in the biosynthesis of flavonoid.The complex role of WRKY in regulating flavonoid biosynthesis highlights its crucial function in this pathway and subsequent stress responses in plants.The WRKY protein modulates flavonoid biosynthesis by controlling the expression of flavonoid biosynthesis genes in various ways.MdWRKY1 and VqWRKY56 can influence the transcription of CHS, while VvWRKY26 can control CHI expression by forming the MBWW complex.PbWRKY75 might enhance the expression of DFR.MdWRKY11, MdWRKY71-L, MdWRKY40, and MdWRKY75 can directly or indirectly control the expression of UFGT.MdWRKY72, MdWRKY11, PpWRKY44, and PyWRKY26 can impact anthocyanin synthesis by forming the MYB-WRKY complex.In addition to positive regulation, MdWRKY41 also has a role in negatively regulating ANR synthesis.All these pathways influence the synthesis of anthocyanin or proanthocyanidin ultimately.Arrows represent positive regulation, and T-bars represent negative regulation.CHS, chalcone synthase; CHI, chalcone isomerase; F3H: flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; ANR, anthocyanin reductase; UFGT, flavonoid 3-O-glycosyl-transferase.

TABLE 1
Mechanism of WRKY on transcriptional regulation of target genes.

TABLE 2
WRKY involved in plant abiotic stresses response.