Function and Mechanism of WRKY Transcription Factors in Abiotic Stress Responses of Plants

Abstract

The WRKY gene family is a plant-specific transcription factor (TF) group, playing important roles in many different response pathways of diverse abiotic stresses (drought, saline, alkali, temperature, and ultraviolet radiation, and so forth). In recent years, many studies have explored the role and mechanism of WRKY family members from model plants to agricultural crops and other species. Abiotic stress adversely affects the growth and development of plants. Thus, a review of WRKY with stress responses is important to increase our understanding of abiotic stress responses in plants. Here, we summarize the structural characteristics and regulatory mechanism of WRKY transcription factors and their responses to abiotic stress. We also discuss current issues and future perspectives of WRKY transcription factor research.

1. Introduction

As a fixed-growth organism, plants are exposed to a variety of environmental conditions and may encounter many abiotic stresses, for example, drought, waterlogging, heat, cold, salinity, and Ultraviolet-B (UV-B) radiation. To adapt and counteract the effects of such abiotic stresses, plants have evolved several molecular mechanisms involving signal transduction and gene expression [1,2]. Transcription factors (TFs) are important regulators involved in the process of signal transduction and gene expression regulation under environmental stresses. TFs can be combined with cis-acting elements to regulate the transcriptional efficiency of target genes by inhibiting or enhancing their transcription [3,4]. Accordingly, plants may show corresponding responses to external stresses via TFs regulating target genes. Although some TF families (MYB, bZIP, AP2/EREBP, NAC) are associated with adversity [2,5], WRKY is the most extensively studied TF family in plant stress responses.

The WRKY family is a unique TF superfamily of higher plants and algae, which play important roles in many life processes, particularly in response against biotic and abiotic stress [6,7]. In 1994, the SWEET POTATO FACTOR1 (SPF1) gene of the WRKY family was first found in Impoea batatas [8]. Later, ABF1 and ABF2 were found in wild Avena sativa, and showed regulatory roles in seed germination [9]. A previous study successively cloned WRKY1, WRKY2, and WRKY3 from Petroselinum crispum, named the WRKY TF, and proved for the first time that WRKY protein can regulate plant responses to pathogens [10]. With an increase in available published genomes, many members of the WRKY TF family have been identified in various species, including 104 from Populus [11], 37 from Physcomitrella patens [12], 45 from Hordeum vulgare [13], 55 from Cucumis sativus [14], 74 from Arabidopsis thaliana [15], 83 from Pinus monticola [16], 81 from Solanum lycopersicum [17], and 102 from Oryza sativa [18]. WRKY TFs exist as gene families in plants, and the number of WRKY TFs varies among species. In plants exposed to abiotic stresses (salt, drought, temperature, and so forth), WRKY family members play important roles in diverse stress responses. In addition, these TFs affect the growth and development of plants [19,20]. Therefore, WRKY TFs have attracted broad attention. Although some reviews on WRKY TFs are available, in this review we focus on the structural characteristics and regulatory mechanisms of WRKY TFs and summarize recent progress in understanding the roles of WRKY TFs during exposure to abiotic stresses such as drought, temperature, salt, and UV radiation.

2. Structural Characteristics of WRKY TFs

The WRKY structure consists of two parts: the N-terminal DNA binding domain and the C-terminal zinc-finger structure [21]. The DNA binding domain sequence of WRKY is based on the heptapeptide WRKYGQK (Figure 1), but there are some differences, such as WRKYGQK, WRKYGKK, WRKYGMK, WSKYGQK, WKRYGQK, WVKYGQK, and WKKYGQK [17,22]. Zinc-finger structures mainly include C₂H₂ type and C₂HC type [23], whereas some exist in the form of CX₂₉HXH and CX₇CX₂₄HXC [17] (Figure 1). According to the number of WRKY domains and the structure of their zinc-finger motifs, WRKY can be divided into groups I, II, and III [23] (Figure 1). Group I usually contains two WRKY domains and one C₂H₂ zinc-finger structure. Those in group II and group III contain only one WRKY domain. The difference is that the zinc-finger structure in group II is C₂H₂ and that in group III is C₂HC [19,21,23] (Figure 1). According to the phylogenetic relationship of the amino acid sequence of the primary structure, group II can be further divided into subgroups a–e [7,23,24]. Evolutionary analyses have shown that the WRKY of group II is not generally a single source, mainly including five categories I, IIa + IIb, IIc, IId + IIe, and III [7,24]. In addition, some WRKY proteins contain a glutamate enrichment domain, a proline enrichment domain, and a leucine zipper structure [25].

3. Regulatory Mechanism of WRKY TFs

WRKY family members have diverse regulatory mechanisms. Briefly, WRKY protein can be effectively combined with W-box elements to activate or inhibit the transcription of downstream target genes. Moreover, it can also bind other acting elements to form protein complexes, which enhances the activity of transcription binding [21].

WRKY TFs can effectively activate the expression of downstream genes by binding conserved W-box cis-acting elements in the downstream gene promoter region [21,26]. There are abundant W-box elements in the self-promoter of most WRKY TFs. Therefore, these WRKY TFs can bind with their own promoters to achieve self-regulation or cross-regulation networks by combining with other WRKY TFs [27]. For example, CaWRKY6 of Capsicum frutescens can activate CaWRKY40 and make the plant more tolerant to high temperature and humidity. Glycine max GmWRKY27 not only inhibits the activity of downstream GmNAC29 promoter by independent inhibition, but also cooperatively interacts with GmMYB174 to inhibit the expression of GmNAC29, thereby increasing drought and salt stress resistances [28]. Moreover, chromatin immunoprecipitation (ChIP) studies have shown that when Petroselinum crispum is infected by pathogenic bacteria, PcWRKY1 promoter can effectively bind to itself and the W-box of PcWRKY3 promoter, and transcriptional activation can be achieved through self-negative feedback regulation and cross-regulation with other WRKY proteins [29]. In addition, WRKY TFs can interact with non-W-box elements. For example, Oryza sativa OsWRKY13 can interact with PRE4 (TGCGCTT) elements [30]. Hordeum vulgare HvWRKY46 and Nicotiana tabacum NtWRKY12 can effectively combine with the sucrose response element SURE [31,32]. These results indicate that there are multiple binding modes between WRKY TFs and structural genes. Different binding patterns and preferences of binding sites allow for the regulation of downstream target genes, providing WRKY TFs with versatile functions in the plant transcriptional regulation network.

4. WRKY TF Involved in Abiotic Stress Responses

When plants sense stress, the corresponding signaling is activated and transferred to the cell interior. Reactive oxygen species (ROS) and Ca²⁺ ions are usually exchanged as the signal transduction in the cell. Protein kinases such as MPKs are subsequently activated to regulate the activities of related TFs. Consequently, the plant presents a stress response [31,32]. In response to abiotic stresses, some WRKY TFs can be rapidly differentially expressed, promoting signal transduction and regulating the expression of related genes [33]. The expression patterns and functional identifications of WRKYs in most studies are generally based on transcriptome analyses, real-time fluorescence quantitative PCR, gene chip analyses, and genetic transformation. Hence, WRKY genes can function effectively in most abiotic stress responses or tolerances in various plants (

Table 1. WRKY transcription factors (TFs) involved in abiotic stress responses in plants.

No.	Gene	Species	Induced by Factors	Function	References
1	AtWRKY25/26	Arabidopsis	Heat	Tolerance to heat	[34]
2	AtWRKY33	Arabidopsis	NaCl, mannitol, H2O2	Tolerance to heat and NaCl, negative regulator in oxidative stress and abscisic acid (ABA)	[33]
3	AtWRKY34	Arabidopsis	Cold	Negative regulator in cold stress	[35]
4	AtWRKY39	Arabidopsis	Heat	Tolerance to heat	[36]
5	AtWRKY53	Arabidopsis	Drought, salt	Reduced drought resistance and H2O2, sensitive to salt	[37,38]
6	AtWRKY57	Arabidopsis	Drought	Tolerance to drought	[39]
7	AtWRKY63	Arabidopsis	ABA	Tolerance to drought, regulated ABA signaling	[40]
8	AtWRKY54	Arabidopsis	Heat	Response to heat stress	[41]
9	POWRKY13	Populus tomentosa	Heat	Response to heat stress	[42]
10	GhWRKY21	Gossypium hirsutum	Drought	Tolerance to drought	[43]
11	GhWRKY25	Gossypium hirsutum	Drought	Tolerance to salt, reduced drought resistance	[44]
12	GhWRKY68	Gossypium hirsutum	Salt, drought	Reduced salt tolerance and drought resistance, positive regulator in ABA signaling	[45]
13	VvWRKY24	Vitis vinifera	Cold	Upregulated expression at all stages of hypothermia	[46]
14	CaWRKY40	Capsicum annuum	Heat	Tolerance to heat	[47]
15	BdWRKY36	Brachypodium distachyon	Drought	Tolerance to drought	[48]
16	FcWRKY70	Fortunella crassifolia	Salt	Tolerance to salt	[49]
17	OsWRKY11	Oryza sativa	Heat, drought	Tolerance to drought and heat	[50]
18	OsWRKY72	Oryza sativa	Drought, NaCl, ABA	Sensitive to salt, drought, sucrose, and ABA	[51]
19	OsWRKY74	Oryza sativa	Pi deprivation, cold	Tolerance to cold and Pi deprivation	[52]
20	OsWRKY76	Oryza sativa	Cold	Tolerance to cold	[53]
21	OsWRKY89	Oryza sativa	ABA, UV-B	Tolerance to UV	[54]
22	GmWRKY13	Soybean	Salt, drought	Sensitive to salt and mannitol, negative regulator in ABA signaling	[55]
23	GmWRKY17	Soybean	Salt	Reduced salt tolerance	[56]
24	GmWRKY54	Soybean	Salt, drought	Tolerance to salt and drought	[55]
25	GmWRKY21	Glycine max	NaCl, drought, cold	Tolerance to cold	[55]
26	ZmWRKY17	Zea mays	ABA, salt	Reduced salt tolerance	[57]
27	TaWRKY2/19	Triticum aestivum	NaCl, drought, ABA	Tolerance to salt and drought	[58]
28	BcWRKY46	Brassica campestris	NaCl, drought, cold	Tolerance to salt and drought	[59]
29	BhWRKY1	Boea hygrometrica	Dehydration, ABA	Tolerance to drought	[60]
30	VpWRKY1	Vitis pseudoreticulata	NaCl, ABA	Tolerance to salt	[61]
31	VpWRKY2	Vitis pseudoreticulata	Cold, NaCl, ABA	Tolerance to salt and cold	[61]
32	VpWRKY3	Vitis pseudoreticulata	Drought, ABA, salicylic acid (SA)	Tolerance to salt	[62]
33	TcWRKY53	Thlaspi caerulescens	Cold, PEG, NaCl	Negative regulator in osmotic stress	[63]
34	NaWRKY3	Nicotiana attenuate	Mechanical damage	Sensitive to mechanical damage	[64]
35	JrWRKY2/7	Juglans regia	Drought, cold	Tolerance to drought and cold	[65]
36	SbWRKY30	Sorghum bicolor	Salt, drought	Tolerance to salt and drought	[66]
37	SbWRKY50	Sorghum bicolor	Salt	Tolerance to salt	[67]
38	IbWRKY47	Ipomoea batatas	Salt	Tolerance to salt	[68]
39	IbWRKY2	Ipomoea batatas	Salt, drought	Tolerance to salt and drought	[69]
40	MdWRKY30	Malus domestica	Salt, osmotic stress	Tolerance to salt and osmotic stress	[70]
41	MdWRKY100	Malus domestica	Salt	Sensitive to salt	[71]
42	SlWRKY81	Solanum lycopersicum	Drought	Reduced drought tolerance	[72]
43	GbWRKY1	Gossypium barbadense	Salt	Tolerance to salt	[73]
44	VbWRKY32	Verbena bonariensis	Cold	Tolerance to cold	[74]
45	PgWRKY33/62	Pennisetum glaucum	Salt, drought	Tolerance to salt and drought	[75]
46	PagWRKY75	Populus alba	Drought	Negative regulator in salt and osmotic stress	[76]

, Figure 2).

4.1. WRKY TFs and Drought Stress

Drought has a major impact on plant growth and development, resulting in a significant decrease in grain and other types of crop yield [77]. Under drought stress, drought-tolerant plants can accumulate oligosaccharides through sucrose metabolism to improve drought resistance. For example, when Arabidopsis is subjected to drought stress, the expression of AtWRKY53 combined with the Qua-Quine Starch (QQS) promoter sequence is rapidly induced, hydrogen peroxide content is reduced, and the glucose metabolism pathway is significantly enhanced, thereby regulating stomatal opening and ultimately affecting drought tolerance [37]. In Boea hygrometrica, BhWRKY1 effectively regulates the expression of the BhGolS1 gene, and the overexpression of BhGolS1 and BhWRKY1 induces the accumulation of raffinose family oligosaccharides (RFOs) in transgenic Nicotiana tabacum, thus improving the ability of seedlings to resist drought [60].

WRKY protein can directly regulate the expression of drought-resistant genes. For example, in sorghum, SbWRKY30 regulates the drought stress response gene SbRD19 by binding with W-box elements of the SbRD19 promoter, and protects plant cells from the damage of reactive oxygen species by improving ROS scavenging capability, enhancing drought tolerance [66]. TaWRKY2 of wheat can bind to STZ and downstream drought-resistant gene RD29B promoter, with a positive regulatory effect on the expression of RD29B [58]. DREB2A regulates the expression of dehydration stress-related genes [78], while TaWRKY19 can bind to DREB2A promoter, ultimately activating the expression of RD29A, RD29B, and Cor6.6 in transgenic Arabidopsis plants [58]. Similarly, Arabidopsis AtWRKY57 positively regulates the expression of RD29A and NCED3 genes by binding their W-box elements in the promoter regions [39]. In addition, WRKY protein can act on other TFs to play regulatory roles in drought tolerance. For example, TcWRKY53 of Thlaspi arvense significantly inhibits the expression of NtERF5 and NterEBp-1 of the AP2/ERF TF family, thus improving plant resistance to drought stress [63].

WRKY TFs also regulate plant tolerance through abscisic acid (ABA) and ROS-related signaling pathways. During drought stress, higher ABA levels were accumulated in plants, and leaf stomata were closed to reduce transpiration rate, thus regulating water balance in plants. ABA accumulation in cells, integrated with a variety of stress signals, regulates the expression of downstream genes, consequently sensing and responding to the adverse environment [40]. Arabidopsis AtWRKY63 has a specific effect on ABA-mediated stomatal closure and other signal transduction pathways, thus affecting the drought response [40]. GhWRKY21 regulates ABA-mediated cotton drought tolerance by promoting the expression of GhHAB [43]. Overexpression of BdWRKY36 in tobacco reduces the accumulation of ROS, activated NtLEA5, NtNCED1, and NtDREB3 in the ABA biosynthetic pathway, and significantly enhances the drought resistance of plants [48]. In Solanum lycopersicum, SlWRKY81 increases the drought tolerance of plants by inhibiting the accumulation of H₂O₂, playing a negative regulation role of stomatal closure [72].

4.2. WRKY TFs and Salt Stress

Salt stress is an important abiotic stress affecting crop productivity, particularly in arid and semiarid regions. WRKY TFs play essential roles in regulating the response to salt stress. To date, a total of 47 WRKY genes have been found to be expressed under salt stress in the wheat genome [79]. STZ is a protein related to ZPT2, which acts as a transcriptional inhibitor to downregulate the deactivation of other transcription factors. GmWRKY54 of Glycine max inhibits STZ expression and responds to salt stress by positively regulating the DREB2A-mediated pathway [55]. FcWRKY70 promotes the upregulation of arginine decarboxylase (ADC) expression, which is heterologously expressed in tobacco, and the content of lemon putrescine is significantly increased, thus enhancing the salt tolerance of plants [49]. The IbWRKY47 gene positively regulates stress resistance-related genes and significantly improves the salt tolerance of Ipomoea batatas [68]. MiR156/SPL modulates salt tolerance by upregulation of Malus domestica salt tolerance gene MdWRKY100 [71]. In Sorghum bicolor, SbWRKY50 could directly bind to the upstream promoter of SOS1 and HKT1 and participate in plant salt response by controlling ion homeostasis [67]. In addition, some WRKY genes function as negative regulation factors involved in salt stress resistance. Arabidopsis RPD3-like histone deacetylase HDA9 inhibits salt stress tolerance by regulating the DNA binding and transcriptional activity of WRKY53 [38]. Chrysanthemum CmWRKY17 overexpressed in Arabidopsis allows the plants to be more sensitive to salt stress. The expression level of stress resistance-related genes in transgenic Arabidopsis is lower than that in wild-type plants, indicating that CmWRKY17 may be involved in negatively regulating the salt stress response in Chrysanthemum [80]. The expression of GhWRKY68 is strongly induced in upland cotton and decreases salt tolerance [45]. In contrast, a high expression level of GhWRKY25 enhances the salt tolerance of upland cotton, while transgenic tobacco shows a relatively weaker tolerance to drought stress [44], indicating that the regulatory effects of different WRKY TFs involved in drought response are different.

Plants can also respond to saline–alkali stress through ABA, H₂O₂, and other signal pathways. In Glycine max, the negative regulatory factor ABI1 in the ABA pathway may be the downstream target gene of GmWRKY13. Genetic transformation experiments in Arabidopsis have shown that overexpression of GmWRKY13 significantly increases the expression of ABI1, but plants show a low tolerance to salt stress [55]. Overexpression of ZmWRKY17 has an inhibitory effect on the sensitivity of exogenous ABA treatment, resulting in a relatively lower tolerance to high levels of salinity [57]. Under salt-induced H₂O₂ and cytosolic Ca²⁺ stimulation, the activity of antioxidant enzymes increases, thus improving the tolerance to high-salinity environments [81]. ABA-induced WRKY gene expression is largely related to salt stress. Exogenous application of ABA and NaCl also induce AtWRKY25 and AtWRKY33 in Arabidopsis [33], OsWRKY72 in rice [51], GbWRKY1 in Verbena bonariensis [73], and VpWRKY1/2 [61] and VpWRKY3 [62] in grape.

4.3. WRKY TFs and Temperature Stress

Both low- and high-temperature stress can reduce crop yield and quality in plants. WRKY TFs play a role in the stress response through different signal transduction pathways. For example, in Verbena bonariensis, VbWRKY32 as a positive regulator, upregulates the transcriptional level of cold response genes, which increases the antioxidant activity, maintains membrane stability, and enhances osmotic regulation ability, thereby improving the survival ability under cold stress [74]. The BcWRKY46 gene of Brassica campestris is strongly induced by low temperature and ABA, activating related genes in the ABA signaling pathway to improve the low-temperature tolerance of plants [59]. CBF TFs regulate the expression of COR, and the overexpressed transgenic lines of CBF1, CBF2, and CBF3 show stronger cold resistance [82]. AtWRKY34 has a negative regulatory effect on the CBF-mediated cold response pathway; it is specifically expressed in mature pollen grains after exposure to low temperatures, resulting in resistance to low temperatures [35]. In addition, plants respond to temperature changes by coordinating organ development in an adverse environment. At low temperatures, rice MADS-Box TF OsMADS57 and its interacting protein OsTB1 synergistically activate the transcriptional regulation of OsWRKY94, preventing tillering by inhibiting transcription of the organ development gene D14 [83].

Due to global climate change, high-temperature stress has attracted significant attention. There is evidence that, to a certain extent, high temperatures will lead to biochemical changes in plants [84]. Thermal stimulation can activate Ca²⁺ channels to maintain a higher intracellular Ca²⁺ concentration, thereby activating calmodulin protein expression and inducing thermal-shock protein transcriptional expression [85]. In Arabidopsis, AtWRKY54 significantly responds to heat shock whereas basic leucine zipper factors (bZIPs) respond to prolonged warming [41]. Overexpression of AtWRKY39 can make plants more heat-sensitive. AtWRKY39 is highly homologous to AtWRKY7, and both of them can effectively bind calmodulin in plants, indicating a similar function [36]. In addition, AtWRKY25, AtWRKY26, and AtWRKY33 can improve tolerance to high-temperature stress in transgenic Arabidopsis by regulating the Hsp101 and Zat10 genes [34]. Plants subjected to heat stress can also activate the oxidative stress response through ethylene [86]. Under high-temperature stress, the expressions of AtWRKY25, AtWRKY26, and AtWRKY33 in Arabidopsis are induced by ethylene, the feedback factor EIN2 is transcriptionally regulated, and the effective activation of ethylene signal transduction contribute to relatively stronger heat resistance. In Oryza sativa, HSP101 promoter can activate the expression of the OsWRKY11 gene. Under heat treatment, the leaves wilted more slowly and the green part of the plant was less damaged, which makes it more heat-resistant [50]. In addition, some noncoding RNAs, such as miR396, play a role in the response to heat stress by regulating its target WRKY6 [87].

4.4. WRKY TFs and Other Abiotic Stresses

WRKY TFs are also involved in oxidative stress, mechanical damage, UV radiation, and other abiotic stresses (Figure 3). FcWRKY40 overexpression can significantly enhance the resistance of transgenic tobacco to oxidative stress [88]. When Arabidopsis is treated with ROS, the expressions of AtWRKY30, AtWRKY40, AtWRKY75, AtWRKY6, AtWRKY26, and AtWRKY45 are significantly upregulated [89]. After mechanical injury, the expression levels of AtWRKY11, AtWRKY15, AtWRKY22, AtWRKY33, AtWRKY40, AtWRKY53 [90] and AtWRKY6 [64] are upregulated. Similarly, NaWRKY3 is strongly expressed in tobacco. By contrast, the sensitivity of transgenic plants is increased when NaWRKY3 is knocked out [64]. In two previous studies, UV-B radiation treatment induced three WRKY genes in Arabidopsis and the OsWRKY89 gene in rice, resulting in a thick waxy substance on the leaf surface and improved tolerance to heat [54,91].

In addition, a single WRKY TF can play multiple roles in different stress responses via various signal pathways and regulatory networks. For example, TaWRKY44 expression in tobacco can improve resistance to drought, salt stress, and osmotic stress [92], while PgWRKY62 and PgWRKY33 in Pennisetum glaucum respond to salt and drought simultaneously [75]. BhWRKY1 protein in Boea hygrometrica binds to the promoter of BhGolS1 and is associated with both low-temperature resistance and drought tolerance [60]. IbWRKY2 can interact with IbVQ4, and drought and salt treatment can induce the expression of IbVQ4, thus improving the tolerance of plants to drought and salt stress [69]. MdWRKY30 overexpression enhances tolerance to salt and osmotic stress in transgenic apple callus through transcriptional regulation of stress-related genes [70]. PagWRKY75 negatively regulates the tolerance of 84 K poplar (Populus alba × P. glandulosa) to salt and osmotic stress by reducing the scavenging capacity of ROS and the accumulation of proline, thus actively regulates the rate of leaf water loss [76].

5. Conclusions and Perspectives

As one of the largest TF families, WRKY plays an important and indispensable role in normal life activities of plants. Over the years, it has been shown that WRKY TFs not only participate in plant growth and development, but also show complex regulatory mechanisms and networks involved in external abiotic stresses. A large number of WRKYs have been functionally characterized in model plants, providing abundant functional references for other plants. Given that crops usually face various stresses and WRKYs play important roles in stress responses, further in-depth studies on WRKY genes in more crops are required. As increasing plant genomes have been sequenced, particularly of economically important crops, the genome-wide identification of WRKY genes will facilitate screening for stress resistance-related functional genes in plants. Moreover, previous studies of WRKY gene functions were largely dependent on transcriptomics and functional predictions, whereas more applications of genetic verification combined with new technologies are accelerating the research progress of WRKY’s novel functions. In addition, characterization of the downstream genes regulated by WRKY TFs or WRKY TF self-regulation will help clarify the regulatory network of abiotic stress responses. Furthermore, noncoding RNAs and epigenetic modifications involved in the regulation of WRKY TFs should be explored in future studies. Ultimately, using WRKY TFs to screen for stress-resistant plant cultivars and improve plant stress resistance will significantly benefit agricultural crop yield and quality in the context of aggravated climate change.