WRKY transcription factors

Abstract

WRKY transcription factors are one of the largest families of transcriptional regulators found exclusively in plants. They have diverse biological functions in plant disease resistance, abiotic stress responses, nutrient deprivation, senescence, seed and trichome development, embryogenesis, as well as additional developmental and hormone-controlled processes. WRKYs can act as transcriptional activators or repressors, in various homo- and heterodimer combinations. Here we review recent progress on the function of WRKY transcription factors in Arabidopsis and other plant species such as rice, potato, and parsley, with a special focus on abiotic, developmental, and hormone-regulated processes.

Keywords: :

• senescence
• seed development
• ROS homeostasis
• biotic stress
• abiotic stress
• phytohormones
• WRKY

Introduction

Reprogramming of a cell in response to external stimuli involves complex changes in gene expression. The activities of a multitude of genes are subject to up- or downregulation and follow defined temporal programs. The perception of an external stimulus leads to the activation of primary response genes. WRKY transcription factors (TFs) are one of the largest families of transcriptional regulators in plants with 72 representatives in Arabidopsis, and more than 100 members in rice, soybean or popular.^1-13 Sixty-eight genes have been identified in Sorghum,¹³ 38 in Physcomitrella patens,¹³ 35 in Sellaginella moellendorffii,¹ 80 in pinus,¹⁴ and about 45 in barley.¹⁵ WRKYs contain the highly conserved amino acid sequence WRKYGQK and the zinc-finger-like motifs Cys(2)-His(2) or Cys(2)-HisCys, and bind to the TTGAC(C/T) W-box cis-element in the promoter of their target genes.^1,2 WRKY TFs are important components of a plant signaling web which regulate many plant processes in response to biotic and abiotic stimuli, but also in response to internal signals which coordinate developmental processes, and include additional DNA-binding and non-DNA-binding proteins as interaction partners.^{1-5,7,9,10,13,16-26} WRKYs can act as activators or repressors, and by doing so, they establish a TF net that participates in many cytoplasmic and nuclear processes including signaling events from organelles and the cytoplasm to the nucleus. Stress responses often involve hormone signaling, therefore, WRKYs are also part of a complex hormone signaling net. The WRKYs can function up- and downstream of hormones, are involved in the antagonistic functions of salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET), control developmental processes via auxins, cytokinins, and brassinosteroids.^{2,18,20,21,25} The SA-mediated signaling pathway is associated with resistance to biotrophic and hemibiotrophic pathogens, while JA/ET signaling often correlates with resistance to necrotrophic pathogens.^27,28 These phytohormones have different effects on WRKY TFs, e.g., WRKY70 acts as an activator of SA-induced genes and as a repressor of JA-responsive genes, thereby integrating signals from these mutually antagonistic pathways.²⁹ There is increasing evidence for an important control of WRKY genes by abscisic acid (ABA)^18,20,30,31 and ET^30,32-34 signaling. ABA represents a key signal to regulate plant growth and development as well as plant responses to abiotic and biotic stress. The WRKY domain in WRKY2, WRKY18, WRKY40, WRKY60, and WRKY63 are also reported in transcriptional regulation of ABA-responsive element binding factors (ABFs/AREBs) through W-box sequences present in the promoters of ABFs/AREBs.³⁵ ABA is involved in drought responses, and ABA signaling components in the nucleus are targets of signals from plastids and mitochondria, thereby participating in retrograde signaling in plant cells. Vanderauwera et al.³⁶ proposed that WRKY15 participates in mitochrondrium-to-nucleus signaling. Finally auxin^{30,31,33,37-41} and cytokinin^42-44 may control plant developmental processes with the help of WRKYs. Phosphate (Pi) limitation is another example of stress which is counteracted by the plant with the help of WRKY6 and WRKY75 TFs.²¹ Besides phytohromones, biotic and abiotic stress responses are often associated with the generation of reactive oxygen species (ROS) in plants.²³ Several ROS-dependent responses are controlled by WRKYs, including the progression of senescence.⁵ Finally, WRKYs actively participate in the control of seed, embryo, microphyle, and endosperm development.^42,43,45,46 Members of the WRKY family have also been reported to regulate major changes in plant transcriptome during early phases of root colonization with arbuscular mycorrhizal fungi.⁴⁷ These observations raise the question how so diverse responses can be controlled by WRKYs. We address the question whether a common theme maybe behind these processes. We summarize the current knowledge on this TF family, with the main focus on abiotic stress responses, as well as control of plant development and hormone regulation, and putative common processes.

WRKY: Structure and Classification

The WRKY name is coined from the highly conserved 60 amino acid long WRKY domains of the TFs, which contain a conserved heptapeptide motif WRKYGQK at the N-terminus and a novel zinc-finger-like motif at the C-terminus. Both heptapeptide sequence and zinc-finger-like motif are required for the high binding affinity of WRKY TFs to the consensus cis-acting elements termed W box (TTGACT/C).

In spite of the presence of the highly conserved W box, DNA binding of WRKY TFs varies mainly due to the different number of DNA binding domains and different features of the zinc-finger-like motifs. Based on these features Eulgem et al.²⁶ classified WRKY proteins into 3 groups:

WRKY proteins with 2 WRKY domains belong to group I, whereas most proteins with one WRKY domain belong to group II. Both group I and group II proteins have the same pattern of potential zinc ligands (C–X4–5–C–X22–23–H–X1–H). WRKY proteins having only one WRKY domain but different patterns of zinc finger motifs are categorized into group III. Instead of a C2–H2 pattern, group III WRKY domains contain a C2–HC motif (C–X7–C–X23–H–X1–C). Group II proteins were further divided into IIa, IIb, IIc, IId, and IIe based on the primary amino acid sequence (Fig. 1). The WRKY domain consists of a 4-stranded β-sheet, with the zinc coordinating Cys/His residues forming a zinc-binding pocket. 72 WRKY genes are present in the Arabidopsis genome and approximately 100 WRKY genes in the rice genomes, respectively.⁴⁸ Out of the 72 Arabidopsis WRKY genes, 32 belong to group I, 26 belong to group II and 14 belong to group III. In spite of different DNA binding domains members of all groups bind sequence-specifically to various W-box elements. The 2 WRKY domains of group I members appear to be functionally distinct. Sequence-specific binding to their target DNA sequences is mediated mainly by the C-terminal WRKY domain. The function of the N-terminal WRKY domain remains unclear.

Figure 1. The consensus WRKY domain for each WRKY group in higher plants. The WRKY motif, the cysteines, and histidines that form the zinc finger are shown in boxes. The 4 β-strands are shown with arrows. I CT and I NT denote the N-terminal and C-terminal WRKY domains from group I WRKY proteins.

WRKY1 is involved in the SA signaling pathway and partially dependent on NPR1. The crystal structure of the C-terminal part of WRKY1 determined at 1.6 A resolution revealed that this domain is composed of a globular structure with 5 β strands, forming an antiparallel β-sheet. A zinc-binding site is situated at one end of the β-sheet, between strands β4 and β5. Based on this high-resolution crystal structure and site-directed mutagenesis, it was shown that the DNA-binding residues of WRKY1 are located at β2 and β3 strands.⁴⁹ Ciolkowski et al.⁵⁰ tested 5 WRKY TF members for their DNA-binding selectivity toward variants of the W-box embedded in neighboring DNA sequences. Their studies demonstrate differences in their binding site preferences which are partially depending on adjacent DNA sequences outside of the W-box core motif. The authors also showed that amino acid substitutions within the DNA-binding region of WRKY11 strongly impinge on its DNA binding activity. Therefore, in spite of the simple binding sequence known for WRKY protein-DNA recognition, more detailed information is necessary to understand the (binding) specificities of the WRKY family members, which might explain the diverse functions of the individual WRKY members.

Origin of WRKY TFs

Besides Arabidopsis, WRKY TFs have been studied in rice, tobacco, oat, cucumber, moss, and other plant species. Further, WRKY-like genes were identified in protists (Giardia lamblia and Dictyostelium discoideum) and green algae such as Chlamydomonas reinhardtii, demonstrating the ancient origin of the gene family. The phylogenetic trees generated from the WRKY domains indicate that the WRKY gene family arose during evolution through gene duplications and that the dramatic amplification of group III WRKY genes in rice is due to tandem and segmental gene duplications, which occurred to a lesser extent in Arabidopsis.^1,2,24 Some of the rice WRKY genes in group III are evolutionarily more active than those in Arabidopsis, and may have specific roles in monocotyledonous plants. Wang et al.⁵¹ showed that purifying selection played a major role during the evolution of this family. Sites relaxed from purifying selection were identified and mapped onto the structural and functional regions of the WRKY1 protein. These analyses will enhance the understanding of the precise role of natural selection to create the functional diversity in WRKY family.⁵¹ More information is required to understand the evolutionary constrains on WRKY gene evolution, and how the functional diversity of the WRKY protein family members can be explained.

Functions of WRKY TFs

Plants devote a large portion of their genome capacity to transcription, with the Arabidopsis and rice genome coding for more than 2100 and 2300 TFs, respectively.⁵² The WRKY gene family has been suggested to play important roles in the regulation of transcriptional reprogramming associated with plant stress responses. Being unable to move, plants encounter numerous biotic and abiotic stresses at different developmental stages which include drought, salinity, temperature changes from freezing to scorching, decreased availability of essential nutrients resulting in nutrient starvation and variable light conditions. To overcome these unfavorable conditions, plants have developed various intricate mechanisms which occur at multiple levels. Recognition of stress cues and transduction of the signals to activate adaptive responses and regulation of stress-related genes are the key steps leading to plant stress tolerance.⁵³ Induction of stress-related genes occurs mainly at the transcriptional level, and modification of the temporal and spatial expression patterns of specific stress-related genes is an important part of the plant stress response.⁵⁴ Interestingly, the strong involvement of the plant-specific WRKY TF family in the stress responses demonstrates that plant developed their own way to deal with these challenges.

In general stress signals are perceived by membrane-bound or soluble receptors and/or activate phytohormone-dependent processes. A large number of information demonstrates the involvement of WRKYs in receptor-mediated processes. In Arabidopsis, small peptides encoded by PROPEP genes act as damage-associated molecular patterns that are perceived by 2 leucine-rich repeat receptor kinases, PEPR1 and PEPR2, to amplify defense responses. WRKY33 binds to the promoter of the 2 receptor kinase genes in a stimulus-dependent manner and regulates their expression.⁵⁵ In addition, besides NB-LRR type R proteins, receptor-like protein (RLP) members participate in plant immunity. Zhang et al.⁵⁶ have demonstrated that WRKY70 functions downstream of SNC2–1D (suppressor of rpr1–1, constitutive 2), a RLP member. In barley, intracellular milder A (MLA) R proteins function in the nucleus and confer resistance against powdery mildrew fungus. Recognition of the avirulence A10 effector by MLA10 induces nuclear associations between the receptor and WRKY TFs.⁵⁷ These 3 examples demonstrate the complexity in this scenario, and the involvement of WRKYs at different regulatory levels. Furthermore, the chloroplast-localized protein magnesium-protoporphyrin IX chelatase H subunit function as an ABA receptor (ABAR). ABAR spans the chloroplast envelope and the cytosolic C-terminus of ABAR interacts with WRKY18, WRKY40, and WRKY60, which function as negative regulators of ABA signaling in seed germination and postgermination growth.⁵⁸ Jiang et al.⁵⁹ have demonstrated that the Arabidopsis 3-ketoacyl-CoA-thiolase-2 (KAT2), an enzyme of fatty acid β-oxidation, is involved in ABA signaling. KAT2 functions downstream of WRKY40 and thus, WRKY40 may link KAT2 with the ABA-mediated signaling. Finally, herbirvory by Spodoptera littoralis activates the synthesis of JA-isoleucine which binds to a complex consisting of the receptor COI1 and JAZ repressors. This results in the activation of WRKY18 and WRKY40 genes.⁶⁰

WRKYs are substrates of calcium-dependent protein kinases (CPK). A point mutation in WRKY28 completely abolishes its phosphorylation by CPK4 and CPK11.⁶¹ Calmodulin (CaM) is a ubiquitous Ca²⁺-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. Park et al.⁶² isolated a cDNA encoding WRKY7 as a CaM-binding TF from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca²⁺-dependent CaM-binding domain (CaMBD) of WRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca²⁺/CaM-dependent enzyme. Furthermore, the CaMBD of WRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family.

Phosphorylation also plays important roles in WRKY processes. Besides phosphorylation of WRKY28 by CPK4 and CPK11,⁶¹ MAP kinase kinase kinase (MEKK) 1 phosphorylates WRKY53 in vitro and phosphorylation increases DNA binding and transcription of a WRKY53-driven promoter construct in vivo. ⁶³Phosphorylation of WRKY33 by 2 pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis.⁶⁴ Xie et al.⁶⁵ demonstrated that PKS2 (SOS2-like protein kinase 5) phosphorylates NPR1 at its C-terminal region which results in the upregulation of WRKY38 and WRKY62. Ueno et al.⁶⁶ showed that MAP kinases phosphorylate rice WRKY45. Phosphorylation of the AD protein (a protein with similarities to HPT kinases) increases its binding to the WRKY53 promoter. These examples demonstrate that phosphorylation is an essential signaling event at, up- and downstream of WRKY TFs and the expression of their genes.

A number of reports points to the importance of epigenetic control of WRKY functions. Regulation of leaf senescence includes epigenetic re-programming via histone methylation at WRKY53.⁶⁷ Also the seed size is controlled by epigenetic and genetic programs which include WRKYs.⁴³ Furthermore, epigenetics controls plant immunity.⁶⁸ Histone deacetylases are involved in the activation of JA/ET-sensitive defense mechanisms. ATP-dependent chromatin remodelers mediate the constitutive repression of the SA-dependent pathway whereas histone methylation at the WRKY40 promoter affects the activation of this pathway. Interestingly, bacterial infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genetic lesions.⁶⁸ The expression of WRKY70 is known to be antagonistically regulated by the SA and JA signaling pathways. Thus, this gene encodes a TF functioning at the crossroad of the 2 pathways. The Arabidopsis homolog of Trithorax, ATX1 activates the expression of WRKY70 and is involved in establishing the trimethylation pattern of histone H3 tail lysine 4 (H3K4me3) residues of its nucleosomes. Chromatin immunoprecipitation analyses demonstrated that WRKY70 is a primary target for the ATX1 histone methylase activity, while the SA-responsive gene, PR1, and the JA-responsive gene, THI2.1, are secondary targets. The finding that PR1 and THI2.1 nucleosomes carry H3K4me3-marks unrelated to their transcription states suggests that the defense-response genes PR1 and THI2.1 keep their nucleosomes in 'actively' modified state, perhaps, in preparation for quick-changes of transcription when needed by the cell. Alvarez-Venegas et al.⁶⁹ proposed that a single epigenetic factor can thereby orchestrate expression of a large number of genes.

Abiotic Stress

Compared with the research on biotic stress, little is known about the involvement of these TFs in abiotic stress responses. The WRKY gene family is induced in response to different abiotic stresses and emerged as an important candidate for imparting stress tolerance. The tight regulation and fine-tuning of WRKY proteins during plant stress responses contribute to the establishment of complex signaling webs and the important roles of WRKY proteins in plant abiotic stress responses make them potential candidates for imparting stress tolerance. Recent studies have demonstrated that many WRKY genes are strongly and rapidly induced in response to certain abiotic stresses, such as wounding, drought or salinity, cold and heat or osmotic stress. A single WRKY protein is often involved in several stress responses, and some of them are even involved in abiotic and biotic stresses. In recent years, numerous groups have demonstrated that manipulation of WRKY TF levels in knockout or overexpressor plants affects specific stress responses. Which stress response is associated with a particular WRKY is puzzling at present. It might be helpful in the future to perform comparative analyses of knockout and overexpressor lines under various stress conditions to pinpoint down the specific roles of the WRKYs in a given stress response.

Drought and Salt Stress

Environmental stresses such as drought and salinity do not only induce imbalances of ionic and osmotic homeostasis in the cell but also impair photosynthesis, cellular energy depletion, and the redox homeostasis. The involvement of WRKYs in regulatory systems that link sensing and signaling of environmental conditions to cellular adaptive responses is much broader than anticipated so far. WRKY TFs are considered as one of the master regulators for molecular reprogramming to enhance stress tolerance of plants. Several WRKYs are reported to be involved in drought and salt stress in Arabidopsis, rice,¹⁹ and other plant species. Many established tolerance mechanisms to drought and salinity stress are related to ABA signaling.

Almost 2000 drought-responsive genes were identified in Arabidopsis under progressive soil drought stress,³³ and several of them code for WRKY TFs. Most of the drought-regulated genes recovered to normal expression levels within 3 h after rewatering. About 2/3 of the drought-responsive genes were also regulated by ABA and the ABA analog PBI425, and the list includes WRKY TF genes. Analysis of promoter motifs confirms that many of the drought-responsive genes are affected by ABA signaling. The endogenous ABA level and that of the ABA catabolites increased under drought stress and they recovered to normal levels 3 h after rehydration. Besides ABA, also other plant hormones such as JA, auxin, cytokinin, ET, brassinosteroids, and gibberellins were affected by the drought stress.³³ Furthermore, there is an extensive cross-talk between responses to drought and other environmental factors including light and biotic stresses, and drought stress affects the hormone homeostasis.³³ Zou et al.⁷⁰ investigated WRKY21 from the creosote bush Larrea tridentata, a xerophytic evergreen C3 shrub thriving in vast arid areas of North America. WRKY21 is highly expressed under drought stress and functions as an activator the ABA signaling pathway. Elevated expression of WRKY57 improved drought tolerance of Arabidopsis by elevation of ABA levels.⁷¹ Transgenic Arabidopsis seedlings overexpressing the grapebine VvWRKY11 showed higher tolerance to water stress induced by mannitol. Thus, VvWRKY11 is involved in the response to dehydration stress.⁷² Consistent with these observations expression of wild soybean WRKY20 in Arabidopsis enhances drought tolerance and regulates ABA signaling.⁷³ Compared with the wild type, GsWRKY20 overexpression lines were more sensitive to ABA in stomatal closure, and exhibited a greater tolerance to drought stress, a decreased water loss rate, and a decreased stomatal density. Babitha et al.⁷⁴ expressed the AtbHLH17 and AtWRKY28 TF genes which are known to be upregulated under drought and oxidative stress, in Arabidopsis. The transgenic lines exhibited enhanced tolerance to NaCl, mannitol, and oxidative stress. Under mannitol stress condition a higher root growth was observed in the transgenic lines. Several downstream target genes were upregulated under various stress conditions in the overexpressor lines and several of the responsive genes had either W-box sequences or bHLH cis elements in their promoter regions. These examples demonstrate that the WRKYs are powerful tools for the generation of drought-resistance plants.

Involvement of WRKY factors in plant salt adaptation was shown for WRKY25 and WRKY33 which increased salt tolerance and ABA sensitivity independently of the SOS-pathway when overexpressed in A. thaliana.⁷⁵ Similarly, overexpression of OsWRKY45 in rice resulted in enhanced salt and drought tolerance, in addition to increased disease resistance. GsWRKY20 from wild soybean was induced by ABA, salt, cold, and drought, suggesting an involvement of this TF in several abiotic stress-related responses. Chen et al.²² analyzed the relationship of WRKY18, -40 and -60 in salt and biotic stress-exposed Arabidopsis seedlings. WRKY18 and WRKY60 have a positive effect on the plant´ ABA sensitivity for inhibition of seed germination and root growth. The same 2 WRKY genes also enhance plant sensitivity to salt and osmotic stress. WRKY40, on the other hand, antagonizes WRKY18 and WRKY60 in the effect on plant sensitivity to ABA and abiotic stress in germination and growth assays. Both WRKY18 and WRKY40 are rapidly induced by ABA, while induction of WRKY60 by ABA is delayed. ABA-inducible expression of WRKY60 is almost completely abolished in the wrky18 and wrky40 mutants. WRKY18 and WRKY40 recognize a cluster of W-box sequences in the WRKY60 promoter and activate WRKY60 expression in protoplasts. Thus, WRKY60 might be a direct target gene of WRKY18 and WRKY40 in ABA signaling. Using a stable transgenic reporter/effector system, both WRKY18 and WRKY60 act as weak transcriptional activators while WRKY40 is a transcriptional repressor in plant cells. These examples demonstrate that the 3 related WRKY TFs form a highly interacting regulatory network that modulates gene expression in both plant defense and stress responses by acting as either transcription activator or repressor.²² Finally, Vanderauwera et al.³⁶ characterized WRKY15 which modulates plant growth and salt/osmotic stress responses in Arabidopsis. WRKY15-overexpressing plants linked an endoplasmic reticulum-to-nucleus communication to a disrupted mitochondrial stress response under salt-stress conditions. This example demonstrates that a WRKY15-mediated salt stress response may be integrated into a signaling network that includes communication between different cellular organelles. Van Aken et al.⁷⁶ showed that AtWRKY40 and AtWRKY63, which are involved in stress responses, modulate the expression of stress-responsive nuclear genes encoding mitochondrial and chloroplast proteins. This provides additional evidence for the involvement of WRKYs in a communication system between the organelles. The crosstalk between the organelles may be more pronounced or relevant when the plants are exposed to (salt) stress. This again reinforces that comparative studies with different wrky knockout and WRKY overexpressor lines might be helpful to understand the complete scenario.

Cold/Heat Stress

In Arabidopsis, 939 cold-regulated genes have been reported with 655 upregulated and 284 downregulated genes.⁷⁷ A large number of early cold-responsive genes encode TFs. Among them are 8 WRKY genes (Table 1).⁷⁷ In particular, the expression of 6 cold-inducible WRKY genes was enhanced in ice1,⁷⁷ a mutant with a lesion in a gene that is strongly induced under cold stress (⁷⁸ and references therein), and the altered gene expression occurred almost exclusively at early time points. This demonstrates that WRKYs are major early cold-regulated genes in Arabidopsis. Luo et al.⁷³ showed that wild soybean WRKY20 is cold inducible and enhances drought tolerance in Arabidopsis and regulates ABA signaling.

Table 1. Names of cold regulated WRKY genes with altered transcription in ice1

Gene name	Locus	Gene ID
WRKY40	261892_at	At1g80840
WRKY33	267028_at	At2g38470
WRKY46	263783_at	At2g46400
WRKY22	255568_at	At4g01250
WRKY18	253485_at	At4g31800
WRKY25	267246_at	At2g30250
WRKY6	264746_at	At1g62300
WRKY32	253603_at	At4g30935

Mature pollen is very sensitive to cold stress in chilling-sensitive plants. Zou et al.⁷⁹ showed that WRKY34 is involved in pollen viability, although the exact mechanism is unclear. Cold treatment increased WRKY34 expression in the wild type, and promoter-GUS analysis revealed that WRKY34 expression is pollen-specific. Enhanced green fluorescent protein-tagged WRKY34 was localized in the nuclei. Pollen harboring the wrky34 allele showed higher viability than pollen with the WRKY34 allele after cold treatment. Further functional analysis indicated that WRKY34 was involved in pollen development regulated by the pollen-specific MIKC* class of MADS-domain TFs under cold stress, and cold-insensitivity of mature wrky34 pollen might be partly attributable to the enhanced expression of transcriptional activator CBFs in the mutants. Thus, the WRKY34 TF negatively mediates cold sensitivity of mature Arabidopsis pollen and might be involved in the CBF signal cascade in mature pollen. This suggests that additional work is required to completely understand the role of WRKYs in cold stress and cold stress adaptation (Table 1)

Arabidopsis WRKY39 provides a nice example for a TF that is induced by heat stress.⁸⁰ WRKY39 is a member of the group II WRKY proteins and responds to both abiotic and biotic stress. Heat-treated seeds and wrky39 knock-down mutants had increased susceptibility to heat stress, showing reduced germination, decreased survival, and elevated electrolyte leakage compared with wild-type plants. In contrast, WRKY39 overexpressing plants exhibited enhanced thermotolerance compared with wild-type plants.⁸⁰ Therefore, WRKYs are involved in both cold and heat acclimation of plants.

Phosphate Limitation

Phosphorus (P), an essential macronutrient required for plant growth and development, makes up about 0.2% of a plant’s dry weight. It is involved in regulation of key metabolic pathways as well as several enzymatic reactions in plants and animals. P is a crucial component of major organic molecules such as nucleic acids, ATP, and membrane phospholipids. It is present in soils in the form of inorganic phosphate (Pi), which has low availability and poor mobility. To cope with Pi limitations, plants have evolved complex adaptive responses that include morphological and physiological modifications to improve their acquisition, use, and remobilization of P.^81-83 Among the molecular determinants of Pi stress responses, TFs are believed to play a critical role in regulating adaptive mechanisms. Several TFs have been reported which are induced during Pi deprivation like WRKY6, -42, and -75.^84-86

Devaiah et al.⁸⁷ identified WRKY75 as an important component of the Pi stress responses. WRKY75 was found to be nuclear localized and induced differentially in the plant during Pi deficiency. Suppression of WRKY75 expression through RNAi silencing resulted in early accumulation of anthocyanin, indicating that the RNAi plants were more susceptible to Pi stress.⁸⁵ WRKY75 is a positive regulator of several Pi starvation-induced genes including phosphatase, Mt4/TPS1-like genes and high affinity Pi transporters.⁸⁶

Chen et al.⁸⁴ studied the role of WRKY6 under low Pi stress conditions. WRKY6 is involved in Arabidopsis responses to low-Pi stress through regulating PHOSPHATE1 (PHO1) expression. PHO1 plays an important role in Pi translocation from root to shoot.⁸⁷ PHO1 is predominantly expressed in the stellar cells of the root and the lower part of the hypocotyls.⁸⁷ Low Pi treatment reduced WRKY6 binding to the PHO1 promoter, which indicates that PHO1 regulation by WRKY6 is Pi dependent and that low Pi treatment may release inhibition of PHO1 expression.⁸⁴ These studies provide important information about the link of WRKYs to nutrient-limitation stress. It remains to be tested whether these responses are specific for Pi, and whether comparable regulatory circuits exist for other essential plant nutrients. Interestingly, although WRKY75 and WRKY6 participate in the Pi-deficiency response, they regulated different target genes. Whether they function in different and independent signaling networks or whether they are connected to each other remains to be determined.

Sugar Sensing and Metabolism

WRKY45 and WRKY65 are involved in regulating genes which respond to carbon starvation.⁸⁸ Three rice WRKY genes are also upregulated in sucrose-starved rice suspension cultures.⁸⁹ Interestingly, an Arabidopsis line overexpressing the rice WRKY72 gene showed increased sensitivity of sugar starvation stress.¹¹ Hammargren et al.⁹⁰ showed that sugar regulates the expression of the Arabidopsis NUCLEOSIDE DIPHSOPHATE KINASE 3a (NDPK3a) gene. NDPK3a is located in mitochondria. Sugar metabolism is intricately connected with mitochondria through the conversion of sugars to ATP, and through the production of carbon skeletons that can be used in anabolic processes. Sugar molecules also take part in signaling cascades. An analysis of the NDPK3a promoter identified 2 boxes that have previously been reported to be involved in sugar signaling in barley by binding to the SUSIBA2 protein. SUSIBA2 is a WRKY TF. T-DNA insertions in WRKY4 and WRKY34 showed a possible involvement of WRKY TFs in sugar induction of NDPK3a. Sugar is also reported to be involved in retrograde signaling to the nucleus.^91,92 Therefore, this regulatory circuit may contribute to couple retrograde signaling to sugar metabolism. Furthermore, a long-standing discussion suggests a link between Pi availability and sugar metabolism in beneficial symbiotic interactions. Since WRKYs are involved in both processes and integrate information from the different organelles involved in sugar metabolism, they might as well have a more general regulatory role in coordinating the cellular metabolism.

Oxidative Stress

Since WRKY TFs are involved in various stress responses, it is not surprising that they also play an important role in oxidative stress. Expression of several WRKY genes is upregulated in response to ROS or ROS-generating stimuli, imbalances in redox homeostasis, as well as endogenous ROS-dependent developmental programs such as senescence.^3,93-96 At least 8 Arabidopsis WRKY genes (WRKY30, -75, -48, -39, -6, -, -22, -53, -8) have been demonstrated to be upregulated in response to H₂O₂ treatments.^22,96-98 Overexpression of several WRKY genes which are upregulated in response to ROS-generating abiotic stresses confer also stress tolerance to plants.

WRKY25 expression is controlled by the transcriptional regulator Zat12, and Zat12 expression, in turn, is transcriptionally enhanced during osmotic, drought, salinity, temperature, oxidative or high-light stress and wounding.^99,100WRKY28 is known to be upregulated under drought and oxidative stress. Babitha et al.⁷⁴ showed that Arabidopsis lines overexpressing the 2 TFs HLH17 and WRKY28 exhibited enhanced tolerance to NaCl, mannitol and oxidative stress. Under mannitol stress higher root growth was observed in these transgenic lines. The performance of the overexpressor lines was also better than the wild-type when they were exposed to gradual long-term desiccation stress. Genes having W-box elements in their promoters showed higher expression levels in the overexpressor lines than in the wild type under oxidative stress conditions. Hence, simultaneous overexpression of the 2 TFs HLH17 and WRKY28 results in upregulation of many downstream target genes and substantially improves the oxidative stress tolerance of the plants.

Scarpeci et al.¹⁰¹ exposed plants to methyl viologen (MV), a superoxide anion propagator in the light, and identified superoxide-responsive genes with unknown function, for TFs and signal transduction components. The W-box sequence was present in the promoters of a number of genes which responded to superoxide. Band shift assays showed that oxidative treatments enhanced the specific binding of leaf protein extracts to this motif. In addition, a GUS reporter gene fused to the WRKY30 promoter, which contains the W-box motif, was induced by MV and H₂O₂. Thus, WRKY TF genes are rapidly activated by superoxide anion and may function as part of the early antioxidant response in Arabidopsis leaves.

Stress also causes perturbation of the ROS homeostasis.³⁶ The ROS-inducible Arabidopsis WRKY15 TF gene modulates plant growth and salt/osmotic stress responses. WRKY15 functions as negative regulator of salt- and osmotic-stress tolerance in Arabidopsis thaliana.³⁶ Overexpression of WRKY15 results in an increased leaf area and a 15–25% increase in plant biomass. Since the cell number did not increase, WRKY15 stimulates cell expansion, and this is caused by an intensified endoreplication. Transgenic plants expression artificial microRNA constructs targeting WRKY15 were smaller than wild-type plants, and contained a decreased leaf cell area.³⁶ Plants with elevated WRKY15 mRNA levels show also increased sensitivity to osmotic and oxidative stress. Expression profiling indicated that the majority of the regulated genes in stress-exposed WRKY15 overexpressors code for mitochondrial proteins, and they are members of the so-called mitochondrial dysfunction regulon.^36,102,103 These genes are likely to participate in the mitochondria-to-nucleus retrograde signaling.³⁶ The authors proposed that WRKY15 might function as a general repressor of genes involved in mitochrondiral retrograde signaling. When exposed to stress, WRKY15 might be released from its cognate W-box elements and replaced by other WRKY TFs. In such a model, WRKYs might act in a network of mutually competing participants with temporal replacement by other WRKY members in a stimulus-dependent manner.^3,36,104

Hormones such as ABA, the stress-responsive JA/ET and SA but also auxin are often coupled to ROS production and alterations in redox homeostasis. Wildermuth and Jones⁴⁰ showed that root growth is inhibited at 10–100 μM SA, concentrations that may be of physiological relevance in endogenous stress signaling and responses to rhizosphere SA. This SA root growth inhibition results primarily from a dramatic reduction in cell elongation and is specific to SA. Structurally similar molecules such as 4-hydroxybenzoate do not inhibit root growth at these concentrations. Root cell elongation is associated with ROS accumulation and auxin signaling. Through the use of ROS specific dyes and auxin reporters, Wildermuth and Jones⁴⁰ showed that root-growth inhibitory SA levels eliminate certain ROS maxima and alter auxin signaling in growing root tips. They further show through analysis of SA signaling mutants that SA-mediated inhibition of root growth does not require the well-established NPR1-SA immune response pathway. Rather, WRKY38, a highly SA responsive TF known to be expressed in the root and to modulate the NPR1 pathway, regulates the SA response. Thus, exogenous SA inhibits root cell elongation, abolishes certain root ROS maxima and alters root auxin signaling, and the SA response involves WRKY38. Another example provides an enzyme catalyzing β-oxidation of fatty acids, 3-ketoacyl-CoA thiolase-2 (KAT2).⁵⁹ KAT2 positively regulates ABA signaling in all the major ABA responses, including ABA-induced inhibition of seed germination and post-germination growth arrest, and ABA-induced stomatal closure and stomatal opening inhibition in Arabidopsis. KAT2 was shown to be important for ROS production in response to ABA, suggesting that KAT2 regulates ABA signaling at least partly through modulating ROS homeostasis. KAT2 may function downstream of WRKY40, and thus WRKY40 may link KAT2 with the ABA receptor ABAR/CHLH-mediated signaling.⁵⁹ Since several WRKYs such as WRKY40 are involved in abiotic and biotic stress responses which induce ROS species or causes imbalances in ROS homeostasis, it remains to be studied whether ROS are the immediate triggers that regulate WRKY gene expression.

Biotic stress responses involve ROS production mainly via plasma-membrane located NADPH oxidases. Exposure of plants to pathogens or PAMPs results in ROS production and the activation of WRKY genes involved in innate immunity, such as WRKY18, -40, and -70. It is generally believed that PAMP-induced receptor activation induces a short signal transduction pathway that activates the plasmamembrane-located NADPH oxidase to produce H₂O₂ in the apoplastic space. Parallel signaling pathways, e.g., via MAP kinase phosphorylation, activate WRKY TFs to induce defense genes. Whether and how these 2 signaling events are connected to each other is unknown. Göhre et al.¹⁰⁵ showed a molecular crosstalk between PAMP-triggered immunity and photosynthesis. Activation of defense by PAMPs leads to a rapid decrease in nonphotochemical quenching (NPQ). Conversely, NPQ also influences several responses of PAMP-triggered immunity. In a mutant impaired in NPQ, apoplastic ROS production is enhanced and defense gene expression is differentially affected. Although induction of the early defense markers WRKY22 and WRKY29 is enhanced, induction of the late markers PR1 and PR5 is completely abolished. Therefore, regulation of NPQ is an intrinsic component of the plant's defense program and affects also WRKY gene expression. Gao et al.¹⁰⁶ showed that WRKY50 and WRKY51 mediate SA- and low- oleic acid (18:1)-dependent repression of JA signaling. Signaling induced upon a reduction in oleic acid levels simultaneously upregulates SA-mediated responses and inhibits JA-inducible defenses, resulting in enhanced resistance to biotrophs but increased susceptibility to necrotrophs. Knockout mutations in WRKY50 and WRKY51 lowered SA levels. Inactivation of the WRKY genes in the ssi2 (suppressor of SA insensitivity2) background (ssi2 wrky50 and ssi2 wrky51) resulted in plants which contained high ROS levels. Thus, the 3 genes participate in maintaining a balanced ROS homeostasis in the plant under stress.

Besides pathogenic plant/microbe interactions, WRKYs also participate in beneficial symbiosis. Brotman et al.¹⁰⁷ reported that during early phases of root colonization by the opportunistic plant symbionts Tricoderma spp., expression of several genes related to osmo-protection and general oxidative stress in roots are upregulated by the fungi. Trichoderma can ameliorate plant growth under conditions of abiotic stress by lowering ameliorating increases in ET levels as well as promoting an elevated antioxidative capacity.

The relationship between ROS produced in response to biotic and abiotic stresses for WRKY expression needs to be investigated. In addition, the spatial distribution of the ROS species, which regulated WRKY genes, need to be analyzed. These studies may contribute to the understanding of the specificity in WRKY functions.

Another intensively studied field is the relationship of WRKYs and ROS during senescence. WRKY53 and WRKY70 are implicated as positive and negative regulators of senescence, respectively. Besseau et al.¹⁰⁸ showed that 2 WRKYs, WRKY54 and WRKY30, are involved in this process. The structurally related WRKY54 and WRKY70 exhibit a similar expression pattern during leaf development and appear to have co-operative and partly redundant functions in senescence, as revealed by single and double mutant studies. These 2 negative senescence regulators and the positive regulator WRKY53 interact independently with WRKY30, as shown by yeast 2-hydrid analysis. WRKY30 was expressed during developmental leaf senescence and could be part of a senescence regulatory network with the other WRKYs. Expression in wild-type and SA-deficient mutants suggests a common but not exclusive role for SA in induction of WRKY30, -53, -54, and -70 during senescence. WRKY30 and WRKY53 but not WRKY54 and WRKY70 are also responsive to additional signals such as ROS, which accumulate during senescence and control senescence progression. The results suggest that WRKY53, WRKY54, and WRKY70 may participate in a regulatory network that integrates internal and environmental cues to modulate the onset and the progression of leaf senescence, possibly through an interaction with WRKY30. Rosenvasser et al.¹⁰⁹ reported on the induction of WRKY6 in the leaves of Pelargonium cuttings during dark-induced senescence. Besides an increase in the ROS level, the senescence-associated protease homolog genes PeSAG12–1 and PeWRKY6–1 were upregulated during senescence.¹⁰⁹ It is not clear at present how these 2 processes are related to each other.

Taken together, a number of independent reports demonstrate that alterations in ROS levels, either in response to biotic or abiotic stress signals or caused by developmental processes, induce various WRKY genes. It remains to be determined how the stress-induced signaling pathways are related to the altered ROS levels in inducing WRKY gene expression. Furthermore, it is also not clear whether the stress stimuli can be replaced by ROS in all instances. ROS with signaling functions are H₂O₂, singlet oxygen (¹O₂), hydroxyl radical (OH·) and superoxide anion radical (O₂^.-) and a given ROS and its location affect its role in signaling.^{11,94,111,112} Whether all of these ROS species induce WRKYs, whether the cellular location—in which the ROS species are produced—is important, and whether there are differences in the response of the individual WRKYs, remains to be studied as well.

Biotic Stress/Wounding

WRKY TFs play an important role in the regulation of early defense-responses associated with pathogen defense. First, pathogen infection or treatment with pathogen elicitors or SA has been shown to induce rapid expression of WRKY genes from a number of plants.^104,113-117 Second, a number of defense-related genes, including the PR genes, contain W-box elements in their promoter regions and these W-box sequences are specifically recognized by WRKY proteins and are necessary for the inducible expression of these genes.^104,113-117 Several studies have provided direct evidence for the involvement of specific WRKY proteins in plant defense responses. For example, WRKY1 has been shown as a transcriptional activator mediating fungal elicitor-induced gene expression by binding to W box elements in parsley. In situ RNA hybridization revealed that WRKY1 is rapidly and locally activated in parsley leaf tissue around fungal infection sites. Transient expression studies in parsley protoplasts showed that a specific arrangement of W box elements in the WRKY1 promoter itself is necessary and sufficient for early activation and that WRKY1 binds to such elements.¹¹⁸ Similarly, WRKY22 and WRKY29 have been shown to be induced by the MAPK pathway involved in plant responses to both bacterial and fungal pathogens, and elevated WRKY29 expression in transiently transformed leaves led to reduced disease symptoms.¹¹⁸

WRKY18, WRKY40, and WRKY60 have been intensively studied and they were shown to be induced in response to biotrophic, hemibiotrophic and necrotrophic fungi.²⁰ Their roles in plant defense against microbial pathogen have been analyzed with various knockout mutant combinations and with constitutive overexpression lines, e.g., after infection with the bacterial pathogen Pseudomonas syringae or the fungal pathogen B. cinerea. Among the 3 single knockout mutants analyzed, only wrky18 exhibited a small reduction in bacterial growth, while wrky40 and wrky60 displayed no significant phenotype after infection of the bacterial pathogen. Two double knockout mutants, wrky18 wrky40 and wrky18 wrky60, on the other hand, supported modestly reduced bacterial growth, while the wrky18 wrky40 wrky60 triple knockout mutant had the most reduced bacterial growth when compared with wild-type plants. Constitutive overexpression of WRKY18 led to constitutive expression of the SA-related PR1 gene and enhanced resistance to P. syringae, its co-expression with WRKY40 or WRKY60 resulted in increased growth of the bacterial pathogen. These examples indicate that WRKY proteins can function as positive regulators of plant defense responses and disease resistance. Other examples for the involvement of WRKYs in innate immunity have been intensively described in various recent reviews.^6-8,13,16

WRKys also participate in other stress responses such as UV-B stress¹¹⁹ and wounding.^22,115 Nicotiana attenuata WRKY3 and WRKY6 responded to wounding, WRKY6 is required for WRK6 elicitation by fatty acid-amino acid conjugates from the larval oral secretions that are released into the ounds during feeding.¹²⁰ Silencing of both genes makes plants highly vunerable to herbivores and reduce Manduca sexta-elicited JA and JA-isoleucine levels.

Beneficial Plant/Microbe Interactions

The establishment of arbuscular mycorrhizal associations causes major changes in the root transcriptome, induced during the pre-, early, and late stages of root colonization. The most important class of regulated genes during the pre-stage of root colonization was associated to plant stress and in particular to the WRKY TF genes. Apparently, arbuscular mycorrhizal establishment requires a mild activation of defense responses in which WRKY TFs are involved.⁴⁷ Gallou et al.⁴⁷ observed 9 WRKY genes which are upregulated during the pre-stage of potato root colonization with Glomus interadices while only one WRKY gene was upregulation at late stages of colonization. No regulation of WRKY is observed at early stage of root colonization. These results suggest an involvement of WRKY genes during the pre-stage of root colonization. It would be interesting to known whether this stage is altered when potato line are used in which the corresponding WRKY genes are silenced or downregulated.

Brotman et al.¹⁰⁷ showed that the plant symbionts Trichoderma spp. colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis roots demonstrated a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and WRKY40, which stimulate JA-signaling via suppression of JAZ repressors and negatively regulate the expression of the defense genes FMO1, PAD3, and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization and reduced root colonization was observed in the wrky18 wrky40 double mutant line, while partial phenotypic complementation was achieved by overexpressing WRKY40 in the wrky18 wrky40 background. On the other hand, increased colonization rate was found in roots of the fmo1 knockout mutant. Trichoderma spp. stimulates plant growth and resistance to a wide range of adverse environmental conditions. Arabidopsis and cucumber (Cucumis sativus L.) plants treated with Trichoderma prior to salt stress imposition show significantly improved seed germination. In addition, Trichoderma treatment affects the expression of several genes related to osmo-protection and general oxidative stress in roots of both plants. The MDAR gene for monodehydroascorbate reductase is significantly upregulated and, accordingly, the pool of reduced ascorbic acid was found to be increased in Trichoderma-treated plants. 1-Aminocyclopropane-1-carboxylate (ACC)-deaminase silenced Trichoderma mutants were less effective in providing tolerance to salt stress, suggesting that Trichoderma, similarly to ACC deaminase producing bacteria, can ameliorate plant growth under conditions of abiotic stress, by lowering ameliorating increases in ET levels as well as promoting an elevated antioxidative capacity. Much more work is required to understand the role of WRKYs in beneficial plant/microbe interactions, in particular, since beneficial microbes promote nutrient uptake under nutrient-limiting conditions, control sugar metabolism and establish systemic resistance responses, all processes in which WRKYs are involved as well.^{39,84-86,90,121,122}

WRKY and Hormone Signaling

Many WRKYs participate in different hormone signaling pathways or their genes are regulated in response to these phytorhormones. For instance, WRKY18, -40 and -60 participate in JA, SA, and ABA signaling.

Role in ABA Signaling

WRKY genes are activators of ABA signaling, a phytohromone which mediates plant responses to abiotic stress.¹ Rushton et al.¹²³ showed that WRKYs act as positive regulators of ABA-mediated stomatal closure and hence drought responses and conversely, many WRKYs are negative regulators of seed germination. In Arabidopsis, WRKYs appear to act downstream of at least 2 ABA receptors: the cytoplasmic PYR ⁄ PYL ⁄ RCAR protein phosphatase 2C-ABA complex and the chloroplast envelope–located ABAR–ABA complex. In vivo and in vitro promoter-binding studies show that the target genes of WRKYs which are involved in ABA signaling include well-known ABA-responsive genes such as ABF2, ABF4, ABI4, ABI5, MYB2, DREB1a, DREB2a, RAB18. Some well-characterized stress-inducible proteins such as RD29A and COR47 are also found in signaling pathways downstream of WRKY TFs.¹²³ ABA signaling is also connected to other phytohromones, but their connetions to WRKYs is less understood. The ArabidopsisWRKY18, -40 and -60 represent nodes in Arabidopsis signaling networks that become activated by numerous stimuli and may integrate signaling from numerous phytohormones including SA, JA, ABA, and gibberellin acid (GA).

SA Signaling and WRKYs

The Arabidopsis WRKY1 was shown to be involved in SA signaling and is partially dependent on NPR1.⁴⁹ Also the heat stress-induced WRKY39 is induced in response to treatment with SA or methyljasmonate and positively regulates the cooperation between the SA- and JA-activated signaling pathways that mediate responses to heat stress.⁸⁰ Kim et al.¹²⁴ showed that WRKY38 and WRKY62, encoding 2 structurally similar type III WRKY TFs, are induced in a NPR1-dependent manner by SA or by virulent P. syringae. Disease resistance and SA-regulated PR1 gene expression are enhanced in the wrky38 and wrky62 single mutants and, to a greater extent, in the double mutants. Overexpression of WRKY38 or WRKY62 reduces disease resistance and PR1 expression. Thus, WRKY38 and WRKY62 function additively as negative regulators of plant basal defense. Rapid pathogen-induced WRKY33 expression does not require SA signaling, but is dependent on PAD4, a key regulator upstream of SA.¹²⁵ WRKY33 is essential for defense toward the necrotrophic fungus Botrytis cinerea.¹²⁶ Loss of WRKY33 function results in inappropriate activation of the SA-related host response and elevated SA levels post infection and in the downregulation of JA-associated responses at later stages. This downregulation appears to involve direct activation of several JA ZIM-domain genes, encoding repressors of the JA-response pathway, by loss of WRKY33 function and by additional SA-dependent WRKY factors. Moreover, genes involved in redox homeostasis, SA signaling, ET-JA-mediated cross-communication, and camalexin biosynthesis were identified as direct targets of WRKY33. Although SA-mediated repression of the JA pathway may contribute to the susceptibility of wrky33 plants to B. cinerea, it is insufficient for WRKY33-mediated resistance. Thus, WRKY33 apparently directly targets other still unidentified components that are also critical for establishing full resistance toward this necrotroph.^126,127 Likewise, the expression profile of VvWRKY11 from grapevine in response to treatment with SA or the pathogen Plasmopara viticola is rapid and transient.⁷² Transgenic Arabidopsis seedlings overexpressing VvWRKY11 showed higher tolerance to water stress induced by mannitol than wild-type plants. These results demonstrated that VvWRKY11 is involved in the response to dehydration stress.⁷² The stress-induced WRKY25 functions as a negative regulator of SA-mediated defense responses to P. syringae.¹²⁸ This is consistent with the finding that WRKY25 is a substrate of Arabidopsis MAP kinase 4, a repressor of SA-dependent defense responses.¹²⁹ The authors showed that MPK4 functions as a regulator of pathogen defense responses, because it is required for both repression of SA-dependent resistance and for activation of JA-dependent defense gene expression. In yeast 2-hybrid experiments the MPK4 substrate MKS1 was isolated. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast 2-hybrid screening revealed that MKS1 interacts with WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant exhibited increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY TFs.¹²⁹ Although these examples demonstrate the involvement of SA in WRKY functions, the role of SA and its integration into the kinase pathways is not completely clear.

Van Verk et al.¹²¹ showed that increased defense against a variety of pathogens in plants is achieved through systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through SA. An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3) plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. The ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays identified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA. These results demonstrate that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively.¹²¹ Recently, Schön et al.¹³⁰ demonstrated that simultaneous mutation of WRKY18 and WRKY40 renders otherwise susceptible wild-type Arabidopsis plants resistant toward the biotrophic powdery mildew fungus Golovinomyces orontii. Resistance in wrky18 wrky40 double mutant plants is accompanied by massive transcriptional reprogramming, imbalance in SA and JA signaling, altered ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) expression, and accumulation of the phytoalexin camalexin. Genetic analyses identified SA biosynthesis and EDS1 signaling as well as biosynthesis of the indole-glucosinolate 4MI3G as essential components required for loss-of-WRKY18 WRKY40-mediated resistance toward G. orontii. The analysis of wrky18 wrky40 pad3 mutant plants impaired in camalexin biosynthesis revealed an uncoupling of pre- from postinvasive resistance against G. orontii. Interestingly, WRKY18 and WRKY40 act as positive regulators in effector-triggered immunity, as the wrky18 wrky40 double mutant was found to be strongly susceptible toward the bacterial pathogen P. syringae DC3000 expressing the effector AvrRPS4 but not against other tested Pseudomonas strains. It appears that G. orontii depends on the function of WRKY18 and WRKY40 to successfully infect Arabidopsis wild-type plants while, in the interaction with P. syringae AvrRPS4, they are required to mediate effector-triggered immunity.¹³⁰

WRKY45 plays also a major role in the SA-induced defense in rice.¹³¹ The nuclear ubiquitin proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient upregulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, transcriptional activity of WRKY45 after SA treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in transcriptional activity of WRKY45.¹³¹ Furthermore, knock-down of the rice WRKY45 severely reduces SA/benzothiadiazole (BTH)-induced resistance to the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv oryzae. Conversely, overexpression of WRKY45 induces extremely strong resistance to both of these pathogens.¹³² A detailed analysis of the transgenic rice plants uncovered a central role of WRKY45 in BTH-induced disease resistance. Furthermore, the SA-activated WRKY45 protein induces the accumulation of its own mRNA. Ueno et al.⁶⁶ showed that a MAPK cascade is involved in this regulation and phosphorylates WRKY45.

Panicle blast 1 (Pb1) is a panicle blast resistance gene derived from the indica rice cultivar “Modan.” Pb1 encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NB-LRR) protein and confers durable, broad-spectrum resistance to M. oryzae races. The Pb1 protein interacts with WRKY45. Pb1-mediated panicle blast resistance is largely compromised when WRKY45 was knocked down in a Pb1-containing rice cultivar. Leaf-blast resistance by Pb1 overexpression was also compromised in WRKY45 knockdown/Pb1 overexpressor rice. Blast infection induced higher accumulation of WRKY45 in the Pb1 overexpressor than in control Nipponbare rice. Overexpression of Pb1-Quad, a coiled-coil domain mutant that had weak interaction with WRKY45, resulted in significantly weaker blast resistance than that of wild-type Pb1. Overexpression of Pb1 with a nuclear export sequence failed to confer blast resistance to rice. These results suggest that the blast resistance of Pb1 depends on its interaction with WRKY45 in the nucleus. In a transient system using rice protoplasts, coexpression of Pb1 enhanced WRKY45 accumulation and increased WRKY45-dependent transactivation activity, suggesting that protection of WRKY45 from ubiquitin proteasome system degradation is possibly involved in Pb1-dependent blast resistance.¹³³ These studies highlight the central role of the rice WRKY45 in panicle blast resistance, and uncovered novel mechanisms of WRKY45 function.

NPR1 regulates PR gene expression by interacting with TGA TFs. NPR1 is essential in balancing the SA- and JA-dependent signal transduction pathways probably through direct or indirect interaction with some WRKY TFs and the NPR1-like protein NPR4. Different reports in different systems also describe an important role of JA in WRKY signaling and function: For instance, overexpression of VvWRKY1 in grapevines induces expression of JA pathway-related genes and confers higher tolerance to the downy mildew.¹³⁴ On the other hand, Rabara et al.¹³⁵ reported that dehydration-induced WRKY genes from tobacco and soybean respond to JA treatments in BY-2 cell culture. To fully understand the role of WRKYs in the SA-JA/ET crosstalk, more experients and different pathosystems need to be analyzed. It is also possible that no common scene for the differernt responses in the differernt pathosystems has been evolved.

Auxin and Cytokinin

Gradients of the plant hormone auxin, which depend on its active intercellular transport, are crucial for the maintenance of root meristematic activity. This directional transport is largely orchestrated by a complex interaction of specific influx and efflux carriers that mediate the auxin flow into and out of cells, respectively. Besides these transport proteins, plant-specific polyphenolic compounds known as flavonols have been shown to act as endogenous regulators of auxin transport. However, only limited information is available on how flavonol synthesis is developmentally regulated. Using reduction-of-function and overexpression approaches in parallel, Grunewald et al.³⁸ demonstrate that WRKY23 is needed for proper root growth and development by stimulating the local biosynthesis of flavonols. The expression of WRKY23 itself is controlled by auxin through the Auxin Response Factor 7 (ARF7) and ARF19 transcriptional response pathway. The authors proposed a model in which WRKY23 is part of a transcriptional feedback loop of auxin on its own transport through local regulation of flavonol biosynthesis.

Grunewald et al.³⁷ also analyzed the development of a multicellular embryo from a single zygote. In higher plants, apical-basal patterning mechanisms are crucial to correctly specify root and shoot stem cell niches that will sustain and drive post-embryonic plant growth and development. The auxin-responsive WRKY23 is expressed from early embryogenesis onwards and the timing and localization of its expression overlaps with the root stem cell niche. Knocking down WRKY23 transcript levels or expression of a dominant-negative WRKY23 version via a translational fusion with the SRDX repressor domain affected both apical-basal axis formation as well as installation of the root stem cell niche. WRKY23 expression is affected by 2 well-known root stem cell specification mechanisms, that is, SHORTROOT and MONOPTEROS-BODENLOS signaling. On the basis of these results, Grunewald et al.³⁷ postulated that a tightly controlled WRKY23 expression is involved in the regulation of both auxin-dependent and auxin-independent signaling pathways toward stem cell specification.

Xu et al.³⁹ analyzed Arabidopsis lines which overexpressed a cotton defense-related gene GbWRKY1. This resulted in modification of the root system by enhanced auxin sensitivity to positively regulate the Pi starvation response. GbWRKY1 from Gossypium barbadense was first identified as a defense-related gene and showed moderate similarity with AtWRKY75 from Arabidopsis. Overexpression of GbWRKY1 in Arabidopsis resulted in attenuated Pi starvation stress symptoms, including reduced accumulation of anthocyanin and impaired density of lateral roots in low Pi stress. Overexpression of GbWRKY1 caused plants constitutively exhibited Pi starvation response including increased development of lateral roots, relatively high level of total P and Pi, high expression level of some high-affinity Pi transporters and phosphatases as well as enhanced accumulation of acid phosphatases activity during Pi-sufficiency. It was speculated that GbWRKY1 may act as a positive regulator in the Pi starvation response as well as AtWRKY75. GbWRKY1 is probably involved in the modulation of Pi homeostasis and participates in the Pi allocation and remobilization but does not accumulate more Pi in Pi-deficient conditions, which was different from the fact that AtWRKY75 influenced the Pi status of the plant during Pi deprivation by increasing root surface area and accumulation of more Pi. The overexpression plants were more sensitive to auxin than wild-type and GbWRKY1 may partly influence the LPR1-dependent (low phosphate response 1) Pi starvation signaling pathway and was putatively independent of SUMO E3 ligase SIZ1 and PHR1 (phosphate starvation response 1) in response to Pi starvation. These examples demonstrate that GbWRKY1 has an influence on auxin-regulated root development as well as Pi acquisition.

Yu et al.³¹ showed that plants can launch resistance mechanisms to stressful environments and acquire a new equilibrium between development and defense. To screen the rice WRKY TF which functions in abiotic stress tolerance and modulates the ABA response, 35S-OsWRKY72 transgenic Arabidopsis plants, whose seed germination was retarded under normal conditions, are more sensitive to mannitol, NaCl, ABA stresses and sugar starvation than vector plants. 35S-OsWRKY72 transgenic Arabidopsis displayed early flowering, reduced apical dominance, lost high temperature-induced hypocotyl elongation response, and enhanced gravitropism response. The expression patterns of 3 auxin-related genes AUX1, AXR1 and BUD1 were significantly altered in rosette leaves and inflorescences of 35S-OsWRKY72 plants, and 2 ABA-related genes ABA2 and ABI4 were induced in 35S-OsWRKY72 seedlings. These results suggest that OsWRKY72 interferes in the signal cross-talk between the ABA signal and auxin transport pathway in transgenic Arabidopsis.

Li et al.³⁰ performed a gene network analysis and functional studies of senescence-associated genes during Arabidopsis leaf senescence. Cytokinin, auxin, nitric oxide as well as Ca²⁺ delay leaf senescence. By contrast, ET, ABA, SA, and JA as well as oxygen promote leaf senescence. WRKY75 and the Cys2/His2-type TF AZF2 are positive regulators of leaf senescence and loss-of-function of WRKY75 or AZF2 delayed leaf senescence. Li et al.³⁰ also found that silencing of a protein phosphatase, AtMKP2, promoted early senescence. Apparently, WRKY75 couples senescence to a phytohromone network that includes auxin.

WRKYs Involved in Developmental Processes

Seed development and others

Several WRKY genes from different plant species are expressed during different stages of seed development. The WRKY gene DGE1 of orchardgrass (Dactylis glomerata) is expressed during somatic embryogenesis.¹³⁶ Similarly, ScWRKY1, a group Ia WRKY protein gene, is strongly and transiently expressed in fertilized ovules at the late torpedo stage in wild potato.¹³⁷ SUSIBA2 is expressed in the endosperm and regulates starch production. Arabidopsis WRKY10, also known as MINISEED3, is expressed in pollen and the globular embryo as well as in developing endosperm from the 2-nuclei stage through the cellularization stage.

WRKYs are found to be involved mainly in seed development. In Arabidopsis, Transparent Testa Glabra 2 (TTG2)/WRKY44 is the first member of the WRKY family with a role in trichome development. WRKY44 effects mucilage and tannin synthesis in the seed coat and also plays an important role in root hair development.^1,2,138 ScWRKY1 which is mainly expressed in leaves has also a specific role during embryogenesis.¹³⁷

Senescence

Leaf senescence is a complex developmental and highly regulated phase that involves both degenerative and nutrient recycling processes. It is characterized by loss of chlorophyll and the degradation of proteins, nucleic acids, lipids, and nutrient remobilization. WRKY TFs play a central role in the regulation of leaf senescence. Expression profiling in Arabidopsis revealed that WRKY TFs are the second largest family of TFs in reprogramming the Arabidopsis genome toward senescence.¹³⁹

Mutual regulation exists between WRKY22, WRKY6, WRKY53, and WRKY70.¹⁴⁰ WRKY22 transcription was suppressed by light and promoted by darkness. Expression of WRKY22 is strongly upregulated by H₂O₂. Dark-adapted WRKY22 overexpressor lines showed an accelerated and knockout line a delayed senescence phenotype, which was reflected in the regulation of senescence-associated genes.¹⁴⁰ This implies an involvement of WRKY22 in abiotic stress and senescence. WRKY22 can influence the expression of its own gene and of WRKY6, WRKY53, and WRKY70, suggesting the involvement of feedback regulatory loops.¹⁴⁰ A well-studied WRKY member involved in senescence, WRKY53, is tightly regulated by largely unknown mechanisms and involved in the cross-talk between senescence, biotic and abiotic stress responses.^5,97 Expression of WRKY53 is induced by H₂O₂ and JA and repressed by SA.⁹⁷ Besides control of senescence, WRKY53 is involved in basal resistance against P. syringae, together with HSPRO2, a protein with similarities to a nematode resistance protein from sugar beet.³² Furthermore, WRKY46 and WRKY70 together with WRKY53 co- ordinates basal resistance to P. synringae, and the 3 TFs appear to have overlapping and synergistic functions.¹⁴¹ In a yeast-2-hybrid screen with WRKY53, Miao and Zentgraf¹⁴² isolated the HECT domain E3 ubiquitin ligase UPL5. UPL5 interacts with WRKY53 via its leucine zipper domain, and was able to use WRKY53 as a substrate for polyubiquitination which led to its degradation. Expression of both genes is regulated antagonistically in response to H₂O₂, JA and plant development. upl5 knockout lines showed the same senescence phenotype as WRKY53 overexpressers. Overexpression of WRKY53 in the upl5 background enhanced the accelerated senescence phenotype of WRKY53 overexpressors. Therefore, UPL5 regulates leaf senescence in Arabidopsis through degradation of WRKY53 and ensures that senescence is executed in the correct time frame.¹⁴² Regulation of leaf senescence in Arabidopsis includes epigenetic re-programming via histone methylation at WRKY53.⁶⁷ A mitogen activated protein kinase kinase kinase (MEKK1) was identified as a protein which binds to the WRKY53 promoter.¹⁴³ The binding is important for the switch of WRKY53 expression from a leaf age dependent to a systemic plant age dependent expression during bolting time. In addition to its promotor-binding activity, MEKK1 was also able to interact with the WRKY53 protein itself. Complex formation of MEKK1 and WRKY53 occurs predominately in the nucleus of Arabidopsis cells. MEKK1 phosphorylates WRKY53 in vitro and phosphorylation increases DNA-binding and transcription of a WRKY53 promoter driven reporter gene in vivo. These results suggest that MEKK1 is a bifunctional protein: it binds to the promoter of the WRKY53 gene regulating the switch from a leaf age dependent to a plant age dependent expression and it phosopharylates WRKY53 in vitro thereby increasing its DNA binding activity.¹⁴³ Furthermore, Miao et al.⁶³ identified a novel DNA-binding protein that contains a transcriptional activation domain and a kinase domain with similarities to HPT kinases, and the novel DNA-binding protein also binds to the upstream region of WRKY53. The activation domain protein (AD protein) of the DNA-binding protein can phosphorylate itself and phosphorylation increases its DNA-binding activity to the WRKY53 promoter region. The AD protein interacted also with MEKK1, however, the AD protein was not phosphorylated by MEKK1. Overexpression and knockout of the gene for this novel transcriptional regulator confirmed that the AD protein is a positive regulator of WRKY53 expression. Expression of the AD protein gene can be induced by H₂O₂ and is repressed by JA treatment, comparable to the regulation of WRKY53.⁶³ Interestingly, the AD protein is also found in plastids.⁶³ Another dual-targeted protein, Whirly1^144,145 acts as a repressor of WRKY53 expression. Whirly1 mutants showed accelerated senescence and enhanced transcription of WRKY53 and of downstream genes WRKY33, SAG101, and SAG12. Transgenic Arabidopsis plants overexpressing Whirly1 targeting the protein either to the plastids or to the nucleus or to both compartments indicates that the nuclear isoform of Whirly1 can directly repress WRKY53 gene expression. Overexpressors where Whirly1 accumulates exclusively in plastids had an enhanced level of WRKY53 transcripts. This suggests that the nuclear form of Whirly1 is an upstream regulator of WRKY53 during leaf senescence,¹⁴⁰ and that the plastid form of Whirly1 is involved in retrograde signaling positively affecting the expression of WRKY53 in the nucleus.¹⁴⁶ More recently, Reusche et al.¹⁴⁷ have demonstrated that the width-inducing pathogen Verticillium longisporum triggers premature plant senescence for efficient host plant colonization. The soil-borne fungus enters the plant via the roots and colonizes the xylem. During later stages of the infection process, the fungal hyphae spread to senescence tissue in the leaves and switches from a biotrophic to a necrotrophic life style. The observed chlorosis induced by the fungus represents a premature senescence which is accompanied by the upregulation of WRKY53.¹⁴⁷ Taken together, regulation of WRKY53 appears to be a central process for reprogramming cells to senescence and the regulatory circuit is imbedded into MEKK1 signaling and retrograde signaling from plastids via the 2 dual-targeted AD protein and Whirly1.

Conclusion and Perspectives

WRKY proteins belong to a plant-specific family of TFs involved in various processes such as pathogen-induced defense programs, extensive metabolic changes associated with the establishment of defense responses or senescence, trichome development, etc. They participate in controlling the expression of a plethora of genes, by operating as positive and negative regulators and in various combinations. They can also be involved in unrelated stress responses; for example, WRKY18 and WRKY40 negatively regulate EDS1 signaling,¹⁴⁸ but positively regulates JA-signaling in response to G. orontii infection. WRKY25 and -33 are involved in biotic and abiotic stress responses; they function as negative regulators after P. syringae infection, but as positive regulators in conferring resistance against NaCl, cold, and heat and oxygenic stress.^{75,128,149,150} One WRKY protein seems to regulate several plant processes at a time and the mechanisms of regulation is not yet clear. Extensive study of these TF families is needed for better understanding of the signaling pathways involved in WRKY-mediated regulation of defense and developmental processes.

Figure 2A gives an overview of important regulatory circuits summarized in this review. The comparison of the individual panels highlights the regulatory ciruits, in which the individual WRKY proteins participate, and how they are connected to each other. The multiple appearance of an individual WRKY protein in different panels demonstrates the complexity of their involvement in the various signaling pathways. While relatively much information is available for the participation of some WRKYs in biotic stress responses, less is now about their involvement in abiotic stress. We are only at the beginning to understand their role in metabolism and their epigenetic control.

Figure 2A. The role of WRKYs and their genes in abiotic stress (A), biotic stress (B), the cross-talk between biotic and abiotic stress (C), metabolism (D), hormone signaling (E), and epigenetic control (F). The gray square boxes with the number refer to the individual WRKY proteins. WRKY genes are in green. The crosses demonstrate inhibiton of expression. A P in a yellow star refers to phosphorylation. Ub, ubiquitin.

Figure 2B

Figure 2B. The role of WRKYs and their genes in abiotic stress (A), biotic stress (B), the cross-talk between biotic and abiotic stress (C), metabolism (D), hormone signaling (E), and epigenetic control (F). The gray square boxes with the number refer to the individual WRKY proteins. WRKY genes are in green. The crosses demonstrate inhibiton of expression. A P in a yellow star refers to phosphorylation. Ub, ubiquitin.

Figure 2C

Figure 2C. The role of WRKYs and their genes in abiotic stress (A), biotic stress (B), the cross-talk between biotic and abiotic stress (C), metabolism (D), hormone signaling (E), and epigenetic control (F). The gray square boxes with the number refer to the individual WRKY proteins. WRKY genes are in green. The crosses demonstrate inhibiton of expression. A P in a yellow star refers to phosphorylation. Ub, ubiquitin.

Figure 2D

Figure 2D. The role of WRKYs and their genes in abiotic stress (A), biotic stress (B), the cross-talk between biotic and abiotic stress (C), metabolism (D), hormone signaling (E), and epigenetic control (F). The gray square boxes with the number refer to the individual WRKY proteins. WRKY genes are in green. The crosses demonstrate inhibiton of expression. A P in a yellow star refers to phosphorylation. Ub, ubiquitin.

Figure 2E

Figure 2E. The role of WRKYs and their genes in abiotic stress (A), biotic stress (B), the cross-talk between biotic and abiotic stress (C), metabolism (D), hormone signaling (E), and epigenetic control (F). The gray square boxes with the number refer to the individual WRKY proteins. WRKY genes are in green. The crosses demonstrate inhibiton of expression. A P in a yellow star refers to phosphorylation. Ub, ubiquitin.

Figure 2F

Figure 2F. The role of WRKYs and their genes in abiotic stress (A), biotic stress (B), the cross-talk between biotic and abiotic stress (C), metabolism (D), hormone signaling (E), and epigenetic control (F). The gray square boxes with the number refer to the individual WRKY proteins. WRKY genes are in green. The crosses demonstrate inhibiton of expression. A P in a yellow star refers to phosphorylation. Ub, ubiquitin.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.