WRKY transcription factor family
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WRKY transcription factors (pronounced ‘worky’) are
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s that bind DNA. They are
transcription factors In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fun ...
that regulate many processes in
plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclu ...
s and algae (
Viridiplantae Viridiplantae (literally "green plants") are a clade of eukaryotic organisms that comprise approximately 450,000–500,000 species and play important roles in both terrestrial and aquatic ecosystems. They are made up of the green algae, which ar ...
), such as the responses to biotic and
abiotic stress Abiotic stress is the negative impact of non-living factors on the living organisms in a specific environment. The non-living variable must influence the environment beyond its normal range of variation to adversely affect the population performan ...
es, senescence,
seed dormancy Seed dormancy is an evolutionary adaptation that prevents seeds from germinating during unsuitable ecological conditions that would typically lead to a low probability of seedling survival. Dormant seeds do not germinate in a specified period of ...
and seed germination and some developmental processes but also contribute to
secondary metabolism Secondary metabolism (also called specialized metabolism) is a term for pathways and small molecule products of metabolism that are involved in ecological interactions, but are not absolutely required for the survival of the organism. These molecu ...
. Like many
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The f ...
s, WRKY transcription factors are defined by the presence of a
DNA-binding domain A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence (a recognition sequence) or have a gener ...
; in this case, it is the WRKY domain. The WRKY domain was named in 1996 after the almost invariant WRKY amino acid sequence at the N-terminus and is about 60 residues in length. In addition to containing the ‘WRKY signature’, WRKY domains also possess an atypical
zinc-finger A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold. It was originally coined to describe the finger-like appearance of a hypothesized struc ...
structure at the C-terminus (either Cx4-5Cx22-23HxH or Cx7Cx23HxC). Most WRKY transcription factors bind to the
W-box The W box is a deoxyribonucleic acid (DNA) cis-regulatory element sequence, (T)TGAC(C/T), which is recognized by the family of WRKY transcription factors. Functionality and conservation of the W-box element across plant species has been shown by ...
promoter element that has a consensus sequence of TTGACC/T. Individual WRKY proteins do appear in the human protozoan parasite
Giardia lamblia ''Giardia duodenalis'', also known as ''Giardia intestinalis'' and ''Giardia lamblia'', is a flagellated parasitic microorganism of the genus '' Giardia'' that colonizes the small intestine, causing a diarrheal condition known as giardiasis. ...
and slime mold Dictyostelium discoideum.


Structural diversity

WRKY transcription factors are denoted by a 60-70 amino acid
WRKY protein domain The WRKY domain is found in the WRKY transcription factor family, a class of transcription factors. The WRKY domain is found almost exclusively in plants although WRKY genes appear present in some diplomonads, social amoebae and other amoebozo ...
composed of a conserved WRKYGQK motif and a zinc-finger region. Based on the amino acid sequence WRKY transcription factors are classified into three major categories, group I, group II, and group III. Group I WRKY proteins are primarily denoted by the presence of two WRKY protein domains, whereas both groups II and III each possess only one domain. Group III WRKY proteins have a C2HC zinc finger instead of the C2H2 motif of group I and II factors. The structure of several plant WRKY domains has been elucidated using crystallography and
nuclear magnetic resonance spectroscopy Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy (MRS), is a spectroscopic technique to observe local magnetic fields around atomic nuclei. The sample is placed in a magnetic fie ...
. As soon as the WRKY domain was characterized, it was suggested that it contained a novel
zinc finger A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold. It was originally coined to describe the finger-like appearance of a hypothesized struct ...
structure and the first evidence to support this came from studies with 2-phenanthroline that
chelates Chelation is a type of bonding of ions and molecules to metal ions. It involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom. These ligands are ...
zinc
ion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conve ...
s. Addition of 2-phenenthroline to gel retardation assays that contained E. coli expressed WRKY proteins resulted in a loss of binding to the
W-box The W box is a deoxyribonucleic acid (DNA) cis-regulatory element sequence, (T)TGAC(C/T), which is recognized by the family of WRKY transcription factors. Functionality and conservation of the W-box element across plant species has been shown by ...
target sequence. The other suggestion was that the WRKY signature amino acid sequence at the N-terminus of the WRKY domain directly binds to the W-box sequence in the DNA of target promoters. These suggestions were shown to be correct by publication of the solution structure of the
C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is ...
WRKY domain of the Arabidopsis WRKY4 protein. The WRKY domain was found to form a four-stranded β-sheet. Soon afterwards, a crystal structure of the C-terminal WRKY domain of the Arabidopsis WRKY1 protein was reported. This showed a similar result to the solution structure except that it may contain an additional β-strand at the N-terminus of the domain. From these two studies it appears that the conserved WRKYGQK signature amino acid sequence enters the major groove of the DNA to bind to the W-Box. Recently, the first structural determination of the WRKY domain complexed with a W-Box was reported. The NMR solution structure of the WRKY DNA-binding domain of Arabidopsis WRKY4 in complex with W Box DNA revealed that part of a four-stranded β-sheet enters the major groove of DNA in an atypical mode that the authors named the β-wedge, where this sheet is almost perpendicular to the DNA helical axis. As initially predicted, amino acids in the conserved WRKYGQK signature motif contact the W Box DNA bases mainly through extensive apolar contacts with thymine methyl groups. These structural data explain the conservation of both the WRKY signature sequence at the N-terminus of the WRKY domain and the conserved cysteine and histidine residues. It also provides the molecular basis for the previously noted remarkable conservation of both the WRKY amino acid signature sequence and the W Box DNA sequence.


History

In 1994 and 1995, the first two reports of WRKY transcription factors appeared. They described newly discovered but as yet ill-defined DNA binding proteins that played potential roles in the regulation of gene expression by sucrose (SPF1) or during germination (ABF1 and ABF2). A third report appeared in 1996 that identified WRKY1, WRKY2 and WRKY3 from parsley. The authors named the new transcription factor family the WRKY family (pronounced ‘worky’) after a conserved
amino acid sequence Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynthe ...
at the N-terminus of the
DNA-binding domain A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence (a recognition sequence) or have a gener ...
. The parsley WRKY proteins also provided the first evidence that WRKY transcription factors play roles in regulating plant responses to
pathogen In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ ...
s. Numerous papers have now shown this to be a major function of WRKY transcription factors. Since these initial publications, it has become clear that the WRKY family is among the ten largest families of transcription factors in higher plants and that these transcription factors play key roles in regulating a number of plant processes including the responses to biotic and
abiotic stress Abiotic stress is the negative impact of non-living factors on the living organisms in a specific environment. The non-living variable must influence the environment beyond its normal range of variation to adversely affect the population performan ...
es, germination, senescence, and some developmental processes.


Evolution

WRKY transcription factor genes are found throughout the plant lineage and also outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa. This patchy distribution suggests that lateral gene transfer is responsible. These lateral gene transfer events appear to pre-date the formation of the WRKY groups in flowering plants, where there are seven well-defined groups, Groups I + IIc, Groups IIa + IIb, Groups IId + IIe, and Group III. Flowering plants also contain proteins with domains typical for both resistance (R) proteins and WRKY transcription factors. R protein-WRKY genes have evolved numerous times in flowering plants, each type being restricted to specific flowering plant lineages. These chimeric proteins contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. Several early reports proposed that a group I WRKY transcription factor was the progenitor of the family. It was thought that a single group I WRKY domain occurred first and then duplicated to form the original ancestral WRKY transcription factor. However, more recent evidence suggests that WRKY transcription factors evolved from a single group IIc-like gene, which then diversified into group I, group IIc, and group IIa+b domains. The original WRKY protein domain has been proposed to have arisen from the
GCM1 Chorion-specific transcription factor GCMa is a protein that, in humans, is encoded by the ''GCM1'' gene. This gene encodes a DNA-binding protein with a gcm-motif ( glial cell missing motif). The encoded protein is a homolog of the ''Drosophila ...
and FLYWCH zinc finger factors. GCM1 and FLYWCH are proposed ancestral proteins base on their crystal structural similarity to the WRKY domain. Both GCM1 and FLYWCH belong to families of DNA-binding factors found in
metazoan Animals are multicellular, eukaryotic organisms in the biological kingdom Animalia. With few exceptions, animals consume organic material, breathe oxygen, are able to move, can reproduce sexually, and go through an ontogenetic stage in ...
. The plant specific NAC transcription factor family also shares a common structural shape and origin with WRKY transcription factors. During plant evolution the WRKY family has dramatically expanded, which is proposed to be a result of through duplication. Some species including Arabidopsis thaliana, rice (
Oryza sativa ''Oryza sativa'', commonly known as Asian rice or indica rice, is the plant species most commonly referred to in English as ''rice''. It is the type of farmed rice whose cultivars are most common globally, and was first domesticated in the Yan ...
), and tomato (
Solanum lycopersicum The tomato is the edible berry of the plant ''Solanum lycopersicum'', commonly known as the tomato plant. The species originated in western South America, Mexico, and Central America. The Mexican Nahuatl word gave rise to the Spanish wor ...
) have WRKY groups which dramatically expanded and diversified in recent evolutionary history. However, differences in expression, not variation in gene sequences, have likely lead to the diverse functions of WRKY genes. Such a model is plausible as WRKY family members are part of numerous phytohormone, developmental, and defense signaling transcriptional networks. Furthermore, W-box elements for WRKY binding occur in promoters of many other WRKY transcription factors indicating not simply a hierarchical rank in gene activation, but also which genes may have arisen later during evolution after initial WRKY regulatory networks were established.


Function

Over the last two decades great effort has been invested in characterizing WRKY transcription factors. The results show that WRKY transcription factors function in a diverse array of plant response, both to internal and external cues.


Plant Development

Studies have demonstrated the function of WRKY transcription factors in plant development. Successful male
gametogenesis Gametogenesis is a biological process by which diploid or haploid precursor cells undergo cell division and differentiation to form mature haploid gametes. Depending on the biological life cycle of the organism, gametogenesis occurs by meiotic di ...
and tolerance to interploidy crosses both require WRKY transcription factors. Embryo and root development also require WRKY transcription factors. WRKYs also contribute to determination of seed size and seed coat color in Arabidopsis. Furthermore, WRKY transcription factors have been shown to play key roles in regulation of developmentally programmed leaf senescence.


Abiotic and Biotic Stresses

One of the most notorious roles of the WRKY transcription factor family is the regulation of plant stress tolerance. WRKYs participate in nearly every aspect of plant defense to abiotic and biotic stressors. WRKYs are known to regulate cold, drought, flooding, heat, heavy metal toxicity, low humidity, osmotic, oxidative, salt and UV stresses. Likewise, WRKY transcription factors play an essential role in plant tolerance to biotic stresses, protecting against innumerable viruses, bacterial and fungal pathogens, as well as insect herbivory. Plants are believed to perceive pathogens via pathogen-associated molecular pattern (PAMP) triggered immunity and effector-triggered immunity. WRKY transcription factors participate in regulating responses to pathogens by targeting PAMP and effector triggered immunity.


Hormone Signaling

WRKY transcription factors function through a variety of plant hormone signaling cascades. Over half of Arabidopsis thaliana WRKY transcription factors respond to salicylic acid treatment. At least 25% of WRKY transcription factors from Madagascar periwinkle (
Catharanthus roseus ''Catharanthus roseus'', commonly known as bright eyes, Cape periwinkle, graveyard plant, Madagascar periwinkle, old maid, pink periwinkle, rose periwinkle, is a species of flowering plant in the family Apocynaceae. It is native and endemic to M ...
) are responsive to
jasmonate Jasmonate (JA) and its derivatives are lipid-based plant hormones that regulate a wide range of processes in plants, ranging from growth and photosynthesis to reproductive development. In particular, JAs are critical for plant defense against herb ...
. Similarly, in grape (
Vitis vinifera ''Vitis vinifera'', the common grape vine, is a species of flowering plant, native to the Mediterranean region, Central Europe, and southwestern Asia, from Morocco and Portugal north to southern Germany and east to northern Iran. There are curr ...
) 63%, 73%, 76%, and 81% or WRKY transcription factors are responsive to salicylic acid, ethylene, abscisic acid, and jasmonate treatment, respectively. In Arabidopsis thaliana, two important WRKY transcription factors are WRKY57 and WRKY70. WRKY57 mediates crosstalk between jasmonate and auxin signaling cascades, whereas WRKY70 moderates signaling between the jasmonate and salicylic acid pathways. Arabidopsis thaliana WRKY23 functions downstream of auxin signaling to positively activate expression of flavonols, which function as
polar auxin transport Polar auxin transport is the regulated transport of the plant hormone auxin in plants. It is an active process, the hormone is transported in cell-to-cell manner and one of the main features of the transport is its asymmetry and directionality ( p ...
inhibitors, to negatively feedback and suppress further auxin responses. Several WRKY transcription factors also respond to gibberellin treatment.


Primary and Secondary Metabolism

Due to difficulty in measuring phenotypes, less is known about the roles of WRKY transcription factors in plant metabolism. The earliest reports identified WRKYs based on their ability to regulate β-amylase, a gene involved in catabolism of starch into sugars. Since then, WRKY transcription factors have also been shown to regulate phosphate acquisition and tolerance to
arsenic Arsenic is a chemical element with the symbol As and atomic number 33. Arsenic occurs in many minerals, usually in combination with sulfur and metals, but also as a pure elemental crystal. Arsenic is a metalloid. It has various allotropes, ...
. Additionally, WRKYs are needed for proper expression of lignin biosynthetic pathway genes, which form products necessary for cell wall and xylem formation. Analysis of WRKY transcription factors from numerous plant species indicates the importance of the family in regulating secondary metabolism. WRKY transcription factors also play a role in regulating pathways for the biosynthesis of pharmaceutically valuable plant-specialized metabolites. Efforts to use WRKY transcription factors to improve production of the valuable anti-malarial drug
artemisinin Artemisinin () and its semisynthetic derivatives are a group of drugs used in the treatment of malaria due to '' Plasmodium falciparum''. It was discovered in 1972 by Tu Youyou, who shared the 2015 Nobel Prize in Physiology or Medicine for her ...
have been successful.


Mode of action

A long-standing question of in the field of transcriptional regulation is how large families of regulators binding a consensus DNA sequences dictate expression of different target genes. The WRKY transcription factor family has long exemplified this problem. Plant species contain numerous WRKY transcription factors which predominantly recognize a conserved cis-element. Only recently has it begun to be revealed how different WRKY transcription factors regulate unique sets of target genes.


Variation in Cis-element Recognition

Early work indicated that the WRKY family could bind W-box (T/A)TGAC(T/A). Later, a barley (
Hordeum vulgare Barley (''Hordeum vulgare''), a member of the grass family, is a major cereal grain grown in temperate climates globally. It was one of the first cultivated grains, particularly in Eurasia as early as 10,000 years ago. Globally 70% of barley pro ...
) WRKY transcription factor, SUSIBA2, was found to bind the Sugar Response Element (TAAAGATTACTAATAGGAA), illustrating some diversity exists in DNA sequence which WRKYs could recognize. Since then, WRKYs have been found to bind a more generic GAC core cis-element with flanking sequences dictating DNA-protein interactions. On the protein side differences in the consensus motif and downstream arginine or lysine residues dictate the exact flanking sequence recognized. Additionally, contrary to early reports, both WRKY domains of group I family members can bind DNA. Implications of these results are still being resolved.


Protein-Protein Interactions

One mechanism for determining WRKY binding activity is by protein-protein interactions. WRKY transcription factors have been found to interact with a variety of proteins, some of which occur by a group specific manner. Recent evidence suggests that VQ protein family is an important regulator of group I and group IIc WRKY transcription factors. VQ proteins appear to bind the WRKY domain, thus inhibiting protein-DNA interactions. At least one WRKY transcription factor, Arabidopsis WRKY57, interacts with jasmonate ZIM-domain (JAZ) and auxin/indole acetic acid (AUX/IAA) repressor of the jasmonate and auxin signaling cascade, respectively, indicating a point of crosstalk between these phytohormones. Other WRKYs interact with
histone deacetylases Histone deacetylases (, HDAC) are a class of enzymes that remove acetyl groups (O=C-CH3) from an ε-N-acetyl lysine amino acid on a histone, allowing the histones to wrap the DNA more tightly. This is important because DNA is wrapped around his ...
. Group IIa WRKY factors form homodimers and heterodimers within the subgroup and with other group II subgroups. Group IId WRKY transcription factors typically possess a domain allowing interaction with calcium bound calmodulin.


Phosphorylation

Protein phosphorylation is a common method to regulate protein activity and WRKY transcription factors are no exception. WRKY gene involved in plant defense, hormone signaling, and secondary metabolism are regulated by phosphorylation via mitogen-activated protein kinase (MAPK) cascades. Additionally, a MAPK can phosphorylate a VQ protein, freeing the WRKY transcription factor for target gene activation. While kinases phosphorylating WRKY transcription factors are known,
phosphatases In biochemistry, a phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol. Because a phosphatase enzyme catalyzes the hydrolysis of its substrate, it is a subcategory of hydrolases. ...
removing phosphate groups have yet to be identified.


Proteasomeal Degradation

Protein degradation via the proteasome is a common feature in plant regulatory networks to limit the duration of activation or repression by transcription factors. WRKY transcription factors have also been found to be regulated by proteasomal degradation mechanisms. In Chinese grapevine (Vitis pseudoreticulata) ERYSIPHE NECATOR-INDUCED RING FINGER PROTEIN1 targets WRKY11 for degradation leading to enhanced powdery mildew resistance. In rice, WRKY45 is degraded by the proteasome although the E3 ubiquitin ligase responsible remains unknown Matsushita, Inoue, Goto, Nakayama, Sugano, et al. (2013) Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program. The Plant Journal. 73(2). 302-313


External links


WRKY family
a
PlantTFDB: Plant Transcription Factor DatabaseWRKY family
at
Plant Transcription Factor Database at University of PotsdamWRKY family
a
SuperfamilyWRKY Transcription Factor Family
a
The Arabidopsis Information ResourceThe Somssich LabThe Shen LabSomssich’s list of WRKY-related publications


References

{{reflist Transcription factors