Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family

Summary Fifty-nine VvWRKY genes were identified. Phylogenetic tree and synteny analysis revealed the specific evolutionary relationship of these genes. Meanwhile, differential expression patterns indicated their possible roles in specific tissues and under different stresses.


Introduction
Transcription factors are proteins that bind to specific DNA sequences in the promoter regions of genes, thereby regulating their transcription. Consequently, transcription factors play pivotal roles in numerous plant signalling and regulatory networks (Hwang et al., 2010). WRKY proteins, which are characterized by a highly conserved domain of about 60 amino acid residues, comprise a class of transcription factors that are known to function as transcriptional activators or repressors in a number of developmental and physiological processes (Eulgem et al., 2000;Rushton et al., 2010Rushton et al., , 2012. The first WRKY gene to be cloned and characterized was from sweet potato (Ishiguro and Nakamura, 1994) and this was followed by studies of WRKY genes from Arabidopsis (Eulgem et al., 2000), rice (Wu et al., 2005), and barley (Mangelsen et al., 2008). Moreover, large-scale genome-wide studies of WRKY genes have been described for Arabidopsis (Dong et al., 2003), rice (Ryu et al., 2006), poplar (He et al., 2012), tomato (Huang et al., 2012), cucumber (Ling et al., 2011), and coffee (Ramiro et al., 2010).
The two most prominent and defining structural characteristics of WRKY proteins are the WRKY domains and the zinc-finger motifs (C-X 4-5 -X 22-23 -H-X 1 -H or C-X 7 -C-X 23 -H-X 1 -C), which provide the basis of their classification into groups I-III (Eulgem et al., 2000). Proteins from group I contain two WRKY domains and a C 2 H 2 zinc-finger motif, while those from groups II and III only contain one WRKY domain and a C 2 H 2 or C 2 HC zinc-finger motif, respectively (Eulgem et al., 2000;Zhang and Wang, 2005). WRKY proteins bind to the W-box ((C/T)TGAC(T/C)) of their target genes (Ciolkowski et al., 2008) as well as a cis-element (SURE), which plays a role in promoting transcription (Sun et al., 2003).
WRKY transcription factors are involved in regulatory processes mediated by various biotic and abiotic stresses (Eulgem and Somssich, 2007;Pandey and Somssich, 2009). For example, defence responses in Arabidopsis to the bacterial pathogen Pseudomonas syringae have been associated with the action of AtWRKY38 and AtWRKY62 (Journot-Catalino et al., 2006;Kim et al., 2008) and the involvement of WRKY genes in the resistance of grapevine to pathogens was suggested by studies using transgenic tobacco lines expressing VvWRKY2, reflecting the important function of WRKY genes in biotic stress tolerance . WRKY genes are also related to abiotic stress induced gene expression: AtWRKY25 and AtWRKY33 show altered expression following either heat (Li et al., 2009;Li et al., 2011) or salt treatments (Jiang and Deyholos, 2009) and the expression of TcWRKY53 from alpine pennygrass (Thlaspi caerulescens) is affected by cold, salt, and polyethylene glycol treatments (Rushton et al., 2010). Studies involving other plants, including rice (Shimono et al., 2011), wheat  and poplar (He et al., 2012), have similarly provided insights into the diversity of WRKY gene function.
Grape (Vitis vinifera L.) is used both as a fresh food commodity and for processed food products such as juice, raisins, and wine. It is cultivated worldwide and has great economic value (Kreamer, 1995;Jaillon et al., 2007) and so there is considerable interest in identifying genes to improve grape horticultural characteristics, such as those that promote stress resistance. In this regard, the various studies of WRKY genes from different plant species mentioned above suggest that members of the grape WRKY gene family (VvWRKY) have considerable potential for contributing to aspects of stress resistance. This work reports the identification of 59 putative VvWRKY genes, together with an analysis of their exon-intron organization and associated gene duplication events in the context of gene evolution, since it has been shown that gene duplication has played a critical role in Arabidopsis and rice WRKY gene family expansion (Cannon et al., 2004). In addition, this work determined the expression profiles of VvWRKY genes in six different tissues and measured their transcript abundance in response to different phytohormone treatments and under various abiotic and biotic stresses. This study provides a foundation for future studies of VvWRKY gene family evolution and function.

Materials and methods
Identification and annotation of grape WRKY genes An Hidden Markov Model profile of the WRKY DNA-binding domain (PF03106) was downloaded from the Pfam protein family database (http://pfam.sanger.ac.uk/) (Finn et al., 2010) and used to identify putative WRKY genes/proteins from the grape genome sequence (http://www.genoscope.cns.f) (Jaillon et al., 2007) using the BLASTP program and default parameters (Ling et al., 2011). All non-redundant gene sequences encoding complete WRKY domains were selected as putative WRKY genes. Expressed sequence tags (ESTs) of each gene sequence from the public V. vinifera EST database were used for further validation. The identified grape WRKY genes were annotated based on their respective chromosome distribution (Ling et al., 2011).
Multiple sequence alignment, phylogenetic analysis, and classification of grape WRKY genes A total of 59 predicted VvWRKY proteins, with amino acids spanning the WRKY core domain, were included in multiple sequence alignments using CLUSTALX version 2.0.12 (Larkin et al., 2007) and Boxshade (http://www.ch.embnet.org/software/BOX_form.html). A further multiple sequence alignment including VvWRKY genes and those from Arabidopsis (AtWRKY) and tomato (Solanum lycopersicum, SlWRKY) was performed using CLUSTALW. A phylogenetic tree based on the alignment was constructed using MEGA 5.0 with the neighbour-joining method and with the bootstrap test replicated 1000 times (Tamura et al., 2011). Based on the multiple sequence alignment and the previously reported classification of AtWRKY genes, the VvWRKY genes were assigned to different groups and subgroups.
Exon-intron structure, tandem duplication, and synteny analysis of grape WRKY genes The exon-intron structures of the grape WRKY genes were determined based on alignments of their coding sequences and their respective full-length sequences (Grape Genome Browser; http:// www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/), while diagrams were obtained from the online program Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.ch). WRKY genes with tandem duplication events were defined as adjacent homologous genes on a single chromosome, while gene duplication events between different chromosomes were characterized as segmental duplications (Liu et al., 2011). The specific physical location of each VvWRKY gene on its individual chromosome therefore determined whether it was regarded as a genes resulting from a tandem duplication event. The syntenic blocks used for constructing a synteny analysis map of the grape WRKY genes, as well as between grape and Arabidopsis WRKY genes, were obtained from the Plant Genome Duplication Database (Tang et al., 2008) and the diagrams were generated by the program Circos version 0.63 (http://circos.ca/).

Plant material and treatments
Grape organs/tissues (roots, tendrils, leaves, inflorescences, fruit, and stems) were obtained from 2-year-old 'Kyoho' (Vitis labrusca × V. vinifera) grape seedlings, which were grown in 12 dm 3 pots in the greenhouse of Northwest A&F University, Yangling, Shaanxi, China (34° 20′ N 108° 24′ E). The third to fifth fully expanded young grapevine leaves beneath the apex were taken for hormone treatments, at which time the shoots of the vines were 25-35 cm long.
Hormone treatments were carried out by spraying leaves with 300 μM abscisic acid (ABA), 100 μM salicylic acid (SA), 50 μM methyl jasmonate (MeJA), and 0.5 g/l ethylene (Eth), and then leaves were sampled at 0.5, 1, 6, 24, and 48 h post treatment (Li et al., 2010). Leaves sprayed with sterile water and similarly harvested were used as a negative control. A salinity stress treatment was carried out by irrigating plants with 2 l of 250 mM NaCl in the pots (Upreti and Murti, 2010) followed by sampling leaves at 1, 6, 24, and 48 h post treatment. Seedlings irrigated with 2 l tap water were used as a negative control. A drought stress treatment was performed by withholding water (Yang et al., 2012) followed by sampling at 24, 48, 96, 144, and 168 h post treatment. Plants were rewatered after 168 h of drought stress and sampled again 48 h later. Grape seedlings grown without drought stress were used as a control.
At each time point of each treatment, six leaves from six separate plants were combined to form one sample, and all of the treatment experiments were performed in triplicate. All these plant samples were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction.

Semiquantitative reverse-transcription PCR analysis
Total RNA samples were extracted from leaves using the EZNA Plant RNA Kit (R6827-01, Omega Bio-tek, USA). First-strand cDNA was synthesized by reverse transcription of 500 ng total RNA using PrimeScript RTase (TaKaRa Biotechnology, Dalian, China). The concentration of the cDNA was adjusted using PCR and the grape Actin1 gene (GenBank accession number AY680701) with the primers F (5′-GATTCTGGTGATGGTGTGAGT-3′) and R (5′-GACAATTTCCCGTTCAGCAGT-3′). Gene-specific primers for each VvWRKY gene were designed using Primer Premier 5.0 and optimized using oligo 7 (Supplementary Table S1 available at JXB online). Semiquantitative reverse-transcription (RT) PCR reactions were conducted using the following profile: initial denaturation at 94°C for 2 min, followed by 30-40 cycles of denaturation at 92°C for 30 s, annealing at 60 ± 5°C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 2 min. PCR products were separated on a 1.5% (w/v) agarose gel with ethidium bromide staining and imaged under UV light for further gene expression analysis. Each reaction was repeated three times and the three independent analyses showed the same trends for each gene and treatment. The expression data from the semiquantitative RT-PCR were collated, analysed, and visualized using the programs GeneSnap and Mev 4.8.1 (Saeed et al., 2006).

Real-time quantitative PCR
Real-time quantitative PCR was conducted using SYBR green (TaKaRa Biotechnology) on an IQ5 real time-PCR machine (Bio-Rad, Hercules, CA, USA) with a final volume of 20 μl per reaction. Each reaction mixture contained 10.0 μl SYBR Premix Ex Taq II (TaKaRa Biotechnology), 1.0 μl cDNA template, 0.8 μl each primer (1.0 μM), and 7.4 μl sterile distilled H 2 O. Each reaction was performed in triplicate. Cycling parameters were 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 30 s. Melt-curve analyses were performed using a program with 95°C for 15 s and then a constant increase from 60°C to 95°C. Gene-specific DNA primers were the same as those used for semiquantitative RT-PCR (Supplementary Table S1). The grape Actin1 gene was used as the internal reference gene. The software IQ5 was used to analyse the relative expression levels using the normalized-expression method (Hou et al., 2013).

Results
Identification of WRKY genes in the grape (V. vinifera L.) genome A total of 61 genes were originally obtained between PF03106 (a Hidden Markov Model profile of WRKY DNA-binding domain) and Grape Genome (12X) with BLASTP. Based on the presence of apparently complete WRKY domains, 59 genes were subsequently selected and annotated as being grape WRKY genes. Genes without a complete predicted WRKY domain were removed (GSVIVT01016690001, GSVIVT01031401001). Further analysis of the protein sequences using the NCBI website revealed that GSVIVT01016690001 shares 63% similarity with a Theobroma cacao GDSL-motif lipase protein, indicating that GSVEVT01016690001 may not belong to the WRKY family. The other putative 59 grape WRKY genes were mapped onto the 19 grape chromosomes and then renamed from VvWRKY1 to VvWRKY59 based on their distributions and relative linear orders among the respective chromosome. VvWRKY4 (GSVIVT01001332001) was located to chromosome 1_random region and VvWRKY59 (GSVIVT01007006001) in an unknown region, and were thus unlike the other 57 VvWRKY genes. At least one EST for 52 VvWRKY genes was found in the public V. vinifera EST database at NCBI and used for further corroboration, while only seven (VvWRKY9, VvWRKY13, VvWRKY 14, VvWRKY 17, VvWRKY 24, VvWRKY 41, VvWRKY 51) were without a corresponding EST. However, in order to perform a systematic and comprehensive analysis of all of the VvWRKY genes, 59 specific primers were designed (Supplementary  Table S1) for the expression analyses and gene confirmation. Detailed information about each VvWRKY gene is showed in Table 1, including the WRKY gene group numbers, gene locus numbers, accession numbers for the full-length sequences at NCBI, chromosome distribution (start sites and end sites), and the length of coding sequences.

Multiple sequence alignment of VvWRKY genes
The most prominent structural feature of WRKY proteins is the WRKY domain, which has been shown to interact with the W-box (C/T)TGAC(T/C), thereby activating a large number of defence-related genes (Eulgem et al., 2000). The WRKY domain consists of a highly conserved hepta-peptide stretch of WRKYGQK at the N-terminus, followed by a zinc-finger motif (Eulgem et al., 2000). A multiple sequence alignment of the core WRKY domain, spanning approximately 60 amino acids of all 59 VvWRKY proteins is shown in Fig. 1. A total of 54 VvWRKY proteins were found to have the highly conserved sequence WRKYGQK, while the others (VvWRKY8, VvWRKY13, VvWRKY14, VvWRKY17, VvWRKY24) vary by a single amino acid. Of these WRKYGKK is the most common, consistent with studies in tomato, being present in four of the five variants, while VvWRKY17 contains a WKKYGQK sequence. One possible consequence of variation in this WRKY domain is an altered binding specificity in the DNA targets, but this remains to be demonstrated. As previously described (Eulgem et al., 2000), the metal-chelating zinc-finger motif (C-X 4-5 -X 22-23 -H-X 1 -H or C-X 7 -C-X 23 -H-X 1 -C) is another important structural characteristic of WRKY proteins. However, some incomplete zinc-finger motifs were also identified, including examples encoded by VvWRKY7, VvWRKY17, and VvWRKY46. Interestingly, in contrast to group-III WRKY in rice (Wu et al., 2005) and barley (Mangelsen et al., 2008), there are no VvWRKY in group-III proteins containing a C-X 7 -C-X 24 -H-X-C zinc-finger motif, perhaps suggesting that this is a feature of monocotyledonous species.

Phylogenetic analysis of WRKY genes from grape, Arabidopsis, and tomato
To further analyse the evolutionary relationships in the VvWRKY gene family and to help in their classification, a total of 210 WRKY genes, comprising 70 from Arabidopsis, 81 from tomato, and 59 from grape, were used to construct a phylogenetic tree (Fig. 2). Based on the number of WRKY domains and the features of the specific zinc-finger motifs, all 59 VvWRKY genes were classified into three main groups, with five subgroups in group II (Eulgem et al., 2000). Twelve grape WRKY genes (VvWRKY4, VvWRKY11, VvWRKY15,VvWRKY18,VvWRKY26,VvWRKY28,VvWRKY35,VvWRKY39,VvWRKY46,VvWRKY57,VvWRKY58,VvWRKY59) with two WRKY domains belong to group I, which have a zinc-finger motif of C-X 4 -C-X 22-23 -H-X 1 -H. The other 40 grape WRKY genes with the zinc-finger structure of C-X 4 -C-X 22-23 -H-X 1 -H were assigned to group II, which comprised 60% of the total number of VvWRKY genes.

Exon-intron organization of VvWRKY genes
Insights into the structures of the VvWRKY genes were obtained through an analysis of the exon/intron boundaries, which are known to play important roles in the evolution of multiple gene families . As shown in Fig. 3, 52 of the 59 VvWRKY genes have two-six exons (seven with two exons, 16 with three exons, nine with four exons, nine with five exons, and 11 with six exons). The fact that VvWRKY46 has 17 exons, VvWRKY18 has 11 exons, VvWRKY58 has nine exons, VvWRKY38 has eight exons, VvWRKY22 and VvWRKY31 have seven exons and VvWRKY7 has only one exon indicates that both exon loss and gain has occurred during the evolution of the WRKY gene family. This may lead to functional diversity of closely related WRKY genes; however, this study noted that VvWRKY genes in the same group usually have a similar number of exons. The number of exons in group I is relatively large, ranging from five to 11 (VvWRKY46 with 17 exons was not included because of the possibility of special variation), while genes in group II have a relatively small number, ranging from one to eight exons, and group III from three to five. This exon pattern similarity may be the consequence of a number of gene duplication events. A further analysis indicated that nearly all VvWRKY genes contained an intron in their respective WRKY domains (Wu et al., 2005;He et al., 2012). The R-type introns are widely exist in majority of WRKY groups (I, IIc, IId, IIe, III), the same cases in rice and Arabidopsis (Eulgem et al., 2000;Wu et al., 2005).

Tandem duplication of VvWRKY genes
Genomic comparison is a method for rapidly transferring available genomic information from a model species to a less-studied species (Lyons et al., 2008;Zhang et al., 2012). The current work analysed the tandem duplication events of the 59 VvWRKY genes on the 19 grape chromosomes (Table 3) according to the methods of Holub (2001), where a chromosomal region within 200 kb containing two or more genes is defined as a tandem duplication event. There were 13 VvWRKY genes (VvWRKY9,VvWRKY10,VvWRKY12,VvWRKY13,VvWRKY14,VvWRKY20,VvWRKY21,VvWRKY24,VvWRKY25,VvWRKY41,VvWRKY42,VvWRKY49,VvWRKY50) clustered into six tandem duplication event regions on grape chromosome 4 (two clusters), 7 (two clusters), 13 (one cluster) and 15 (one cluster) (Table 3). Chromosomes 4 (cluster 1 and cluster 2) and 7 (cluster 3 and cluster 4) had two clusters respectively, indicating a hot spot of WRKY gene distribution. Besides the tandem duplication events, 15 segregation duplication events were also identified ( Fig. 4 and Supplementary Table S3), indicating that some VvWRKY genes were possibly generated by gene duplication. Moreover, the segregation duplication events can also provide a reference for the WRKY gene evolutionary relationship and functional prediction.

Synteny analysis of VvWRKY genes
A substantial number of WRKY genes from the model plant Arabidopsis have been systematically investigated (Eulgem et al., 2000;Dong et al., 2003) and so the current work performed a synteny analysis of Arabidopsis and grape WRKY genes (Fig. 5) to determine whether this might provide some functional insights. A total of 66 pairs of syntenic relations were identified, including 50 AtWRKY genes and 39 VvWRKY genes. Three AtWRKY genes (AtWRKY6, AtWRKY31, AtWRKY36) and nine VvWRKY genes (VvWRKY6, VvWRKY9, VvWRKY21, VvWRKY27, VvWRKY31, VvWRKY33, VvWRKY38, VvWRKY49, VvWRKY54) were found to be associated with at least three synteny events and interestingly, of these 12 genes, six were in group IIb, including three AtWRKY genes and three VvWRKY genes (VvWRKY31, VvWRKY38, VvWRKY54). This may indicate a high conservation of group-IIb WRKY genes, which may in turn suggest a fundamental function in plant development.
Detailed results from the comparative analysis are shown in Supplementary Table S4. The large number of synteny events suggests that many WRKY genes arose before the divergence of the Arabidopsis and grape lineages.

Expression patterns of VvWRKY genes in different tissues
To assess the potential functions of VvWRKY genes during grape development, this study investigated the expression patterns of all 59 VvWRKY genes in six organs/tissues (roots, tendrils, leaves, inflorescences, fruit, and stems). Nearly half of the VvWRKY genes showed no significant organ/tissue related differences in expression, but some clear spatial differences were noted (Fig. 6). For example, VvWRKY40 and VvWRKY45 were expressed at high levels in roots and VvWRKY42 and VvWRKY52 expression were particularly high in leaves. No VvWRKY gene was specifically associated with inflorescences or fruit. The WRKY genes which showed no significant expression difference between tissues are likely to play a more ubiquitous role in grape.

Expression profiles of VvWRKY genes in response to drought, salt, and powdery mildew infection
The ability of plants to tolerate a variety of abiotic and biotic stresses is an essential adaptive feature in changing environments. Moreover, the identification and functional analysis of genes involved in biotic and abiotic stress signal transduction pathways is of considerable interest in the context of enhancing agricultural productivity. In order to obtain insights into the potential roles of all 59 VvWRKY genes in stress associated gene expression, this study used 2-year-old 'Kyoho' (V. labrusca × V. vinifera) seedlings growing in pots in the greenhouse of Northwest A&F University that were exposed to salt and drought stress and monitored expression using semiquantitative RT-PCR, 'Shang-24' was used for powdery mildew inoculation. A total of four VvWRKY genes that could be associated with salt stress (VvWRKY16, VvWRKY25, VvWRKY28, VvWRKY35), drought stress (VvWRKY3, VvWRKY25, VvWRKY28, VvWRKY35), and powdery mildew inoculation (VvWRKY19, VvWRKY27, VvWRKY48, VvWRKY52), based on semiquantitative RT-PCR analysis ( Fig. 7A and B and Supplementary Figs S1-S3) were selected for further analysis and validation using real-time quantitative PCR ( Fig. 8A and B). In most cases a similar result was seen, including the four genes that were induced by drought stress and powdery mildew inoculation and three genes (VvWRKY16, VvWRKY25, VvWRKY35) by salt stress treatment. Only one gene (VvWRKY28) showed no significant difference in the real-time quantitative PCR. Based on the semiquantitative RT-PCR data (Fig. 7A), VvWRKY genes tend to be downregulated to a greater degree by salinity stress than by drought stress. VvWRKY12, VvWRKY14,VvWRKY15,VvWRKY26,VvWRKY28,VvWRKY31,VvWRKY32,VvWRKY39,VvWRKY46,and VvWRKY48, which all showed clear downregulation by the salt stress treatment, were upregulated to varying degrees by the drought stress treatment, indicating distinctly different regulatory networks existence. The reaction time for the expression pattern changes was another focus of this study and some genes, such as VvWRKY1 and VvWRKY51, showed altered expression at an early time point (1 h under salt stress, 24 h under drought stress), while others, including VvWRKY57 and VvWRKY59 showed upregulated expression at a relatively late time (48 h under salt stress, 144 or 168 h under drought stress). On the other hand, some genes showed an early downregulation but subsequent upregulation (VvWRKY2, VvWRKY27, VvWRKY49): for example, VvWRKY2 was downregulated at 1 h after the onset of the salt stress treatment, but was upregulated after 48 h for example.

Expression profiles of VvWRKY genes to hormone treatments
Plant hormones such as ABA, SA, MeJA, and ethylene have well-established roles in modulating plant signalling networks (Fujita et al., 2006). In this study, hormone treatments resulted in a wide variety of VvWRKY gene expression profiles ( Fig. 7C and Supplementary Figs S4-S7). A total of 31 VvWRKY genes showed different degrees of downregulation by the ABA treatment while 14 were upregulated. Similarly, 19 VvWRKY genes were downregulated and 18 were upregulated expression following SA treatment. However, the expression profiles resulting from MeJA and Eth treatments were distinct from those modulated by ABA and SA and a substantially greater number of upregulated genes were observed: 37 were upregulated by MeJA and 11 were downregulated, while 37 were upregulated and six were downregulated by Eth treatment. These expression variations indicate that the VvWRKY gene family possible is collectively regulated by a broad set of hormonal signals.

Discussion
Many WRKY family genes play important roles in diverse plant developmental and physiological processes (Du and Chen, 2000;Eulgem et al., 2000), including embryogenesis (Lagace and Matton, 2004), seed coat and trichome development (Johnson et al., 2002), and leaf senescence (Miao and Zhetgraf, 2007;Zhou et al., 2011), as well as various plant abiotic and biotic stress responses. This current study describes the identification of 59 VvWRKY genes from grape, together with an analysis of their structure, evolutionary history, and expression pattern diversity with respect to biotic and abiotic stresses.

Identification and annotation of VvWRKY genes
The publicly available collection of V. vinifera ESTs were used to confirm the identity of the 59 VvWRKY genes that were initially identified amongst the 30 434 annotated grape genes using BLASTP. Of the 59 VvWRKY genes, seven were without supporting EST data, indicating that they may not be expressed during grape development. However, semiquantitative RT-PCR analysis was used to confirm that all 59 VvWRKY genes, including the seven genes without EST support, were indeed expressed and had putative functions in various aspects of grape biology, indicating all these 59 VvWRKY genes were putative WRKY genes in grape.

Structural conservation and divergence of VvWRKY genes
Comparative genomic analysis is an effective method for studying gene structures and so this study assessed the conserved structural domains of the predicted grape WRKY proteins. Multiple sequence alignments revealed that five VvWRKY proteins (VvWRKY8, VvWRKY13, VvWRKY14, VvWRKY17, VvWRKY24) had sequence variations in their WRKY domain and three (VvWRKY7, VvWRKY17, VvWRKY46) had variations in their zinc-finger motif. In previous studies of Arabidopsis AtWRKY transcription factors, it was found that the binding-site preferences of the WRKYGQK motif depend on the DNA sequences adjacent to the TTGACY core motif (Ciolkowski et al., 2008). In the genes recognized by the proteins with a variation to the WRKY domain, the TTGACY core motif may exhibit an altered structure and function, and so WRKY genes without the WRKYGQK motif may recognize binding sequences other than the W-box element ((C/T) TGAC(C/T)). In tobacco, the NtWRKY12 protein, which contains a WRKYGKK motif, recognizes the downstream binding sequence TTTTCCAC, which is substantially different from the W-box (van Verk et al., 2008). Moreover, the soybean WRKY proteins GmWRKY6 and GmWRKY21, which have a WRKYGKK motif, do not bind normally to the W-box (Zhou et al., 2008). It therefore seems that variations in the WRKYGQK motif influence the normal interaction of WRKY genes with downstream target genes, and so it would be interesting to investigate the functions and binding specificities of VvWRKY8, VvWRKY13, VvWRKY14, VvWRKY17, and VvWRKY24. Moreover, as far as is known, nothing is known about the effect of zinc-finger motif changes. It is possible that the variations of the WRKY domain and zinc-finger motif could influence the classification of the VvWRKY genes reported here.   One example of this is VvWRKY17, which has a divergent zincfinger sequence, leading to a nebulous classification in the phylogenetic tree. It remains to be determined whether the observed sequenced variations in the conserved domains affect the function or the expression patterns of the regulated gene targets. Exon-intron structural diversification also plays an important role in the evolution of many gene families and exonintron gain or loss may be caused by the rearrangement and fusions of different chromosome fragments Guo et al., 2013). The current study provides an example of such diversification in the form of a WRKY gene (VvWRKY7) with only one exon, while other genes in the same phylogenetic group (group IIb) have four or five exons. Moreover, VvWRKY46 has as many as 17 exons, while VvWRKY4, which is similar to VvWRKY17, only has six exons.

The evolutionary relationship of VvWRKY genes
The size of the grape WRKY gene family (59) is small compared to that of other experimental model plants such as Arabidopsis (72) and rice (96). The WRKY genes from grape, Arabidopsis, rice, and tomato align within distinct phylogenetic groups (Table 2) and it is apparent that variations in the number of WRKY genes in group III are the primary cause of the diversity of WRKY gene family size. Interestingly, previous studies described group-III WRKY genes as being a newly defined group, as well as being the most dynamic group with respect to gene family evolution (Zhang and Wang, 2005). Therefore, a key role of group-III WRKY genes in plant evolution may exist.
Gene duplication events play a major role in genomic rearrangements and expansions (Vision et al., 2000) and are defined as either tandem duplications, with two or more genes located on the same chromosome, or segmental duplications, with duplicated genes present on different chromosomes (Liu et al., 2011). The large number of gene duplication events for grape (Figs 4 and 5) will help aid future gene function prediction and evolution analysis. Whole-genome duplication events (γ, β, α) are a common phenomenon in angiosperms (Zhang and Wang, 2005) and often lead to gene family expansion (Cannon et al., 2004). Ling et al. (2011) reported that in cucumber (Cucumis sativus) CsWRKY family, a divergence generated in the number of group-III WRKY genes resulted from different style of duplication events that occurred after the divergence of the eurosids' groups I and II (110 Mya). For some species in the eurosids' group I (cucumber, soybean, and grape), the number of group-III WRKY genes is small, which may be caused by a different pattern of duplication events. This idea is consistent with the current results concerning the group-III VvWRKY genes. The group-III AtWRKY genes with normal tandem duplication events (AtWRKY63, AtWRKY64, AtWRKY66, AtWRKY67) show evidence of large-scale duplication. According to Zhang (2003) not all duplication events are stable, but instead can be fixed or lost in the population due to selection pressure and evolution. If the duplication events happens at a favourable time and in genes that are highly expressed, they will most likely be retained, but other duplication events that are not useful to the organism because of functional redundancy, or even a negative effect on plant development, will either be deleted from the genome or become very will diverge. The group-III WRKY gene divergence may reflect such a gene evolutionary selection.

VvWRKY genes function in abiotic and biotic stresses
There is considerable evidence that WRKY genes play crucial roles in responses to abiotic and biotic stress-induced defence signalling pathways (Qiu et al., 2004;Chen et al., 2012;Ishihama and Yoshioka, 2012). From an applied perspective, the identification of WRKY genes with potential value in stress resistance improvement of grape would likely benefit from targeting such genes that are part of abiotic and biotic stressresponse networks. The semiquantitative RT-PCR expression profiles generated in this study (Fig. 7) revealed different expression patterns (upregulation and downregulation) for each VvWRKY gene under specific treatments, thus providing a useful resource for future gene expression and functional analyses. Dong and coworkers (2003) showed that nearly 70% of the AtWRKY genes are differentially expressed in response to microbial infection or SA treatment. Consistent with these previous studies, the current results show that 70-90% of VvWRKY genes are differentially expressed following various abiotic and biotic stress treatments, highlighting the extensive involvement of WRKY genes in environmental adaptation.
The roles of many Arabidopsis WRKY genes in plant abiotic stress responses have been extensively studied recently. For example, AtWRKY25 and AtWRKY33 regulate plants adaptation to salinity stress through an interaction with their upstream or downstream target genes (Jiang and Deyholos, 2009). VvWRKY26 shares 76% sequence similarity with AtWRKY25 and 71% similarity with AtWRKY33, but in the current study, the change in expression profile of VvWRKY26 as a consequence of the various treatments applied was not apparent. It is possible that although VvWRKY26, AtWRKY25, and AtWRKY33 are segregation duplication genes, their function may differ in different plant species. The presence or absence of the regulatory elements in the duplicated genes can have an important consequence on subsequent divergence of gene function for an explanation.
VvWRKY1  and VvWRKY2  (named in this work VvWRKY53 and VvWRKY4, respectively) have also been isolated and functionally analysed. VvWRKY2 is known to activate the promoter of VvC4H, which is involved in the lignin biosynthesis pathway and cellwall formation (Guillaumie et al., 2010), and it likely plays an important role in resistance or tolerance to biotic and abiotic stress in plants. However, VvWRKY4 appears not to respond to powdery mildew inoculation, suggesting that it may involve in resistance other abiotic and biotic stresses other than powdery mildew. On contrary, VvWRKY53 is significantly upregulated at 24 h post inoculation, which appears to share similar inoculation response with VvWRKY1 as reported earlier , thus suggesting that VvWRKY53 may play a role in eliciting resistance response during early stage of infection.
The temporal and spatial diversification of WRKY gene expression is widespread, which is important for gene function analysis. The VvWRKY genes VvWRKY40, VvWRKY45, VvWRKY42, and VvWRKY52 had a higher expression in certain grape organs/tissues suggesting divergent roles for these genes in grape development. With regard to temporal diversification, this work considered the response time to the different treatments since previous studies had shown that some WRKY genes respond to drought stress at an early stage, such as GsWRKY18, which peaked at 0.5 h after drought stress treatment (Ling et al., 2011). The current data show the opposite pattern, since about half of the VvWRKY genes (VvWRKY3 and VvWRKY35) selected for real-time quantitative PCR showed a peak of expression at 144 h after treatment and one (VvWRKY28) peaked at 168 h after drought stress treatment, indicating that a certain response time is needed for VvWRKY genes to respond to drought stress. The upregulation of the expression of some WRKY genes in response to powdery mildew inoculation showed a similar delay in response time, but shorter than that caused by the drought stress treatment (12 h post treatment).
Phytohormones are critically important in coordinating regulatory networks and the signal transduction pathways associated with external cues. ABA is known to function in signalling in some stressful environments , and SA, JA, and Eth play important roles in biotic stresses (Fujita et al., 2006), while MeJA responds to some biotic stresses and wounding . The current results show that the majority of VvWRKY genes showed significantly upregulated expression following treatment with MeJA and Eth, while ABA and SA treatments had the opposite effect. It has been reported that AtWRKY33 can act as a positive regulator in Eth-mediated defence signalling against necrotrophic pathogens and as a negative regulator in SA-mediated responses for some biotrophic bacterial pathogens (Zheng et al., 2006), which is the same trend seen in this study.
In conclusion, WRKY gene expression is influenced by a broad range of abiotic and biotic stress resistances and also responds to hormonal signals, indicating that they play a key role in signal transduction in plant resistance regulation. The role of WRKY genes on abiotic and biotic stress regulatory networks is systematic and complex, both between two WRKY proteins (Xu et al., 2006) and their downstream or upstream targets (Jiang and Deyholos, 2009).