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Journal of Experimental Botany logoLink to Journal of Experimental Botany
. 2014 Feb 7;65(6):1513–1528. doi: 10.1093/jxb/eru007

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

Chunlei Guo 1,2,*, Rongrong Guo 1,2,*, Xiaozhao Xu 1,2, Min Gao 1,2, Xiaoqin Li 1,2, Junyang Song 1,2, Yi Zheng 3, Xiping Wang 1,2,†
PMCID: PMC3967086  PMID: 24510937

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.

Key words: Evolution, expression profile analysis, grape (Vitis vinifera L.), phylogenetic analysis, synteny analysis, WRKY genes.

Abstract

WRKY proteins comprise a large family of transcription factors that play important roles in plant defence regulatory networks, including responses to various biotic and abiotic stresses. To date, no large-scale study of WRKY genes has been undertaken in grape (Vitis vinifera L.). In this study, a total of 59 putative grape WRKY genes (VvWRKY) were identified and renamed on the basis of their respective chromosome distribution. A multiple sequence alignment analysis using all predicted grape WRKY genes coding sequences, together with those from Arabidopsis thaliana and tomato (Solanum lycopersicum), indicated that the 59 VvWRKY genes can be classified into three main groups (I–III). An evaluation of the duplication events suggested that several WRKY genes arose before the divergence of the grape and Arabidopsis lineages. Moreover, expression profiles derived from semiquantitative PCR and real-time quantitative PCR analyses showed distinct expression patterns in various tissues and in response to different treatments. Four VvWRKY genes showed a significantly higher expression in roots or leaves, 55 responded to varying degrees to at least one abiotic stress treatment, and the expression of 38 were altered following powdery mildew (Erysiphe necator) infection. Most VvWRKY genes were downregulated in response to abscisic acid or salicylic acid treatments, while the expression of a subset was upregulated by methyl jasmonate or ethylene treatments.

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., 2010, 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–X4–5–X22–23–H–X1–H or C–X7–C–X23–H–X1–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 C2H2 zinc-finger motif, while those from groups II and III only contain one WRKY domain and a C2H2 or C2HC 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 (Mzid et al., 2007). 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 (Wu et al., 2008) 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 dm3 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 25–35cm 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 0.5g/l ethylene (Eth), and then leaves were sampled at 0.5, 1, 6, 24, and 48h 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 250mM NaCl in the pots (Upreti and Murti, 2010) followed by sampling leaves at 1, 6, 24, and 48h 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 168h post treatment. Plants were rewatered after 168h of drought stress and sampled again 48h later. Grape seedlings grown without drought stress were used as a control.

Samples of the powdery mildew fungus (Erysiphe necator) were used to inoculate young leaves of V. quinquangularis ‘Shang-24’ (Northwest A&F University, Yangling, Shanxi, China; Wang et al., 1995). Leaves were harvested at 6, 12, 24, 48, 72, 96, and 120h post inoculation and uninoculated leaves served as a negative 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 500ng 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 2min, 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 2min. 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 H2O. 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.

Table 1.

Grape WRKY genes and accessionsCDS, coding sequence; NG, no group; NSG, no subgroup, ORF, open reading frame.

Group Subgroup Gene ID Gene locus ID Accession no. CDS (bp) ORF (aa) Chromosome Start site End site Full length
I VvWRKY4 GSVIVT01001332001 CBI36506.3 1308 435 1_random 297 660 312 015 14 356
VvWRKY59 GSVIVT01007006001 CBI33229.3 1653 550 Unknown 29 694 308 29 699 639 5332
VvWRKY46 GSVIVT01011472001 CBI22264.3 2670 889 14 29 916 400 29 925 511 9112
VvWRKY58 GSVIVT01014854001 CBI39865.3 1869 622 19 10 665 036 10 669 055 4020
VvWRKY15 GSVIVT01019109001 CBI17638.3 1461 486 4 16 664 476 16 666 741 2266
VvWRKY35 GSVIVT01023600001 CBI36524.3 1500 499 11 7 835 815 7 846 137 10 323
VvWRKY18 GSVIVT01024624001 CBI15865.3 1713 570 6 8 290 025 8 295 109 5085
VvWRKY28 GSVIVT01025562001 CBI32633.3 1317 438 8 14 033 451 14 039 562 6112
VvWRKY39 GSVIVT01030046001 CBI28412.3 1095 364 12 9 116 731 9 122 807 6077
VvWRKY26 GSVIVT01030258001 CBI18092.3 1542 513 8 9 796 056 9 798 907 2852
VvWRKY11 GSVIVT01035965001 CBI21139.3 1593 530 4 6 569 931 6 576 637 6707
VvWRKY57 GSVIVT01037775001 CBI26736.3 1659 552 19 7 760 186 7 767 468 7283
II a VvWRKY30 GSVIVT01015952001 CBI25166.3 837 278 9 16 094 219 16 096 253 2035
a VvWRKY9 GSVIVT01035884001 CBI21068.3 789 262 4 5 247 592 5 248 886 1295
a VvWRKY10 GSVIVT01035885001 CBI21069.3 861 286 4 5 265 806 5 268 041 2236
b VvWRKY54 GSVIVT01008046001 CBI15258.3 1818 605 17 6 316 168 6 320 317 4150
b VvWRKY45 GSVIVT01011356001 CBI22167.3 1419 502 14 28 923 786 28 926 499 2714
b VvWRKY31 GSVIVT01012682001 CBI23209.3 1533 510 10 618 603 621 252 2650
b VvWRKY2 GSVIVT01020060001 CBI32009.3 1785 594 1 10 977 206 10 982 423 5218
b VvWRKY22 GSVIVT01028244001 CBI37053.3 1440 479 7 4 899 927 4 903 175 3249
b VvWRKY40 GSVIVT01029688001 CBI19998.3 1473 490 12 13 065 135 13 099 628 34 494
b VvWRKY38 GSVIVT01030453001 CBI28821.3 1497 498 12 5 678 707 5 681 171 2465
b VvWRKY56 GSVIVT01037686001 CBI26664.3 1491 496 19 6 882 419 6 884 988 2570
c VvWRKY53 GSVIVT01008553001 CBI15677.3 456 151 17 922 600 925 171 2572
c VvWRKY3 GSVIVT01010525001 CBI27681.3 570 189 1 21 460 123 21 461 397 1275
c VvWRKY1 GSVIVT01012196001 CBI27268.3 852 283 1 628 682 633 595 4914
c VvWRKY47 GSVIVT01018300001 CBI16682.3 687 228 15 11 498 970 11 504 729 5760
c VvWRKY37 GSVIVT01020864001 CBI22108.3 936 311 12 878 934 881 289 2356
c VvWRKY33 GSVIVT01021397001 CBI30827.3 960 319 10 4 894 476 4 896 340 1865
c VvWRKY24 GSVIVT01022245001 CBI21522.3 582 193 7 17 794 379 17 797 240 2862
c VvWRKY25 GSVIVT01022259001 CBI21534.3 681 226 7 17 958 306 17 960 930 2625
c VvWRKY49 GSVIVT01026969001 CBI40411.3 606 201 15 18 940 954 18 942 146 1193
c VvWRKY21 GSVIVT01028147001 CBI36970.3 909 302 7 4 200 160 4 202 241 2082
c VvWRKY44 GSVIVT01033063001 CBI21329.3 549 182 14 25 479 103 25 481 683 2581
c VvWRKY13 GSVIVT01033194001 CBI24019.3 471 156 4 9 399 944 9 400 803 860
c VvWRKY14 GSVIVT01033195001 CBI24020.3 306 101 4 9 409 805 9 411 286 1482
c VvWRKY16 GSVIVT01034968001 CBI22862.3 930 309 5 530 084 531 773 1690
c VvWRKY8 GSVIVT01035426001 CBI20684.3 501 166 4 1 209 585 1 211 712 2128
d VvWRKY19 GSVIVT01000752001 CBI17951.3 855 284 7 381 035 383 836 2802
d VvWRKY7 GSVIVT01001286001 CBI31897.3 318 105 2 4 989 461 4 989 778 318
d VvWRKY55 GSVIVT01009441001 CBI19480.3 960 319 18 8 391 930 8 393 726 1797
d VvWRKY23 GSVIVT01022067001 CBI21376.3 843 280 7 16 322 549 16 324 116 1568
d VvWRKY36 GSVIVT01029265001 CBI17876.3 840 279 11 17 821 900 17 823 266 1367
d VvWRKY12 GSVIVT01033188001 CBI24014.3 804 267 4 9 363 169 9 365 026 1858
d VvWRKY43 GSVIVT01036223001 CBI35175.3 915 304 14 8 753 538 8 756 300 2763
e VvWRKY32 GSVIVT01021252001 CBI30716.3 837 278 10 3 008 687 3 010 451 1765
e VvWRKY5 GSVIVT01019419001 CBI34393.3 972 323 2 512 163 513 533 1371
e VvWRKY34 GSVIVT01021765001 CBI31090.3 1266 421 10 10 755 760 10 759 820 4061
e VvWRKY50 GSVIVT01026965001 CBI40407.3 1047 348 15 18 957 231 18 958 817 1587
e VvWRKY20 GSVIVT01028129001 CBI36956.3 729 242 7 4 044 128 4 045 807 1680
e VvWRKY51 GSVIVT01028823001 CBI22627.3 549 182 16 18 360 079 18 360 711 633
NSG VvWRKY29 GSVIVT01034148001 CBI30536.3 900 299 8 14 828 040 14 830 056 2017
NG VvWRKY17 GSVIVT01025491001 CBI16558.3 366 121 6 364 396 365 387 992
III VvWRKY6 GSVIVT01019511001 CBI34466.3 1029 342 2 1 228 314 1 229 702 1389
VvWRKY48 GSVIVT01027069001 CBI40493.3 1083 360 15 18 191 021 18 193 489 2469
VvWRKY52 GSVIVT01028718001 CBI22547.3 1095 364 16 19 477 141 19 479 868 2728
VvWRKY27 GSVIVT01030174001 CBI18028.3 996 331 8 10 843 756 10 846 082 2327
VvWRKY42 GSVIVT01032661001 CBI25345.3 867 288 13 1 719 393 1 720 884 1492
VvWRKY41 GSVIVT01032662001 CBI25346.3 927 308 13 1 716 836 1 718 836 2001
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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–X4–5–X22–23–H–X1–H or C–X7–C–X23–H–X1–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–X7–C–X24–H–X–C zinc-finger motif, perhaps suggesting that this is a feature of monocotyledonous species.

Fig. 1.

Fig. 1.

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Multiple sequence alignment of the WRKY domain among V. vinifera WRKY genes. Red indicates conserved WRKY amino acid domains; green indicates zinc-finger motifs; dashes indicate gaps. ‘N’ and ‘C’ indicate the N-terminal and C-terminal WRKY domain of a specific WRKY gene (this figure is available in colour at JXB online).

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–X4–C–X22–23–H–X1–H. The other 40 grape WRKY genes with the zinc-finger structure of C–X4–C–X22–23–H–X1–H were assigned to group II, which comprised 60% of the total number of VvWRKY genes. The 40 group-II VvWRKY genes are unevenly distributed amongst the five subgroups: group IIa (three: VvWRKY9, VvWRKY10, VvWRKY30), group IIb (eight: VvWRKY2, VvWRKY22, VvWRKY31, VvWRKY38, VvWRKY40, VvWRKY45, VvWRKY54, VvWRKY56), group IIc (15: VvWRKY1, VvWRKY3, VvWRKY8, VvWRKY13, VvWRKY14, VvWRKY16, VvWRKY21, VvWRKY24, VvWRKY25, VvWRKY33, VvWRKY37, VvWRKY44, VvWRKY47, VvWRKY49, VvWRKY53), group IId (seven: VvWKY7, VvWRKY12, VvWRKY19, VvWRKY23, VvWRKY36, VvWRKY43, VvWRKY55), and group IIe (six: VvWRKY5, VvWRKY20, VvWRKY32, VvWRKY34, VvWRKY 50, VvWRKY51). In contrast to group I, group-II genes have only one WRKY domain. Instead of the C2H2 pattern, group-III genes contain a C2HC zinc-finger motif (C–X7–C–X23–H–X1–C) and six of the 59 VvWRKY genes (VvWRKY6, VvWRKY27, VvWRKY41, VvWRKY42, VvWRKY48, VvWRKY52) belong to this group. Finally, VvWRKY17 and VvWRKY29 do not group within any of the other group-II subgroups, possibly because of their apparently incomplete structures. Detailed information about the classification of the genes and the WRKY domains, as well as the profile of zinc-finger motifs can be found in Table 1 and Supplementary Table S2, respectively.

Fig. 2.

Fig. 2.

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Phylogenetic tree of WRKY genes among grape (red), Arabidopsis (blue), and tomato (black). Filled circle lines are used to cluster the genes with similar structures and functions (this figure is available in colour at JXB online).

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 (Zhang et al., 2012). 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).

Fig. 3.

Fig. 3.

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Genomic organization of grape WRKY genes. (A) Unrooted phylogenetic tree built on the basis of 59 complete WRKY-domain proteins in grape; details of clusters are shown in different colours. (B) Exon–intron structure of grape WRKY genes; blue indicates untranslated 5′- and 3′-regions; green indicates exons; black indicates introns (this figure is available in colour at JXB online).

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 200kb 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.

Table 3.

Tandem duplication events in the 59 VvWRKY genes

Cluster number Gene ID Chromosome Start site End site
1 VvWRKY9 4 5 247 592 5 248 886
VvWRKY10 4 5 265 806 5 268 041
2 VvWRKY12 4 9 363 169 9 365 026
VvWRKY13 4 9 399 944 9 400 803
VvWRKY14 4 9 409 805 9 411 286
3 VvWRKY20 7 4 044 128 4 045 807
VvWRKY21 7 4 200 160 4 202 241
4 VvWRKY24 7 17 794 379 17 797 240
VvWRKY25 7 17 958 306 17 960 930
5 VvWRKY41 13 1 716 836 1 718 836
VvWRKY42 13 1 719 393 1 720 884
6 VvWRKY49 15 18 940 954 18 942 146
VvWRKY50 15 18 957 231 18 957 231
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Fig. 4.

Fig. 4.

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Chromosome distribution and synteny analysis of grape WRKY genes. Chromosomes 1–19 are shown with different colours and in a circular form. The approximate distribution of each VvWRKY gene is marked with a short red line on the circle. Coloured curves denote the details of syntenic regions between grape WRKY genes (this figure is available in colour at JXB online).

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.

Fig. 5.

Fig. 5.

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Synteny analysis of grape and Arabidopsis WRKY genes. The chromosomes of grape and Arabidopsis are depicted as a circle. The approximate distribution of each AtWRKY gene and VvWRKY gene is marked with a short red line on the circle. Coloured curves denote the details of syntenic regions between grape and Arabidopsis WRKY genes (this figure is available in colour at JXB online).

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.

Fig. 6.

Fig. 6.

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Expression pattern of 59 VvWRKY genes in organs/tissues of ‘Kyoho’ (V. labrusca × V. vinifera); Actin1 (GenBank accession number AY680701) was used as an internal control. Lanes: 1: root, 2: tendril, 3: leaf, 4: inflorescence, 5: fruit, 6: stem. The experiments were repeated three times and the results were consistent.

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.

Fig. 7.

Fig. 7.

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Expression profiles of 59 VvWRKY genes. The results of semiquantitative RT-PCR were quantified using the Gene Tools software, and the relative expression levels of VvWRKY genes under treatments compared to the controls were used for hierarchical cluster analysis with MeV 4.8.1. The colour scale represents relative expression levels, with red as increased transcript abundance and green as decreased transcript abundance (A) Expression profiles of VvWRKY genes under biotic stress treatments, salinity, and drought (original results shown in Supplementary Figs S1 and S2). (B) Expression profiles of VvWRKY genes under biotic stress treatment, powdery mildew (original results shown in Supplementary Fig. S3). (C) Expression profiles of VvWRKY genes following four hormone treatments (abscisic acid (ABA), salicylic acid (SA), methyl jasmonic acid (MeJA), and ethanol (Eth)) (original results shown in Supplementary Figs S4–S7). The experiments were repeated three times and the results were consistent (this figure is available in colour at JXB online).

Fig. 8.

Fig. 8.

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Real-time quantitative PCR expression levels of selected VvWRKY genes following salinity stress, drought stress treatments, powdery mildew inoculation, and various hormone treatments (abscisic acid (ABA), salicylic acid (SA), methyl jasmonic acid (MeJA), and ethanol (Eth)). The expression levels were normalized to 1h (salt stress treatment), 24h (drought stress treatment), 6h (powdery mildew inoculation), and 0.5h (hormone treatments) CK sample, respectively. Mean values and SDs were obtained from three biological and three technical replicates. Asterisks indicate the corresponding gene significantly up- or downregulated under the differential treatment by t-test (*P<0.05, **P<0.01). (A) Expression levels of selected VvWRKY genes under abiotic stresses (salt and drought). (B) Expression levels of selected VvWRKY gene under biotic stress (powdery mildew). (C) Expression levels of selected VvWRKY genes under hormone treatments (this figure is available in colour at JXB online).

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 (1h under salt stress, 24h under drought stress), while others, including VvWRKY57 and VvWRKY59 showed upregulated expression at a relatively late time (48h under salt stress, 144 or 168h 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 1h after the onset of the salt stress treatment, but was upregulated after 48h for example.

The association of the VvWRKY genes with biotic stress was investigated using infection with powdery mildew, a severe disease worldwide (Zhang et al., 2012). The semiquantitative RT-PCR based expression profiles (Fig. 7B) of VvWRKY4, VvWRKY11, VvWRKY12, VvWRKY23, VvWRKY24, VvWRKY30, VvWRKY31, VvWRKY32, VvWRKY33, VvWRKY38, VvWRKY40, VvWRKY42, VvWRKY43, VvWRKY46, VvWRKY47, and VvWRKY51 indicated a potentially negative effect on grape powdery mildew resistance, while VvWRKY7, VvWRKY8, VvWRKY9, VvWRKY13, VvWRKY14, VvWRKY25, VvWRKY26, VvWRKY27, VvWRKY34, VvWRKY35, VvWRKY36, VvWRKY37, VvWRKY39, VvWRKY41, VvWRKY44, VvWRKY45, VvWRKY48, VvWRKY49, VvWRKY50, VvWRKY52, VvWRKY55, and VvWRKY58 showed the opposite pattern. VvWRKY12, VvWRKY23, VvWRKY40 VvWRKY43, and VvWRKY46 showed significant downregulation during early infection (6–72h or to 96h), while VvWRKY34, VvWRKY35, and VvWRKY36 were upregulated during a later stage (24–120h), possibly indicating a relationship between expression pattern and responding time.

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 zinc-finger 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 exon–intron gain or loss may be caused by the rearrangement and fusions of different chromosome fragments (Xu et al., 2012; 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.

Table 2.

The number of WRKY genes in each phylogenetic group from Arabidopsis, rice, grape, and tomatoVvWRKY17, VvWRKY29, SlWRKY26, SlWRKY27, and SlWRKY49 are not included, since they could not be placed.

Gene Phylogenetic group
I IIa IIb IIc IId IIe III
AtWRKY 13 4 7 18 7 9 14
OsWRKY 15 4 8 15 7 11 36
VvWRKY 12 3 8 15 7 6 6
SlWRKY 15 5 8 16 6 17 11
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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 stress-response 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 (Marchive et al., 2007) and VvWRKY2 (Mzid et al., 2007) (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 cell-wall 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 24h post inoculation, which appears to share similar inoculation response with VvWRKY1 as reported earlier (Marchive et al., 2007), 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 0.5h 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 144h after treatment and one (VvWRKY28) peaked at 168h 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 (12h post treatment).

WRKY genes that are components of plant biotic stress regulatory networks have a complex response pattern. For example, Arabidopsis AtWRKY33 (Lippok et al., 2007) and AtWRKY18 (Chen and Chen, 2002) can positively modulate defence-related gene expression and improve disease resistance, while some negative regulatory elements may prevent the overexpression of AtWRKY33 and AtWRKY18 and are detrimental to plant growth. Moreover, other WRKY genes, such as AtWRKY7 (Kim et al., 2006) and AtWRKY48 (Xing et al., 2008) have an immediate negative effect in the plant defence response. In these current studies, although VvWRKY4, VvWRKY11, VvWRKY12, VvWRKY23, VvWRKY24, VvWRKY30, VvWRKY31, VvWRKY32, VvWRKY33, VvWRKY38, VvWRKY40, VvWRKY42, VvWRKY43, VvWRKY46, VvWRKY47, and VvWRKY51 may have a negative effect on grape powdery mildew resistance and VvWRKY7, VvWRKY8, VvWRKY9, VvWRKY13, VvWRKY14, VvWRKY25, VvWRKY26, VvWRKY27, VvWRKY34, VvWRKY35, VvWRKY36, VvWRKY37, VvWRKY39, VvWRKY41, VvWRKY44, VvWRKY45, VvWRKY48, VvWRKY49, VvWRKY50, VvWRKY52, VvWRKY55, and VvWRKY58 may have a positive effect, the possible interactions between two or more genes and regulatory mechanisms remain to be resolved.

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 (Huang et al., 2008), and SA, JA, and Eth play important roles in biotic stresses (Fujita et al., 2006), while MeJA responds to some biotic stresses and wounding (Huang et al., 2008). 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).

Supplementary material

Supplementary data are available at JXB online.

Supplementary Table S1. Primers of 59 VvWRKY genes used for semiquantitative RT-PCR and real-time quantitative PCR.

Supplementary Table S2. WRKY domains and the characteristics of the zinc-finger motif of 59 VvWRKY genes.

Supplementary Table S3. The synteny regions between grape WRKY genes.

Supplementary Table S4. The synteny regions between grape and Arabidopsis WRKY genes.

Supplementary Fig. S1. Expression profiles of 59 VvWRKY genes under salinity stress treatment analysed using semiquantitative RT-PCR.

Supplementary Fig. S2. Expression profiles of 59 VvWRKY genes under drought stress treatment analysed using semiquantitative RT-PCR.

Supplementary Fig. S3. Expression profiles of 59 VvWRKY genes under powdery mildew (Erysiphe necator) inoculation analysed using semiquantitative RT-PCR.

Supplementary Fig. S4. Expression profiles of 59 VvWRKY genes under ABA treatment analysed using semiquantitative RT-PCR.

Supplementary Fig. S5. Expression profiles of 59 VvWRKY genes under SA treatment analysed using semiquantitative RT-PCR.

Supplementary Fig. S6. Expression profiles of 59 VvWRKY genes under MeJA treatment analysed using semiquantitative RT-PCR.

Supplementary Fig. S7. Expression profiles of 59 VvWRKY genes under Eth treatment analysed using semiquantitative RT-PCR.

Supplementary Data
supp_65_6_1513__index.html (931B, html)

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31272136), 948 Project from the Ministry of Agriculture of China (2012-S12), and the Program for Innovative Research Team of Grape Germplasm Resources and Breeding (2013KCT-25).

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