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

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 ). 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 ; 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 ), rice (Wu ), and barley (Mangelsen ). Moreover, large-scale genome-wide studies of WRKY genes have been described for Arabidopsis (Dong ), rice (Ryu ), poplar (He ), tomato (Huang ), cucumber (Ling ), and coffee (Ramiro ).

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 ). 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 ; Zhang and Wang, 2005). WRKY proteins bind to the W-box ((C/T)TGAC(T/C)) of their target genes (Ciolkowski ) as well as a cis-element (SURE), which plays a role in promoting transcription (Sun ).

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 ; Kim ) 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 ). WRKY genes are also related to abiotic stress induced gene expression: AtWRKY25 and AtWRKY33 show altered expression following either heat (Li ; Li ) 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 ). Studies involving other plants, including rice (Shimono ), wheat (Wu ) and poplar (He ), 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 ) 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 ). 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 ) and used to identify putative WRKY genes/proteins from the grape genome sequence (http://www.genoscope.cns.f) (Jaillon ) using the BLASTP program and default parameters (Ling ). 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 ).

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 ) 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 ). 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 ). 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 ) 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 ). 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 ) 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 ). 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 ).

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 ).

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

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 ). The WRKY domain consists of a highly conserved hepta-peptide stretch of WRKYGQK at the N-terminus, followed by a zinc-finger motif (Eulgem ). 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 ), 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 ) and barley (Mangelsen ), 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.

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 ). 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.

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 ). 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 ; He ). 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 ; Wu ).

Fig. 3.

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 ; Zhang ). 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

Fig. 4.

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 ; Dong ) 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.

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.

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.

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.

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 ). 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 ). 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 ), including embryogenesis (Lagace and Matton, 2004), seed coat and trichome development (Johnson ), and leaf senescence (Miao and Zhetgraf, 2007; Zhou ), 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 ). 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 ). Moreover, the soybean WRKY proteins GmWRKY6 and GmWRKY21, which have a WRKYGKK motif, do not bind normally to the W-box (Zhou ). 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 ; Guo ). 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

Gene duplication events play a major role in genomic rearrangements and expansions (Vision ) 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 ). 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 ). Ling 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 ; Chen ; 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 ) and VvWRKY2 (Mzid ) (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 ), 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 ), 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 ). 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 ) 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 ) and AtWRKY48 (Xing ) 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 ), and SA, JA, and Eth play important roles in biotic stresses (Fujita ), while MeJA responds to some biotic stresses and wounding (Huang ). 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 ), 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 ) 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.