Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera
© Wang et al.; licensee BioMed Central Ltd. 2014
Received: 25 December 2013
Accepted: 11 April 2014
Published: 22 April 2014
WRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown.
We identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress.
We identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape.
KeywordsWRKY transcription factor family Grapevine Biotic and abiotic stress Cold stress
Plants have a variety of defense mechanisms to protect themselves from adverse environmental effects. Families of transcription factors are involved in these processes by functioning to reorganize gene expression patterns. The WRKY family is among them and plays key roles in modulating genes expression during plant defense in response to pathogens [1, 2]. The WRKY transcription factors were first identified in sweet potato (SPF1) as DNA binding proteins . Two similar genes (ABF1 and ABF2) were found in wheat during germination . Subsequently, Rushton et al.  reported the identification and characterization of WRKY1, WRKY2 and WRKY3 from parsley (Petroselinum crispum) and proposed these genes belong to a gene family. This gene family was named WRKY due to a conserved region (WRKYGQK) that was identified in the N-terminal amino acid sequence of all the members [4, 5]. Further studies showed that the conserved WRKY domain had other forms such as WRKYGKK and WRKYGEK , or the WRKY domain could be replaced by WKKY, WKRY, WSKY, WIKY, WRIC, WRMC, WRRY or WVKY [7, 8].
According to variation in WRKY domain and a zinc finger motif in the C-terminus, WRKY proteins were divided into four groups [9, 10]. WRKY proteins with two WRKY domains composed group I. Groups II and III were characterized by a single WRKY domain. Group II WRKY proteins were further subdivided into five or more subgroups based on short conserved structural motifs while group III proteins contained a variant zinc-finger which ends with HXC. Finally, group IV WRKY proteins contained the WRKY domain, but lack a complete zinc-finger structure in the C-terminus. WRKY proteins usually functioned as transcriptional regulators via binding to W-boxes (TTGACC/T) in the promoter regions of down-stream genes and clusters of W-boxes had an amplified effect [3–5, 11–15]. However, some other studies have found that some WRKY proteins bind to the PRE4 element (TGCGCTT), SURE element (TAAAGATTACTAATAGGAA) or SURE-like element and the WK box (TTTTCCAC) .
WRKY proteins have been found to play essential roles in pathogen defense in response to bacteria [16, 17], fungi [18, 19], and viruses [20, 21]. Evidence also supported that WRKY transcription factors were involved in modulating gene expression in plants during abiotic stresses such as cold [22, 23], salt [24, 25] and drought [26–28]. Besides roles in response to biotic and abiotic stress, WRKY proteins were also implicated in processes that modulate plant developmental processes such as morphogenesis of trichomes and embryos, senescence, dormancy, and metabolic pathways .
Grape is one of the most important fruit crops worldwide. The productivity of grapevines is largely limited by disease pressure and stressful fluctuations in environmental conditions. Due to their essential role in the early response to pathogens and abiotic stresses, several WRKY genes were intensively studied in grape. VvWRKY1 and VvWRKY2, isolated from grape (V. vinifera cv. Cabernet Sauvignon) berries, were found to potentially participate in defending against fungal pathogens [18, 29]. VvWRKY1 was found involving in enhanced protection against Botrytis cinerea by transactivating the VvLTP1 promoter , and VvWRKY2 may regulate lignification and response to biotic or abiotic stresses in grapevine . VpWRKY1 and VpWRKY2, isolated from Chinese wild V. pseudoreticulata, may contribute to resistance to powdery mildew (Erysiphe necator) and tolerance to salt and cold stresses in grape . VpWRKY3 was found to be involved in pathogen defense and also interact with the salicylic acid, ethylene, and abscisic acid signal pathways . Transgenetic Arabidopsis plants expressing VvWRKY11, isolated from ‘Beifeng’, an interspecific cultivar of V. thunbergii × V. vinifera, showed increased dehydration tolerance . Its homologous gene, VpWRKY11, was found to serve as a negative regulator of disease resistance . Although several individual WRKY genes have been identified in grapevine, the WRKY gene family in grapevine remains wholly uncharacterized.
Based on our previous transriptome analysis, we found that some WRKY genes respond to cold stress in different patterns in V. amurensis (a cold hardy grapevine species) and V. vinifera cv. Muscat Hamburg . VvWRKY14 (GSVIVT01015952001) and VvWRKY12 (GSVIVT01012682001) were found up-regulated over 30 fold in V. amurensis after being subjected to cold stress but up-regulated to a lesser extent in V. vinifera. In contrast, the expression of VvWRKY43 (GSVIVT01030258001) was up-regulated in V. vinifera (26 fold) while expression remained low in V. amurensis. These different gene expression patterns in response to cold stress may be contribute to the distinctive cold hardiness between the two species. To further characterize how WRKY genes respond to freezing stress of grapevine, we initiated this study to identify the entire WRKY gene family in grapevine based on the published 12× V. vinifera cv. Pinot noir (PN40024) genome sequences . A phylogenetic tree was constructed for identified WRKY proteins and the gene expression patterns in different tissues of V. vinifera were detected by RT-PCR. WRKY genes responding to biotic and abiotic stresses were cross-evaluated by using public gene-chip databases. Additionally, real time RT-PCR was used to detect the expression level of VvWRKYs under cold treatment and exogenous ABA. A comparative analysis was conducted to identify VvWRKYs that may participate in cold signal transduction pathways in V. vinifera using microarray data in public databases, our previously reported transcriptome data and qRT-PCR analysis conducted in this study.
Identifying of WRKY transcription factors in V. viniferagenome
Identified WRKY genes in 12× V. vinifera ‘Pinot Noir’ genome
The putative genome location of each VvWRKY in the grape genome was shown in Additional file 1: Figure S1. Fifty-eight of the VvWRKYs could be mapped to 18 of the 19 grape chromosomes, with no VvWRKYs found on chromosome 3. VvWRKY4 was putatively located on the ‘Chromosome Unknown’. WRKY transcription factors were not evenly distributed across the chromosomes of the grape genome. There were most abundant on Chromosome 4 (8 VvWRKYs) and chromosome 7 (7 VvWRKYs) and least abundance on Chromosome 5 and 18 (1 VvWRKY).
Categorization of VvWRKYs basis on conserved WRKY domains
RT-PCR based transcription levels detection of VvWRKYsin different tissues
Gene-chip based expression analysis of 26 VvWRKYsunder various stresses
Although we identified WRKY transcription factors from the V. vinifera genome, functions for these genes in response to abiotic and biotic stress remain unknown. Using microarray results from publically available data, it was possible to find gene expression data from multiple experimental conditions for several of the grapevine WRKY genes. We carefully checked the genes on the ‘GeneChip Vitis vinifera (Grape) Genome Array’ (Affymetrix) and a total of 26 VvWRKYs were found on this chip. Microarray data related to salinity, water-deficit, PEG, cold, ABA and pathogen stresses were downloaded and their corresponding probes and the CV (coefficent of variation of the corresponding treatment means) of these genes in each of the microarray experiments were listed in Additional file 3: Table S1. If the expression of a probe set (gene) is affected by some of the treatments in an experiment, it shows a higher CV (more fluctuation); and vice versa. According to the data, the CV of 20 of the 26 VvWRKYs were over 5% in at least one experiment. The highest CV appeared in VvWRKY57 (up to 36%) associated with compatible viral diseases in berry experiment in V. vinifera cv. Cabernet Sauvignon. VvWRKY03, 06, 08, 28, and 55 responded to both abiotic and pathogens stresses while VvWRKY21, 39, 48 seemed to respond primarily to pathogens stresses.
Real-time RT-PCR based expression analysis of VvWRKYs under cold treatment in V. vinifera
Exogenous ABA induced accumulation of VvWRKYs in V. Vinifera
To illustrate how the VvWRKYs respond to ABA and whether the cold stress related VvWRKYs may participate in the ABA-dependent cold signal pathway, ABA treated grapevine apices were examined using qRT-PCR. VvWRKY12, 29, and 46 were excluded from this experiment due to their higher Ct value (Figure 4D and Additional file 6: Figure S4). Among the 55 VvWRKYs we detected, twelve VvWRKYs were expressed over 2-fold greater within 2 h of exogenous ABA treatment (Figure 4D). After statistical analyses of qRT-PCR results, 7 of them were evaluated to significantly change during exogenous ABA treatments. Transcripts of VvWRKY35 showed the greatest increase in expression at 0.5 h after ABA treatments. Six other genes showed increases in expression 1 h after exogenous ABA treatment (Figure 4D).
When the data from the cold and ABA experiments were compared, 6 of 7 genes (VvWRKY, 19, 28, 35, 42, 50 and 55) that were up-regulated during exogenous ABA treatment were also up-regulated under cold treatment (Figure 4A and B, marked by underline). Two genes (VvWRKY55, 28) were greatly up-regulated, over 10 fold. The expression levels of the rest of the 44 VvWRKYs were lower than 2-fold and not significantly changed during exogenous ABA treatments (Additional file 6: Figure S4).
Identification of candidate cold-stress related VvWRKYs
WRKY family in grape
Considering the important roles that WRKY transcription factors play during plant development and in response to various stresses, it is not surprising that we identified so many family members in grapevine. Previously, 74 WRKY genes were found in Arabidopsis, 55 in cucumber , 102 in rice , 47 in castor bean , 86 in Brachypodium distachyon and 136 in maize . Here we identified 59 candidate WRKY proteins in V. vinifera and categorized them into four groups.
Group I WRKY proteins
When compared with WRKY family groups, WRKYs in primitive plant ancestors Giatdia lamblia, Dictyostelium discoideum and Chlamydomonas reinhardtii closely resembled Vitis group I [7, 38]. In our study, two domains of VvWRKYs in group 1 were closely related. A BLASTp search of EuGene.1100010359 from an ancient alga species (Ostreococcus sp. RCC809) which has a single WRKY domain allowed us to identify 9 corresponding WRKY homologs in grape and 8 of these belonged to group I by MAP VIEW (Plant Genome Duplication Database) . These data support the hypothesis that the dual WRKY domains present in members of group I may be derived from a single WRKY domain duplication [6, 7].
Group II WRKY proteins
Group II was divided into three parts: subgroup IIa + IIb, subgroup IIc and subgroup IId + IIe (Figure 1). Subgroup IIa + IIb belong to the same clade and is sister to the WRKYs in group I. Interestingly, the presumed function of CTWDs in group I for sequence-specific DNA binding  were more similar to the single WRKY domain members in group II and III than to the NTWDs of group I, This result may indicate that subgroup IIa + IIb evolved from group I by domain structure loss of the group I NTWD.
Group III WRKY proteins
Group III in the phylogenetic tree was most closely related to the very large subgroup IIc, which was separately into four clades and seemed to indicate an expansion of the gene family. A thorough search of the Plant Transcription Factor Database (http://planttfdb.cbi.pku.edu.cn) indicated that the earliest evolutionary occurrences of group III genes were those found in ferns (Selaginella moellendorffii). There was no evidence of any sequenced plant species that only contain members of group I and III but we found in some species with only members of group I and II, for example in mosses (Physcomitrella patens) , and some gymnosperms (Pinus taeda) (http://planttfdb.cbi.pku.edu.cn). We speculated that group III may have evolved from group II, particularly IIc. As group III WRKYs in Arabidopsis responded to diverse biotic stresses , group III members may indicate adaptation of early plants to the stressful conditons associated with the colonization of land and subsequent increase in biotic pathogen pressures.
Group IV WRKY proteins
We found that group IV WRKY proteins, which were characterized by the loss of the zinc-finger domain, were in the same clade as subgroups IId + IIe. VvWRKY02 and VvWRKY57 were duplicated gene pairs according to a whole genome analysis of grapevine gene duplications . This might suggest an origin of group IV from subgroups IId + IIe. Group IV proteins were considered non-functional due to the loss of the zinc-finger domain . However, these genes of group IV can be found in all higher plant species as well as in algaes (Bathycoccus prasinos: Bathy17g02050). Furthermore, some genes were expressed in rice (OsWRKY56)  as were two genes identified in this study (VvWRKY02 and VvWRKY29). Therefore, it remains questionable whether group IV WRKYs have biological function in plants.
VvWRKYsparticipate in development and stress-related signal pathways
WRKY genes were found to be expressed in many tissues and seem to be involved in regulating plant developmental and physiological processes. Transcriptomic analysis of senescence in the flag leaf of wheat demonstrated that WRKY transcription factors are greatly up-regulated during the senescence process . OsWRKY78 was found to be up-regulated in elongating stems and knockdown mutations in this gene cause plants to produce a semi-dwarf and small seed phenotype caused by reducing cell length . Moreover, the transcription of GhWRKY15 was observed abundant in the roots and stems of tobacco and transgenic overexpression lines of these plants displayed faster elongation at the earlier shooting stages . Here the expression of 15 VvWRKYs (Figure 2) can be detected in all grape tissues we used, which may indicate its fundamental roles in different cell-types in grape. Similar to expression patterns observed in other plant species, VvWRKYs were found to be expressed in young tissues such as young leaf, shoot apex, tendril and young fruit.
Several numbers of VvWRKYs were found activated in more than one type of stress condition (Figure 3 and also Additional file 3: Table S1). VpWRKY3, homologous to VvWRKY55 was observed to be up-regulated in response to many different sources of stress, including pathogen exposure, salicylic acid, ethylene, cold and drought stress . VvWRKYs that were up-regulated in response to more than two types of stresses (e.g. pathogen and drought) supported the occurrence of cross-talk between signal transduction pathways in response to different stress conditions in plants .
Phylogenetic relationships between VvWRKY genes suggested that there may be conserved responses of these genes to salt exposure, PEG and cold-stress (Figure 3). All members of group IId clustered into one clade with similar expression pattern during these three stress conditions, suggesting the function of these VvWRKY proteins may relate to the structures of WRKY domains. Subgroup IId was identified as a novel CaM-binding transcription factor family in plants and their conserved structural motif was a Ca2+-dependent CaM-binding domain . Thus the placement of the WRKYs in the phylogenetic tree may also help to predict function of new members that belong to certain gene family.
VvWRKYsthat participate in the cold related signal transduction in grape
Three different experimental methods were combined to robustly analyze the response of VvWRKY genes to cold stress (Figure 5 and Additional file 4: Table S2). Results from qRT-PCR demonstrated the greatest number of cold stress-related VvWRKYs (36) while gene-chip based methods identified the least, 10 VvWRKYs. This difference may be attributed to the method used but is also likely due to differences in the treatment conditions between experiments. During Digital Gene Expression profile (DGE) analysis , plant material was obtained from 4 h cold treatment at 4°C, whereas in our pRT-PCR experiment, we used samples collected at several different time periods (at 8 h, 24 h and 48 h after cold treatment at 4°C). Additionally, multiple matched tags were excluded from the final analysis performed by Xin et al. , which may have reduced the number of identified cold related VvWRKYs. Finally, gene-chip based methods may bias results due to a lower number of genes with corresponding probes related to the WRKY proteins (only 26 WRKY). By integrating the data from different methods, we obtained more reliable results and a total of 15 candidate cold tolerance VvWRKYs (Figure 5) were identified during our investigation.
According to previous studies, the transcriptional control of plant responses to cold stress can be divided into ABA-dependent and ABA-independent signal pathways . The results of our study also indicated that 15 putative cold stress-related VvWRKYs can be divided into two groups according to their responses to exogenous ABA. Three VvWRKYs (VvWRKY28, 42 and 55) may participate in an ABA-dependent signal pathway and other 12 in ABA-independent pathway. WRKY transcription factors have been identified as key components in the ABA signaling pathways [8, 53]. In rice, OsWRKY24, 51, 71 and 72 are induced by (ABA) in aleurone cells. OsWRKY24 and 45 were functional as negative regulators in ABA induction of the HVA22 promoter-beta-glucuronidase construct, while OsWRKY72 and 77 synergistically interacted with ABA to activate this reporter construct . It is still unknown how WRKYs participate in the cold stress-related signal pathway and what relationship these genes have with C-repeat Binding Factor genes (CBFs), which are critical transcription factors responsible for cold tolerance in plant .
The reliability of the identified 15 cold–related VvWRKYs was also supported by homologous genes in other species. STHP-64, which showed high similarity with VvWRKY43, was not present in leaves until November and December in Solanum dulcamara. WRKY38, a homolog gene of VvWRKY14, was transiently accumulated when leaves and roots were exposure to low temperature in barley . BcWRKY46 showed higher similarity with VvWRKY33 and responded to low temperatures in Pak-choi. Constitutive expression of BcWRKY46 reduced the freezing susceptibility in transgenic tobacco . The transcription level of VvWRKY55 was up-regulated robust under cold treatment. Its homolog gene, WRKY71 was found in banana with a similar expression pattern . All these VvWRKYs mentioned above were confirmed by at least two set of experiment methods, which provided appropriate candidates to illustrate the roles of WRKY protein under low temperature-related signal pathways in grape.
Although low-temperature related WRKYs were isolated in several species, the mechanism of how WRKYs respond to cold signals and regulate the expression of downstream genes is still largely unknown. Further work is needed to elucidate the function of these important genes in low-temperature related signal pathways. Previously we reported the different expression patterns of WRKYs in V. amurensis, a cold-hardness species. The WRKY genes identified here from V. vinifera may accelerate the functional analysis of this gene family in V. amurensis. The comprehensive analysis of cold stress-related WRKYs in two different Vitis species with contrasting cold hardiness phenotypes would certainly help to illustrate the function of WRKY genes in conveying cold hardiness in grapevine.
In summary, a total of 59 VvWRKYs in the V. vinifera genome were identified. The VvWRKYs were unevenly distributed in 18 of the 19 chromosomes. WRKY domain based phylogenetic analysis allowed categorizing 59 VvWRKYs into four large groups. A majority of VvWRKYs were found expressed in more than one tissue in V. vinifera. Gene-chip based data analysis suggested that a subset of VvWRKYs was activated in respond to diverse biotic and abiotic stresses. The transcription level of 36 VvWRKY genes changed over 2 fold after cold induction. A comparative analysis of qRT-PCR results, gene-chip based data and transcriptome analysis allowed us to identify 15 VvWRKYs that show identical expression patterns during cold treatment at least in two kinds of analyses. These studies not only increase our knowledge of WRKY family, but also provide candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape.
Identification of WRKYgenes in the grape genome
Candidate WRKY proteins were identified from the 12X V. vinifera cv. Pinot noir genome (quasi-homozygous line PN40024, http://www.phytozome.net). Full-length amino acids sequences of all WRKY proteins in Arabidopsis thaliana (http://www.arabidopsis.org/) were used as query sequences. A BLASTp search was performed and E-value of e−6 was used as the threshold . Candidate WRKY proteins were manually confirmed  by searching for WRKY domains in the candidate amino acids sequences using SWISS-MODEL (http://swissmodel.expasy.org/) and the results were shown in Table 1.
Phylogenetic analysis of WRKY family
Multiple alignments of the amino acid sequences of 73 WRKY domains from V. vinifera were performed using CLUSTALW by MEGA5.1 . Twelve Arabidopsis WRKY domains from different WRKY groups were used as references to categorize the WRKY proteins from grape. The GenBank accession numbers of those AtWRKYs are AtWRKY01: ABJ17102, AtWRKY11: AEE85928.1, AtWRKY14: AAP21276.1, AtWRKY18: AAM78067, AtWRKY21: AAB63078.1, AtWRKY27: ABH04558, AtWRKY28: AEE84006, AtWRKY31: AEE84546.1, AtWRKY38: AED93044.1, AtWRKY41: AEE82969, AtWRKY43: AEC10646.1, AtWRKY45: ABD57509.1, AtWRKY49: AAQ62425.1. The parameters used during alignment were: protein weight matrix: Gonnet series; negative matrix: on; gap open penalty: 10; gap extension penalty: 0.20; delay divergent sequences: 30; residue-specific gap penalties: on; hydrophilic penalties: on; gap separation distance: 0; end gap separation penalty: on. An unrooted phylogenetic tree was constructed using Neighbor-Joining (NJ) methods and bootstrapped with 1,000 iterations to help identify WRKY protein groups.
‘Muscat Hamburg’ (V. vinifera) was obtained from the Institute of Botany, the Chinese Academy of Sciences. Tissues of young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits were collected from the vineyard in July, 2012. Cold and exogenous ABA treatment experiments were performed on tissue culture seedlings of ‘Muscat Hamburg’ according to Li et al. . Briefly, seedlings were cultured on 1/2 B5 medium with 30 g/L of sucrose in a growth chamber under 16-h light/8-h dark photoperiod at 26°C. Cold treatments were performed in another growth chamber with the same parameters except for temperature (4°C). Seedlings with five well developed leaves were used and the shoot apex with one well developed leaf was collected at 0 hour (h, used as control), 8 h, 24 h and 48 h. Seedlings with five well developed leaves were transplanted in 1/2 B5 nutrient solution. Exogenous ABA treatments were performed after one week under normal culture conditions with 100 μM ABA and the shoot apex with one well developed leaves were collected at 0 h (used as control), 0.5 h, 1 h and 2 h after treatments. Three independent replicates were collected for each time point and frozen in liquid nitrogen. Samples were then stored at - 80°C for the following RNA isolation.
Expression patterns analysis of VvWRKYsby RT-PCR
Total RNA was isolated from collected samples using Plant Total RNA Isolation kit (Tiandz Inc; Beijing, China). RNase-free DNase (RQ1, Promega) was used to degrade DNA from total RNA. cDNA was synthesized by the SuperScript III Reverse Transcriptase (Invitrogen) with Oligo(dT)18 (Promega) according to the manufacturer’s instructions. Primer pairs (Additional file 7: Table S3) for VvWRKYs were designed by Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) and tested by NCBI Primer BLAST. Two genes, β-actin (GenBank accession: EC969944; sense primer: 5′-CTTGCATCCCTCAGCACCTT-3′; antisense primer: 5′-TCCTGTGGACAATGGATGGA-3′) and malate dehydrogenase gene (MDH; GenBank accession: EC921711; sense primer: 5′-CCATGCATCACCCACAA-3′; antisense primer: 5′-GTCAACCATGCTACTGTCAAAACC-3′) were used as positive control for RT-CR . Three biological replicate and 35 cycles for each reaction were performed. PCR products were detected by agarose gel electrophoresis with 2.5% gel concentration.
Gene-chip based expression pattern analysis of VvWRKYs
We explored the expression profiles of VvWRKYs using publically available data from the 16 k Affymetrix V. vinifera gene-chip stored at PLEXdb (Plant Expression Database)  to explore the response of VvWRKYs during biotic and abiotic stresses in grape. The different studies and datasets that were included in these analyses were: A) a short term abiotic stress experiment in ‘Cabernet Sauvignon’ , B) a long-term salt and water stress study ; C) a study examining gene expression associated with compatible viral diseases in grapevine cultivars ; D) an experiment designed to examine the powdery mildew-induced transcriptome in a susceptible grapevine ‘Cabernet Sauvignon’ ; E) the complimentary dataset of the powdery mildew-induced transcriptome of a resistant grapevine ‘Norton’ ; F) a study of gene expression in grapevine in response to Bois noir infection ; G) a study of the grape skin transcriptome of berries grown on an exogenous abscisic acid treated vine ; H) the complimentary dataset of the grape skin transcriptome in the berries cultured in vitro and treated with exogenous ABA ; and lastly, I) a gene expression study associated with compatible viral diseases in the berry . In our comparative analysis, we divided these experiments into either abiotic or biotic stresses related datasets. For each microarray experiment, the Affymetrix MAS5.0 normalized data were used for calculations of the fold change of differentially expressed genes. Probe sets corresponding to the putative VvWRKYs were identified at PLANEX (http://planex.plantbioinformatics.org) and completed via PLEXdb blast tool. Comparisons of WRKY expression level from gene-chip data for the short term abiotic stress treatment in ‘Cabernet Sauvignon’ was performed using Cluster 3.0 and JavaTreeview.
Total RNA was isolated from cold and exogenous ABA treated shoot apices following the cDNA synthesis methods mentioned above. Synthesized cDNA was diluted 1:10 with ddH2O, and the quantitative RT-PCR reaction mixture contained 5 μl of 2 × SYBR Green I Master Mix (Roche, USA), 2.6 μL ddH2O, 0.2 μL of 10 μM solution of each primer and 2 μL diluted template cDNA. Reaction specificities for each primer pair was tested using qRT-PCR melting curve analysis. The experiment was carried out using a StepOnePlus real-time PCR Instrument (Applied Biosystems). Transcription levels of each VvWRKY was normalized against the average of β-actin, MDH (as mentioned above) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH: CB973647; sense primer: 5′-TTCTCGTTGAGGGCTATTCCA-3′; antisense primer: 5′-CCACAGACTTCATCGGTGACA-3′) . Each sample had three biological and two technical replicates to ensure the accuracy of results, and RNA samples with the same reverse-transcription (without Reverse Transcriptase) and dilution procedure were used as negative controls. The Ct values and the real-time PCR efficiencies were obtained using Lin-RegPCR  and the normalized relative quantities and standard errors for each sample were calculated by qbaseplus . The relative expression level of each VvWRKY in different templates was calculated based on normalized relative quantities. We used One-Way ANOVA analysis to test the impact of timing of cold treatment. When the effects were significantly different, we examined the difference between treatments using post hoc multiple comparisons (LSD, p < 0.05). All data analyses were conducted using IBM SPSS Statistics 20.
The authors thank Professor Yuepeng Han (Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Chinese Academy of Sciences) for critical review of this manuscript. This work was supported by the National Natural Science Foundation of China (NSFC Accession No.: 31130047, 31000902).
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