Genome and transcriptome analysis of the grapevine (Vitis vinifera L.) WRKY gene family

Abstract

The plant WRKY gene family represents an ancient and complex class of zinc-finger transcription factors (TFs) that are involved in the regulation of various physiological processes, such as development and senescence, and in plant response to many biotic and abiotic stresses. Despite the growing number of studies on the genomic organisation of WRKY gene family in different species, little information is available about this family in grapevine (Vitis vinifera L.). In the present study, a total number of 59 putative grapevine WRKY transcription factors (VvWRKYs) were identified based on the analysis of various genomic and proteomic grapevine databases. According to their structural and phylogentic features, the identified grapevine WRKY transcription factors were classified into three main groups. In order to shed light into their regulatory roles in growth and development as well as in response to biotic and abiotic stress in grapevine, the VvWRKYs expression profiles were examined in publicly available microarray data. Bioinformatics analysis of these data revealed distinct temporal and spatial expression patterns of VvWRKYs in various tissues, organs and developmental stages, as well as in response to biotic and abiotic stresses. To also extend our analysis to situations not covered by the arrays and to validate our results, the expression profiles of selected VvWRKYs in response to drought stress, Erysiphe necator (powdery mildew) infection, and hormone treatments (salicilic acid and ethylene), were investigated by quantitative real-time reverse transcription PCR (qRT-PCR). The present study provides a foundation for further comparative genomics and functional studies of this important class of transcriptional regulators in grapevine.

Figure 1

Chromosomal distribution of VvWRKY genes. Chromosome numbers are provided at the top of each chromosome together with the approximate size. Paralogous chromosome segments resulting from whole-genome duplication or fusion events are shown in the same colour. Genes belonging to clusters are indicated in green font. The 11 synteny blocks are linked by lines. VvWRKY58 and VvWRKY59, which were not assigned to any chromosome bacause on the PN40024 12X V1 prediction, are not reported in the figure.

Figure 2

Structures of VvWRKY genes. Exon–intron composition of VvWRKY genes. Names of genes are indicated on the left. Exons, represented by black or red boxes, were drawn to scale. Dashed lines connecting two exons represent an intron. Intron phases 0, 1 and 2 are indicated by numbers 0, 1 and 2, respectively. WRKY domains in VvWRKY proteins are marked in red.

Figure 3

Phylogenetic tree of WRKY domains based on an alignment of grapevine and Arabidopsis. The consensus unrooted phylogenetic tree generated after an alignment of deduced grapevine and Arabidopsis WRKY domains at N-terminus. The phylogenetic tree was generated with ClustalX 2.1 and using the NJ method. The phylogenetic tree was inferred using MEGA 5.0 software. Reliability of the predicted tree was tested using bootstrapping with 1000 replicates. Numbers at the nodes indicate how often the group to the right appeared among bootstrap replicates. Branch lines of subtrees are coloured indicating different WRKY subgroups. NJ, neighbour-joining.

Figure 4

Phylogenetic tree of deduced VvWRKY domains associated with the motif compositions in the amino-acid sequences. Consensus phylogenetic tree generated after alignment of deduced VvWRKY domains (both N-terminal and C-terminal domains) with ClustalX 2.1 using the NJ method. The phylogenetic trees were constructed using MEGA 5 software. Reliability of the predicted tree was tested using bootstrapping with 1000 replicates. Numbers at the nodes indicate how often the group to the right appeared among bootstrap replicates. Subtrees branch lines are coloured indicating different WRKY subgroups. Group I N-terminal WRKY domains are indicated with the suffix N after the name, whereas Group I C-terminal WRKY domains are indicated with the suffix C. The motif composition related to each VvWRKY protein is displayed on the right-hand side. The motifs, numbered 1–20, are displayed in different coloured boxes. The sequence information for each motif is provided in Table 2. NJ, neighbour-joining.

Figure 5

Expression profiles of the grapevine VvWRKY genes in different grapevine organs, tissues and developmental stages. (a) Expression of VvWRKY genes in the V. vinifera cv ‘Corvina’ atlas (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=lfcrxesyciqgsjoandacc=GSE36128). Data were normalized based on the mean expression value of each gene in all tissues analysed. Genes were hierarchically clustered based on average Pearson’s distance metric and ‘average linkage’ method. Yellow and blue boxes indicate high and low expression levels, respectively, for each gene. (b) qRT-PCR validation of VvWRKY expression in roots, stems, leaves and shoot tips obtained from 4-week-old in vitro explants of the V. vinifera PN40024 genotype. Transcripts were normalized to the expression of the actin gene. The mean±s.d. of three biological replicates are presented. Asterisks indicate that the corresponding gene was significantly up- or downregulated in a given organ (*p≤0.05, **p≤0.01). Bud-AB, bud after burst; Bud-B, Bud burst; Bud-W, winter bud; Bud-L, latent bud; Bud-S, bud swell; Flower-F, flowering; Flower-FB, flowering begins; FS, fruit set; Inflorescence-Y, young inflorescence with single flowers separated; Inflorescence-WD, well-developed inflorescence; Leaf-FS, mature leaf; Leaf-S, senescing leaf; Leaf-Y, young leaf; MR, mid-ripening; R, ripening; PFS, post fruit set; Stem-G, green stem; Stem-W, woody stem; Tendril-FS, mature tendril; Tendril-WD, well-developed tendril; Tendril-Y, young tendril; V, véraison.

Figure 6

Expression profiles of VvWRKY genes in response to abiotic stresses. Expression of VvWRKY genes in the V. vinifera cv ‘Cabernet Sauvignon’ (microarray) or 4-week-old in vitro explants of V. vinifera PN40024 genotype (qRT-PCR) in response to cold (cold), drought (WS) and salt stress (SS). Microarray analysis presented in a and b. qRT-PCR data presented in c. Microarray data were downloaded from the NCBI GEO datasets (GSE31594 and GSE31677), processed as log₂ of the ratio between treated and untreated samples and graphically represented with MeV software. (a) V. vinifera cv ‘Cabernet Sauvignon’ plants grown in a hydroponic drip system were treated with 120 mM salt (SS), polyethylene glycol (PEG), cold (5 °C) or left untreated. Shoots with leaves were collected at 0, 1, 4 and 8 h for all treatments, and at 24 h for all treatments except cold (GEO series GSE31594). (b) Potted V. vinifera cv ‘Cabernet Sauvignon’ vines in the greenhouse were exposed to a water-deficit stress (WC) by withholding water or a salt stress (SS) by watering plants with a saline solution for 16 days. Non-stressed, normally watered plants served as the control for both treatments. Shoot tips were harvested every 4 days (0, 4, 8, 12 and 16 days) (GEO series GSE31677). (c) qRT-PCR expression analysis of VvWRKYs in V. vinifera PN40024 genotype subjected to a drought treatment. Transcripts were normalized to the actin gene expression. The mean±s.d. of three biological replicates are presented. Asterisks indicate that the corresponding gene was significantly up- or downregulated in a given organ (*p≤0.05, **p≤0.01). MeV, Multi Experiment Viewer.

Figure 7

Expression profiles of VvWRKY genes in response to biotic stresses. (a) V. vinifera cv ‘Cabernet sauvignon’ and V. aestivalis cv ‘Norton’ plants were grown in an environmental chamber and inoculated with Erysiphe necator conidiospores (PM). Inoculated leaves were harvested at 0, 4, 8, 12, 24 and 48 h after inoculation (GEO series GSE6404). (b) Field-grown plants of V. vinifera cv ‘Chardonnay’ and ‘Incrocio Manzoni’ naturally infected with Bois Noir phytoplasma (BN), compared to healthy samples (GEO series GSE12842). (c) V. vinifera cv ‘Cabernet Sauvignon’ was infected with GLRaV-3 during véraison and the ripening stages of berry development (GEO series GSE31660). (d) qRT-PCR expression analysis of VvWRKY genes in Pinot PN40024 genotype infected with powdery mildew. Transcripts were normalized to actin gene expression. The means±s.d. of three biological replicates are presented. Asterisks indicate that the gene was significantly up- or downregulated in a given organ (*p≤0.05, **p≤0.01). GLRaV-3, grapevine leaf roll-associated virus-3.