Genome-wide characterization of WRKY gene family in Helianthus annuus L. and their expression profiles under biotic and abiotic stresses

WRKY transcription factors play important roles in various physiological processes and stress responses in flowering plants. Sunflower (Helianthus annuus L.) is one of the important vegetable oil supplies in the world. However, the information about WRKY genes in sunflower is limited. In this study, ninety HaWRKY genes were identified and renamed according to their locations on chromosomes. Further phylogenetic analyses classified them into four main groups including a species-specific WKKY group. Besides, HaWRKY genes within the same group or subgroup generally showed similar exon-intron structures and motif compositions. The gene duplication analysis showed that five pairs of HaWRKY genes (HaWRKY8/9, HaWRKY53/54, HaWRKY65/66, HaWRKY66/67 and HaWRKY71/72) are tandem duplicated and four HaWRKY gene pairs (HaWRKY15/82, HaWRKY25/65, HaWRKY28/55 and HaWRKY50/53) are also identified as segmental duplication events, indicating that these duplication genes were contribute to the diversity and expansion of HaWRKY gene families. The dN/dS ratio of these duplicated gene pairs were also calculated to understand the evolutionary constraints. In addition, synteny analyses of sunflower WRKY genes provided deep insight to the evolution of HaWRKY genes. Transcriptomic and qRT-PCR analyses of HaWRKY genes displayed distinct expression patterns in different plant tissues, as well as under various abiotic and biotic stresses, which provide a foundation for further functional analyses of these genes. Those functional genes related to stress tolerance and quality improvement could be applied in marker assisted breeding of the crop.


Introduction
The WRKY gene family is considered as one of the largest transcription factor (TF) family in higher plants [1], which basically contain an approximate 60-residue DNA-binding domain, named as WRKY domain, with a highly conserved heptapeptide motif WRKYGQK and a C 2 H 2 -or C 2 HC-type of zinc-finger motif included. Both the heptapeptide motif and zinc-finger motif are needed for binding of WRKY TFs to the cis-acting element W-box (C/T)TGAC (C/T) [2,3]. WRKY gene family can be classified into three main groups (I-III), based on the number of WRKY domains and the structure of their zinc-finger motifs [4]. The group I WRKY proteins consist of two WRKY domains, whereas groups II and III contain only one. The group II and III WRKY proteins are distinguished by the type of zinc-finger motif, with a C-X 4-5 -C-X 22-23 -H-X 1 -H type of motif in group II and a C-X 7 -C-X 23 -H-X 1 -C type in group III [5]. The first WRKY gene was cloned and identified from sweet potato, encoding a 549 amino acid protein called SPF1 (SWEET POTATO FACTOR1) [6]. Since then, a large number of WRKY genes have been discovered from different plants. Functional analyses showed that WRKY genes are associated with various aspects of physiological processes, including seed dormancy and germination, root development, leaf senescence, modulation of flowering time, plant nutrient utilization etc [7]. The knockout mutant of AtWRKY2 resulted in hypersensitivity of Arabidopsis to ABA during seed germination and post-germination, early growth, suggesting that AtWRKY2 mediates seed germination and post-germination development [8].
Overexpression of OsWRKY31 in rice inhibited plant lateral root formation and elongation, and also affected the transport process of auxin [9]. AtWRKY12, AtWRKY13 and AtWRKY71 are three main genes regulating Arabidopsis flowering time, with AtWRKY12 and AtWRKY13 working antagonistically under short daylight conditions [10], and AtWRKY71 accelerating flowering [11]. It has been also documented that 12 WRKY genes are involved in leaf senescence in Arabidopsis and rice, as their mutants inhibited or promoted leaf senescence to different extents [7].
In addition to plant growth and development, WRKY genes also participate in modulation of plant tolerance to abiotic and biotic stress. Qiu and Yu [12] reported that overexpression of OsWRKY45 in Arabidopsis significantly increased the expression level of PR genes and ABA/ stress regulated genes, thus contributed to the enhancement of disease resistance and salt and drought tolerance of the plant. GmWRKY54 from soybean, which was confirmed in a DNA binding assay that could interact with the W-box, conferred salt and drought tolerance to transgenic Arabidopsis, possibly through the regulation of DREB2A and STZ/Zat10 [13]. In tobacco, overexpression of grape VvWRKY2 reduced the susceptibility to fungal pathogens like Botrytis cinerea, Pythium spp. and Alternaria tenuis [14]. The WRKY1 in tobacco could be phosphorylated by a salicylic acid-induced protein kinase (SIPK), resulting in enhanced DNAbinding activity to a W-box sequence from the tobacco chitinase gene CHN50, and subsequently formation of hypersensitive response-like cell death [15]. Thus, WRKY genes may be involved in mitigating the damage caused by stresses, through interacting with the cis-element W-box and activating downstream plant defense signaling [2].
Common sunflower (Helianthus annuus L.) is grown throughout the world as an industrial crop for edible oil. It is the fourth important oilseed crop which contributes to 12% of the edible oil produced globally. However, sunflower production has been threatened by different stresses, among which drought and salinity are two major abiotic constraints [16]. Moreover, parasitic weed Orobanche cumana is a new emerged biotic issue worldwide [17]. WRKY transcription factors are involved in regulation of plant tolerance to both abiotic and biotic stresses. Thus, it is of great interest to characterize a WRKY gene family in sunflower and identify their functions under different stresses.
The WRKY gene family has been well studied in sunflower. Giacomelli et al. [18] have identified a total number of 97 WRKY genes in the Asteraceae family, while only 26 of them belong to H. annuus, and this identification was all based on EST database. The publication of reference genome will provide an opportunity to reveal the organization, expression and evolutionary traits of common sunflower WRKY gene family at the genome-wide level. Badouin et al. [19] reported a high-quality reference for the sunflower genome (3.6 gigabases), with 17 chromosomes and 52,232 protein-coding genes on them. Guo et al. [20] identify 112 sunflower WRKY genes from this reference genome and Liu et al. [21] have extended this family to 119 members. In the current study, another sunflower database from a different sunflower genotype was used to search WRKY genes as support and addition to the previous works. A total of 90 HaWRKY genes were identified, among which 89 had corresponding genes in the updated sunflower WRKY gene family [21], whereas the rest one was newfound (S3 Table). The 90 WRKY genes could be classified into four main groups, including an extra WKKY group. Analyses on exon-intron organization, motif composition, gene duplication, chromosome distribution, phylogenetic relationship and gene synteny were further conducted to systemically characterize these common sunflower WRKY genes. Additionally, the expression patterns of HaWRKY genes in different plant tissues and in responses to different abiotic and biotic stresses were also recorded, to identify the implication of specific WRKY genes in different biological processes. The present findings provide a foundation for future research on functional characterization of WRKY genes in common sunflower.

Phylogenetic analysis and gene structure
Multiple sequence alignment based on WRKY domain sequences were conducted by clustal W analysis with default parameters. A neighbor-joining (NJ) tree was constructed in MEGA 5.2 with the following criteria: Poisson model, pairwise deletion, and 1000 bootstrap replications. Further maximum likelihood (ML) analysis of WRKY gene family from sunflower and Arabidopsis was conducted, to confirm the reliability of the result. The intron-exon structures of sunflower WRKY genes were analyzed by comparing predicted coding sequences with their corresponding full-length sequences using the online tool Gene Structure Display Sever (GSDS, http://gsds.cbi.pku.edu.cn/) [28]. The MEME online program (Multiple Expectation Maximization for Motif Elicitation) version 4.11.1 (http://meme-suite.org/index.html) was used to identify conserved motifs in the sunflower WRKY proteins [29].

Chromosomal distribution and gene duplication
Multiple Collinearity Scan toolkit (MCScanX) was adopted to analyze the gene duplication events with default parameters [30]. PAL2NAL v14 was subsequently used to calculate dN and dS, with a dN/dS ratio of 1 indicative of neutral selection [31].

Plant materials, growth conditions and treatments
Seeds of both O. cumana and two sunflower cultivars JY207 and TK0409 were provided by the Institute of Plant Protection, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Hohhot, China. For abiotic stresses, common sunflower cultivar TK0409 was used. The seeds were germinated and grown in peat moss according to our previous study [32]. At the four-leaf stage, seedlings with uniform size were selected to expose to NaCl (0, 150, and 300 mM) for salt stress and polyethylene glycol-6000 (PEG-6000, 0, 10% and 20% w/v) for simulated drought stress, respectively. The treatment concentrations were selected based on our preliminary experiments [32]. All the plants were placed in a growth chamber with light intensity ranging from 250 to 350 μmol m -2 s -1 , temperature at 16-20˚C, and relative humidity at approximately 55-60%. After another one week, roots and leaves of the sunflower seedlings were sampled for RNA isolation. For biotic stress, root parasitic weed Orobanche cumana were applied to common sunflower cultivars TK0409 (susceptible) and JY207 (resistant). 200 mg of O. cumana seeds were homogeneously mixed with 0.5 kg of the peat and vermiculite (1:1, v/v) substrate. Sunflowers were grown in the substrate containing O. cumana seeds as mentioned above. All the plants were placed in a growth chamber with 20˚C at the daytime and 14˚C at night, photoperiod for 14 h, and an irradiance of 300 μmol m −2 s −1 . Three weeks after inoculation, sunflower roots were collected for experiments. In order to avoid tissue contamination of O. cumana, sunflower roots that were over 1 cm adjacent to the interaction site with O. cumana were harvested. Each treatment was replicated three times.

Gene expression analysis
To determine the expression profiles of the HaWRKY genes under natural conditions, the transcriptomic data from 10 sunflower tissues, including bract, corolla, leaf, ligule, ovary, pollen, seed, stamen, stem, and style, were downloaded from the "Gene Expression Browser" of Sunflower Genome Database. Data were transformed by log 2 (FPKM+1). For biotic stress, the transcriptomic data were from our previous work [33]. Genes with false discovery rate (FDR) less than 0.01 and fold change more than 2 were considered differentially expressed between infected and non-infected sunflower. The expression profile heat-maps were generated using HemI 1.0 software.
For abiotic stress, TaKaRa MiniBEST Plant RNA Extraction Kit (Takara Bio, Kyoto, Japan) was used to extract total RNA from sunflower leaves and roots. The quantity and quality of RNA samples were assessed by agarose gel electrophoresis and using a Nanodrop 2000 Spectrophotometer for A260/A280 ratio. 200 ng of total RNA was reverse transcribed by using TaKaRa PrimeScript TM RT reagent Kit with gDNA Eraser. The gene-specific primers of sunflower for qRT-PCR amplification were designed by using Primer Premier 5.0 software and provided in S1 Table. SYBR Premix Ex Taq II (Tli RNaseH Plus, TaKaRa) in CFX96TM Real-Time PCR detection System (Bio-Rad, Hercules, CA, USA) was used to conduct the qRT-PCR experiments. The PCR conditions consisted of pre-denaturation at 95˚C for 30 s, 40 cycles of denaturation at 95˚C for 5 s, and annealing and extension at 58˚C for 30 s. The default setting was used for the melting curve stage. The 2 −ΔΔCt method with three replications was performed for analysis and the ACT2 was selected as the reference gene. Genes with relative expression fold change (stress/control) � 2 and � 0.5 were considered significantly (Tukey's HSD test, P < 0.05) up and down-regulated, respectively. Ethical approval. This article does not contain any studies with human participants or animals performed by the authors.

Identification of the WRKY genes
A total of 104 candidate WRKY genes were predicted from H. annuus, among which 14 were removed after domain check and the rest 90 were named as HaWRKY1-HaWRKY90 (S2 Table). In comparison to the results of Guo et al. [20] and Liu et al. [21], one gene (HaWRKY51) in our study was found not included in the previous works, whereas the rest had their corresponding genes. The length of these genes ranged from 388 bp (HaWRKY5) to 8445 bp (HaWRKY49), with molecular weight (MW) from 10.48 to 74.25 kDa. The isoelectric point (pI) of these proteins ranged from 4.81 (HaWRKY86) to 10.44 (HaWRKY53) (S2 Table).

Phylogenetic analysis of WRKY family members
Phylogenetic analysis of WRKY family members of H. annuus and A. thaliana was conducted based on WRKY domain. The common sunflower WRKY domains were divided into four large groups (Figs 1 and 2), corresponding to group I, II and III in Arabidopsis [2] and an extra WKKY group. In contrast, Guo et al. [20] and Liu et al. [21] just distributed WRKY genes into group I, II and III. Among 90 HaWRKY family members, group II accounts for the largest part with 48 HaWRKY proteins, followed by group I with 18 proteins and group III with 17 proteins (Fig 1). There were 7 HaWRKY proteins in group WKKY, with C-X 5 -C-X 23 -H-X-H type zinc-finger motifs (Fig 2), which is not found in Arabidopsis (Fig 1). In addition, each group could be divided into several subgroups. Proteins with two WRKY domains were assigned as the N-terminal and the C-terminal WRKY domains according to their locations on protein. The proteins grouped either in N-terminal or C-terminal WRKY domains, usually followed by C 2 H 2 -type zinc-finger motifs (C-X 4 -C-X 22-23 -H-X-H), were classified as group I, with 16 identified as N-terminal WRKYs (I N) and 15 C-terminal (I C), and among them 13 members contained two WRKY domains (Fig 2). Group II of HaWRKY family could be clustered into five subgroups, with 4 in IIa, 10 in IIb, 13 in IIc, 11 in IId and 10 in IIe (Fig 1, S2 Table). 17 members of HaWRKYs in group III contained the C-X 7 -C-X 23 -H-X-C type zincfinger motifs (Fig 2, S2 Table), and were classified as subgroup IIIa. There were no HaWRKY proteins found in subgroup IIIb, not as that in Arabidopsis (Fig 1).

Gene structure and motif composition of WRKY family members
The distributions of exons and introns on HaWRKY genes were investigated via GSDS program, to gain further insight into the structure diversity of the WRKY family in sunflower. As shown in Fig 3B, almost half number (43) of the HaWRKY genes had three exons, followed by 17 with two exons, 17 with four exons, 12 with five exons and 1 with seven exons. Genes within same groups generally shared similar structures, such as group IIIa, in which all HaWRKY genes possessed three exons and two introns. Most of the WRKY domains spanned an exonexon junction, whereas HaWRKY genes with two WRKY domains in the group I at least had one complete domain within one exon, except HaWRKY1. Further analyses on introns indicated that HaWRKY genes only with phase-0 introns (between two consecutive codons) were clustered into group IIa and IIb, and only with phase-2 introns (between the second and third nucleotide of a codon) into the group IId, IIe and IIIa. The phase-1 introns (between the first and second nucleotide of a codon) were widely distributed among these groups, except group IIa.
Motif structures on HaWRKY proteins were constructed via MEME program. As exhibited in Fig 3C, HaWRKY

Evolution of group III HaWRKY genes
In order to understand the evolution of common sunflower group III WRKY genes, a phylogenetic tree of group III WRKY proteins from two monocots (rice and maize) and three dicots (sunflower, Arabidopsis and grape) was constructed. All the group III WRKY family members were divided into 10 clades as shown in Fig 4. WRKY proteins from closer species were clustered into same clades. Most proteins from dicots gathered in clade 1 and 3, whereas monocots MEME analysis was also conducted to search the conserved motifs of group III WRKY proteins from five species. Proteins within same clades usually displayed similar motif structures, indicating potential functional similarities among WRKY proteins. Motifs 1 and 7 were WRKY domains. Interestingly, motif 1 was found in all clades, whereas motif 7 was unique to clades 9 and 10, two clades only containing rice proteins, implying that motif 1 might have common function among different species, while motif 7 might play specific roles in rice and contribute to the divergence of group III WRKY genes. Motifs 1, 10 and 18 were specific to dicots. In contrast, motifs 12 and 19 were only observed in monocots. These motifs might be also important to the divergence of WRKY genes.

Chromosomal location and synteny of HaWRKY genes
HaWRKY genes are distributed unevenly on 17 chromosomes (S2 Table, Fig 5A). Chromosomes Ha10 and Ha15 both have 13 HaWRKY genes as the largest groups, whereas there was no HaWRKY gene observed on chromosomes Ha2. No correlation between chromosome length and HaWRKY gene number could be determined.
Two or more genes located within 200 kb on same chromosome is defined as a tandem duplication event [34]  HaWRKY50/53) are also identified (Fig 4). These results indicated that tandem and segmental duplication possibly contributes to the diversity and expansion of HaWRKY gene families. The dN/dS ratio of these duplicated gene pairs were calculated to understand the evolutionary constraints. The synonymous substitution rates (dS) of all segmental and tandem duplicated HaWRKY gene pairs were higher than non-synonymous substitution rate (dN) as shown in Table 1, indicating that HaWRKY gene family probably went through strong purifying selection during evolution.
Dual syntenies of common sunflower with Arabidopsis and rice were also conducted. A total of eight HaWRKY genes showed syntenic relationship with those in Arabidopsis, composing 9 orthologous pairs, whereas only one HaWRKY gene was collinear with one in rice ( Fig  5B and 5C). Similarly, more collinear gene pairs were observed between sunflower and Arabidopsis than rice, as sunflower is phylogenetically closer to Arabidopsis. HaWRKY16 was associated with two Arabidopsis genes and HaWRKY25 and HaWRKY65 are syntenic with a same Arabidopsis gene. HaWRKY25 is also found to be syntenic with a rice gene, indicating that these orthologous pairs might occur before the divergence of monocots and dicots.

Transcriptomic pattern of HaWRKY genes from different tissues
The transcriptome data of HaWRKY genes of different sunflower tissues were downloaded from Sunflower Genome Database. 20 of the 90 identified HaWRKY genes weren't expressed in all ten tissues (Fig 6A), which might be pseudogenes, have special temporal and spatial expression patterns or express in other tissues. The expression patterns of HaWRKY genes in sunflower were organ-specific, as bract, corolla, ligule, ovary, seed and stamen, which are related to flower, were clustered into a big group, and leaves and stem were in another group (Fig 6A). Most of the HaWRKY genes didn't express in pollen (Fig 6A). In general, the expression levels of HaWRKY genes in bract, ligule, leaves and stem were higher than that in other tissues (Fig 6B). HaWRKY17/22/79/81 displayed highest transcript abundances across all tissues except pollen and were clustered into a group, whereas the expression levels of HaWRKY23/31/37/40/68/84 were extremely low in all tested tissues (Fig 6A). The expression patterns of some genes were tissue-specific, for example, HaWRKY73 was abruptly induced only in leaves, HaWRKY3 in stem, HaWRKY11 in style, etc.

Profiles of HaWRKY genes under abiotic and biotic stress
Twenty-three HaWRKY genes which were highly induced in different tissues of common sunflower (except in pollen) were selected to test the reactions of different WRKY genes to different abiotic stresses. Generally, HaWRKY genes were inhibited in sunflower leaves after treatment of PEG and NaCl with different concentrations, whereas HaWRKY29/30 at 150 mM NaCl and HaWRKY48/89 at 20% PEG were significantly (P < 0.05) up-regulated by 46%/ 140% and 70%/51% compared with control, respectively. But no any significant (Tukey, P<0.05) changes of HaWRKY55/57 were observed in response to abiotic stresses in all comparisons (Fig 7A). Similarly, in roots as shown in Fig 7B,   HaWRKY52 and HaWRKY89 were up-regulated by PEG, and 10% PEG significantly (P < 0.05) increased by 152% and 179% compared with control. In contrast, Most of the HaWRKY genes were significantly (P < 0.05) up-regulated after treatment with NaCl in roots, as compared to the control. Among them, the transcript levels of 14  In order to understand the role of sunflower WRKY gene family against biotic stress, transcription levels of two contrasting common sunflower cultivars (TK0409, susceptible; JY207,   (Fig 8), suggesting these genes might partly contribute to the resistance of sunflower against O. cumana.

Discussion
The WRKY transcription factor family is considered to be involved in diverse stress responses, developmental and physiological processes in plants. Systematical characterization of WRKY genes in several species has been studied, including Arabidopsis, rice, tomato, maize etc. Sunflower WRKY genes have been well characterized benefiting from the release of its reference genome in previous studies [20,21]. However, there are two sunflower genome database available ("HanXRQr1.0" and "HA412.v1.1.bronze") assembled from different sunflower genotypes. Guo et al. [20] and Liu et al. [21] have used the "HanXRQr1.0" database for retrieval of WRKY genes. In our study, we used "HA412.v1.1.bronze" database to search WRKY genes as support and addition to the previous works. Indeed, we found 89 WRKY genes which have corresponding genes in the results of Guo et al. [20] and Liu et al. [21], and we discovered another WRKY gene (HaWRKY51) which was neglected in their works. Multiple protein sequence alignments revealed domain variations in common sunflower WRKY family. In comparison to the results of Guo et al. [20] and Liu et al. [21] that classify sunflower WRKY proteins into three normal groups, an extra WKKY group with 7 proteins was identified in our study based on the phylogenetic analysis, including HaWRKY 60 with WKKYGQK, HaWRKY33/50/53/54 with WKKYGEK, and HaWRKY51 with WKKYGKK (Fig 2). Interestingly, this group has been also found in Helianthus exilis, Helianthus petiolaris and Helianthus tuberosus [19], indicating that the WKKY variation is common in the Asteraceae. In addition, WKKYGQK, WKKYGKK and WKKYGEK are also observed in different legumes, but with low frequencies [35]. However, there are no more reports about WKKY group in other plant species. Although the WRKYGQK is highly conserved in most WRKY domains, variation in the core sequence has been documented [35]. In our studies, mutations happened to R and Q sites, while the others were conserved. WRKYGKK and WRKYGEK are the most frequently occurring variants of the core sequence in most plant species [35]. As the WRKYGQK core sequence can interact with the W-box to activate downstream genes, the variations in this motif might influence the function of downstream target genes [13,36]. Thus, further investigations on functions and binding specificities of these sunflower proteins with mutated WRKY motifs might provide deep insight into this transcription factor family.
A domain loss is common in the WRKY gene family in plants, which is recognized as a divergent force for expansion of this gene family [3,37]. In the current study, 5 domain loss events were found in group I, suggesting a potential cause of the diversity of WRKY genes in this group. Tandem and segmental duplication events also played a pivotal role in the expansion of WRKY gene family [4]. Five pairs of tandem and four pairs of segmental duplicated genes were identified in the present study, with five pairs in group IIIa, two pairs in group IId, one pair in group IIb and one pair in group IIc. This result indicated that tandem and segmental duplication events might contribute to the amplification of sunflower WRKY genes in these groups, as compared to those of Arabidopsis.
The origin of WRKY genes from group III appears to have occurred prior to the divergence of monocots and dicots, and then numerous duplications and diversifications happened after that event [38]. In order to explore how the WRKY group III gene family evolved, a phylogenetic tree of WRKY group III proteins from sunflower with two dicots (Arabidopsis, grape) and two monocots (rice, maize) was constructed, which divided the 17 group III HaWRKYs into three clades. WRKY proteins from closer species appeared to be clustered together. Both monocots and dicots proteins occurred in many clades, suggesting group III WRKY genes diversified before the monocot-eudicot split. In addition, clade 7 contained group III WRKY proteins from all 5 species, which tended to form monocot-and dicot-specific subclades, implying that group III WRKY genes evolved separately after the divergence of monocots and dicots.
It is well known that WRKY genes play essential roles in plant growth and development [7]. Li et al. [39] reported that AtWRKY13 functioned in stem development, as a weaker stem phenotype was observed and lignin-synthesis-related genes were repressed in Arabidopsis wrky13 mutants. In the current study, the orthologous of AtWRKY13 in sunflower, HaWRKY28 displayed high expression levels in stem, indicating that these genes might also act in stem development in sunflower. Overexpression of WRKY15 exhibited an increased leaf area of Arabidopsis, which implied that AtWRKY15 seemed to be involved in leaf growth [40]. HaWRKY79, which is the orthologous of AtWRKY15, were highly induced not just in leaves, but across all tissues, indicating that this gene might be constitutive in sunflower plant growth and development. In contrast, HaWRKY7 was specifically expressed in leaves, suggesting its role in sunflower leaf growth. Among all WRKY genes in sunflower, only HaWRKY30 displayed a high level of expression in pollen, with other WRKY genes extremely low expressed. Interestingly, two pollen-specific regulators in Arabidopsis, AtWRKY34 and AtWRKY2, have phylogenetically close relationship with this sunflower WRKY gene, indicating that HaWRKY30 might be associated with pollen developmental modulation.
In addition to their role in plant growth and development, WRKY TFs also play pivotal roles in various stress responses, providing an important basis for genetic improvement of crops. Drought and salinity, both of which can cause plant cellular dehydration [41], are two major constraints to sunflower production [42]. Responses of plants to drought and salinity usually result in accumulation of reactive oxygen species (ROS) and abscisic acid (ABA) [41,43], which activate downstream WRKY genes [7].
Overexpression of a membrane-localized cysteine-rich receptor-like protein kinase, CRK5 in Arabidopsis, led to increase of ABA sensitivity and promotion of stomatal closure, and subsequent enhancement of plant drought tolerance. Knockout of AtWRKY18, AtWRKY40 and AtWRKY60 significantly increased the expression of CRK5, suggesting negative regulation of these three genes on CRK5 [44]. In our study, the relative expression levels of two orthologous of AtWRKY40, HaWRKY74 and HaWRKY81, were recorded in sunflower roots and leaves. Expression levels of both two genes decreased as the concentrations of PEG increased in sunflower leaves, while in sunflower roots, two genes were induced under low concentration of PEG and inhibited under high concentration. It has also been reported that AtWRKY46, AtWRKY54, and AtWRKY70 are implicated in promotion of BR-regulated plant growth and inhibition of drought response, as reduced BR-regulated growth and higher survival rates under drought stress was observed in their triple mutant [45]. HaWRKY9 and HaWRKY22, which were phylogenetically close to AtWRKY46, were both repressed under PEG treatments in sunflower roots and leaves. These results are suggesting that sunflower probably enhanced drought tolerance via down-regulating specific WRKY genes and subsequently activating downstream signal pathways. The increase of ABA level caused by drought usually induces high expression of AtWRKY57, which binds to W-box in the promoter region of the downstream response genes. HaWRKY57, the orthologous of AtWRKY57, displayed a high expression level under PEG treatment in sunflower roots. Interestingly, the increase of ROS level caused by salinity also activates AtWRKY57, and consistently, HaWRKY57 was highly expressed under treatment of 300 mM NaCl in sunflower roots. These results indicated that HaWRKY57 might share similar functions with AtWRKY57 in sunflower under drought and salinity. AtWRKY15 is another WRKY gene induced by ROS, but will make Arabidopsis more susceptible to osmotic stress and oxidative stress [40]. In our study, HaWRKY79, the orthologous of AtWRKY15, was significantly suppressed in sunflower roots under treatment of NaCl, implying their similar roles in conferring salt tolerance.
The parasitic weed Orobanche cumana is a new emerged threat to sunflower production worldwide. Previous studies proposed that O. cumana deployed effectors in sunflower to suppress host defense responses and resistant sunflower cultivars recognized effectors with the help of R proteins to activate effector-triggered immunity. WRKY family has been found to be involved in the microbe-associated molecular pattern-triggered immunity, PAMP-triggered immunity or effector-triggered immunity [7]

Conclusion
In this study, we identified 90 WRKY genes from Helianthus annuus L. and characterized their structure, duplication, chromosomal distribution, phylogenetic tree, followed by tissue-differential gene expression and differential expression in response to biotic and abiotic stress. Supporting information S1