Genome-Wide Identification, Evolution and Expression Analysis of the Grape (Vitis vinifera L.) Zinc Finger-Homeodomain Gene Family

Plant zinc finger-homeodomain (ZHD) genes encode a family of transcription factors that have been demonstrated to play an important role in the regulation of plant growth and development. In this study, we identified a total of 13 ZHD genes (VvZHD) in the grape genome that were further classified into at least seven groups. Genome synteny analysis revealed that a number of VvZHD genes were present in the corresponding syntenic blocks of Arabidopsis, indicating that they arose before the divergence of these two species. Gene expression analysis showed that the identified VvZHD genes displayed distinct spatiotemporal expression patterns, and were differentially regulated under various stress conditions and hormone treatments, suggesting that the grape VvZHDs might be also involved in plant response to a variety of biotic and abiotic insults. Our work provides insightful information and knowledge about the ZHD genes in grape, which provides a framework for further characterization of their roles in regulation of stress tolerance as well as other aspects of grape productivity.


Introduction
Transcription factors (TFs) have been shown to play important roles in the regulatory networks of numerous developmental processes in plants, for instance, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs can regulate fruit color [1,2].Transcription factors can either activate or repress target genes through direct binding to gene regulatory elements or motifs.Accordingly, different TF families evolve unique DNA-binding domains that confer their binding specificity.One of the well characterized domains is the homeodomain (HD) that is composed of about 60 amino acids [3].Several HD-containing proteins/TFs are known to play crucial roles in plant or animal development [4][5][6][7] but plants evolve specific HD-Zip TF family [8] that bear a unique leucine zipper domain at the C-terminal end.Zinc fingers serve as important motifs in many regulatory proteins [9,10], typically consisting of two pairs of conserved cysteine and/or histidine residues that associated with zinc ions [11].Zinc fingers can be divided into different categories according to the nature, number, and spacing pattern of the zinc-binding residues.For instance, C 2 H 2 , C 2 C 2 and C 3 H zinc fingers interact with one zinc ion, while the recently identified plant RING finger and the animal Lin-11/Isl-1/Mec-3 (LIM) domain interact with two zinc ions [12][13][14][15][16].It has been shown that many Zn-finger TFs and the C 2 H 2 -type protein factors are involved in various stress signaling pathways [17][18][19][20].
A cluster of zinc finger-homeodomain (ZHD) proteins were first discovered in Flaveria as a regulator of the gene encoding C4 phosphoenolpyruvate carboxylase (PEPCase) [21].Since then, ZHD proteins have been identified and characterized in many plants, including Arabidopsis thaliana [22], Oryza sativa [23], Glycine max [24] and Triticum aestivum [25].In Arabidopsis, the first described example of a zinc finger (ZF) HD protein is involved in the regulation of floral development [26], and an Arabidopsis ZHD protein (AtZHD1) was subsequently reported to bind specifically to the promoter of Early Response To Dehydration Stress 1 (ERD1).The expression of AtZHD1 is inducible by dehydration, salt stress and abscisic acid (ABA) [27].To date, 14 Arabidopsis ZHD genes have been identified [26,28].The function of ZHD genes in other plants has also been elucidated recently.For instance, soybean GmZF-HD1 and GmZF-HD2 directly up-regulate a gene coding for calmodulin isoform 4 (GmCaM4) upon pathogen stimulation [24].Four rice ZHD genes have also been implicated in gene regulation [23].
Although the function of several ZHD genes have been elucidated in Arabidopsis and other model species, their roles in fruit crops including grape remains largely unknown.Grape is one of the most economically important perennial fruit crops worldwide and has been extensively studied at the physiological and developmental levels.Grape was one of the first fleshy fruit species to have its genome fully sequenced [29].With the release of the grape genome it is now possible to perform a genome-wide identification and characterization of the family.Here we report the identification of 13 ZHD genes using the grape genome sequence, their classification, and phylogenetic and syntenic analyses.We also present their expression profiles in six different tissues/organs, and describe the dynamics of the corresponding transcript levels in response to different phytohormone treatments and various abiotic and biotic stresses.

Genome-Wide Identification of V. vinifera ZHD (VvZHD) Genes
To identify ZHD genes in the grape genome, ZF and HD domains were used as a query to search the GenBank non-redundant protein database and the Grape Genome Database with the BLASTP program.After removing redundant sequences, we identified 13 putative VvZHD proteins with E values less than 0.01 (Table 1), all of which contain a complete HD and a ZF domain.All the genes were mapped to specific chromosomes and were named VvZHD1-13 based on their order on the chromosomes (Figure 1).A multiple sequence alignment of the predicted full length protein sequences was carried out using the DNAMAN software to determine the domain structures of the VvZHD proteins.Two conserved domains were found (the zinc finger domain and homeodomain) in all the VvZHD proteins (Figure 2A) and they showed a high degree of sequence conservation within each class (Figure 2B).

Expansion Patterns of the Grape ZHD Gene Family
Segmental and tandem duplications are known to be the main causes leading to gene family expansions [30].In the present study, we identified two tandemly duplicated ZHD genes (VvZHD5 and VvZHD6 located on grape chromosomes 12) (Figure 1).We also examined the segmentally duplicated blocks within the grape genome and identified two pairs of grape ZHD genes, VvZHD2/8 and VvZHD3/11, which are associated with segmental duplications (Figure 1).

Evolutionary Relationship of Grape and Arabidopsis ZHD Genes
To explore the origins and evolutionary dynamics of the grape ZHD protein family, we performed a comparative synteny analysis between the grape and Arabidopsis genome sequences.Given that Arabidopsis ZHD genes have been well characterized [28], the knowledge generated in Arabidopsis could help determine the origin and diversification of ZHD genes in the grape.A large-scale syntenies containing 7 grape and 11 Arabidopsis ZHD genes were identified (Figure 1).Several grape orthologues including VvZHD1, VvZHD7 and VvZHD8 displayed synteny location with Arabidopsis AtZHD14, AtZHD3, AtZHD9, respectively.Interestingly, we also found that a single grape gene syntenically corresponded to multiple Arabidopsis genes such as VvZHD2-AtZHD10/AtZHD11, VvZHD10-AtZHD1/AtZHD2, VvZHD11-AtZHD5/AtZHD6/AtZHD13 and VvZHD3-AtZHD5/AtZHD6/ AtZHD7, suggestive of preferential gene deletion occurred in certain syntenic locations during grape genome evolution.

Phylogenetic Analysis of Grape ZHD Genes
Phylogenetic analyses of ZHD gene sequences from Arabidopsis and other plant species have previously been reported [28] and our result was in agreement with the earlier studies in general (Figure 3).The major clades of angiosperm ZHD genes have been shown to fall into class I to VII [28] while the VvZHD genes belong to classes I-III and V-VII (Figure 3).Class I contains two VvZHD genes (VvZHD10 and 13) and two AtZHDs share high levels of sequence similarity even outside the ZF and HD domains.Similarly, Class II includes two VvZHD genes (VvZHD4 and 7) as well as those from a wide range of angiosperms.Class III has two VvZHD genes (VvZHD3 and 11) that are fairly conserved at the N-terminal regions, whereas sequences in Class IV are not.Class IV ZHD genes encode proteins that are rich in glutamine (Q) at the C-termini, but there are no VvZHD genes in this clade.Class V includes two VvZHD genes (VvZHD2 and 8) and the central region of the encoded ZF domain in the corresponding proteins typically has more residues rather than are found in other ZHD proteins.Class VI, which has four VvZHD genes (VvZHD1, 5, 6 and 9) has the best supported major clade in the phylogenetic tree and the constituent sequences are highly divergent from the other classes (Figure 3).VvZHD12 belongs to Class VII and shares no apparent sequence similarity with other ZHDs outside the ZF and HD domains.

Sequence Analysis of Grape ZHD Genes
Phylogenetic relation among the 13 VvZHD proteins was analyzed and presented in Figure 4A.VvZHD1 appears to distantly relate to the rest of the genes.To delineate the evolutionary relationships among the VvZHD genes, we determined the distribution of the conserved domains in the encoded proteins (Figure 4B) and they all shared the following basic structure with ZF and HD domains (Figure 4B).Actin1 was used as the internal control.

Expression Profiles of Grape ZHD Genes in Different Organs
To gain insights into the potential functions of the grape ZHD genes during development, we performed semi-quantitative RT-PCR analysis in various tissues.Figure 4C shows that all of the 13 genes were expressed in at least one of the six tissues but with various specificities.Most of the genes, including VvZHD1, 3-6, 8 and 12, were expressed in all tissues examined, while the remainders showed tissue-specific expression.However, ten VvZHD genes that were constitutively expressed in all organs exhibited notable variation in expression levels, with VvZHD3 displaying high levels of expression in stems, leaves and flowers, but much lower expression in the roots, fruits and tendrils.

Expression Patterns of Grape ZHD Genes under Different Stress Conditions and Following Exogenous Hormone Treatments
Different approaches have been developed to improve plant stress tolerance, including manipulating and reprogramming the expression of endogenous stress-related genes.Therefore, identification and functional characterization of potential stress-related genes provides fundamental information for future improvement of plant stress tolerance.In the present study, we investigated the response of grape ZHD genes to various abiotic and biotic stress conditions, as well as hormone treatments.

Abiotic Stresses
To determine whether VvZHD genes respond to osmotic stress in leaves, semi-quantitative RT-PCR was performed to assess their transcript levels under salt and drought stress conditions.As shown in Figure 5, the expression of five VvZHD genes (VvZHD1, 3, 6, 8 and 12) was differentially regulated by both treatments.In particular, the transcript level of VvZHD11 was gradually down-regulated under drought stress, but increased after re-hydration, while VvZHD5 did not respond to the treatment.VvZHD6, 7 and 13 were up-regulated in response to salt stress, but down-regulated by drought stress.The opposite response of salt stress was observed for VvZHD2, 3 and 9. VvZHD10 was transiently up-regulated in response to salt stress and then declined, but increased gradually with time under drought stress.VvZHD5 did not respond to either treatment.The consistent results were obtained for five randomly selected VvZHD genes by real-time RT-PCR under the same treatments (Figure 6).

Biotic Stress
The responses of the 13 VvZHD to powdery mildew infection were investigated by semi-quantitative RT-PCR.More than half of 13 VvZHDs responded to the infection, with VvZHD2, 3, 5-7 and 13 being up-regulated and VvZHD1, 4, 8 and 12 being down-regulated (Figure 5).The transcript abundance of VvZHD6 in infected leaves peaked at 48 h, but that of VvZHD5 increased sharply at 6 h and remained constant thereafter till it decreased at 96 and 120 h.These results were further validated by real-time quantitative RT-PCR (Figure 6).

Hormone Treatments
We also evaluated the effects of ABA, salicylic acid (SA), methyl jasmonate (MeJA), and ethylene (Eth) on the VvZHD gene expression since SA, MeJA and Eth are involved in plant response to biotic stresses [31,32].Analysis of transcript levels from "Kyoho" grape leaves sprayed with SA showed that seven VvZHD genes (VvZHD3, 5, 6 and 10-13) were up-regulated to different degrees upon treatment, whereas 2 VvZHD genes (VvZHD4 and 9) were down-regulated.The transcript level of VvZHD2 was high at the first three sampling points following SA treatment, but declined thereafter, while VvZHD7 and 8 expressions remained unchanged (Figure 5).Following treatment with MeJA, four VvZHD genes (VvZHD1, 3, 6 and 13) exhibited increased expression (Figure 5), while five genes (VvZHD2, 5, 8, 9 and 12) were down-regulated.VvZHD4 and 7 transcript abundance was low at the first two sampling points after MeJA treatment, but subsequently increased.No obvious changes were noted in the expression of the other two VvZHD genes (VvZHD10 and 11) analyzed (Figure 5).As was the case with the Eth treatment, most VvZHD genes also showed altered expression in leaves following treatment (Figure 5).Of these, VvZHD4-6 and 13 were induced by, whereas VvZHD2 was down-regulated.The VvZHD7 transcript level was high at the first three sampling times, but decreased thereafter.
ABA is known to play a central role in plant responses to various abiotic stresses [33] and analysis of transcript levels from "Kyoho" leaves treated with exogenous ABA indicated that three VvZHD genes (VvZHD4, 5 and 13) were up-regulated at different times following treatment (Figure 5).For example, the transcript levels of VvZHD4 and 13 were induced at 1 h after treatment, while those of VvZHD1, 5 and 6 peaked at 1 h after treatment, and VvZHD2 transcript abundance at 12 h post-treatment.Three of the analyzed genes (VvZHD3, 9 and 10) exhibited decreased transcript levels, whereas expression of VvZHD8 remained unchanged.These results were also supported by quantitative real-time RT-PCR analysis (Figure 6).

Discussion
ZHD genes, which play importantly regulatory roles in plants, are critical for both development and responses to environmental changes.However, little is known about the grape ZHD genes, and their evolution and function, even though grape is one of the most important fruit crops worldwide and various forms of stress have posed serious negative impacts on the production and quality of grape crops worldwide.Accordingly, in this study we aimed to identify and characterize the structure, evolution, expression and stress-related responses of the grape ZHD gene family.

Evolution of Grape VvZHD Genes
ZHD genes encode a family of specific TFs in all major groups of land plants, including various seed plants, the seedless vascular plant Selaginella, and the nonvascular plant Physcomitrella, but are absent in fungi, yeast, the chlorophyte green algae Chlamydomonas and Volvox, and prokaryotes, which suggests that although plant ZHD genes may be derived from a common ancestor, and appeared after the divergence of land plants, many have undergone lineage specific differentiation [28].In this study, a total of 13 genes were identified in the grape genome and subjected to phylogenetic analysis.The plant ZHD genes were clustered into seven distinct groups (CI-CVII), as well as a cluster that does not form a well-supported group (Figure 3).The plant ZHD gene family has diversified through evolution and our work suggests that the family has expanded during angiosperm evolution.Angiosperm ZHD genes form six well supported clades (Classes I-VI) and one weakly supported clade (Class VII) (Figure 3) and the grape ZHD genes were clustered into six of the seven groups (CI, CII, CIII, CV, CVI and CVII).Furthermore, most VvZHD genes were more closely related to Arabidopsis ZHD genes, which is consistent with the fact that grape and Arabidopsis are eudicots and diverged more recently from a common ancestor.

Expansion and Synteny of the VvZHD Gene Family
It is known that tandem, segmental and whole genome duplications have played significant roles in the evolution of many organisms [34].Based on comprehensive analysis of gene chromosomal locations, lengths, structures and sequence similarities we determined that one pair of VvZHD genes, (VvZHD5 and 6) likely arose by tandem duplication.Similarly, genome duplications also contributed to the expansion of the ZHD gene family in other plants, such as the three whole-genome duplication events in the Arabidopsis genome [35].More than half of the Arabidopsis ZHD genes are thought to be generated by genome duplication [28,36]; however, since the grape genome has apparently not undergone any recent whole genome duplication events [29], segmental and tandem duplications would probably account for most of the expansions, although there is debate on the exact nature and timing of these events in grape [29,37].In summary, tandem and segmental duplications have likely played critical roles in the expansion and evolution of the VvZHD gene family, resulting in their quantitative, structural and functional diversification.
Genomic comparison is a relatively rapid and effective way to transfer genomic knowledge acquired in one taxon, whose genome structure, function and/or evolution is better characterized, to a less-studied organism [38].Thus, the putative functions of VvZHD genes can be inferred from comparisons with their respective orthologs in the model plant Arabidopsis.In this study, synteny analysis of the grape and Arabidopsis genomes indicated that three pairs of ZHD genes (VvZHD1-AtZHD14, VvZHD7-AtZHD3 and VvZHD8-AtZHD9) are located in syntenic genomic regions (Figure 1).The situation with other genes was more complex, including four cases of one VvZHD gene corresponding to multiple AtZHD genes, and although six VvZHD genes could not be mapped to any syntenic blocks, we could not conclude that these genes did not share a common ancestor.Since the lineages that led to grape and Arabidopsis have undergone multiple rounds of significant chromosomal rearrangement and fusions, followed by selective gene loss, the identification of chromosomal syntenies can be somewhat obscured [39].It may therefore be deduced that some of the grape and Arabidopsis ZHD genes arose from a common ancestor, while others did not.The first Arabidopsis ZHD gene to be characterized, AtZHD1, was found to be involved in the floral transition [26] and now the list of Arabidopsis ZHD genes that have been functionally characterized includes AtZHD1 [27], AtZHD5 [22] and AtZHD12 [26].
Based on such studies we can infer possible functions.For example, VvZHD11 is a homolog of AtZHD5 that functions as regulating floral architecture and leaf development.

Spatial Expression Patterns of VvZHD Genes in Various Grape Tissues
There are a couple of resemblances in the spatial expression models of VvZHD genes and those from other plant species.For instance, the AtZHD10 and VvZHD8 proteins are closely related and this is also reflected by their presence within the same phylogenetic group (V) (Figure 3).AtZHD10 is expressed preferentially in inflorescence and seed tissues [28], while the closely related VvZHD8 is highly expressed in leaves, flowers and stems.Members of groups I and III also appear to have similar expression patterns to each other.Hu et al. reported that AtZHD9 was expressed in all tested tissues/organs [28].This is similar to the expression patterns observed for VvZHD8, an orthologous gene with AtZHD9 (Figure 1).It therefore seems that members of individual ZHD groups and orthologous pairs have similar expression features, again indicating that there may be conserved functionality amongst members of the same groups and orthologous genes.

VvZHD Proteins Play Important Roles in a Range of Biological Processes
To gain further insights into the potential roles of the VvZHD genes, we analyzed their spatiotemporal expression patterns, as well as their responsiveness to various types of abiotic and biotic stresses and hormone treatments.Our results revealed that the VvZHD genes exhibit diverse expression patterns in six organs, indicating widespread roles in plant development.Genes that showed higher expression levels in one organ than in others may be more important.One such example is VvZHD6, which are expressed at high levels in fruits and thus may play a role in their development.
We also showed that five of the VvZHD genes exhibited substantial increases in their levels of expression in response to dehydration and/or high salinity (Figure 5), two of which (VvZHD1 and 12) were up-regulated by both treatments.This aligns with a previous report that the Arabidopsis ZHD1 gene is induced by dehydration, salt stress and ABA [27].Moreover, AtZHD1 can interact with NAM/ATAF1, 2/CUC2 (NAC) proteins and the simultaneous overexpression of AtZHD1 and AtNAC genes improved Arabidopsis drought stress tolerance [27].Our results suggest that, like a number of ZHD genes previously described, the VvZHD genes may play important roles in protecting grape from damage caused by various types of abiotic stress.
Since plant hormones such as ABA, SA, MeJA and Eth have been reported to be involved in stress tolerance, we also analyzed the responsiveness of 13 VvZHD genes to phytohormone treatments (Figure 5).Our results showed varying patterns of differential expression in response to the plant hormones, which correlates well with previous findings from other plant species.For instance, Eth, SA, and MeJA coordinately play a critical role in biotic stress signaling upon pathogen infection [40], while ABA is extensively involved in responses to various biotic and abiotic stresses, including pathogen infection [41], water-deficit, cold, and osmotic stress [42].Arabidopsis, AtZHD1 is up-regulated by ABA [43][44][45] and similarly, we found that VvZHD4 and 13 exhibited increased expression after treatment with exogenous ABA.Besides, the expression of TaZHD1 also correlates with Eth, ABA and MeJA obviously [25].Taken together, our analyses of the responses of VvZHD genes to different plant hormone treatments revealed potentially important targets for increasing the resistance of grape to stress conditions, and will provide the basis for guiding future studies of the functions of VvZHD genes and their associated signal transduction networks.

Identification and Annotation of Grape ZHD (VvZHD) Genes
The HMM (Hidden Markov Model) profile of the ZF domain (Accession No. PF04770) was downloaded from the Pfam database [46].The HD domain was determined based on previous reports, as it is not present in Pfam [21].These domains were then used as a query using profile hidden Markov models to search the GenBank non-redundant protein database [47] and the Grape Genome Database [48] using the BLASTP program.All hits with an E value <0.01 were collected and the domains were manually checked in each identified VvZHD gene.

Determination of Chromosomal Location and Synteny Analysis
The VvZHD genes were positioned on the grape pseudo-molecules available at the Grape Genome Database (12 X).Tandemly duplicated VvZHD genes were defined as adjacent to homologous ZHD genes on the grape chromosomes or within a sequence distance of 50 kb [49], with no more than one intervening gene [50].For synteny analysis, we used MCScan algorithm for detection of synteny and collinearity [51].Synteny blocks within the grape genome and between the grape and Arabidopsis genomes were downloaded from the Plant Genome Duplication Database, and those containing VvZHD genes were identified (Original results shown in Tables S1 and S2).

Sequence Alignments, Phylogenetic Analysis and Domain Location of VvZHD Genes
The 13 VvZHDs were aligned using ClustalX, which can provide an integrated system for performing multiple sequence and profile alignments and analyzing the results [52] (The Percent identity of alignment shown in Table S3).To compare and define subgroups, we integrated conserved ZF and HD domains into this dataset.Phylogenetic trees were constructed with the MEGA 5.0 software using the neighbor-joining (NJ) method, and the bootstrap test was replicated 1000 times [53].The 13 VvZHD genes with the complete ZF and HD domains were used in this study.Pfam domains and signal peptides were predicted using SMART (European Molecular Biology Laboratory, Heidelberg, Germany) [54,55].The diagram of protein structures was constructed using DOG 1.0 software (Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science &Technology of China, Hefei, China) [56,57].

Abiotic, Hormone and Biotic Stress Treatment
For abiotic stress treatments, two year-old "Kyoho" grape seedlings planted in pots were irrigated with 2 dm 3 250 mM NaCl [58,59].Following treatments for 1, 6, 12, 24 and 48 h, the fully unfolded young leaves were collected.Drought stress was induced by withholding water from "Kyoho" seedlings as previously described, with minor modification [60,61].Briefly, young leaves of the seedlings were harvested at 24, 48, 72, 96, 120, 144 and 168 h post treatment.Subsequently, the stressed plants were re-watered to soil saturation and leaves were collected at 48 h after re-watering.For salt and drought stress, plants watered every three days were used as controls.
Hormone treatments were conducted by spraying young leaves with 100 µM SA [62,63] or 100 µM ABA [58,64] followed by sampling at 1, 6, 12, 24 and 48 h post-treatment.Leaves sprayed with sterile distilled water at the same time points were collected as the control.
Pathogen infection was carried out by inoculating the young leaves of "Shang-24" with powdery mildew as previously described with minor modifications [65].Prior to inoculation, leaves were sprayed with sterile water, and leaves were harvested at 6, 12, 24, 48, 72, 96 and 120 h post-inoculation (Hpi).Control plants were sprayed with sterile water and not inoculated.
At each time point of each treatment, nine leaves from three separate plants were pooled and immediately frozen in liquid nitrogen and stored at −80 °C until use.

Semi-Quantitative RT-PCR and Real-Time PCR Analysis
Total RNA was extracted according to Zhang et al. [66], and treated with 10 units of RNase-free DNase I (TaKaRa Bio Inc., Dalian, China) to remove genomic DNA contamination.For each sample, 1 µg of total RNA was used to synthesize first-strand cDNA using SuperScriptII reverse transcriptase (Invitrogen).For the following experiments, the reverse transcription products were diluted six times.The concentration of the cDNA was adjusted according to a PCR product corresponding to the grape Actin1 gene (GenBank Accession number AY680701), amplified with the primers F (5'-GAT TCT GGT GAT GGT GTG AGT-3') and R (5'-GAC AAT TTC CCG TTC AGC AGT-3').Gene-specific primers were designed for the 13 VvZHD genes including the complete ZF and HD domains (Table S4).For semi-quantitative reverse transcription-PCR (RT-PCR), a 20 µL reaction volume included 1 µL of cDNA template, 1.6 µL of gene-specific primers (1.0 µM), 9.8 µL PCR Master Mix (Tiangen Biotech Co. Ltd., Beijing, China) and 7.6 µL sterile distilled water.The PCR parameters were 95 °C for 3 min, followed by 25-35 cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 25 s, and a final step at 72 °C for 2 min.Each PCR reaction was replicated three times.The results of semi-quantitative RT-PCR were quantified using the Gene Tools software, and the log-transformed values of the relative transcript abundance of VvZHD genes under abiotic, hormone and biotic stress treatment compared to the control were used for hierarchical cluster analysis with Genesis software.
Quantitative real-time PCR analysis was conducted with an IQ5 real-time PCR instrument (Bio-Rad, Hercules, CA, USA).Each reaction was carried out in triplicate with a reaction volume of 20 µL containing 1.6 µL of gene-specific primers (1.0 µM), 1.0 µL of cDNA, 10 µL of SYBR green (TaKaRa Bio Inc., Dalian, China), and 7.4 µL sterile distilled water.The PCR parameters were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Relative expression levels were analyzed with the IQ5 software using the Normalized Expression method (the gene expression results were also normalized to the mock controls, respectively) and a student t-test performed using the SPSS software (SPSS 17.0 ® , Chicago, IL, USA).

Conclusions
ZHD genes encode a family of transcription factors that are exclusively present in plants.While significant progress has been made in identifying and characterizing ZHD genes in a number of plant species, little equivalent information is available for fruit crops.In this study, we identified a total of 13 VvZHD genes in the grape genome and developed a nomenclature based on their chromosome location, and demonstrated that segmental duplication has likely contributed to the expansion and evolution of the VvZHD gene family.Comparative synteny analysis between grape and Arabidopsis showed that several grape and Arabidopsis ZHD genes were located in syntenic regions, suggesting that they have common ancestors.Expression analysis of the VvZHD genes suggested that all 13 genes were functional and that a few may have specific roles in particular organs.Finally, many VvZHD genes were also found to be responsive to various abiotic and biotic stresses and hormone treatments.These studies provide a resource for future studies of VvZHD genes and may promote research aimed at revealing their role in grape stress tolerance.The results provide the basic information of the grape VvZHD genes, which should facilitate further research aiming at revealing the potential important functions of VvZHD genes in grape.

Figure 2 .
Figure 2. Alignment ZF and HD domains of VvZHD proteins.(A) A multiple sequence alignment of the ZF (Ia and Ib) and HD (II) domains of the grape ZHD proteins.The two conserved structures (Zinc-Finger and Homeodomain) are indicated; (B) Sequence logo of the ZF and HD domains of the grape ZHD proteins.The overall height of each stack represents the degree of conservation at this position, while the height of the individual letters within each stack indicates the relative frequency of the corresponding amino acids.

Figure 4 .
Figure 4. Phylogenetic relationships, primary protein structures and expression patterns of VvZHD genes.(A) Neighbor-joining un-rooted phylogenetic tree; (B) Schematic representation of protein length and domain location; (C) Expression analysis of VvZHD genes in various "Kyoho" (V.labrusca × V. vinifera) organs by semi-quantitative RT-PCR.Actin1 was used as the internal control.

Figure 5 .
Figure 5. Expression profile of 13 VvZHD genes under salinity and drought stress treatments, powdery mildew inoculation and various hormone treatments (SA, MeJA, Eth, ABA) analyzed by semi-quantitative RT-PCR.Blocks with different colors indicate the transcript accumulation relative to the respective control: up-regulated (red), down-regulated (green).(A) Expression profile of VvZHD genes under biotic stress treatments, salinity and drought (original results shown in Figure S1); (B) Expression profile of VvZHD genes under biotic stress treatment, powdery mildew (original results shown in Figure S1); (C) Expression profile of VvZHD genes following four hormone treatments: SA, MeJA, Eth and ABA (original results shown in Figure S2).

Figure 6 .
Figure 6.Effect of salt (A); drought (B); powdery mildew (PM) (C); salicylic acid (SA) (D); methyl jasmonate (MeJA) (E); ethylene (Eth) (F) and abscisic acid (ABA) (G) on the expression of VvZHD gene expression in leaves was investigated by qRT-PCR.The grape actin1 gene was used as the reference gene.Error bars represent SD (n = 3).The gene expression results were normalized to the mock controls, respectively.Asterisks indicate levels of significance of differential expression (t-test: * p ≤ 0.05).

Table 1 .
The grape ZHD gene family.CDS, coding sequence.Synteny analyses of VvZHD genes and ZHD genes between grape and Arabidopsis.Grape and Arabidopsis ZHD genes are indicated by vertical orange lines.Colored bars denote syntenic regions.