Genomic Organization, Phylogenetic Comparison and Differential Expression of the SBP-Box Family Genes in Grape

Background The SBP-box gene family is specific to plants and encodes a class of zinc finger-containing transcription factors with a broad range of functions. Although SBP-box genes have been identified in numerous plants including green algae, moss, silver birch, snapdragon, Arabidopsis, rice and maize, there is little information concerning SBP-box genes, or the corresponding miR156/157, function in grapevine. Methodology/Principal Findings Eighteen SBP-box gene family members were identified in Vitis vinifera, twelve of which bore sequences that were complementary to miRNA156/157. Phylogenetic reconstruction demonstrated that plant SBP-domain proteins could be classified into seven subgroups, with the V. vinifera SBP-domain proteins being more closely related to SBP-domain proteins from dicotyledonous angiosperms than those from monocotyledonous angiosperms. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologs of several grape SBP genes were found in corresponding syntenic blocks of Arabidopsis. Expression analysis of the grape SBP-box genes in various organs and at different stages of fruit development in V. quinquangularis ‘Shang-24’ revealed distinct spatiotemporal patterns. While the majority of the grape SBP-box genes lacking a miR156/157 target site were expressed ubiquitously and constitutively, most genes bearing a miR156/157 target site exhibited distinct expression patterns, possibly due to the inhibitory role of the microRNA. Furthermore, microarray data mining and quantitative real-time RT-PCR analysis identified several grape SBP-box genes that are potentially involved in the defense against biotic and abiotic stresses. Conclusion The results presented here provide a further understanding of SBP-box gene function in plants, and yields additional insights into the mechanism of stress management in grape, which may have important implications for the future success of this crop.


Introduction
Transcription factors, which are proteins that bind DNA in a sequence-specific manner and regulate gene expression by activating or repressing the transcription of downstream target genes, are found in virtually all living organisms and play an essential role in regulatory networks of many important developmental processes. SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box genes encode a family of plant-specific transcription factors [1,2] that contain a highly conserved DNAbinding domain termed the SBP domain. This domain is an assembly of approximately 76 amino acid residues that are involved in both DNA-binding and nuclear localization, and features two zinc-binding sites [2,3]. SBP-box genes were first identified in Antirrhinum majus (AmSBP1 and AmSBP2) based on the ability of their encoded proteins to interact with the promoter region of the floral meristem identity gene SQUAMOSA [2].
In Arabidopsis, the first SBP-box gene identified was SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3), which was found to be involved in the floral transition. Similar to the AmSBP1 and AmSBP2 proteins from A. majus, AtSPL3 recognizes a conserved motif in the promoter region of the Arabidopsis floral meristem identity gene APETALA1 (AP1), which is an ortholog of SQUAMOSA [4]. To date, sixteen SBP-box genes have been identified in the Arabidopsis genome [1,5], of which some have been shown to function in regulation of plant development, ranging from sporogenesis [6], shoot development [7], flowering [8], plastochron formation [9], vegetative and reproductive phase transitions [10,11], leaf development [12], fertility [13], to plant hormone signaling [14] and copper homeostasis [15].
In recent years, knowledge concerning the functions of SBP-box genes in plant species other than Arabidopsis has also begun to accumulate and highlights the diverse roles of these proteins in plant development. For example, a novel SBP-box gene, BpSPL1, from silver birch (Betula pendula) was shown to be capable of binding a cis-element of BpMADS5, which is a close homolog of the Arabidopsis FRUITFULL gene, and indicated a role for birch SBPbox genes in the regulation of flower development [16]. Similarly, AmSBP1 from A. majus has recently been implicated in flowering initiation through the activation of meristem identity genes [17]. In contrast, a tomato SBP-box gene has been found to be a critical factor in fruit ripening [18], while the maize tasselsheath4 (tsh4) SBPbox gene has been shown to regulate bract development and to be a necessity in branch meristem initiation and maintenance [19]. Conversely, in the unicellular alga, Chlamydomonas reinhardtii, an SBP-domain protein has been determined to regulate nutritional copper signaling [20]. Finally, of the 19 putative SBP-like genes identified and characterized in the rice (Oryza sativa) genome, more than half were shown to be highly expressed in young panicles, suggesting their putative roles in these organs [21].
In recent years, an increasing number of microRNAs (miRNAs) have been found to play a crucial role in the regulation of gene function in plants, and these numbers are still accumulating at present. MiRNAs are small RNA molecules (20-24 nucleotides in length) that can cause the degradation of mRNAs or repress translation by binding to the transcripts of their target genes, of which approximately half encode transcription factors [22], and forming an RNA-induced silencing complex. As a gene family encoding transcription factors, more than half of the SBP-box genes identified to date have been found to be targeted by miR156/157. In rice, for instance, 11 of the 19 SBP-box genes have been revealed to be putative targets of OsmiR156 [21], while 10 of 15 SBP-box gene family members in tomato were found to carry putative miR156/157-response elements [23].
Unfortunately, despite an increasing body of physiological and biochemical data, the biological role of the SBP-box transcription factor-encoding gene family remains elusive. Although grapevine (V. vinifera) is one of the most important perennial fruit crops worldwide, there is little information concerning SBP-box gene, or the corresponding miR156/157, function in this species [24]. In the present study, we first systematically performed a genome-wide identification of SBPbox genes in the V. vinifera genome and then conducted further gene classification through an examination of exon-intron structure, gene phylogeny and synteny analysis. Since various types of stresses can cause significant losses in grape yield and reduce berry quality, we also endeavored to investigate expression patterns of grape SBP-box genes under various abiotic and biotic stresses, as well as in response to particular phytohormone treatments. This was carried out by both mining publicly available microarray datasets and through the examination of transcription levels in various grape organs and at different stages of fruit development. The results obtained from this study provide a foundation for additional evolutionary and functional characterization of SBP-box genes in plants, and further our understanding of stress management strategies utilized in grape.

Identification of Grape SBP-box Genes in the V. vinifera Genome
Previously, a subset of SBP-box genes were identified in grape; however, the results were incomplete and included a number of genes that lacked a complete SBP-box [24].Therefore, in an effort to identify the entire collection of putative SBP-box genes in the grape genome, we searched the GenBank non-redundant protein database, as well as the Grape Genome Database (12X) (http:// www.genoscope.cns.fr), with a profile Hidden Markov Model (pHMM) of the SBP domain(PF03110). Following the removal of redundant sequences, we identified 20 putative protein sequences. However, two of these sequences (GSVIVT01020050001 and GSVIVT01020051001) contained no zinc-finger motif and were thus excluded from further analysis. Therefore, we determined that at least 18 putative SBP-box genes were present in the V. vinifera genome, which we termed VvSBP1 to VvSBP18 (to maintain simplicity we also used this nomenclature when referring to orthologs in other grape species) based on their chromosomal order. In this study, to determine or validate the predicted exonintron structures,the complete open reading frames of all the grape SBP genes were isolated from cDNA of V. quinquagularis 'Shang-24' with gene-specific primers (Table S1, Fig. S1). Amplified products were sequenced and corroborated the expression of all predicted grape SBP genes. Moreover, the sequences of SBP genes amplified were consistent with those published in GenBank.
In addition, in accordance with findings in other species, we also found that 12 of the 18 grape SBP genes identified in this study contained sequences that were complementary to miR156/157, with a maximum of one to three mismatches to the mature VvmiR156/157 sequences (Fig. 1).

Phylogenetic Analysis of SBP-domain Family Genes
In an effort to gain further insight into the evolutionary relationship between SBP-box genes in various species of plants, we selected 128 SBP-box genes from nine species, including seven from the green alga C. reinhardtii, fourteen from the moss P. patens, eighty from dicotyledonous angiosperms (Arabidopsis, Vitis spp., Solanum spp., A. majus and poplar), as well as twenty-seven from monocotyledonous angiosperms (rice and maize) (Table S2), and constructed a phylogenetic tree based on the encoded amino acid sequences of their highly conserved SBP domains (76 aa) using the neighbor-joining algorithm (Table S3). The resulting unrooted phylogenetic tree suggested that the plant SBP-domain family is evolutionarily diverse (Fig. 2), with the 128 plant SBP sequences tested classified into seven subgroups. Interestingly, the seven proteins from green alga were grouped into the same clade (Group 7), while those from land plants were grouped into the remaining clades (Group 1-Group 6).
As expected, SBP-domain proteins from grape generally exhibited closer relationships to SBP-like proteins from dicotyledonous angiosperms than to those from monocotyledonous angiosperms. These results suggest that while plant SBP-box genes may be derived from a common ancestor, a number of them may have undergone further differentiation in monocotyledon and dicotyledon lineages. Interestingly, the miR156/157-targeted SBPlike genes, including sequences from rice, Arabidopsis and grape, were distributed into only three of the subgroups (Groups 2, 3 and 4).

VvmiR156 Target Sites and Gene Structure of Grape SBP Genes
To compare the 18 grape SBP genes directly, a phylogenetic tree was constructed based on the full-length cDNAs (Fig. 3A) and their exon-intron structures were predicted (Fig. 3B). In general, the topology of the resulting phylogenetic tree was similar to that constructed with the 128 SBP-domain sequences from nine plant species (Fig. 2). In addition, the majority of grape SBP genes in the same group bore a similar number of exons, as well as length of open reading frames (ORFs). For example, all genes within Group 1 were made up of more than ten exons and 800 corresponding amino acid residues. Conversely, genes in Group4 all contained two exons and fewer than 210 corresponding amino acid residues, while genes in Groups2 and 3 had three and four exons, respectively, with the exception of VvSBP13, which contained seven exons. Interestingly, VvSBP6 exhibited differences in the two phylogenetic analyses, which was the sole sequence classified into Group 5 in the multi-species tree ( Fig. 2) but was clustered into Group 1 in the V. vinifera-specific tree (Fig. 3). However, it is noteworthy that the exon-intron structure of this gene was similar to that of other members of Group 1.
Furthermore, both the locations of miR156/157 target sites and composition of encoded SBP domains were compared in each of the grape SBP genes to gain further insight into their evolutionary relationship with one another. Of the twelve grape SBP genes containing a miR156/157 target site, Group 4 members (with the exception of VvSBP4) bore this site within their 39 UTRs (Fig. 3B), which is similar to AtSPL3, AtSPL4 and AtSPL5 in Arabidopsis. In addition, grape SBP proteins also contained a highly conserved SBP-domain bearing two zincbinding sites (zinc finger 1 and zinc finger 2) and a nuclear localization signal (NLS). All SBP-domain proteins in Groups 1-4, as well as Group 6, possessed a zinc finger 1 (CR4CR13HCR2H) and zinc finger2 (CR2CR3HR11CR6H) of the C2HCH type, while VvSBP6 contained a zinc finger 1 (CR4CR13HCR2C) of the C2HC2 type, as was also the case for other SBP-domain proteins within Group 5 (Fig. S2).

Expansion Patterns and Distribution of Grape SBP and miR156 Genes in the Grape Genome
According to available annotation information, the 18 grape SBP genes were dispersed on all grape chromosomes except for chromosomes 2, 3, 6, 9, 13 and 16. Three grape SBP genes were present on chromosome 1, two on chromosomes 5, 15 and 17, respectively, and one on each of the remaining chromosomes. In addition, the nine VvmiR156 genes were found to be distributed on 6 of the 19 grape chromosomes: two were present on chromosomes 4, 11 and 14, respectively, and one was present on chromosomes 12, 17 and 19, respectively. Interestingly, as was the case for the grape SBP genes, none of the VvmiR156/157 genes were present on chromosomes 2, 3, 6, 9, 13 or 16 (Fig. 4).
Moreover, we also examined duplicated blocks within the grape genome and found that nine grape SBP genes (VvSBP2/VvSBP15, VvSBP3/VvSBP12, VvSBP5/VvSBP7, and VvSBP9/VvSBP11/ VvSBP18) were located in six pairs of duplicated genomic regions (Fig. 4, Table S4). In addition, according to their chromosomal positions, theVvSBP13/VvSBP14 genes were present as a single tandem duplication.

Evolutionary Relationship between SBP-box Family Genes of Grape and Arabidopsis
Genomic comparison is a relatively rapid method to transfer genomic knowledge acquired in one taxon to another that has been less well-studied. However, the degree to which genome synteny can facilitate cross-species analyses of gene function depends upon both the conservation of gene order and content, as well as the frequency with which similar traits have a common genetic basis in different species [25]. Since Arabidopsis is an important model plant species and the functions of most Arabidopsis SBP-box genes have been well-characterized, we analyzed a comparative synteny map between grape and Arabidopsis genomes in order to provide further insight into the functions of grape SBP-box genes. With regards to grape to Arabidopsis SBPbox gene correspondences, the syntenies were unambiguous and included the following ortholog pairs: VvSBP1-AtSPL6; VvSBP2-AtSPL11; VvSBP6-AtSPL7; VvSBP7-AtSPL1; VvSBP8-AtSPL9,15; VvSBP9-AtSPL3; VvSBP11,18-AtSPL4,5; VvSBP12-AtSPL13; and VvSBP17-AtSPL14 (Fig. 5, Table S4).

Expression Profiles of Grape SBP-box Genes in Different Organs and Stages of Fruit Development
To increase our understanding of the function of SBP-box genes in grape development, we investigated their expression profiles in various organs and at different stages of fruit development in V. quinquagularis 'Shang-24' via semi-quantitative RT-PCR with transcript-specific primers (Table S5). In general, the expression patterns of the 18 SBP-box genes could be classified into two types according to the presence or lack, of a miR156/157 target site (Fig. 6). In the case of genes lacking a miR156/157 target site, including VvSBP4, VvSBP5, VvSBP6, VvSBP7, VvSBP14 and VvSBP17, there tended to be little or no variation inexpression in anyof the tissues tested. In contrast, genes containing a miR156/157 target site, includingVvSBP1, VvSBP2, VvSBP3, VvSBP8, VvSBP9, VvSBP10, VvSBP11, VvSBP12, VvSBP13, VvSBP15, VvSBP16 and VvSBP18, were expressed at relatively higher levels in leaves, stems and tendrils compared to the reproductive tissues analyzed. Furthermore, the majority of these 12 grape SBPbox genes with miR156/157 target sites also exhibited the highest levels of expression in the early stages of fruit development, which gradually decreased or even vanished during the fruit ripening process.

Expression Profiles of Grape SBP-box Genes in Response to Various Types of Stresses
Plants undergo continuous exposure to various biotic and abiotic stresses in their natural environment. To improve the ability of a plant to tolerate stresses and improve disease resistances, it is necessary to identify and functionally characterize the genes that are involved in biotic and abiotic stress signaling pathways. Unfortunately, previous studies of SBP-box genes have focused mainly on plant growth and development, with only a handful having been shown to play a role in stress responses. Therefore, in the present study, we investigated the responses of grape SBP-box genes to various abiotic and biotic stress conditions, as well as hormone treatments, by mining publicly available grape microarray datasets. A total of 16 experiments containing 359 hybridizations from grape genome arrays were obtained and subjected to manual curation, and 68 comparisons between numerous distinct experimental conditions were constructed. We identified 11 grape SBP-box transcripts corresponding to 12 probe sets on the array, including VvSBP2, VvSBP3, VvSBP4, VvSBP5, VvSBP6, VvSBP7, VvSBP8, VvSBP10, VvSBP11, VvSBP15 and VvSBP17. A heat map representation of the expression profiles of these genes is shown in Figure 7, which reveals that several of the grape SBP-box genes were highly responsive to each of the different stresses applied individually.
Following cold treatment (5uC), two genes (VvSBP3 and VvSBP5) demonstrated increased expression, while two (VvSBP4 and VvSBP7) exhibited decreased expression (Table S6). In contrast, following heat stress, we found that none of the grape SBP-box genes displayed any significant changes in expression levels (Table  S6).
To further investigate the responses of the grape SBP-box genes to abiotic stresses, we performed quantitative real-time RT-PCR (qRT-PCR) assays to test the expression of all 18 SBP-box genes in the leaves of Chinese wild V. quinquangularis clone 'Shang-24' upon short-term salinity (1-48 h) treatment (Fig. 8). Genes with expression levels altered by more than two-fold were considered for subsequent analyses. The results obtained showed that two genes (VvSBP13 and VvSBP18) were strongly induced (over fourfold), and four genes (VvSBP4, VvSBP8, VvSBP10 and VvSBP17) were also moderately up-regulated (over two-fold) in response to short-term salinity stress. In contrast, three genes (VvSBP9, VvSBP14 and VvSBP16) were significantly down-regulated (over four-fold) and two genes (VvSBP3 and VvSBP12) were moderately down-regulated (over two-fold) by this same treatment. The results obtained with qRT-PCR here were largely consistent with the array results.
Developmental and environmental cues. Photoperiod influences several aspects of plant growth, and is an important environmental cue for synchronizing plant growth, flowering, and dormancy with changing seasons [26]. Intriguingly, we found that all 11 grape SBP-box genes on the array were induced during long photoperiods in both V. riparia and V. spp. 'Seyval', indicating that the expression of SBP-box genes could be regulated by photoperiod (Table S6).
Biotic stress. Diverse diseases, such as downy mildew, powdery mildew, Bois Noir phytoplasma, and those of viral origin, have a serious impact on grapevine productivity and fruit quality. Plasmopara viticola is the causal agent of downy mildew, which is one of the most catastrophic and baffling diseases of grapevine worldwide [27]. Our microarray data mining revealed that only VvSBP7 exhibited a decreased expression upon P. viticola infection in a line that was highly resistant to this disease (Rpv12/ Rpv2+). Conversely, in both partially resistant (Rpv1+/Rpv22) and susceptible (Rpv12/Rpv22) lines, none of the SBP-box genes displayed significant alterations in expression levels (Table S6).
Similarly, no significant changes in the expression levels of any of the 11 grape SBP-box genes analyzed were observed upon infection with powdery mildew in either the disease-resistant V. aestivalis 'Norton' or the disease-susceptible V. vinifera 'Cabernet sauvignon'. In contrast, the expression of VvSBP3 was significantly decreased after infection with Bois Noir phytoplasma in the grape cultivar 'Manzoni', which is moderately resistant to this disease, while in the highly susceptible 'Chardonnay', both VvSBP4 and VvSBP17 were significantly up-regulated and one gene (VvSBP11) was down-regulated. Lastly, the expression of VvSBP5, VvSBP6, VvSBP7, VvSBP10 and VvSBP17 were all up-regulated, while VvSBP4 was down-regulated, in ripening berries of V. vinifera cv. 'Cabernet Sauvignon' when infected with leaf roll-associated closeterovirus-3 (GLRaV-3), which is one of the most widespread viruses among the more than 40 different viruses known to infect grapevines [28]. However, none of the grape SBP-box genes showed any significant changes in expression in response to GLRaV-3 at veraison (Table S6).
Hormone treatment. Plant hormones such as salicylic acid (SA), jasmonates (JA), ethylene (ET) and abscisic acid (ABA) play important roles in regulating developmental processes and signaling networks involved in plant responses to a wide range of biotic and abiotic stresses [29]. Analysis of expression data from red-skinned 'Crimson Seedless' grape (V. vinifera) cell-suspension cultures [30] indicated that the majority of grape SBP-box genes analyzed here were differentially expressed upon both JA and methyljasmonate (MeJA) treatment, with the exception of VvSBP8, VvSBP11 and VvSBP15. Among the JA-responsive genes, two (VvSBP3 and VvSBP6) were up-regulated, while six (VvSBP2, VvSBP4, VvSBP5, VvSBP7, VvSBP10 and VvSBP17) were downregulated. In the skin of grape berries treated with exogenous ABA [31], which is known to play a central role in the response of plants to various types of abiotic stresses, four of the 11 SBP genes analyzed exhibited altered levels of expression. Among them, three (VvSBP4, VvSBP3 and VvSBP7) were up-regulated, while one (VvSBP6) was down-regulated (Table S6). To provide further insight into the responses of the grape SBPbox genes to signaling molecules, we carried out qRT-PCR assays to test the expression of all 18 SBP-box genes in the leaves of Chinese wild V. quinquangularis clone 'Shang-24' upon MeJA, ethylene (ET) and SA treatments (Fig. 9). Genes with expression levels altered by more than two-fold were considered for subsequent analyses.
Six hours following MeJA treatment, five genes (VvSBP1, VvSBP6, VvSBP9, VvSBP11 and VvSBP15) exhibited significant increases in expression, while eight genes (VvSBP2, VvSBP3, VvSBP4, VvSBP7, VvSBP13, VvSBP14, VvSBP16, VvSBP17 and VvSBP18) displayed significant decreases in their expression levels (Fig. 9B). The results obtained were consistent with the array results in that VvSBP2, VvSBP3, VvSBP4, VvSBP7 and VvSBP17 were significantly down-regulated by MeJA, and VvSBP6 was moderately up-regulated by this same hormone, while the expression of the remaining three genes (VvSBP5, VvSBP8 and VvSBP10) did not appear to be obviously altered by MeJA treatment.
With the exception of six genes (VvSBP1, VvSBP2, VvSBP5, VvSBP7, VvSBP8 and VvSBP14) that did not exhibit any significant alterations in expression levels in response to SA, the expression levels of the remaining 12 grape SBP-box genes were significantly modified by this hormone (more than a two-fold increase or decrease). Of these, six genes (VvSBP4, VvSBP6, VvSBP10, VvSBP11, VvSBP15 and VvSBP17) were up-regulated and six (VvSBP3, VvSBP9, VvSBP12, VvSBP13, VvSBP16 and VvSBP18) were down-regulated (Fig. 9A).

SBP-box Genes
It has been proposed that SBP-box genes encode a family of plant-specific transcription factors [1] as no SBP-box homologs have been identified in other kingdoms to date. Phylogenetic analyses demonstrated that all SBP-box genes from land plants were clustered into six groups (Groups 1-6), while the seven SBPlike genes identified from green alga fell into their own separate group (Group7, Fig. 2), which was consistent with previous findings [5]. These results imply that SBP-box genes arose after the divergence of plants and animals, but before the divergence of green algae from the last common ancestor of land plants.
Duplication at both the gene and genomic levels is one of the primary driving forces of genomic variation and gene family expansion [32,33], contributing to the origin of biological novelty during evolution [34]. Gene duplications in angiosperms have been reported in many transcription factor families, such as the AP2, MADS, and DOF families [35][36][37], and two tandem SBPbox gene duplications have been reported in Arabidopsis (AtS-PL13A/AtSPL13B and AtSPL10/AtSPL11) [38]. In the present study, we also identified a tandem duplication in the grape SBP  genes (VvSBP13/VvSBP14, Fig. 4). Moreover, several segmental duplications of SBP-box gene pairs in Arabidopsis (AtSPL10/ AtSPL11, AtSPL4/AtSPL5, and AtSPL1/AtSPL12) and rice (OsSBP10/OsSBP5, OsSBP11/OsSBP4, and OsSBP12/OsSBP3) have been identified [39][40][41][42]. Similarly, an examination of duplicated blocks within the grape genome indicated that nine of the grape SBP-box genes (VvSBP2/VvSBP15, VvSBP3/VvSBP12, VvSBP5/VvSBP7, and VvSBP9/VvSBP11/VvSBP18) were located in six pairs of duplicated genomic regions (Fig. 4, Table S4). Taken together, our results demonstrate that both segmental and tandem duplications have played important roles in the expansion of the grape SBP-box gene family during their evolution.
Comparative genomic analysis across different taxa allows the transfer of functional information from a taxon for which there is a better understanding of genome structure, function and/or evolution to another less well-studied taxon [43]. In this study, we  Table S6. Log2-based fold changes were used to create the heat map. Differences in gene expression are shown in color according to the scale. doi:10.1371/journal.pone.0059358.g007 Figure 8. Expression levels of grape SBP-box genes following short-term salinity treatment in the leaves of V. quinquangularis 'Shang-24'. Grape Actin1 was used as an internal control for qRT-PCR and fold changes were used to indicate expression levels in treated leaves compared to negative controls, which were set to 1 or 21 depending on the expression trends. Mean values and SDs were obtained from three technical and three biological replicates. doi:10.1371/journal.pone.0059358.g008 found that the majority of grape and Arabidopsis SBP-box genes were located in syntenic regions of the two genomes (Fig. 5, Table  S4). To date, several important and divergent biological processes regulated by SBP-box genes have been reported in Arabidopsis, including sporogenesis [6], leaf development [44], vegetative and reproductive phase transitions [4], response to copper and fungal toxins [15,45] and plant hormone signaling [14]. Together with our expression data, these results will help infer the probable functions of grape SBP-box genes.

Expression Profiles of Grape SBP-box Genes and their Potential Functions in Diverse Grape Tissues and Developmental Stages
SBP-box genes have been identified in numerous plants including green algae, moss, silver birch, snapdragon, Arabidopsis, rice, maize and tomato. To date, these genes have been found to play critical roles in regulating flower and fruit development, as well as various other physiological processes; however, their roles in grapevine have remained unclear. Therefore, in this study, we predicted the functions of the grape SBP-box genes in both vegetative and reproductive growth phases based on their Arabidopsis counterparts in syntenic regions of the two genomes (Fig. 5) and homology to genes of other species.
In general, semi-quantitative RT-PCR results demonstrated that the grape SBP-box genes exhibited distinct expression patterns. However, four pairs of grape SBP-box genes (VvSBP2/ VvSBP15, VvSBP3/VvSBP12, VvSBP5/VvSBP7, and VvSBP11/ VvSBP18) sharing very high sequence and exon-intron structure similarity in duplicated genomic regions (Fig. 3), also showed similar expression patterns (Fig. 6). Although VvSBP11 exhibited a higher level of expression than VvSBP18 in most grape tissues and organs, both of these Group 4 genes had elevated expression levels in inflorescences and during the early stages of fruit development, which then gradually decreased during veraison (Fig. 6). These results suggest that these two genes may play a role in flower and fruit development. This assumption is corroborated by the fact that the Arabidopsis AtSPL5 gene, which is the ortholog of VvSBP11/VvSBP18, was reported to be involved in the flowering process. In addition, the snapdragon gene AmSBP1, as well as the Arabidopsis genes AtSPL3 and AtSPL4, were also clustered into Group 4 in our phylogenetic analysis. Intriguingly, AmSBP1 andAtSPL3 have also been reported to bind cis-elements in the promoters of the floral organ identity genes SQUA and AP1, respectively [2], and AtSPL3/4/5, as well as AmSBP1, have all been implicated in the vegetative phase change and floral induction [2,4,10].
Interestingly, grapeVvSBP2 and VvSBP15, which are located in duplicated genomic regions, were not classified into the same subgroup in our phylogenetic analysis (Fig. 2). However, they did exhibit similar expression patterns in the various grape developmental phases tested. For example, the expression of these two genes was elevated in vegetative organs (leaves, stems and tendrils), and gradually diminished throughout the fruit maturation process (Fig. 6). It is worth noting that the expression of VvSBP15 and VvSBP2 waned completely in fruits at 50 and 80 days after flowering, respectively. These findings suggest that these two genes may play a regulatory role in leaf initiation and during different stages of grape fruit ripening. In addition, grape VvSBP8 and VvSBP13, which were distributed into the same group as VvSBP2 (Group 3), both shared similar trends during the fruit maturation process. Therefore, we speculate that the Group 3 grape SBP-box genes might function in both vegetative and reproductive stages, a hypothesis that is further supported by previous studies. For instance, AtSPL9 and AtSPL15, which are orthologs of grape VvSBP8, are quite active in the vegetative shoot apex and play a role in the juvenile-to-adult phase transition [11]. In addition, other members of Group 3 have also been implicated in both vegetative and reproductive processes. Indeed, AtSPL10/ 11/2 are involved in the development of lateral organs, shape of cauline leaves and number of trichomes on cauline leaves and flowers [46], while OsSPL14 promotes panicle branching in the vegetative stage and increased rice crop yield in the reproductive stage [47]. In addition, ZmSBP6 (tasselsheath4) has been found to be involved in inflorescence development in maize while an SBP-box gene from tomato (CNR) plays a role in fruit ripening [18,19].
In contrast, the Group 2 grape genes VvSBP3 and VvSBP12 were expressed in all organs analyzed and grape members of this group were homologous to AtSPL13, teosinte glume architecture 1 (tga1) and OsSPL6. The Arabidopsis AtSPL13 gene has been shown to affect the initiation of the first true leaves [44], maize tga1is involved in ear glume development [48], and OsSPL16 controls grain size, shape and quality in rice [49]. Therefore, we hypothesize that the grape Group 2 SBP-box genes may provide functions in controlling characteristics of leaf and/or fruit.
The three groups of SBP-box genes discussed above (Groups 2, 3, and 4), with the exception of SBP4, all contain a miR156/157 target site. This miRNA family is highly conserved in plants, with homologs having been identified in a large number of angiosperms, ferns, lycopods and mosses [50][51][52]. Previously, nine members of the miR156/157 family, termed VvmiR156a to VvmiR156i, which are highly conserved in plants and are thought to interact with numerous SBP-box genes, were identified in the V. vinifera genome [53][54][55][56]. To date, miR156/157 target sites were found in 10 Arabidopsis [57], 11 rice [21] and 10 tomato [23] SBPbox genes. Indeed, 12 of 18 grape SBP-box genes contained a miR156/157 target site in the V. vinifera genome (Fig. 1). In most cases, miR156/157-regulated SBP-box genes tend to play a role in the control of phase change and reproductive development [58,59]. It has been shown that the grape VvmiR156f gene is expressed at the highest levels in mature berries, followed by inflorescences, ripening berries, green berries and leaves [53]. Our experimental results, which were consistent with the expression patterns of miR156/157-targeted genes in tomato and rice [21,23], indicated that the miR156/157-targeted grape SBP-box genes were generally expressed in a similar fashion to the VvmiR156f gene in all tissues tested (Fig. 6). This provides yet another example of the mutual relationship between miR156/157 and SBP-box genes.
In contrast to the grape SBP-box genes discussed above, Group 1 and Group 5 genes did not contain a miR156/157 target site and were all expressed ubiquitously and constitutively, with little or no variation in any of the tissues analyzed (Fig. 6). These results indicate that grape genes from these two groups may have functions that are distinct from the miR156/157-targeted SBP-box genes in Groups 2, 3 and 4. Figure 9. Expression levels of grape SBP-box genes following SA(A), MeJA(B), and ET (C) treatment in the leaves of V. quinquangularis 'Shang-24'. Grape Actin1 was used as an internal control for qRT-PCR and fold changes were used to indicate expression levels in treated leaves compared to negative controls, which were set to 1 or 21 depending on the expression trends. Mean values and SDs were obtained from three technical and three biological replicates. doi:10.1371/journal.pone.0059358.g009 Grape SBP-box Genes are Responsive to Abiotic and Biotic Stresses Transcriptional control of stress-responsive genes is a crucial means by which plants respond to a range of abiotic and biotic stresses and research carried out in recent years has been productive in identifying transcription factors that are important for regulating these types of responses [60]. To date, several important and divergent biological processes regulated by SBPbox genes have been reported; however, only a small number of these genes have been shown to play a role in the response to stresses. For example, in Arabidopsis, the expression of an SBP-box gene has been found to be responsive to various types of biotic and abiotic stresses through interactions with genes involved in the defense response pathway [61]. In addition, AtSPL14 has been found to be involved in programmed cell death and plays a role in sensitivity to fumonisin B1 [45]. In grape, we found that VvSBP17, which is the orthologof AtSPL14, was up-regulated after infection with Bois Noir in the susceptible V. vinifera cultivar 'Chardonnay'. Furthermore, grape VvSBP7 exhibited down-regulated expression upon P. viticola infection in a highly resistant line. Similarly, VvSBP17, VvSBP7 and VvSBP5 all exhibited significant increases in expression levels following infection with GLRaV-3 in ripening berries. Intriguingly, these three genes all belonged to the same subgroup (Group 1), which insinuates that the Group 1 grape SBPbox genes may play a key role in the response to pathogen infection in grape. Moreover, our microarray data mining revealed that both VvSBP4 and VvSBP11 from Group 4, as well as VvSBP3 from Group 2, also displayed significant alterations in expression levels after infection with pathogens (Fig. 7, Table S6).
Among the diverse grape SBP-domain proteins, the SBP domain of VvSBP6 showed the highest similarity to that of AtSPL7 from Arabidopsis, which regulates Cu deficiency responses [15]. Moreover, the SBP-box gene, Cu response regulator 1 (Crr1), from C. reinhardtii fulfills a very similar role [20]. Therefore, it is possible that VvSBP6, which is the most likely AtSPL7 ortholog and single grape representative in Group 5, is also an important regulator of copper homeostasis in this species. In addition, VvSBP6 also exhibited responsiveness to abiotic stress, being upregulated by drought, PEG treatment and salinity (Fig. 7, Table  S6).
The existence of multiple defense strategies and complex signaling networks in plants has led to their enhanced defense capacity. Induced defense responses are regulated by a network of interconnected signal transduction pathways in which the hormonal signals SA, JA, ET and ABA, can coordinately activate the transcription of various defense-related genes [62]. In this sense, these plant hormones provide a critical function in signaling networks involved in plant responses to a wide range of biotic and abiotic stresses. To date, the only report of an SBP-box gene exhibiting responsiveness to biotic stress signaling hormones has come from AtSPL2, which was found to be repressed in transgenic Arabidopsis overexpressing the JASMONATE CARBOXYL METHYLTRANSFERASE gene (AtJMT) [63]. In this study, we determined that the expression of the majority of grape SBP-box genes was significantly modified by SA, MeJA and/or ET treatment (Fig. 8, Table S6). It is worth noting that grape SBP genes responsive to pathogen or virus infection (VvSBP3, VvSBP4, VvSBP5, VvSBP6, VvSBP7, VvSBP10, VvSBP11 and VvSBP17) were also responsive to at least one of the three hormone treatments. SA, JA and ET have been reported to be involved in the defense not only during pathogen infection but also during salt stress. The ameliorative effects of SA to salt tolerance have been well documented in many crops such as bean [64], mung bean [65] and mustard [66]. JA can also activate plant defense mechanisms and provide protection to salinity stress [67][68][69][70][71]. A recent study suggests that ethylene signaling may be required for salt tolerance and promote salt tolerance in Arabidopsis [72,73]. This study suggests that grape SBP genes may be involved in the response to salt stress dependent on MeJA and ET molecular signals. However, their roles in salt stress are equivocal. Furthermore, ABA is extensively involved in responses to abiotic stress, such as water-deficit, cold and osmotic stress [74], and also acts as a negative regulator of disease resistance in many cases [75]. In our study, five of the 11 grape SBP-box genes analyzed were also found to be regulated by ABA (Fig. 7, Table S6). Taken together, our analyses indicate that SBP-box genes have a variety of functions in grape and play important roles in both biotic and abiotic stresses, possibly dependent on SA, MeJA, ET and ABA molecular signals.

Identification and Annotation of Grape SBP-box Genes
To identify members of the SBP-box gene family in grape, previously identified Arabidopsis SBP sequences were first submitted to the Pfam database (http://pfam.sanger.ac.uk) [76] to obtain the domain architecture of this family. SBP motifs were found to be represented by Pfam accession number PF03110. Searches for each domain within the Grape Genome Database (12X; http:// www.genoscope.cns.fr) were performed using HMMER [77] with an E-value of ,1e 25 . To confirm results obtained using the HMMER algorithm, protein motifs were also queried against the Pfam database.

Sequence Alignments and Phylogenetic Analyses
Multiple alignments of SBP-domain protein sequences derived from 128 SBP-box genes from 9 plant species (Table S2) were performed using the ClustalW program [78]. Phylogenetic trees were constructed using the MEGA 4.0 software and the maximum parsimony (MP) method, with a bootstrap test that was replicated 1000 times [79].

Exon/Intron Structure Analysis of Grape SBP-box Genes
The exon/intron structures of the grape SBP genes were determined from alignments of their coding sequences with corresponding genomic sequences using the est2genome program [80]. A diagram of exon/intron structures was obtained using the online Gene Structure Display Server (GSDS: http://gsds.cbi.pku. edu.ch), which depicts both exon position and gene length.

Tandem Duplication and Synteny Analysis
Tandem duplications of SBP genes in the V. vinifera genome were predicted by determining their physical locations on individual chromosomes. Tandemly duplicated genes were defined as adjacent homologous genes on a single chromosome, with no more than one intervening gene. For synteny analysis, syntenic blocks within the V. vinifera genome, as well as between the grape and Arabidopsis genomes, were downloaded from the Plant Genome Duplication Database [81] and those containing grape and Arabipidopsis SBP-box genes were identified.

Expression Analysis of Grape SBP-box Genes
Affymetrix grape microarray data were downloaded from ArrayExpress [82] and PLEXdb [83] databases. A total of 16 experiments were used for our gene expression analyses (Table  S6). For each microarray experiment, the methods utilized for normalization and to adjust background, as well as detection calls, P-value calculation and adjustment have been described pre-viously [84]. Genes with adjusted p-values (FDR) less than 0.05 were considered to be differentially expressed genes. Hierarchical clustering of the expression profiles of the grape SBP-box genes was performed using dChip [85].

Plant Material and Treatments
Grape tissues, including leaf, stem, tendril, inflorescence, and fruit at 20, 35, 50, 65, 80 and 95 days after flowering (daf), were obtained from V. quinquangularis 'Shang-24', which had been maintained in the grape germplasm resource orchard of Northwest A&F University, Yangling, China (34u209, 108u249E). Grape seedlings were grown in pots under greenhouse conditions. When shoots of vines were 25-35 cm in length, the third to fifth fully expanded young grapevine leaves beneath the apex were selected for SA, MeJA and ET treatments. Hormone treatments were conducted by spraying leaves with 100 mM SA, 0.5 g/L ET or 50 mM MeJA followed by sampling at 0, 0.5, 1, 3, 6, 12, 24 and 48 h post-treatment as described previously [86]. Grape leaves sprayed with sterile water were collected as the control. Salinity treatments were carried out by irrigating with 2 dm 3 250 mMNaCl. Grapevine leaves were sampled at 0, 1, 3, 6, 12, 24 and 48 h post-treatment. Leaves of grape irrigating with water were collected as the control.

Isolation of Complete Open Reading Frames of Grape SBP Genes
For each SBP gene, a pair of gene-specific primers (Table S1) were designed to amplify the predicted ORF with cDNA templates prepared from leaves of V. quinquangularis 'Shang-24'. Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China) was used to amplify the grape SBP genes with the following cycling profile: 94uC for 3 min, 25 to 30 cycles at 94uC for 30 s, 58uC for 30 s and 72uC for 2 or 3 min; and extension at 72uC for 10 min. The amplified products were cloned into pGEM-Teasy vector (Promega, Madison, WI, USA) and transformed into E.coli strain DH5a. The positive clones, characterized by blue/white screening, were sequenced at TaKaRa Biotechnology.

Quantitative Real-time RT-PCR Analysis
Total RNA was extracted from the leaves of V. quinquangularis 'Shang-24' using an improved SDS/phenol method described previously [87]. Residual DNA was removed via treatment with DNaseI (Promega, Madison, WI, USA). RNA purity was ascertained by determining the A 260 /A 280 ratio, and RNA integrity was examined through electrophoresis on a 1% agarose gel. Concentrations of total RNA were measured using an ultraviolet spectrophotometer (V-550, JASCO, Japan) at 260 nm. First-strand cDNA synthesis was carried out using 1 mg DNase-treated total RNA and a mixture of Poly dT and random hexamer primers (PrimeScript TM RTase, TaKaRa Biotechnology, Dalian, China). Gene-specific primers were designed for all 18 grape SBP-box genes (Table S5). Quantitative RT-PCR was conducted using SYBR green (Takara Biotechnology) on an IQ5 real time PCR machine (Bio-Rad, Hercules, CA, USA). Each reaction was done in triplicates with a volume of 25 ml. Cycling parameters were 95uC for 30 s, 40 cycles of 95uC for 5 s, and 60uC for 30 s. For dissociation curve analysis, a program including 95uC for 15 s, followed by a constant increase from 60uC to 95uC, was included after the PCR cycles. The grape Actin1 (GenBank Accession number AY680701) was amplified with primers F (59-GAT TCT GGT GAT GGT GTG AGT-39) and R (59-GAC AAT TTC CCG TTC AGC AGT-39) as an internal control. Relative expression levels were analyzed using the IQ5 software and the normalized-expression method. A one-sided paired t-test was performed using Sigma Plot 11.0 (Ashburn, VA, USA) to assess significant differences between the control and each treatment.

Semi-quantitative RT-PCR Analysis
Total RNAs were extracted from leaf, stem, tendril, inflorescence, and fruit at 20, 35, 50, 65, 80 and 95 daf of V. quinquangularis 'Shang-24' as described above. Grape SBP and Actin1 specific primer sequences were the same as those used for qRT-PCR. Cycling parameters were as follows: 94uC for 3 min, 25 cycles at 94uC for 30 s, 60uC for 30 s and 72uC for 30 s, with a final elongation step at 72uC for 10 min. PCR products were subsequently separated on a 1.2% (w/v) agarose gel, then stained with ethidium bromide and photographed under UV light.