Gene cloning and expression analyses of WBC genes in the developing grapevine seeds

TANG Yu-jin, WANG Qian, XUE Jing-yi, LI Yan, , LI Rui-min, Steve Van Nocker, WANG Yue-jin,ZHANG Chao-hong

1 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northwest Region), Ministry of Agriculture/State Key Laboratory of Crop Stress Biology for Arid Areas/College of Horticulture, Northwest A&F University, Yangling 712100, P.R.China

2 College of Life Sciences, Northwest A&F University, Yangling 712100, P.R.China

3 Department of Horticulture, Michigan State University, East Lansing 48824, USA

1. Introduction

Wine (Vitis vinifera) and table grapevines (Vitishybrids) are among the most popular and extensively cultivated fruit in the world, with 2015 production of over 7.5×106ha (http://www.oi v.int/public/medias/4587/oiv-noteconjmars2016-en.pdf). Seedless grapevines are preferred by consumers and are classified into two types based on pollination and fruit setting characteristics: parthenocarpy and stenospermocarpy. Parthenocarpy is that the ovules develop into fruit naturally or artificially without fertilization(V. vinifera cv. Concord seedless,V. vinifera cv. Black Corinto,V. vinifera cv. Sultana andV. vinifera cv. Black Corinth). Stenospermocarpic types exhibit normal floral structure (Wang and Horiuchi 1990) and while pollination and fertilization occur normally, fertilized embryos exhibit various degrees of ovule abortion, leading to little to no perceptible trace of seed (Halbrooks and Mortensen 1988) (V.vinifera cv. Delight,V.vinifera cv. Thompson Seedless,V.vinifera cv. Monukka,V.vinifera cv. Autumn Seedless andV.vinifera cv. Flame Seedless). In addition, stenospermocarpic grapevines are valuable research tools because their ovule abortion trait is completely hereditary and is not influenced by various environmental factors (Ramnninget al. 2000).However, our knowledge about the seedless mechanism of stenospermocarpic grapevines is still limited.

Transport of nutrients, metabolic compounds and signaling molecules across membranes is essential for cellular function. The ATP-binding cassette (ABC)transporters are responsible for import and export of substrates across the membrane in an ATP dependent manner (Nuruzzamanet al. 2014; Andolfoet al. 2015).ABC family is one of the largest and oldest protein families discovered so far, and is found widely in archaea, bacteria and eukaryotes (Kimet al. 2013; Andolfoet al. 2015; Zhaet al. 2015). The typical ABC transporter contains both transmembrane domains (TMD) and ATP-binding domains,which are also called nucleotide-binding domains (NBD)(Zhaet al. 2015). The structures of TMDs are generally not conserved (Smartand Fleming 1996; Van and Tampe 2004; Rea 2007). In contrast, NBDs contain six highly conserved motifs, the ABC signature motif (otherwise known as C motif), Walker A, Walker B, H-loop, D-loop, and Q-loop(Higgins and Linton 2004). Full-size ABC transporters contain two NBDs and two TMDs and can function as monomers, whereas half-size ABC transporters function as hetero- or homo-dimers (Tarret al. 2009). In plants, ABC transporters were classified into nine subfamilies based on their domain organization (Hwanget al. 2016). The ABCG subfamily is the largest family in plants (Crouzetet al.2006)and they are a kind of ‘reverse’ ABC transporters with NBDs in front of TMDs (Verrieret al. 2008). ABCG subfamily contains white-brown complex (WBC) and pleiotropicdrug resistance (PDR) transporters. WBC tranporters, also called half-sized ABCGs, have only one NBD and one TMD while the PDR tranporters (full-sized ABCGs) have two NBDs and TMDs (Verrieret al. 2008; Hwanget al. 2016). Evidence has emerged that different WBCs can dimerize in different combinations to transport similar but distinct substrates,providing a potential mechanism for their relatively broad substrate specificity (McFarlaneet al. 2010).

The ABCG transporters are important for plants development and have been studied extensively. Some of them were found to transport phytohormone (Kuromoriet al.2010; Kretzschmaret al. 2012; Zhanget al.2014; Xieet al.2015): Many ABCG transporters also participated in pollen development (Kuromoriet al. 2010; Quilichiniet al. 2010; Xuet al. 2010; Choiet al. 2011; Douet al. 2011; Kuromoriet al.2011a; Zhuet al. 2013; Yimet al.2016; Zhaoet al. 2016),and the formation of hydrophobic and protective extracellular barriers, including lignin, suberin, sporopollenin, cutin, and waxes (Pighinet al.2004; Birdet al. 2007; Luoet al. 2007;Panikashviliet al.2007, 2010, 2011; Ukitsu 2007; Samuelset al. 2008; McFarlaneet al. 2010; Chenet al. 2011; Budaet al. 2013; Liet al. 2013; Fabreet al. 2016; Hwanget al.2016). Many ABCG transporters may play an essential role in resistance to biotic and abiotic stresses. (Kuromoriet al.2011b; Ruoccoet al. 2011; Banasiaket al. 2013; Crouzetet al. 2013; Muhovskiet al. 2014; Ezakiet al. 2015; Shibataet al. 2016).

Whole-genome sequencing ofgrapevineprovides the opportunity to identify and characterize members of theVvWBCgene family. However, our current understanding of these transmembrane transporters in grapevine is still limited. To date, only a whole-genome survey of ABC transporter family has been performed in grapevine (Cakir and Kilickaya 2013). To reveal the molecular characteristics ofVvWBCs and the connection betweenVvWBCs and the seedless trait of stenospermocarpic grapevines, we identified 30VvWBCgenes and analyzed these using bioinformatics approaches. We also cloned 20 full-length complementary DNAs (cDNAs) fromV.vinifera cv. Pinot Noir andV. vinifera cv. Thompson Seedless. The expression profiles ofVvWBCs were analyzedviaquantitative RT-PCR(qRT-PCR) and semi-quantitative RT-PCR (sqRT-PCR)technologies. Our findings would provide insight into the connection between stenospermocarpic grapevines and theVvWBCgenes and lay a foundation for further researches of the agronomic traits of stenospermocarpic grapevines.

2. Materials and methods

2.1. Plant materials

V.vinifera cv. Thompson Seedless, also known as Sultanina or white Kishmish, is the main source of stenospermocarpic seedlessness in table grape breeding programs (Adam-Blondonet al. 2001).V.vinifera cv. Pinot Noir, one of the world’s great red wine varieties, is a seeded grape cultivar.Thompson Seedless and Pinot Noir plants were maintained in the Grape Germplasm Repository of Northwest A&F University in Yangling, Shaanxi, China, under natural environmental conditions and standard management procedures. Embryos at 10, 15, 20, 25, 30, 35, 40, and 45 days after full-bloom (DAF) were dissected and stored using methods described previously (Zhanget al.2013).Leaf, root, flower, stem, pericarp, floral bud, and tendril tissues were sampled from Pinot Noir and treated as described above.

2.2. ldentification and isolation of grapevine WBCs

All 28AtWBCopen reading frame (ORF) translations from theArabidopsisInformation Resource (http://www.arabidopsis.org/) were utilized as queries to search for grapevineWBCgenesin GenBank (https://www.ncbi.nlm.nih.gov/genbank/)using the BLASTP Program, and all hits with anE＜0.01 were collected. Subsequently, we employed the obtained sequences to scan the Grape Genome Database (http://www.genoscope.cns.fr/externe/Genome Browser/Vitis/) with 12x sequence coverage to confirm whether the sequences were redundant. Next, we queried the NCBI (https://www.ncbi.nlm.nih.gov/) with BLASTP Program to ensure that the sequences contained the ABC signature motif, Walker A, Walker B, H-loop, D-loop, and Q-loop conserved motifs.The ORFs were identified using DNASTAR Software.Primers for amplifying genes were designed using Primer Premier 6.0 (Appendix A). An improved SDS-based method(Zhanget al.2003) was employed to isolate total RNA from ovules at each stage in two grapevine cultivars and from the leaf, root, flower, stem, pericarp, floral bud, and tendril tissues of Pinot Noir. Synthesis of first-strand cDNAs and the cloning of full-length cDNAs were conducted with methods described previously (Liet al.2015). ProtParam (http://web.expasy.org/protp aram/) was used to calculate various parameters of genes, including the molecular weight (MW)and isoelectric point (pI).

2.3. Structures and positions of WBCs in grapevine chromosomes

The genomic DNA sequences and chromosomal locations of theWBCgenes were obtained through BLAST searches of each sequence against a draft grapevine genome(http://www.Genoscope.cns.fr/externe/GenomeBrowser/Vitis/). The intron/exon organizations and predicted protein locations of theWBCgenes were analyzedviaFGENESH-C (http://linux1.softberry.com/berryphtml?top ic=fgenes_c&group=programs&subgroup=gfs).

2.4. Phylogenetic analysis and multiple sequence alignments of grapevine WBCs

Multiple sequence alignment analysis was performed using ClustalX2 Software (Dublin University, Ireland)(alignment parameters are as follows: gap opening=10,gap extension=0.2, delay divergent sequences=30%, and DNA transition weight=0.5). DNAMAN Software (Lynnon Biosoft, USA) was employed to analyze sequence similarity.MEGA 6.0 Software (Tamuraet al. 2013) was used to build a phylogenetic tree using minimum-evolution method with the LG with frequencies (Freqs.)+Gamma Distributed Model(LG+F+G), bootstrap method and 1 000 replications.

2.5. Synteny analysis of grapevine WBCs

We downloaded synteny blocks between the grapevine andArabidopsisand within the grapevine genome from the Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication) and identified those syntenic blocks that includedWBCs. Visualization of blocks was performed using Circos Software (Krzywinskiet al.2009).

2.6. sqRT-PCR expression analysis of VvWBCs

Gene-specific primer pairs were designed forVvWBCs(Appendix B). The cDNAs from leaf, root, flower, stem,pericarp, floral bud, tendril andthe mixed cDNAs of ovules at eight developmental stages of Pinot Noir were used as templates. sqRT-PCR analysis was performed using a previously described method with a slight modification (Liet al.2015). The reaction system mixture (20 μL) consisted of 10 μL 2×EsTaqMasterMix (Dye) (Beijing ComWin Biotech Co., Ltd., China), 1.0 μL cDNA templates, 0.8 μL of forward and reverse primers (10 μmol L–1), respectively, and 7.4 μL double distilled water (ddH2O). The amplification was carried out in an Eppendorf Mastercycler Gradient Thermocycler(Type 5331, Eppendorf AG, Hamburg, Germany) using the following program: 94°C for 5 min, 35 cycles at 94°C for 30 s, 57°C for 30 s, and 72°C for 30s, with a final extension of 72°C for 3 min. AnActingene was utilized as an internal control gene and the results were evaluated through agarose gel electrophoresis.

2.7. qRT-PCR expression analysis of VvWBCs

Gene-specific primer pairs were designed forVvWBCs with each primer pair located near the 3´ untranslated regions(UTR) and anActingene was used as an internal control(Appendix C). The reactions were performed with the iQ5 RT-PCR machine (Bio-Rad, USA) and utilized SYBR Green I(TaKaRa, Japan). The reaction systems and conditions for qRT-PCR make reference to methods described previously with a slight modification (Zhanget al.2012). The PCR reaction mixture (21.0 μL) consisted of 1 μL cDNA templates,0.8 μL of forward and reverse primers (10 μmol L–1),10.5 μL SYBR PremixExTaqTMII (2×), ddH2O to 21 μL.The reactions were performed with the iQ5 and RT-PCR machine (Bio-Rad, USA) using the following cycling parameters: 95°C for 3 min, followed by 45 cycles of 95°C for 5 s and 60°C for 30 s, with a final extension at 72°C for 30 min. iQ5 Software (BioRad, USA) was employed to analyze the relative expression levels of eachVvWBC via2-ΔΔCTmethod. All analyses were performed in triplicate and the experimental results obtained were expressed as means±standard deviations. The significance of expression level differences between two grapevines was assessed with independent samplet-test. Microsoft Excel (Microsoft, USA)was used to analyze the results and Origin 8.0 (OriginLab,USA) was used to create figures.

3. Results

3.1. ldentification and isolation of VvWBCs

To identify WBC transporters inVitisby a homology-based approach, we used the peptide sequence of all 28WBCgenes fromArabidopsisas queries to search grapevine sequences (predicted genes) cataloged in GenBank. This approach resulted in the identification of 31 sequences.Two genes,VvWBC11.5andVvWBC11.8, had been assigned to the same locus on chromosome 7 and were distinguished only by a deletion of 450 nucleotides at the 5´terminus. Since this deletion would result in the truncation of the protein and removal of the NBD, we assumed thatVvWBC11.5andVvWBC11.8represent the same gene.Thus, the grapevine genome contains 30VvWBCfamily members (Appendix D) and this result is in line with previous research (Cakir and Kilickaya 2013).

We isolated 20 full-length cDNAs fromV.viniferacv.Thompson Seedless andV.vinifera cv. Pinot Noir using PCR and primers designed to amplify individual gene family members, and determined their nucleotide sequences.Sequences data ofVvWBCgenes from this article can be found in the GenBank data libraries and the accession number been shown in the Table 1. The length of the predicted ORFs ranged from ～1.8 (VvWBC8) to ～2.4 kb(VvWBC1), and the length of the encoded polypeptides ranged from 598 to 801 aa, with predicted molecular masses ranging from ～66.9 to ～90.3 kDa. All of the proteins were predicted to localize to membranes, consistent with their presumed function as transporters.

3.2. Chromosome mapping and synteny analysis of VvWBCs

Based on their annotated chromosomal locations, the 30VvWBCs are distributed widely in theV.viniferagenome (Appendix E). We identified two loci containing tandem arrangement of genes:VvWBC11.1,VvWBC11.4,VvWBC11.3andVvWBC11.2onchromosome 3;VvWBC15.4,VvWBC15.1,VvWBC15.2, andVvWBC15.3on chromosome 6.

To gain new insight into the origin and evolution of theVvWBCs, we preformed synteny analysis within the grapevine genome and between the grapevine andArabidopsisgenomes(Fig. 1), two orthologous gene pairs and two paralogous gene pairs were identified as follows:VvWBC28.2-AtWBC28,VvWBC3-AtWBC3,VvWBC28.1-VvWBC28.2andVvWBC10.1-VvWBC10.2. It suggests that these pairs of genes descended from one evolutionary ancestor. Meanwhile, the situation with other genes was more complex, including two cases of a single grapevineVvWBCgene syntenically corresponded to multipleArabidopsisgenes (VvWBC20-AtWBC1,VvWBC20-AtWBC16,VvWBC20-AtWBC2,VvWBC20-AtWBC20,VvWBC6-AtWBC1,VvWBC6-AtWBC16), and two cases of a singleArabidopsis WBCgene syntenically corresponded to multiple grapevine genes (AtWBC1-VvWBC20,AtWBC1-VvWBC6;AtWBC16-VvWBC6, AtWBC16-VvWBC20). These genes constitute a closely related orthologous groups. The cause of this phenomenon might be chromosomal rearrangement, fusions, and selective gene loss occurred in certain syntenic locations during grapevine genome evolution (Zhanget al.2012).

3.3. Structures and phylogenetic analysis of WBCs in grapevine and Arabidopsis

We assessed the intron/exon organization ofWBCs in grapevine (Fig. 2). Based on this analysis, thesegenes can be divided into nine types: one exon (VvWBC4,VvWBC5,VvWBC6,VvWBC10.1,VvWB20andVvWBC23),two exons (VvWBC1andVvWBC10.2), four exons(VvWBC9), five exons (VvWBC21andVvWBC25), eight exons (VvWBC7,VvWBC11.1,VvWBC11.2,VvWBC11.3,VvWBC11.4andVvWBC11.6), nine exons (VvWBC11.7,VvWBC11.8,VvWBC15.1,VvWBC15.2,VvWBC15.3,VvWBC15.4,VvWBC22.1,VvWBC26andVvWBC28.2),10 exons (VvWBC22.2), 11 exons (VvWBC3) and 15 exons(VvWBC28.1).

In order to better understand the evolutionary relationship ofWBCs between grapevine andArabidopsis, we also performed phylogenetic analyses (Fig. 2). It can be seen that these genes form four large clades, with each clade containingWBCs from both grapevine andArabidopsis.

3.4. Multiple sequence alignments of WBCs

The nucleotide sequence similarity matrix revealed that the similarity of these 30VvWBCORF nucleotide sequences range from 35.8 to 90.6%.VvWBC11.4andVvWBC11.1have the highest similarity and the lowest similarity appear betweenVvWBC3andVvWBC4(Appendix F).

Fig. 1 Synteny analyses of VvWBC genes in grapevine and Arabidopsis. The approximate location of each gene on grapevine or Arabidopsis chromosomes is indicated. Lines represent syntenic regions between grapevine and Arabidopsis chromosomes.

Alignment of grapevine andArabidopsisWBCs protein sequences showed that most of the proteins have structural similarities consisting of ABC signature motif, Walker A,Walker B, H-loop, D-loop, and Q-loop conserved motifs. The ABC signature motif resides in the area between Walker A and Walker B, which is a typical secondary structure of ABC transporters (Appendix G).

3.5. Expression profile of grapevine WBCs

We assessed the expression of the grapevineWBCs in eight diverse structures of Pinot Noir (leaf, root, flower, stem,pericarp, floral bud, tendril ovule)viasqRT-PCR (Fig. 3).The results demonstrated that these genesexhibit distinct expression patterns in these structures. Of the 30 genes,26 were expressed to detectable levels in at least one of the eight structures. It is worth noting that expression of someVvWBCs showed tissue and organ specificity. For example,VvWBC15.3was primarily detected in floral bud,andVvWBC11.4andVvWBC11.6were detected only in root.In contrast,VvWBC26was detected in all eight samples,suggesting a ubiquitous function.

Expression of 12 genes was detected in ovules (Fig. 3).We utilized qRT-PCR to clarify the expression patterns of these 12 genes during ovule development in seeded(Pinot Noir) and seedless (Thompson Seedless) varieties.As shown in Fig. 4, many of these genes show distinct expression profiles between the seeded and seedless varieties. For example, the expression ofVvWBC20in both Pinot Noir and Thompson Seedless wasrelatively low during early ovule development (approximately 10 to 25 days after full bloom). In Pinot Noir, expression increased sharply until 45 DAF, whereas in Thompson Seedless, expression remained low.VvWBC11.8andVvWBC15.4all exhibited low expression in Pinot Noir relative to Thompson Seedless.

4. Discussion

Fig. 2 Phylogenetic analysis of grapevine and Arabidopsis white-brown complex (WBC) proteins and gene intron/exon structures.The four best supported clades are denoted with Roman numerals (I-IV). A, phylogenetic analysis. B, gene intron/exon structures.

The gene structures ofWBCs showed both conservation and divergence in our study. On one hand, it became obvious that almost allWBCsin the same phylogenetic clade shared similar exon/intron structures (Fig. 2). For instance,all ofWBCs exceptVvWBC1 andVvWBC 10.2within Clade I,whether grapevine orArabidopsis, showed a structure consisting of only one exon. These results showed that theWBCmembers are, to some extent, conserved among plants. On the other hand, the structures of some genes in the same branch may differ. To take an example, the gene structures ofAtWBC28,AtWBC24,VvWBC28.2andVvWBC28.1from Clade IV were very different:VvWBC28.1had many more (six) exons than that ofVvWBC28.2. Exon/intron diversification of gene family members has played an important role in the evolution of multiple gene families and exon/intron gain or loss is one of three main types of mechanism (Xuet al.2012). Based on the results presented here, it is easy to conclude thatdivergence of exon/intron structure is an essential component of the expansion ofVvWBCs gene family. As a matter of fact, exon/intron gain/loss is also a potentially way to generate functionally distinct paralogs (Xuet al.2012).

Whole genome duplication has not yet been documented for grapevine. Segmental and tandem duplications are two major mechanisms for gene family expansion (Jaillonet al.2007). Tandem gene duplication frequently occurs in chromosome recombination hotspots, and is a way to form a series of functionally similar paralogous genes in a“head-tail” arrangement (Fanget al.2012). In this study, we found two groups of tandem genes (Appendix E). The intron and exon organizations within these two groups of genes were similar. Genes within these two groups have relatively high similarity. All of the results suggest that they represent two pairs of tandem genes. Thus, tandem duplications contributed to the expansion of grapevineWBCfamily. It was reported that functions of duplicated genes change rarely, and changes always happened in regulatory systems(Wapinskiet al.2007). We can see that these tandemly duplicatedgenes have relatively high nucleotide sequence similarity (Appendix E) but their tissue-expression profiles are different from those of their tandem genes (Fig. 4). More interestingly, we can see thatVvWBC15.3andVvWBC15.4exhibited different expression patterns during ovule development in seeded and seedless grapevines varieties.Divergence of expression patterns within duplicated genes may result from the following reasons: multiple regulatory networks, mutations ofcis-regulatory elements (Smithet al.2006), mutations affecting the regulatory networks (Wanget al. 2007;Xinget al.2007) and epigenetic mechanisms(Rapp and Wendel2005; Chen and Ni2006). The factors governing the diversity in the transcript levels of these two pairs of duplicated genes remain unknown. Further functional analyses of theVvWBCgene family could address whether these duplicatedVvWBCgenes have changed when they acquire different expression patterns.

Fig. 3 The tissues or organ-specific expression analyses of white-brown complex (WBC) genes in grapevine. Expression of WBCs analyzed by semi-quantitative RT-PCR (sqRT-PCR)in root, stem, leaf, tendril, alabastrum, flowers, pericarp, pulp,and ovule tissues of Pinot Noir. ACTIN gene was utilized as the control.

In the stenospermocarpy phenotype, the flower structure is normal, and pollination and fertilization occur normally.Thus, the seedless phenotype may due to the abortion of fertilized embryos during ovule development (Halbrooks and Mortensen 1988). During the seed development stage,the endosperm not only provides the embryo with nutrition,but also forms a scaffold around the embryo. There are many studies indicating that deficiencies in cutin and cuticular wax syntheses have effects on seed and embryo development (Baudet al.2003; Bonaventureet al.2003;Chenet al.2005). Earlier studies showed that many ABCG familymembers participate in transport of aliphatic polymer precursors for wax and cutin biosynthesis (Pighinet al.2004; Birdet al.2007; Luoet al.2007; Panikashviliet al.2007, 2010; Le Hiret al.2013).AtWBC20, an orthologous gene ofVvWBC20, is involved in transportation of wax and suberin precursors within the root (Yadavet al.2014). In our research,VvWBC20was found to be specifically expressed in the ovule and weakly expressed in floral bud (Fig. 4).We also found thatVvWBC20was differentially expressed in later ovule developmental stages (after 30 DAF, close to the timing of endosperm abortion at 31 DAF) between seeded and seedless grapevines. At the same time, three differential expressed genes (VvWBC11.1,VvWBC11.8andVvWBC15.4) were detected between seeded and seedless grapevines nearly throughout the development of ovules. EspeciallyVvWBC11.8has a very large difference in expression between seeded and seedless grapevines at 20–25 days after full bloom (just before the timing of endosperm abortion at 31 DAF). According to the previous research,AtWBC11participated in the cuticle formation inArabidopsis thaliana(McFarlaneet al. 2010).AtWBC11,VvWBC11.1, andVvWBC11.8are in the same phylogenetic clade, so they may have the similar function. The grape seeds are composed of embryo, endosperm and seed coat which contain a cuticle, an epidermis, three layers inner integument and two layers outer integument. It is of great value to identify the structure differences between the seeds from Pinot Noir and seed traces from Thompson Seedless,especially structures related to cutin and cuticular wax syntheses. Further research such asin situhybridization analysis could be conducted to see whether these candidate genes are expressed in above structures and have different expression profiles at the cellular level in seeded and seedless grape seeds. Wax and suberin composition analysis also can be performed in seeds to see whether there is any difference between seedless and seeded grape.Gene silencing and overexpression experiments are needed to reveal the impact of different expression level of these candidate genes on ovule development and the function of these differentially expressed genes. Whether the lower expression level ofVvWBC11.1, andVvWBC11.8influenced the cuticle formation in seeds and lead to seed abortion in seedless grapevines is still a mystery. But there is no doubt that different relative expression levels of these genes would inevitably changed their substrates transportation across membranes and further affect the metabolic activity during growth and development of seeds.

Fig. 4 Expression analyses of white-brown complex (WBC) genes during ovule development in Pinot Noir and Thompson Seedless.A-L are the relative expression level of VvWBC3, VvWBC4, VvWBC5, VvWBC6, VvWBC7, VvWBC11.1, VvWBC11.7, VvWBC11.8,VvWBC15.3, VvWBC15.4, VvWBC20, and VvWBC25, respectively, during ovule development in Pinot Noir and Thompson Seedless.The grapevine Actin gene was utilized as the reference gene. The gene expression results were normalized to the mock controls,respectively. Error bars represent SD (n=3). Asterisks indicate levels of significance (t-test; ＊, P≤0.05; ＊＊, P≤0.01).

5. Conclusion

In summary, we identified 30WBCgenes from the grape genome and cloned full-length cDNAs for 20 of them.Bioinformatic analyses such as chromosome mapping,multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed onVvWBCgenes. Tissue or organ-specific expression analysis showed that 12VvWBCgenes are expressed in the developing ovules. Further expression analyses revealed that four of 12 ovule-expressedVvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Our experiment presented a new standpoint about the mechanism of stenospermocarpic seedless phenotype and provided a useful reference for the further study ofVvWBCs.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31372023), and the earmarked fund for China Agricultural Research System (CARS-30-yz-7).

Appendicesassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

Adam-Blondon A F, Lahogue-Esnault F, Bouquet A, Boursiquot J M, This P. 2001. Usefulness of two SCAR markers for marker-assisted selection of seedless grapevine cultivars.Vitis,40, 147–155.

Andolfo G, Ruocco M, Di Donato A, Frusciante L, Lorito M, Scala F, Ercolano M R. 2015.Genetic variability and evolutionary diversification of membrane ABC transporters in plants.BMC Plant Biology,15, 51.

Banasiak J, Biala W, Staszków A, Swarcewicz B, Kepczynska E, Figlerowicz M, Jasinski M. 2013. AMedicago truncatulaABC transporter belonging to subfamily G modulates the level of isoflavonoids.Journal of Experimental Botany,64,1005–1015.

Baud S, Guyon V, Kronenberger J, Wuilleme S, Miquel M,Caboche M, Lepiniec L, Rochat C. 2003. Multifunctional acetyl-CoA carboxylase 1 is essential for very long chain fatty acid elongation and embryo development inArabidopsis.The Plant Journal,33, 75–86.

Bird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu X, Yephremov A, Samuels L. 2007.Characterization ofArabidopsisABCG11/WBC11, an ATP binding cassette(ABC) transporter that is required for cuticular lipid secretion.The Plant Journal,52, 485–498.

Bonaventure G, Salas J J, Pollard M R, Ohlrogge J B. 2003.Disruption of theFATBgene inArabidopsisdemonstrates an essential role of saturated fatty acids in plant growth.The Plant Cell,15, 1020–1033.

Buda G J, Barnes W J, Fich E A, Park S, Yeats T H, Zhao L,Domozych D S, Rose J K. 2013. An ATP binding cassette transporter is required for cuticular wax deposition and desiccation tolerance in the mossPhyscomitrella patens.The Plant Cell,25, 4000–4013.

Cakir B, Kilickaya O. 2013. Whole-genome survey of the putative ATP-binding cassette transporter family genes inVitis vinifera.PLoS ONE,8, 11.

Chen G, Komatsuda T, Ma J F, Nawrath C, Pourkheirandish M,Tagiri A, Hu Y G, Sameri M, Li X, Zhao X, Liu Y, Li C, Ma X,Wang A, Nair S, Wang N, Miyao A, Sakuma S, Yamaji N,Zheng X, Nevo E. 2011. An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice.Proceedings of the National Academy of Sciences of the United States of America,108, 12354–12359.

Chen X, Goodwin S M, Liu X, Bressan R A, Jenks M A. 2005.Mutation of the RESURRECTION1 locus ofArabidopsisreveals an association of cuticular wax with embryo development.Plant Physiology,139, 909–919.

Chen Z J, Ni Z F. 2006. Mechanisms of genomic rearrangements and gene expression changes in plant polyploids.BioEssays,28, 240–252.

Choi H, Jin J Y, Choi S, Hwang J U, Kim Y Y, Suh M C, Lee Y.2011. An ABCG/WBC-type ABC transporter is essential for transportof sporopollenin precursors for exine formation in developing pollen.The Plant Journal,65, 181–193.

Crouzet J, Roland J, Peeters E, Trombik T, Ducos E, Nader J, Boutry M. 2013. NtPDR1, a plasma membrane ABC transporter fromNicotiana tabacum, is involved in diterpene transport.Plant Molecular Biology,82,181–192.

Crouzet J, Trombik T, Fraysse A S, Boutry M. 2006. Organization and function of the plant pleiotropic drug resistance ABC transporter family.FEBS Letters,580, 1123–1130.

Dou X Y, Yang K Z, Zhang Y, Wang W, Liu X L, Chen L Q, Zhang X Q, Ye D. 2011. WBC27, an adenosine tri-phosphatebinding cassette protein, controls pollen wall formation and patterning inArabidopsis.Journal of Intergrative Plant Biology,53, 74–88.

Ezaki B, Takahashi K, Utsumi K, Higashi A. 2015. A halftype AvABCG1 transporter derived fromAndropogon virginicusL. confers aluminum tolerance.Environmental and Experimental Botany,118, 21–31.

Fabre G, Garroum I, Mazurek S, Daraspe J, Mucciolo A,Sankar M, Humbel B M, Nawrath C. 2016. The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle inArabidopsis.New Phytologist,209, 192–201.

Fang L, Cheng F, Wu J, Wang X W. 2012. Gene retention following whole genome duplication and tandem duplication inBrassica rapa.Biothchnology Bulletin,11, 9–14. (in Chinese)

Halbrooks M C, Mortensen J A. 1988. Effects of gibberellic acid on berry and seed development in ‘Orlando Seedless’grape.HortScience,23, 409.

Higgins C F, Linton K J. 2004. The ATP switch model for ABC transporters.Nature Structural & Molecular Biology,11,918–926.

Hwang J U, Song W Y, Hong D, Ko D, Yamaoka Y, Jang S, Yim S, Lee E, Khare D, Kim K, Palmgren M, Yoon H S, Martinoia E, Lee Y. 2016. Plant ABC transporters enable many unique aspects of a terrestrial plant’s lifestyle.Molecular Plant,9,338–355.

Jaillon O, Aury J M, Noel B, Policriti A, Clepet C, Casagrande A,Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D,Poulain J, Bruyère C, Billault A,et al. 2007. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.Nature,449, 463–465.

Kim Y, Park S Y, Kim D, Choi J, Lee Y H, Lee J H, Choi W.2013.Genome-scale analysis of ABC transporter genes and characterization of the ABCC type transporter genes inMagnaporthe oryzae.Genomics,101, 354–361.

Kretzschmar T, Kohlen W, Sasse J, Borghi L, Schlegel M,Bachelier J B, Reinhardt D, Bours R, Bouwmeester H J, Martinoia E. 2012. A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching.Nature,483, 341–344.

Krzywinski M, Schein J, Birol İ, Connors J, Gascoyne R,Horsman D, Jones S J, Marra M A. 2009. Circos: An information aesthetic for comparative genomics.Genome Research,19, 1639–1645.

Kuromori T, Ito T, Sugimoto E, Shinozaki K. 2011a.Arabidopsismutant ofAtABCG26, an ABC transporter gene, is defective in pollen maturation.Journal of Plant Physiology,168,2001–2005.

Kuromori T, Miyaji T, Yabuuchi H, Shimizu H, Sugimoto E,Kamiya A, Moriyama Y, Shinozaki K. 2010. ABC transporter AtABCG25 is involved in abscisic acid transport and responses.Proceedings of the National Academy of Sciences of the United States of America,107, 2361–2366.

Kuromori T, Sugimoto E, Shinozaki K. 2011b.Arabidopsismutants ofAtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility.The Plant Journal,67, 885–894.

Le Hir R, Sorin C, Chakraborti D, Moritz T, Schaller H, Tellier F, Robert S, Morin H, Bako L, Bellini C. 2013. ABCG9,ABCG11 and ABCG14 ABC transporters are required for vascular development inArabidopsis.The Plant Journal,76, 811–824.

Li L, Li D, Liu S, Ma X, Dietrich C R, Hu H C, Zhang G, Liu Z, Zheng J, Wang G, Schnable P S. 2013. The maizeglossy13gene, clonedviaBSR-Seq and Seq-walking encodes a putative ABC transporter required for the normal accumulation of epicuticular waxes.PLoS ONE,8, e82333.

Li X Q, Yin X G, Wang H, Li J, Guo C L, Gao H, Zheng Y, Fan C H, Wang X P. 2015.Genome-wide identification and analysis of the apple (Malus×domesticaBorkh.)TIFYgene family.Tree Genetics & Genomes,11, 808.

Luo B, Xue X Y, Hu W L, Wang L J, Chen X Y. 2007. An ABC transporter gene ofArabidopsis thaliana,AtWBC11, is involved in cuticle development and prevention of organ fusion.Plant and Cell Physiology,48, 1790–1802.

McFarlane H E, Shin J J, Bird D A, Samuels A L. 2010.ArabidopsisABCG transporters, which are required for export of diverse cuticular lipids, dimerize in different combinations.The Plant Cell,22, 3066–3075.

Muhovski Y, Jacquemin J M, Batoko H. 2014. Identification and differential induction ofABCGtransporter genes in wheat cultivars challenged by a deoxynivalenol producingFusarium graminearumstrain.Molecular Biology Reports,41, 6181–6194.

Nuruzzaman M, Zhang R, Cao H Z, Luo Z Y. 2014. Plant pleiotropic drug resistance transporters: Transport mechanism, gene expression, and function.Journal of Integrative Plant Biology,56, 729–740.

Panikashvili D, Savaldi-Goldstein S, Mandel T, Yifhar T, Franke R B, Höfer R, Schreiber L, Chory J, Aharoni A. 2007.TheArabidopsisDESPERADO/AtWBC11 transporter is required for cutin and wax secretion.Plant Physiology,145, 1345–1360.

Panikashvili D, Shi J X, Bocobza S, Franke R B, Schreiber L, Aharoni A. 2010. TheArabidopsisDSO/ABCG11 transporter affects cutin metabolism in reproductive organs and suberin in roots.Molecular Plant,3, 563–575.

Panikashvili D, Shi J X, Schreiber L, Aharoni A. 2011. TheArabidopsisABCG13 transporter is required for flower cuticle secretion and patterning of the petal epidermis.New Phytologist,190, 113–124.

Pighin J A, Zheng H, Balakshin L J, Goodman I P, Western T L,Jetter R, Kunst L, Samuels A L. 2004.Plant cuticular lipid export requires an ABC transporter.Science,22, 702–704.

Quilichini T D, Friedmann M C, Samuels A L, Douglas C J.2010. ATP-binding cassette transporter G26 is required for male fertility and pollen exine formation inArabidopsis.Plant Physiology,154, 678–690.

Ramnning D W, Emershad R L, Tarllo R. 2000. A Stenospermocarpic, seedlessVitis vinifera×Vitis rotundiyoliahybrid developed by embryo rescue.HortScience,35,732–734.

Rapp R A, Wendel J F. 2005. Epigenetics and plant evolution.New Phytologist,168, 81–91.

Rea P A. 2007. Plant ATP-binding cassette transporters.Annual Review of Plant Biology,58, 347–375.

Ruocco M, Ambrosino P, Lanzuise S, Woo S L, Lorito M,Scala F. 2011. Four potato (Solanum tuberosum) ABCG transporters and their expression in response to abiotic factors andPhytophthora infestansinfection.Journal of Plant Physiology,168, 2225–2233.

Samuels L, Kunst L, Jetter R. 2008. Sealing plant surfaces:Cuticular wax formation by epidermal cells.Annual Review of Plant Biology,59, 683–707.

Shibata Y, Ojika M, Sugiyama A, Yazaki K, Jones D A, Kawakita K, Takemoto D. 2016. The full-size ABCG transporters Nb-ABCG1 and Nb-ABCG2 function in pre- and post-invasion defense againstPhytophthora infestansinNicotiana benthamiana.The Plant Cell,28, 1163–1181.

Smart C C, Fleming A J. 1996.Hormonal and environmental regulation of a plant PDR5-like ABC transporter.The Journal of Biological Chemistry,271, 19351–19357.

Smith A D, Sumazin P, Xuan Z Y, Zhang M Q. 2006. DNA motifs in human and mouse proximal promoters predict tissue-specific expression.Proceedings of the National Academy of Sciences of the United States of America,103, 6275–6280.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013.MEGA6: Molecular evolutionary genetics analysis version 6.0.Molecular Biology and Evolution,30, 2725-2729.

Tarr P T, Tarling E J, Bojanic D D, Edwards P A, Baldán A.2009.Emerging new paradigms for ABCGtransporters.Biochimica et Biophysica Acta(BBA)- Molecular and Cell Biology of Lipids,1791, 584–593.

Ukitsu H. 2007. Cytological and biochemical analysis ofCOF1,anArabidopsismutant of an ABC transporter gene.Plant and Cell Physiology,48, 1524–1533.

Van D D C, Tampe R. 2004.How do ABC transporters drive transport.Biological Chemistry,385, 927–933.

Verrier P J, Bird D, Burla B, Dassa E, Forestier C, Geisler M, Klein M, Kolukisaoglu U, Lee Y, Martinoia E, Murphy A, Rea P A, Samuels L, Schulz B, Spalding E J, Yazaki K, Theodoulou F L. 2008. Plant ABC proteins: A unified nomenclature and updated inventory.Trends in Plant Science,13, 151–159.

Wang D Y, Sung H M, Wang T Y, Huang C J, Yang P, Chang T, Wang Y C, Tseng D L, Wu J P, Lee T C,Shih M C, Li W H. 2007. Expression evolution in yeast genes of single-input modules is mainly due to changes in transacting factors.Genome Research,17, 1161–1169.

Wang J W, Horiuchi S. 1990. A histological study on the seedlessness in ‘Himrod seedless’ grape.Journal of the Japanese Society for Horticultural Science,59, 455–462.

Wapinski I, Pfeffer A, Friedman N, Regev A. 2007. Natural history and evolutionary principles of gene duplication in fungi.Nature,449, 54–61.

Xie X, Wang G, Yang L, Cheng T, Gao J, Wu Y, Xia Q. 2015.Cloning and characterization of a novelNicotiana tabacumABC transporter involved in shoot branching.Physiologia Plantarum,153, 299–306.

Xing Y, Ouyang Z Q, Kapur K, Scott M P, Wong W H. 2007.Assessing the conservation of mammalian gene expression using high-density exon arrays.Molecular Biology and Evolution,24, 1283–1285.

Xu G X, Guo C C, Shan H Y, Kong H Z. 2012. Divergence of duplicate genes in exon-intron structure.Proceedings of the National Academy of Sciences of the United States ofAmerica,109, 1187–1192.

Xu J, Yang C S, Yuan Z, Zhang D S, Gondwe M Y, Ding Z W,Liang W Q, Zhang D B, Wilson Z A. 2010. The ABORTED MICROSPORES regulatory network is required for postmeiotic male reproductive development inArabidopsis thaliana.The Plant Cell,22, 91–107.

Yadav V, Molina I, Ranathunge K, Castillo I Q, Rothstein S J,Reed J W. 2014. ABCG transporters are required for suberin and pollen wall extracellular barriers inArabidopsis.The Plant Cell,26, 3569–3588.

Yim S, Khare D, Kang J, Hwang J U, Liang W Q, Martinoia E, Zhang D B, Kang B H, Lee Y. 2016. Postmeiotic development of pollen surface layers requires twoArabidopsisABCG-typetransporters.Plant Cell Reports,35, 1863–1873.

Zha W J, Li S H, Zhou L, Chen Z J, Liu K, Yang G C, Hu G,He G C, You A Q. 2015.Molecular cloning and expression profile of an ATP-binding cassette (ABC) transporter gene from the hemipteran insectNilaparvata lugens.Genetics and Molecular Research,14, 2654–2664.

Zhao G C, Shi J X, Liang W Q, Zhang D B. 2016. ATP binding cassette G transporters and plant male reproduction.Plant Signaling and Behavior,11, e1136764.

Zhang C H, Gong P J, Wei R, Li S X, Zhang X T, Yu Y H, Wang Y J. 2013. The metacaspase gene family ofVitis viniferaL.:Characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes.Gene,528, 267–276.

Zhang J J, Wang Y J, Wang X P, Yang K Q, Yang J X. 2003.An improved method for rapidly extracting total RNA fromVitis.Journal of Fruit Science,20, 178–181. (in Chinese)

Zhang K, Novak O, Wei Z, Gou M, Zhang X, Yu Y, Yang H, Cai Y, Strnad M, Liu C J. 2014.ArabidopsisABCG14 protein controls the acropetal translocation of root-synthesized cytokinins.Nature Communication,5, 3274.

Zhang Y C, Gao M, Stacy D S, Zhang J F, Wang H, Wang X P.2012. Genome-wide identification and analysis of the TIFY gene family in grape.PLoS ONE,7, e44.

Zhu L, Shi J X, Zhao G C, Zhang D B, Liang W Q. 2013.Post-meiotic deficient anther1 (PDA1) encodes an ABC transporter required for the development of anther cuticle and pollen exine in rice.Journal of Plant Biology,56, 59–68.