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Scientia Horticulturae 130 (2011) 708–714

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h o me page: www.elsev ier .com/ locate /sc ihor t i

olecular cloning and characterization of a novel gene encoding an EF-handalcium-binding protein related to fruit seedlessness of grapevine

hengyi Wanga,c,d, Yihe Yub,c,d, Chaohong Zhangb,c,d, Weirong Xub,c,d, Yuejin Wangb,c,d,∗

College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, PR ChinaCollege of Horticulture, Northwest A & F University, Yangling, Shaanxi 712100, PR ChinaKey Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Yangling, Shaanxi 712100, PR ChinaState Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A & F University, Yangling, Shaanxi 712100, PR China

r t i c l e i n f o

rticle history:eceived 7 April 2011eceived in revised form 27 July 2011ccepted 29 July 2011

a b s t r a c t

EF-hand calcium-binding protein (CBP) gene plays a critical role during embryonic development in ani-mals, but it remains unclear whether this gene possesses similar function in plants. In this study, anovel CBP gene was isolated from Vitis vinifera cv. Thompson Seedless and designated as VvCBP1. Using acombination of sequence structural investigation, expression pattern analysis of different embryo devel-opment in grapevine, and silencing assay of VvCBP1 homolog in tomato, the functional role of VvCBP

eywords:F-hand calcium-binding proteinmbryo abortioneedlessnessobacco rattle virus (TRV)itis viniferavCBP1

gene in embryo development of seedless grapevine was studied. Structural investigations showed thatVvCBP1 is 907 bp in length, and contains two EF-hand motifs. Expression pattern of this gene is differentin embryo between seedless and seeded grapevine species. Moreover, the silencing of homology geneled to the seeds number significantly decreased in tomato. These data demonstrate clearly that VvCBP1plays an important role in embryo development of seedless grapevine.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Seedlessness is an economically important quality trait inrapevine. Seedless grapevines are of great interest for fresh fruitonsumption and raisin production worldwide. Two different typesf seedlessness have been observed in grape genetic resources:arthenocarpy and stenospermocarpy (Stout, 1936). In stenosper-ocarpic varieties, pollination and fertilization occur as normal,

ut the embryo and/or endosperm abort two to four weeks afterertilization (Ledbetter and Ramming, 1989). It has been previ-usly reported that the property of seedlessness was controlledy a single or a few recessive genes (Bouquet and Danglot, 1996;

piegel-Roy and Sahar, 1990; Stout, 1936), although the possibil-ty of dominant control has also been suggested (Roytchev, 1998;tout, 1937).

Abbreviations: CBPs, calcium-binding proteins; EST, expressed sequence tag;ACE, rapid amplification of cDNA end; RT-PCR, reverse transcription polymerasehain reaction; SCAR, sequence characterized amplification region; SSH, suppres-ion subtractive hybridization; TRV, tobacco rattle virus; VIGS, virus-induced geneilencing.∗ Corresponding author at: College of Horticulture, Northwest A & F University, 3aicheng Road, Yangling, Shaanxi 712100, PR China. Tel.: +86 29 87082803;ax: +86 29 87082803.

E-mail address: wangyj@nwsuaf.edu.cn (Y. Wang).

304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2011.07.029

In previous studies, marker-assisted selections for seedlessnessin grapevine have been well-documented (Doligez et al., 2002;Lahogue et al., 1998; Yang et al., 2005a). Many of them displayedquantitative characteristics (Doligez et al., 2002; Lahogue et al.,1998), while the SCAR marker selection was reported to be a domi-nant allele (Yang et al., 2005b). In recent years, numerous essentialgenes for seed and embryo development have been intensivelystudied, many of which remain uncharacterized and are currentlyannotated to encode proteins with unknown functions (Albert et al.,1999; Griffith et al., 2007; Kwong et al., 2003; Stone et al., 2001;Tzafrir et al., 2004). In addition, the molecular and cellular mecha-nisms of embryo abortion and seedlessness during grapevine berrydevelopment remain to be uncovered. A recent differential expres-sion analysis indicated that a chloroplast chaperonin 21 (ch-Cpn21)gene from Vitis vinifera cv. Thompson Seedless, identified by silenc-ing assays in tobacco and tomato fruits, was essential for grapeseed development (Hanania et al., 2007). Subsequently, the samegroup has isolated a ubiquitin extension gene S27a using similarapproach, whose overexpression resulted in abnormal plant regen-eration and inhibited shoot development. Moreover, S27a silencingin embryogenic callus led to cell necrosis and callus death (Hana-

nia et al., 2009). Evidence from these results suggests that the genefunctions in regulation of developing organs of grapevine.

EF-hand calcium-binding proteins (CBPs) consist of large pro-tein family genes, which are found in a number of plant species.

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revious studies on EF-hand CBPs indicated that the family mem-er can sensor the cellular Ca2+ concentration and transduct thea2+ signals to trigger downstream response (Gifford et al., 2007). Itas also been reported that EF-hand CBPs were involved in variousevelopmental processes including root, floral organ and reproduc-ive development (Capoen et al., 2009; Dumas and Gaude, 2006;aothien et al., 2005; Li et al., 2007; Poulter et al., 2008; Yoont al., 2006), hormone regulated cellular activities (cell divisionnd elongation, stomatal closure/opening) (Kim et al., 2006; Lanterit al., 2006), biotic and abiotic stress-induced defense responsesAlbrecht et al., 2003; Chung et al., 2004; Kobayashi et al., 2007;ecourieux et al., 2006; Pandey et al., 2007; Yang and Poovaiah,000).

In our previous study, a cDNA library of V. vinifera cv. Thomp-on Seedless related to embryo abortion at different developmenttages was constructed, and 3908 EST sequences were obtained.

combination of sequence analysis and RT-PCR assay revealedhat one EST sequence (GenBank accession no. GR905727) pos-esses EF-hand calcium-binding protein domain and displayed aifferent expression profile from seeded grapevine species. Thisnding promotes us to further investigate the structure and func-ional characterization of the gene. Our results demonstrated thathe full-length cDNA, designated VvCBP1, possesses two EF-handalcium-binding motifs, which shares high similarity with that ofhe tomato. Moreover, using tobacco rattle virus (TRV)-mediatedirus-induced gene silencing (VIGS) technology, we silenced theomato homolog of VvCBP1 in tomato fruits, which led to theumber of transformation tomato seed significantly decrease. Theesults suggest that VvCBP1 may be tightly associated with fruiteedlessness of grapevine.

. Materials and methods

.1. Plant materials and growth conditions

V. vinifera cvs. Thompson Seedless and Pinot Noir were cul-ivated in the Grape Repository of Northwest A & F University,angling, Shaanxi, China under the natural environment with nor-al management. Embryos of V. vinifera cvs. Thompson Seedless

nd Pinot Noir were sampled after full-bloom stage of 15 d, 20 d,5 d, 30 d, 35 d, 40 d, 45 d, and 50 d during embryo development,nd stored at −80 ◦C before use.

Lycopersion esculentum cv. Micro-Tom used for VIGS were grownn the greenhouse under the condition of 25/20 ◦C and 70% humidity

ith a 14/10 h light/dark period.

.2. Cloning the full-length cDNA of VvCBP1 and sequencenalysis

A cDNA library of V. vinifera cv. Thompson Seedless berryt different stages of embryo development was previously con-tructed and a total of 3908 high quality ESTs were generatedZhang, 2007). Bioinformatic analysis indicated that one of theseSTs sequence possesses EF-hand calcium-bind protein, and furtherxpression analyses displayed a different expression profile fromeeded grapevine in embryo development. Gene-specific primersVvCBP1-5′: 5′-CAAC CACTTCTCTATGTACTCTTCTAC-3′; VvCBP1-R:′-CAAGCCCCACGGAG GCTCCAGCCACC-3′) were designed to theST sequence to amplify full-length cDNA from V. vinifera cv.hompson Seedless. Total RNA was isolated from each sample stagerom V. vinifera cvs. Thompson Seedless and Pinot Noir according

o the method previously described (Zhang et al., 2003), and cDNAynthesized using PrimeScript reverse transcriptase kit (TaKaRa,alian, China) according to the manufacture’s instructions. RACE

echniques were employed to obtain full-length cDNA sequence

urae 130 (2011) 708–714 709

using the SMART RACE cDNA Amplification Kit (Clontech). The PCRproducts was cloned into pMD18-T vector (TaKaRa) and sequenced.

The nucleotide sequence analysis was performed usingBLASTN and BLASTX programs of NCBI (http://www.ncbi.nlm.nih.gov/BLAST). Chromosomal location of VvCBP1 gene was pre-dicted by BLAT server at the Genoscope Genome Browser(http://www.genoscope.cns.fr/blat-server/cgi-bin/vitis/webBlat).The protein conserved domain was analyzed using Smart(http://smart.emblheidelberg.de/smart/change mode.pl) andExPASy (http://au.expasy.org/tools/). Amino acid sequencealignment and phylogenetic tree were generated by ClustalW(http://www.ebi.ac.uk/Tools/clustalw2/index.html). Thesequences used for bioinformatics analysis are Lycopersion escu-lentum (GenBank accession no. BT014117), Arabidopsis thaliana(NP 196037), Populus trichocarpa (XP 002308014), Medicago trun-catula (ACJ84993), Ricinus communis (XP 002534120), Saccoglossuskowalevskii (XP 002737488), Oryza sativa (NP 001066106), P.trichocarpa (XP 002308014), Juglans nigra (ACN39566), Zea mays(NP 001147282), Pisum sativum (CAB63845), Sorghum bicolor(XP 002450240), Picea sitchensis (ABR17289) and Glycine max(ACU14400).

2.3. Construction of silencing plasmids

To produce the VIGS vector for knock-down of VvCBP1homolog in tomato (LeCBP1, accession no. BT014117), a 167 bpfragment of LeCBP1 corresponding to 269–435 nt was ampli-fied using the primers 5′-TCCCGTTCCGTCTCCCTAC G-3′ and5′-CCTTCGGTGGTTTGTTCGGATC-3′. The resultant PCR productswere confirmed by DNA sequencing and cloned into pTRV2 to gen-erate pTRV2-LeCBP1.

2.3.1. Virus infection by Agrobacterium-mediated infiltrationFor VIGS assay, pTRV1, pTRV2 and pTRV2-LeCBP1 were respec-

tively transformed into Agrobacterium tumefaciens strain GV3101by electroporation. Briefly, single colony harboring each of theconstruct was isolated and grown overnight in 50 ml LB mediumsupplemented with the appropriate antibiotics (28 ◦C, 200 rpm).Cells were harvested, resuspended in infiltration media (10 mMMgCl2, 10 mM MES pH 5.6, 200 mM acetosyringone) and adjusted toOD600 of 1.0. Subsequently, pTRV1 was mixed (1:1 v/v) either withpTRV2 or with pTRV2-LeCBP1 in which the former was control. Mix-tures were incubated in a dark incubator at RT for 3 h with gentleagitation. The cultures were then infiltrated into the carpopodiumof tomato fruits (10 d after pollination) that still attached to theplant using 1 ml needleless syringe (Fu et al., 2005).

2.3.2. RT-PCR analysisTotal RNA were extracted from grapevine embryos or tomato

fruit samples according to the protocol previously described (Zhanget al., 2003). Reverse transcription was carried out with 2.5 �g oftotal RNA after DNase treatment using PrimeScript Reverse Tran-scriptase (TaKaRa). Semi-quantitative RT-PCR was performed usingPrimeScriptTM RT-PCR Kit (TaKaRa). Grapevine GAPDH gene (Gen-Bank accession no. EF192466) was used as internal control. Primersfor VvCBP1 and GAPDH were 5′-GCTGAGAGAGGCACTGATGAG-3′

and 5′-GC AATAAGAAATGGCAAGACG-3′, and 5′-GGAGCTGAATTTGTTGTTGAG-3′; 5′-CATTAACTCCAACAACG-AACATAGG-3′), respectively.

To determine the transcriptional level of LeCBP1 in tomatofruit, a pair of primers (5′-ATCAGGATGGGAGTGGGTTA-3′ and 5′-CAGCAATTCCAGGA TAGAAGGT-3′) was used. Tomato Ubiqutin

gene (GenBank accession no. AK246708) was used as internalcontrol with specific primers (5′-ACTGATTTCTCTCCTCCAGAC-3′;5′-GAGTGCCCTAATGCTGAGTGCG-3′). RT-PCR products were sep-arated on 1.2% agarose gels, EtBr stained, and quantified by

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ensitometry using Bio-imaging Systems software (SYNGENE,ambridge, UK). The experiments were conducted three times

ndependently.

.3.3. PCR Southern blot hybridizationTo confirm the accumulation of TRV virus in tomato fruit, we

etected the TRV-R2 vector in reverse transcript of RNA using PCR-outhern blot hybridization. The specific probe was labeled withigoxigenin, using a random priming method according to the man-facturer’s instructions (DIG-DNA Labeling Kit, Roche Diagnostics,ermany). The cDNA from the silenced tomato fruit was ampli-ed with the primers as followed, 5′-TACGACGAACCAAGGG-3′ and′-TGCGAAACTCAAATGCT-3′. The products were separated on 1.0%garose gels, and then transferred to a nylon membrane (Hybond-, Amersham, UK). Eventually, the membranes were pre-incubated

n a buffer containing the substrates nitroblue tetrazolium (NBT,40 �g/ml, Roche Diagnostics) and 5-bromo-4-chloro-3 indolylhosphate (BCIP, 170 �g/ml, Roche Diagnostics) for 5 min, and then

ncubated for 5 h in the dark in the same buffer.

. Results

.1.1. Identification of VvCBP1 gene

Based on the obtained partial EST sequence, a RACE-PCR methodas used to isolate the full-length of VvCBP1 gene. The result indi-

ated that the full-length cDNA of VvCBP1 is 907-bp with a 35-bp′ untranslated sequence and a 41-bp 3′ untranslated region (Gen-ank accession no. HQ214155). The predicted amino acid encodeswo EF-hand motifs (Fig. 1A). In Grape Genome Browse, the VvCBP1as localized on chromosome 8 of grapevine (Fig. 1A). Sequence

lignment of VvCBP1 deduced amino acid sequence with previ-usly reported EF-hand calcium-binding proteins of other speciesre presented in Fig. 1B. VvCBP1 shares 97% similarity with homologn L. esculentum (accession no. BT014117), 93% with P. trichocarpaomolog (accession no. XP 002308014) and A. thaliana calcium-inding EF-hand family of proteins (accession no. NP 196037),espectively. Phylogenetic tree analysis showed that they could belassified into three subfamilies (Fig. 1C). S. kowalevskii occupiedhe first subfamily, G. max and M. truncatula belong to the secondranch, and the rest constitute a third subfamily.

.2. Expression of VvCBP1 gene during the embryo development

It has been previously reported that V. vinifera cvs. Thompsoneedless and Pinot Noir possess the similar florescence (Jaillon et al.,007; Velasco et al., 2007). However, our observation indicated thathe embryo of Pinot Noir formed the seeds 40 d after full-bloom,hereas the embryo of Thompson Seedless was aborted (Fig. 2). To

xamine the expression of VvCBP1 in developing embryos of botheedless and seeded varieties, the total RNAs extracted from thembryos at different developmental stages were used for RT-PCRnalysis. As shown in Fig. 2, the expression of VvCBP1 displayed dif-erent patterns during embryo development of Thompson Seedlessnd seeded Pinot Noir. The transcript levels of VvCBP1 of Pinot Noiremained stable with only minor variation during embryo devel-pment. In contrast, the transcript in Thompson Seedless showedignificant fluctuation with peak expression at 30 d and minor peak

t 40 d after full-bloom. At the late stage of embryo developmentn Thompson Seedless, there was almost no VvCBP1 expression.hese results indicated that VvCBP1 might play an important rolen grapevine embryo development.

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3.3. Silencing of VvCBP1 homolog in tomato by TRV-VIGS resultedin reduced seeds

To investigate the function of VvCBP1 in embryo developmentindirectly, we observed the knock-down effects of LeCBP1 in tomatousing TRV-mediated VIGS. The Agrobacterium strains containingeither pTRV-R1 or pTRV-R2-LeCBP1 were mixed and infiltrated intothe carpopodium of young tomato fruit about 10 d after pollina-tion. After 4 weeks infiltration, we found the TRV-R2 in the tomatofruit infiltrated with the Agrobacterium containing pTRV constructs,but not in the controls (Fig. 3A and B). We then assessed the tran-script level of LeCBP1 by RT-PCR analysis with the primers specificto the 3′ terminal of LeCBP1. As expected, the LeCBP1 transcripts intomato fruit infiltrated with the Agrobacterium mixture containingboth pTRV-R1 and pTRV-R2-LeCBP1 constructs were significantlydecreased compared to wild type or infiltrated controls (Fig. 3C).After 4 weeks infiltration, the tomato fruits were carved to exam-ine the phenotype of the seeds. As shown in Fig. 3D, the numberof and size of the seeds in tomato fruit with silenced expressionof LeCBP1 was obviously reduced, and some seeds were stuntedand shriveled. These results indicated that LeCBP1 might play animportant role in seed development of tomato fruit.

4. Discussion

Seedless grapes have high commercial value and seedlessnessis one of the principle objectives of grapevine breeding. Most seed-lessness in grapevine is considered as a result of stenospermocarpy(Korkutal, 2005), which may involved in a combinative effect ofmultiple genes (Bouquet and Danglot, 1996). So far, genetic mech-anism of stenospermocarpic seedless grapes still remains elusive.Previous studies in grapevine seedlessness were mainly focusedon the isolation of the genes involved in stenospermocarpy. Amongthem, suppression subtractive hybridization (SSH) was widely usedto isolated the candidate genes related to seedlessness that weredifferentially expressed during flower development both in seededand seedless lines (Hanania et al., 2007, 2009). In our previousstudy, a cDNA library of V. vinifera cv. Thompson Seedless fruit atdifferent stages of embryo development was constructed (Zhang,2007), of which one of the EST sequences was identified withEF-hand calcium-binding protein domain and displayed differentexpression profile in embryo, compared to the seeded grapevinespecies. Based on the findings, we further cloned the full-lengthcDNA and investigate its functional role in grapevine embryo devel-opment.

Some studies have suggested the protein with the EF-handmotif plays a critical role during embryonic development in ani-mals (Gersdorff et al., 2006; Webb and Miller, 2003). In plants,several EF-hand calcium-binding protein, including calmodulins(CaMs) (Gawienowski et al., 1993; Zielinski, 2002), Ca2+-dependentprotein kinases (CPKs) (Harmon et al., 2000), Calcineurin B-likeprotein/salt-overly-sensitive 3 family (CBLs/SOS3) (Kudla et al.,1999; Liu and Zhu, 1998), Ca2+-binding protein in pollen (Doligezet al., 2002), have been well-documented in A. thaliana. How-ever, the function of EF-hand calcium-binding protein remainsunclear in plant embryo development process. Here, we conducteda functional analysis of a grapevine EF-hand calcium-binding pro-tein involved in embryo development. As is known, the complexgenetic background of V. vinifera and the low-efficiency of thegrapevine genetic transformation system limit to obtain the func-tional information of the target gene in grapevine. However, the

sequence homologies between grapevine and tomato provided ussome information on the possible function of the grapevine EF-hand calcium-binding protein. Therefore, we applied TRV-VIGSapproach to analyze the function of LeCBP1 in tomato, which may

S. Wang et al. / Scientia Horticulturae 130 (2011) 708–714 711

Fig. 1. Sequence analysis of the EF-hand calcium-binding protein sequence of the V. vinifera Thompson Seedless (VvCBP1). (A) Schematic representation of VvCBP1 primarystructure and loci in the grapevine genome. (B) Amino acid sequence of alignment of VvCBP1 and the representative closely related proteins from plant species. Vv, Vitisvinifera; Le, Lycopersion esculentum; At, Arabidopsis thaliana; Pt, Populus trichocarpa. (C) Phylogenetic analysis of VvCBP1 and closely related proteins. The tree was generatedbased on the deduced amino acids of the 14 proteins using the Clustal method of MegAlign program.

712 S. Wang et al. / Scientia Horticulturae 130 (2011) 708–714

Fig. 2. RT-PCR analysis the expression of the VvCBP1 transcripts in grapevine. (A) The phenotype of the V. vinifera cv. Thompson seedless embryo 40 d after full-bloom. (B)The phenotype of the V. vinifera cv. Pinot Noir embryo 40 d after full-bloom. (C) The VvCBP1 transcripts expression in 8 stages in V. vinifera cv. Thompson Seedless. (D) TheVvCBP1 transcripts expression in 8 stages in V. vinifera cv. Pinot Noir. GAPDH was used as an internal control. Quantitative analysis of the VvCBP1 transcripts accumulationwas performed by densitometry using Bio imaging Systems software (Syngene) for 3 independent experiments. Each value was normalized to the GAPDH controls. Error barsindicate SD. Lines 1–8 indicated after full-bloom 15 d, 20 d, 25 d, 30 d, 35 d, 40 d, 45 d, and 50 d.

Fig. 3. Silencing of VvCBP1 homolog in Lycopersion esculentum based on TRV-VIGS. (A) Detected the TRV-R2 sequence in transformed plants by RT-PCR. (B) PCR-SouthernBlot tested the TRV-R2 vector in transformed tomato. (C) RT-PCR detected the efficiency of silence of the target gene. (D) The phenotype of the transformed tomato fruit.M: DL2000 DNA marker, I: L. esculentum wild type, II: L. esculentum treated with suspension solution, III: L. esculentum treated with TRV-R1 and TRV-R2 empty vector, IV: L.esculentum treated with TRV-R1 and TRV-LeCBP1. LeUbi was the internal control.

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ndirectly reflect the function of VvCBP1 in grapevine. Using thesetrategies, Hanania et al. (2007, 2009) have studied the functions ofh-Cpn21 and S27a of grapevine, Liu et al. (2004) have investigatedhe function of the Nicotiana benthamiana DEF ortholog (NbDEF).hese studies indicated the feasibility of this method in analyz-ng the function of the homologous gene. However, we noted thathe TRV-VIGS could not completely silence the target gene, whichtill showed a weak expression after gene silencing (Fig. 3). Moremportantly, we observed that there was a significantly differenthenotype between the silenced plants and control demonstrated.hese results indicated a critical function of VvCBP1 in embryoevelopment.

In conclusion, we report that the isolation and functional analy-is of a novel EF-hand calcium-binding protein gene, VvCBP1, from. vinifera cv. Thompson Seedless. Expression analysis illustratedhat VvCBP1 displays a different expression pattern from that of thembryo in the seeded cultivar. Moreover, VIGS result demonstratedhat silencing the tomato homolog of VvCBP1 can significantlyecrease the seed number of tomato fruits. These results suggestedhat the VvCBP1 is essential for embryo development and embryobortion in grapevine.

cknowledgments

We are grateful to Prof. Dinesh-Kumar (Yale University) for hisermission to use the pTRV1 and pTRV2 vectors. We thank Ph.D.iaofeng Cui (Shanghai Institutes for Biological Sciences, Chinesecademy of Sciences) for critical review and comments on theanuscript. This work was supported by the National Public Benefit

Agricultural) Research Foundation of China (grant no. 200903044)nd the earmarked fund for Modern Agro-industry Technologyesearch System (grant no. Nycytx-30-zp-06).

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