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Cloning and Characterization of WOX4 Gene from Vitis vinifera L. Involved in Stem Cell Regulatio

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Available online at、fl,、fI,、fI,.sciencedirect,com 一神 椁 Agricultural Sciences in China 2011,10(12):1861-1871 静 ScienceDirect December 20 l1 Cloning and Characterization of ene from Vitis vinifera L。Involved — in Stem Cell Regulation DAI RuI ,J1N Hai—peng1 ,WANG Zeng1 ,Avihai Per12,XU Hai-ying3,ZHANG Wen ,CHEN Shang一、Ⅷ and MAHui—qinl 1 College ofAgriculture and Biotechnology,China Agricultural University,Beijing 100193,P.R.China 2Department ofFruit Tree Breeding and Molecular Genetics,Agricultural Research Organization,Bet—Dagan 50250,Israel Institute ofForestry and Fruit Tree Sciences,Beijing Academy ofAgricultural Sciences,Beijing 100093 P.R.China Key Lab ofFunctional Dairy Science,Ministry ofEducation ofChina,Municipal Government ofBeijing/College ofFood Science and Nutritional Engineering,China Agricultural University,Beijing 1 00083,P.R.China Abstract Wuschel-related homeobox(wox)genes play essential,speciifc,and sometimes redundant roles in plant embryo development,shoot and root meristem maintenance,and plant development.Though much information was quickly gained with members of the WOX gene family of Arabidopsis,monocotyledonous crops,and gymnospermous conifers, liRle is known about perennial woody plants.In this study,we isolated the first WOX gene family member from grape(Vitis vinifera L.cv.Cabemet Sauvignon),and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI—TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wusche1.Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco.A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines.Besides the expression in the vegetative shoot apical meristem(SAM)of grape shoot tips,VvWOX4 is expressed in dormant winter buds,inflorescence,young leaves,and tendril tips,but not in root tips.In young leaves,the expression of VvWOX4 is strongly upregulated by wounding,and also by plant growth regulators such as 2 mg L 2,4一D,l mg L一 NAA and 1 mg L。。BAP treatments,while downregulation was monitored by 1 mg L i IBA treatment.and there was no response to O.5 mg L~GA3 treatment. Together,our results revealed the first member of grape WOX gene family and indicated different roles and regulation of WOX4 in the perennial woody crop grapevine. Key words:expression characteristics,gene cloning,plant hormone induced expression,Vitis vinifera L.,WOX4 Arabidopsis;it is expressed in a small group of ceils of shoot apical meristem(SAM)and plays an important role in SAM maintenance.Moreover,its activity is re— WUSCHEL(wus)related homeobox(wox)genes quired for plant growth,floral induction,and somatic constitute a crucial family in plant embryonic patteming, embryogenesis(Mayer et a1.1 998,Zuo et a1.2002). stem—cell maintenance,and organ formation(van der At least 15 members are found inArabidopsis WOX Graaff et a1.2009).WUS was originally identiifed from gene family,they are subtly and differentially expressed Received 16 September,2010 Accepted 31 March,2011 。 Correspondece MA Hu-qin,Tel/Fax:+86-10-62731157,E—mail:hqma@cau.edu.cn;CHEN Shang-wu,Tel/Fax:+86-10-62738663,E-mail:swchen@cau.edu.cn These authors contribute equally to this work. O2011,CAAS Allfights reserved PublishedbyElsevierLtd doi:l0 1016/S1671-2927(11160186—7 1 862 in a lineage—specific manner in the cells ofArabidopsis embryos,and marked in embryonic pattern formation, though a redundant effect was found among different members(Haecker et a1.2004;Deveaux et a1.2008). Wuschel and WOX are divided by the character of homeodomain(HD)amino acid sequence,speciifcally. It is 4 1 amino acid long in WOX proteins,whereas it is 42 amino acid in WUS orthologues due to an extra Y residue(Nardmann et a1.2007).Besides the common and unknown function of WUS—box,WUS and WOXs are of little conservation in the other part of the se— quence(Palovaara and Hakeman 2008). WOX genes are also isolated from economically im— portant crops and trees。WOX2 ofPicea abies is highly expressed during somatic embryo development and declines as the embryo matures(Palovaara and Hakman 2008).WOX3 in rice is involved in leaf development; the expression is excluded from the shoot apical met- istem(Dai et a1.2007).WOX5 is reported to play a role similar to WUS in shoot meristem,and induced expression with plant hormone auxin is reported in Mec ̄cago truncatula(Imin et a1.2007).WOX9 is re- po ̄ed to act in the central zone of SAM where it main— tains the stem—cell population by positively regulating the activity of WUS(Wu eta1.2005).Though hte func— tion is largely unclear in the majority of WOXmembers (Nardmann et a1.2009),it is known that they express asymmetrically and may be involved in the cell differ— entiation process. Compared with Arabidopsis,mOnOcOtyled0nOus crops,and gymnospermous conifers,there is little in- formation available for WOXs of dicotyledon woody plants.Grapes are one of the most important fruit crops in the world and as such,there is great economical importance tied to cultivation aspects such as the shoot and root apical meristems formation,germination of winter and summer buds,adventitious root formation ofcuttings,in vitro somatic embryo initiation and plantlet regeneration or direct organogenesis under the plant growth regulator treatments,which are all tightly con— nected with expression of the regulatory genes in the basic cellular process.However,information about grape WOXgene family and the function ofdifferent members in the up—-mentioned economic important pro・- cess remains largely unknown.In this research,we have cloned the first grape WOX gene(designated as DAIRu et al Vv WOX4),and heterologously overexpressed it in to— bacco which remarkably increased the regeneration of shoot meristems on transgenic tobacco calli,thus con— ifnning its morphological development regulatory ufnc— tion in plant.The expression of VvWOX4 in grapevine was found actively in growing shoot tips and quiescent winter buds,wounding inducible,and with multi—plant hormones regulated paRems. MATERIALS AND METHODS Cloning and sequence analysis of VvWOX Total DNAfrom shoot tips ofgrapevine r vinlieFo L. cv.Cabernet Sauvignon)was isolated using a CTAB method(Porebski et a1.1 997).TWo primers.i…e for. ward primer r5 一ATGGGAAGCATGAAGGTGCA TCAG11_3 and backward primer f5,-TCATCTGCCT TCCGGGTGCA ACGGG一3 1.were designed to clone putative 0 according to conserved sequences of a玎d the grape genome sequences(Jaillon et a1.2007; Velasco et a1.2007).PCR ampliifcation and gene splic— ing by overlap extension(SOE)method,which is a PCR—based method of recombining DNA sequences without reliance on restriction sites and of directly gen— erating mutated DNA rfagments in vitro rHorton et a1. 1 989)were used to synthesize the uf11一length gene and delete the introns.The PCR products were cloned into pMD一1 9一T vector(TaKaRa.Japan)and sequenced.The DNA sequence and the amino sequence were blasted on the web browser htto://blast.ncbi.nlm.nih.gov. HeterOgen0us expression and validation of VvWO× PcR primers designed with two restriction enzyme cutting sites I and Xho I(primer.F.5 一CCATA TGGGAAGCATGAAGGTG.3 :primer-B。5"-CCCTC GAGTCATCTGCCTTCCGGGT_3 、were used to clone the intron free 口 from the splicing by overlap extension fSOE product.The final prokaryotic ex— pression vector pET-3 lb(+ 一VvW0X was仃ansformed into Escherichia coli BL2 1(DE3)/pLysS for 0 protein expression with 0.5 mmol L~isopropy—D—D— thiogalactoside(IPTG)as inducer in Luria—Bertani fLB、 @2011 CAAS All ifghts reserved Published by Elsevier Ltd Cloning and Characterization of WOX4 Gene from Vitis vinifera L.Involved in Stem Cell Regulation l 863 medium.The recombinant protein was extracted from E.coli,separated by SDS—PAGE and stained by and berries(5 0 d after flower.DAF)using a CTAB method(Iandolino et a1.2004).Total RNA concentra. tion was measured in triplicates before and after dilu— tion to l mg mL~.Potential genomic DNA contamina— Coomassie brilliant blue(Candiano et a1.2004).The target band of VvWOXwas cut from SDS—PAGE and prepared according to our previously reported method don in the resulting RNA samples was removed by rteat— ment with RNase—free DNase I.First eDNA strands were synthesized using the SuperScriptTM Reverse Tran— (Zhang et a1.2008),and further identiifed by matrix assisted laser desorption ionization—time of light mass fspectroscopy(MALDI—TOF MS)and peptide mass ifn- scriptase RT-PCR Kit(Invitrogen.USA).For semi— gerprinting(PMF)Mascot. A rabbit anti-AtWUS polyclonal antibody was pre— pared and veriifed according to the procedure described by Wang et a1.(2009).This antibody(1:l 000 dilution) was used as first antibody and goat anti—rabbit IgG conjugated with horseradish peroxidase(HRP)r 1:1 000 dilution)as secondary antibody(Sigma,USA)for West— ern blots. Tobacco transformation and phenotypic observation The () PcR products(with 5,-GATCCATGGG AAGCATGAAG GTGCATCA一3 and 5 r_TAGTCAGATC TACGCCTTCCGGGTGCA.3 as primers and S0E product as the template)were inserted into PMD—l 9一T vector.and transformed into E coli DH5tx competent cells.The isolated plasmid and plant expression vector pCAMBIA1 302 were digested with Nco I and egl II, respectively.After overnight ligation,the products were transformed into E coli DH5 Oc competent cells.Finally. the plasmid,i.e.,plant expression vector pCAMBIA— VvWOX4一GFP was transformed into Agrobacterium tumefaciens strain EHA1 05.Transformation oftobacco leaf discs was performed according to Horsch et a1. r l 985).The transformed tissue and plantlets were se 1ected by 1 0 mg L~hygromycin,and further identified by PCR and I PCR.The phenotype was either di— rectly observed with stereo..microscope or after a stan.. dard treatment procedure with scanning electron mi— croscope(Hitachi S一3400N,Japan). RNA preparation fr0m grape tissue and semi— quantitative RT-PCR Total RNA was extracted rfom ifeld grown grape shoot tips,dormant winter buds,young inflorescence,young leaves(about 1 cmx 1 cm in size),tendril tips,root tips, quantitative RT—PCR(sqRT—PCR).primers 5 一 ACATCCAGGT GGGACGC一3 and 5"-CGCTTCTGCT TCTGTCTC.3 were used ofr Vv rC .and 5"-TGGCCT TCGGGATCGGAGTAA一3 and 5 一ATCCCTGGTC GGCATCGTTTA1'.3 were used for 1 8S rRNA. respectively. The sqRT-PCR cycling parameters were set as ofllows:50。C f0r 2 min.1 cycle;95。C for 5 min.1 cycle;95。C for 15 s。49。C for 30 s,72。C for 30 s 3l cycles;and 72。C for 8 min,1 cycle,using RealMasterMix(Tiangen,China).All sqRT-PCR am— pliifcations were perofrmed in triplicates ofr each RNA sample. Wound and plant hormone induced VvWOX4 expression Leaves of tissue cultured graDe plantlets were cut from the petiole,two extra cuts were made on the midrib with a sharp razor.Leaves were soaked in half strength liquid MS medium containing either 0.5 mg L一 gibberellic acid (GA ),1 mg L~6-benzylaminopurine(BAP),2 mg L。 2,4一dichIorophenoxyacetic acid(2,4一D),1 mg L一 1一naphthlcetic acid(NAA)or 1 mg L~indole一3一butyric acid(IBA1 serially ofr 10 min.For each rteatment 15 leaveswereusedandhalfstrengthliquidMSwihtoutplnat hormonewastaken asthe contro1.Afterblotting drythe surface,leaveswereputonhalfsrtengthMS solidmedium, and samples were taken at 2,12,24,and 72 h after hte rteatments.RNAextractionand semi—quantitativeRT-PCR were carried out as mentioned above. RES U LTS Cloning and sequence analysis of VvWOX4 The cloned DNA sequence was 1 1 62 bp including wto @2011.CAAS All dghtsre9erved PublishedbyElsevierLtd Cloning and Characterization of WOX4 Gene from Vitis vinifeFa L.Involved in Stem Cell Regulation 1 869 regulators are widely used.Phytohormones such as auxin and cytokinin are important for meristem regula— tion(Leyser 2003).Synthetic auxin 2,4一D is widely used in the stem cell formation related tissue culture processes such as the induction of grape embryogenic calli rfom diferentiated anther cells(Perl et a1.1 995). The upregulation of Arabidopsis WOX5 was recorded 4 h after 20 ̄tmol L。 IAA or 20 gmol L~2,4一D treat— ment fGonzali et a1.2005).In our results,a l 0一min treatment with a roughly half dose of 2,4一D as used by Gonzali et a1.(2005)obviously increased expression of in a whole period of 2 to 72 h.while the effect Of NAA was less remarkable.For cytokinins. upregulation Of was found when the shoot apex was treated with 1 mmol L~BAP.with the highest in— duction level found at 24 h after the treatment.simulta— neously downregulated CL l was recorded(Lindsay Pf a1.2006).However.it must be borne in mind that the BAP cOncentratiOn used by the authors was hun— dreds of times higher than the normal dose added in grape tissue culture,thus here we demonstrate that BAP at a concentration of 4.44 gmol L~upregulated the ex- pression of Vv lo .The downregulation of Vv was found under 4.92 gmol L0 IBA.IBA is rfequently used to prompt grape root formation,and the downregulation of Vv could be regarded as a new balance between the growth of the aerial part and the root.which is in agreement with the fact that the ex. pression of was not found in the root tips of grapevine.Differentially expressed by plant growth regulators was previously reported by Imin et a1. (2007).where Medicago truncatula WoX5 was upregulated by 1 0 gmol L‘ NAA and downregulated by 4 gmolL一 BAP. In our results wounding remarkably increased the expression of .There was no report on the direct relationship between wounding and the expres— sion of or ra in plants.Signals arose by wound— ing such as jasmonic acid,ethylene,ABA,and others were suggested to mediate wound—-activated gene ex・・ pressions(Leon et a1.200 1).Apart from a controlled expression,wounding mediated expression can be also initiated by the senescence hormone signaling pathway, or by the gene promoter region located on the cis-・ele-・ ment determined pathway,such as the D 7 gene in aerial and subterranean organs(Nishiuchi et a1.1 999). The detailed and exact process of VvWOX4 on the wounding response certainly requires further investigation. CONCLUSION In this research we cloned the first WoX gene family member VvWOX4 from the perennial woody plant,the grapevine( vinifeFa L.).The identity of the gene was validated by Western blotting and tobacco transformation where the overexpression of transformants demons ̄ated a similar phenotype to that Of .The expression Of D was limited in the aerial part of grapevines,with higher expression in shoot tips,dormant winter buds,and inflorescence, while lower expression was detected in young leaves and tendril tips.The upregulation of VvWOX4 by 2,4一 D,NAA,and BAP,and the downregulation by IBA were in line with the root meristem exclusive expression pattern.The character of both the aerial parts and the plant hormone induced expression of Vv WOX4 sug— gests further research on its role in not only grapevine winter and summer bud formation,but also shoot and lateral shoot growth,inflorescence development,and somatic embryogenesis. 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