基因启动子甲基化导致红梨产生芽变

The red sport of ‘Zaosu’pear and its red-striped pigmentation pattern are associated with demethylation of the PyMYB10promoterMinjie Qian a ,1,Yongwang Sun a ,1,Andrew C.Allan c ,d ,Yuanwen Teng a ,⇑,Dong Zhang a ,b ,⇑aDepartment of Horticulture,The State Agricultural Ministry Key Laboratory of Horticultural Plant Growth,Development &Quality Improvement,Zhejiang University,Hangzhou 310058,Zhejiang Province,China bCollege of Horticulture,Northwest A&F University,Yangling 712100,Shaanxi Province,China cPlant and Food Research,Mount Albert Research Centre,Auckland 1142,New Zealand dSchool of Biological Sciences,University of Auckland,Auckland 1020,New Zealanda r t i c l e i n f o Article history:Received 1March 2014Received in revised form 9July 2014Available online xxxx Keywords:PearPyrus hybrid Rosaceae AnthocyaninDNA demethylation Red skin mutation Striped patterna b s t r a c t‘Zaosu’pear,a hybrid of Pyrus pyrifolia and Pyrus communis ,is a popular cultivar developed in China.‘Zaosu Red’is a bud sport of ‘Zaosu’with red shoots,young leaves,and fruit.After grafting of ‘Zaosu Red’,reverse mutations in some branches lead to a loss of colour in leaves and stems.Also,the mature fruit of ‘Zaosu Red’exhibits two phenotypes;fully red and striped.The aim of this study was to establish the mechanism of the red colour mutation in ‘Zaosu’and the striped pigmentation pattern in fruit of ‘Zaosu Red’.The accumulation of anthocyanins and transcript levels of the genes PpUFGT2and PyMYB10were highly correlated.The open reading frames (ORF)and promoter regions of these two key genes were cloned and compared between ‘Zaosu’and its bud sports,but no sequence differences were found.The R2R3MYB,PyMYB10,can activate expression of genes encoding enzymes of the anthocyanin biosynthetic pathway.A yeast one-hybrid assay showed that PyMYB10was associated with the À658to À172bp frag-ment of the PpUFGT2promoter,probably via a MYB binding site (MBS)located at À466bp.The PyMYB10promoter had lower methylation levels in anthocyanin-rich tissues,indicating that the red bud sport of ‘Zaosu’pear and the striped pigmentation pattern of ‘Zaosu Red’pear are associated with demethylation of the PyMYB10promoter.Ó2014Elsevier Ltd.All rights reserved.1.IntroductionAnthocyanins are water-soluble pigments that are responsible for most of the red,purple,and blue coloration of flowers and fruit in various plant species,for example apple (Malus Âdomestica Borkh.)(Espley et al.,2007;Telias et al.,2011),grape (Vitis vinifera L.)(Hichri et al.,2011;Kobayashi et al.,2004),maize (Zea mays L.)(Cocciolone and Cone,1993),common morning glory (Ipomoea purpurea )(Park et al.,2007)and European pear (Pyrus communis L.),(Dussi et al.,1995),respectively.These pigments also play important roles in plant disease resistance (Nicholson and Hammerschmidt,1992)and protection against ultraviolet radia-tion (Bieza and Lois,2001),and have been shown to have antioxi-dant and other bioactive properties in experiments on human cell lines (Veeriah et al.,2006).Anthocyanins are derived from a branch of the flavonoid path-way and a number of anthocyanin biosynthetic genes have been isolated,including PAL (phenylalanine ammonia-lyase),CHS (chal-cone synthase),CHI (chalcone isomerase),F3H (flavanone 3-hydroxylase),DFR (dihydroflavonol 4-reductase),ANS (anthocyani-din synthase),and UFGT (UDP-glucose:flavonoid 3-O -glucosyl-transferase)(Jaakola,2013).In pear,the two predominant anthocyanin compounds are cyanidin-3-galactoside (1)and cyani-din-3-arabinoside (2)(Fig.1)(Dussi et al.,1995),and most antho-cyanin biosynthetic genes have family members,which have been isolated for PpPAL (2members),PpCHS (4members),PpCHI (3members),PpDFR (2members),and PpUFGT (2members)(Qian et al.,2014;Yu et al.,2012).Three transcription factors,R2R3-MYB,bHLH (basic helix–loop–helix),and the WD40repeat-con-taining protein orthologous to TRANSPARENT TESTA GLABRA1(TTG1),activate expression of genes encoding their biosynthetic steps by forming the MYB-bHLH-WD40complex,which regulates anthocyanin biosynthesis at the transcriptional level (Broun,2005;Gonzalez et al.,2008)./10.1016/j.phytochem.2014.08.0010031-9422/Ó2014Elsevier Ltd.All rights reserved.⇑Corresponding authors.Address:Department of Horticulture,The State Agri-cultural Ministry Key Laboratory of Horticultural Plant Growth,Development &Quality Improvement,Zhejiang University,Hangzhou 310058,Zhejiang Province,China (Y.Teng).Tel./fax:+8657188982803.E-mail addresses:ywteng@ (Y.Teng),afant@ (D.Zhang).1These authors contributed equally to this work.Sequence mutations in the coding(Hichri et al.,2011)or pro-moter regions(Kobayashi et al.,2004)of genes related to anthocy-anin biosynthesis can disrupt anthocyanin accumulation and cause colour changes in plant organs.Such mutations include:nucleotide substitutions,deletions,and insertions,and transposon insertions and deletions.Such changes can also affect the transcription fac-tors which control anthocyanin levels.In tobacco,flowers of plants over-expressing the grape transcription factor VvMYB5b showed stronger red pigmentation than that offlowers of plants with a mutated VvMYB5b,which has a single amino acid change within the R2domain(Hichri et al.,2011).In the common morning glory (Ipomoea purpurea),whiteflowers result from a number of muta-tions including:a transposon insertion into a bHLH(Park et al., 2007);a nucleotide insertion into a WD40;or a nucleotide deletion from a MYB(Chiu et al.,2010).A retrotransposon insertion in the promoter region of VvmybA1was responsible for pigmentation pattern in the white-skinned grape cultivars‘Italia’and‘Muscat of Alexandria’(Kobayashi et al.,2004).In apple,a striking pheno-type with red leaves and red fruitflesh is the result of a rearrange-ment in the upstream regulatory region of MYB10;this generates an auto-regulatory locus.The auto-regulation is the result of the protein MYB10further up-regulating expression of MYB10,leading accumulation of anthocyanin(Espley et al.,2009).DNA methylation also underlies the appearance of plant colour variants.A study of red-skinned‘Max Red Bartlett’pear and its green-skinned sport established that hyper-methylation of the PcMYB10promoter is associated with the green-skinned pheno-type(Wang et al.,2013).In the apple cultivar‘Ralls’,there is a higher level of DNA methylation in the promoter region of MdMYB1 in the striped red phenotype than in the blushed sport(Xu et al., 2012).The variegated colour of the maize pericarp is also due to differential DNA methylation in the enhancer region(5Kb upstream of the transcription start site)of p1,a gene involved in phlobaphene biosynthesis(Sekhon and Chopra,2009).The striped phenotypes of both maize and apple are due to differential DNA methylation levels in the promoter regions of key MYB transcrip-tion factors(Cocciolone and Cone,1993;Telias et al.,2011).Fur-thermore,the extensive methylation and transcriptional silencing of the Lcyc gene causes radially symmetricalflowers in a Linaria vulgaris mutant(Cubas et al.,1999).‘Zaosu’pear,a hybrid of‘Pingguoli’(Pyrus pyrifolia Nakai)‘Mishirazu’(Pyrus communis L.),is cultivated extensively in North China.Recently,‘Zaosu Red’pear,a bud sport of‘Zaosu’, with young red shoots,leaves,and fruit,was discovered in Shaanxi Province,China(Fig.2).After the grafting of‘Zaosu Red’,some branches with young green shoots and leaves(no fruit set),here designated as‘Green Zaosu Red’,were found(Fig.2).A suppression subtractive hybridization library and proteomic analysis of‘Zaosu’and‘Zaosu Red’was made,but this did not explain the genetic basis of these variations(Hu et al.,2012;Liu et al.,2011).DNA methylation is known to contribute to the red-colour-loss muta-tion of the European pear‘Max Red Bartlett’(Wang et al.,2013). This raised the possibility that a similar mutation process underlies the red colour mutation in‘Zaosu’.Previous research has also shown that Chinese pears show pigmentation patterns that differ substantially from those of European pear at different develop-ment stages(Huang et al.,2009;Steyn et al.,2005),under bagging treatment,and in various postharvest irradiation conditions(Qian et al.,2013).Also,the mature fruit of‘Zaosu Red’exhibits two phe-notypes:fully red and striped.The aim of this study was to investigate the molecular mecha-nism underlying the colouration of the red sport of‘Zaosu’,as well as that responsible for the striped pattern of the fruit skin.Antho-cyanin levels and expression of genes related to anthocyanin bio-synthesis in different pear organs were analysed,and a hierarchical clustering analysis was conducted.The sequences of coding and promoter regions of PpUFGT2and PyMYB10were com-pared,and the interaction between PyMYB10and the promoter region of PpUFGT2was evaluated using a yeast one-hybrid(Y1H) assay.Also,DNA methylation levels in the PyMYB10promoter region were analysed.These analyses established the molecular mechanism for colouration in these lines of Chinese pear.2.Results2.1.Anthocyanin concentration in different tissues/organsTissues of‘Zaosu Red’contained high concentrations of antho-cyanin,with6.3mg100gÀ1in young leaves,8.7mg100gÀ1in fruitlets,and6.1mg100gÀ1in mature fruit(Fig.3).Anthocyanin concentrations were lower in tissues of‘Zaosu’and‘Green Zaosu Red’,with0.5mg100gÀ1and0.9mg100gÀ1in young leaves, respectively(Fig.3).No anthocyanin was detected in peels of fruit-lets and mature fruit of‘Zaosu’(Fig.3).For the mature fruit of ‘Zaosu Red’,peels of the fully red phenotype showed the highest anthocyanin concentration(16.1mg100gÀ1).The anthocyanin concentration in red-stripe enriched peel(11.0mg100gÀ1)was also much higher than that in green-stripe enriched peel(3.2mg 100gÀ1)(Fig.3).2.2.Transcript levels of genes related to anthocyanin biosynthesis in different tissues and organsGenes related to anthocyanin biosynthesis in pear have been isolated(Qian et al.,2014;Yu et al.,2012),and most genes are part of a small gene family.Transcript levels of genes PpPAL1,PpPAL2, PpCHS1,PpCHS2,PpCHI1,PpCHI2,PpCHI3,PpF3H,PpDFR1,and PpANS were significantly higher in young tissues than in mature tissues,regardless of the anthocyanin concentrations in these tis-Fig.1.Structures of cyanidin-3-galactoside(1)and cyanidin-3-arabinoside(2).sues(Supplementary Fig.1).By comparison,the transcript levels of PpUFGT2and PyMYB10were most strongly correlated with antho-cyanin accumulation.Higher mRNA levels of these two genes were detected in anthocyanin-rich tissues including young leaves and fruitlets,mature fruit,peels of the fully red fruit,and the red-stripe enriched peel of‘Zaosu Red’fruit,when compared with containing little anthocyanin(young leaves of‘Zaosu’andZaosu Red’,fruitlets and mature fruit of‘Zaosu’,and green-stripe enriched peel of‘Zaosu Red’fruit)(Fig.4).2.3.Hierarchical clustering analysisMost genes involved in anthocyanin biosynthesis together,and higher expression levels of these genes detected in young tissues(Fig.5).PpUFGT2and PyMYB10 clustered into one group because of the strong correlationtheir transcript levels and anthocyanin accumulationAnother cluster included PpCHS3and PpDFR2,as their expression levels were relatively low in all plant tissues(Fig.5).Becauseits unique transcription profile,PpCHS4clustered alone(Fig.parison of coding and promoter regions of PpUFGT2 PyMYB10in different tissues and organsThe coding and promoter regions of PpUFGT2and PyMYB10 were cloned and sequenced from‘Zaosu’,‘Zaosu Red’and‘Green Zaosu Red’.The promoter region of PpUFGT2was isolated using the Genome Walking Kit as this sequence has not yet been reported.The obtained sequence was designated as proUFGT2Tissues and organs of‘Zaosu’pear,‘Zaosu Red’pear and‘Green Zaosu Red’pear.Young shoots of(A)‘Zaosu’,(B)‘Zaosu Red’,and(C)‘Green Zaosu Red’;Fruitlet and(E)‘Zaosu Red’;Mature fruit of(F)‘Zaosu’,(G)Fully red and(H)red-striped phenotypes of‘Zaosu Red’fruit.(For interpretation of the references to colour legend,the reader is referred to the web version of this article.)Anthocyanin concentrations in different tissues of‘Zaosu’,‘Zaosu Red’,andZaosu Red’pears.Zs-YL,young leaves of‘Zaosu’pear;ZsR-YL,young leaves ofRed’pear;GzsR-YL,young leaves of‘Green Zaosu Red’pear;Zs-fruitlet,peel of‘Zaosu’pear;ZsR-fruitlet,fruitlet peel of‘Zaosu Red’pear;Zs-MF,fruit peel of‘Zaosu’pear collected in2011;ZsR-MF,mature fruit peel ofRed’pear collected in2011;ZsR(GS)-peel,green-striped peel of‘Zaosu Red’in2012;ZsR(RS)-peel,red-striped peel of‘Zaosu Red’collected in2012;(FR)-peel,fully red peel of‘Zaosu Red’collected in2012.Values arestandard error of three replicates.Transcript levels of PpUFGT2and PyMYB10in different tissues of‘Zaosu’‘Zaosu Red’pear and‘Green Zaosu Red’pear.Zs-YL,young leaves of‘Zaosu’ZsR-YL,young leaves of‘Zaosu Red’pear;GzsR-YL,young leaves of‘Greenpear;Zs-fruitlet,fruitlet peel of‘Zaosu’pear;ZsR-fruitlet,fruitlet peel of‘Zaosupear;Zs-MF,mature fruit peel of‘Zaosu’pear collected in2011;ZsR-MF,mature fruit peel of‘Zaosu Red’pear collected in2011;ZsR(GS)-peel,green-stripedof‘Zaosu Red’collected in2012;ZsR(RS)-peel,red-striped peel of‘Zaosucollected in2012;ZsR(FR)-peel,fully red peel of‘Zaosu Red’collected inValues are mean±standard error of three replicates.(GenBank accession number KF822631).Three MYB-binding(MBS)potentially involved in activation of expression were foundproUFGT2,as well as a number of light-responsive elementsincluding Sp1,an I-box,a GT1-motif,a GAG-motif,and a G-boxTable1).The sequences of the open reading frames(ORFs)PpUFGT2and PyMYB10were1482-bp and735-bp long,respec-tively.The lengths of the cloned promoter sequences of PpUFGT2clustering analysis of anthocyanin levels,and log2transcription levels of anthocyanin structural and regulatory genes.Clustering expressed in tissues of‘Zaosu’pear,‘Zaosu Red’pear,and‘Green Zaosu Red’pear using TIGR MeV v.3.0.1and Pearson’sZs-YL,young leaves of‘Zaosu’pear;ZsR-YL,young leaves of‘Zaosu Red’pear;GzsR-YL,young leaves of‘Green Zaosu Red’fruitlet peel of‘Zaosu Red’pear;Zs-MF,mature fruit peel of‘Zaosu’pear collected in2011;ZsR-MF,mature fruit peel green-striped peel of‘Zaosu Red’collected in2012;ZsR(RS)-peel,red-striped peel of‘Zaosu Red’collected in2012;ZsR2012.Red boxes indicate higher levels of expression;green boxes indicate lower levels of expression.Colour brightness to colour scale at the top of thefigure.(For interpretation of the references to colour in thisfigure legend,the readerThis was associated with higher transcript levels of PyMYB10and higher anthocyanin concentrations compared with those in red-stripe enriched peel and green-stripe enriched peel (Figs.3,4and 7).There was a significantly higher methylation level of CNG and CNN in the À587bp to À277bp region in green-stripe enriched peel than in red-stripe enriched peel (Fig.7).3.DiscussionBecause of their similar genetic backgrounds but contrasting pigmentation patterns,bud mutants have proven to be excellent material to study the molecular mechanisms of anthocyanin bio-synthesis.Bud mutants have been used in,amongst others,apple (Ben-Yehudah et al.,2005;Xu et al.,2012),grape (Azuma et al.,2009;Kobayashi et al.,2004),and European pear (Thomas et al.,2010;Wang et al.,2013).Previous research has shown that the red-colour-loss mutation in European pear ‘Max Red Bartlett’was because of DNA methylation in the PyMYB10promoter (Wanget al.,2013).In contrast,the mutation in ‘Zaosu’with green fruit skin to a red morph was associated with DNA de-methylation in the promoter of PyMYB10.Thus,DNA methylation/de-methylation can inhibit/activate the transcription of PyMYB10,subsequently suppressing/inducing the expression of key structural genes involved in anthocyanin biosynthesis (e.g.,UFGT ).The results of this study show that the striped pigmentation pattern in the mature fruit of ‘Zaosu Red’correlated well with DNA methylation patterns in the PyMYB10promoter.This finding is consistent with those of other studies on striped pigmentation in apple (Telias et al.,2011),and maize (Cocciolone and Cone,1993).The transcript levels of PpUFGT2were strongly correlated with anthocyanin accumulation (Figs.4and 5).UFGT is one of the last enzymes in the anthocyanin biosynthetic pathway,catalysing the formation of stable anthocyanins by adding sugar moieties to the anthocyanin aglycone.UFGT is regarded as the rate-limiting step in anthocyanin biosynthesis (Montefiori et al.,2011).In litchi (Litchi chinensis ),among six genes encoding anthocyanin biosyn-thetic steps,only UFGTs showed transcript levels that significantlyTable 1Cis -acting elements within the promoter region of PpUFGT2.Motif Strand Distance from ATG Sequence Putative functionABRE À609CCTACGTGGC Abscisic acid responsivenessÀ320TACGTG AE-box À664AGAAACAALight-responsive module AT1-motif +533AATTATTTTTTATT Light-responsive module Box 4+815ATTAAT Light responsivenessCGTCA-motif +238CGTCA Element involved in MeJA-responsiveness G-Box+320CACGTA Element involved in light responsiveness GAG-motif +100GGAGATG Light-responsive element GT1-motif +722GGTTAA Light-responsive element+255GGTTAATHD-Zip 1+103CAAT(A/T)ATTG Differentiation of palisade mesophyll cells I-box À100cCATATCCAAT Light-responsive element PyMYB10and promoter of PpUFGT2as determined by Y1H assay.Diagrams of the PpUFGT2promoter deletion indicated by black triangles.Numbers under dotted line indicate locations of MBSs,and numbers on the right Each fragment of the UFGT promoterwas ligated to pAbAi vector to generate pAbAi-bait plasmids.AD-MYB and then transformants were screened on selective synthetic dextrose medium lacking Leu/AbA.An control.M.Qian et al./Phytochemistry xxx (2014)xxx–xxx5correlated with the pericarp anthocyanin content(Wei et al., 2011).In‘Max Red Bartlett’pear and its green-skinned sport, PcUFGT expression was consistent with anthocyanin accumulation (Wang et al.,2013).Our results suggest that PpUFGT2plays a dom-inant role in anthocyanin biosynthesis in‘Zaosu’,‘pears.R2R3MYB transcription factors have been implicated in the regulation of anthocyanin biosynthesis in various plant species including pear(Feng et al.,2010),apple(Espley et al.,2009, 2007;Telias et al.,2011),grape(Azuma et al.,2009;Kobayashi et al.,2004),and Chinese bayberry(Niu et al.,2010).Specific R2R3MYBs were reported to activate the promoter regions of anthocyanin biosynthetic genes such as DFR(Niu et al.,2010), and UFGT(Wang et al.,2013),thereby promoting anthocyanin accumulation.In Chinese sand pear,strong up-regulation of PyMYB10was detected during anthocyanin biosynthesis at differ-ent developmental stages(Feng et al.,2010),under bagging treat-ment(Qian et al.,2013),and under postharvest irradiation(Qian et al.,2013;Zhang et al.,2012).3.1.An absence of sequence variations in PpUFGT2and PpMYB10 between pear sportsSequence variations in the coding region of genes related to anthocyanin biosynthesis can alter colour phenotypes.In Chinese bayberry,when compared with the MrMYB1allele in the red–pur-ple cultivar‘Biqi’,the MrMYB1d allele in the white and red cultivars ‘Shuijing’and‘Dongkui’have a1-bp deletion at nucleotide30of the coding region.This suggested that a non-sense mutation in the MYB1protein might be responsible for an absence or low expression of MYB1in white and red fruit,respectively(Niu et al.,2010).In tobacco,plants expressing the grape VvMYB5b dis-played a significantly stronger red pigmentation inflowers than plants over-expressing a mutated VvMYB5b,which had a single amino acid change in the R2domain(Hichri et al.,2011).Sequence variations in promoter regions are also common.Such variations in promoter sequences of transcriptional regulators can explain some of the pigmentation mutations in grape(Kobayashi et al.,2004) and apple(Espley et al.,2009).However,in the present study,there were no sequence variations in the coding and promoter regions of PpUFGT2and PyMYB10when comparing‘Zaosu’,‘Zaosu Red’and ‘Green Zaosu Red’pears(Supplementary Figs.2–5).3.2.PyMYB10associates with promoter region of PpUFGT2The transcription of UFGT is regulated by R2R3MYBs in Arabid-opsis(Shan et al.,2009),Chinese bayberry(Niu et al.,2010),apple (Espley et al.,2007),and pear(Feng et al.,2010;Qian et al.,2013; Wang et al.,2013;Zhang et al.,2012).The specific MYB binds to the MYB binding site(MBS)motif in the promoter region of UFGT to activate its expression.An analysis of the sequence of proUFGT2 revealed three MBS motifs(Table1).MBS motifs were also detected PpPAL1,PpCHS1,PpCHI1,PpF3H,PpDFR1and PpANS(data not shown).The yeast one-hybrid(Y1H)assay showed that PyMYB10can activate the transcription in vivo from the sequence located atÀ658toÀ172bp of proUFGT2,which contains a MBS site atÀ466bp(Fig.6).This suggests that PyMYB10binds to this MBS site to regulate the expression of PpUFGT2.In‘Max Red Bartlett’pear,six MBS motifs were found in the promoter region of UFGT, and two of them were able to associate with PcMYB10(Wang et al.,2013).DNA methylation may affect gene expression by interfering with the RNA-polymerase transcription complex or by preventing binding of additional factors needed for transcription(Telias et al.,2011).In the present study,there were higher transcript lev-els of PyMYB10in anthocyanin-rich tissues,but no sequence vari-ations in the ORF and promoter sequences among the different colour phenotypes.Further analyses established that the methyla-tion levels of CG,CNG,and CNN in theÀ1206bp toÀ892bp region of PyMYB10promoter,as well as the methylation levels of CNG and CNN in theÀ587bp toÀ277bp region,were significantly lower in anthocyanin-rich tissues than in tissues with low levels of antho-cyanin(Fig.7).This result suggested that the lower methylation levels in these regions allowed increased PyMYB10transcription, resulting in greater accumulation of anthocyanin.In‘Honeycrisp’apple,different transcript levels of MYB10in red versus green stripes were inversely associated with methylation levels in the promoter region,but the observed methylation differ-ences are modest(Telias et al.,2011).By comparison,striped pig-mentation pattern in‘Zaosu Red’is more obvious,and the methylation differences are more significant(Figs.2and7).In European pear‘Max Red Bartlett’and its green-skinned sport,a high methylation level of PcMYB10promoter regions(-1,649to À1,218bp andÀ911toÀ604bp)was responsible for the green-skinned phenotype(Wang et al.,2013).In‘Zaosu Red’the methyl-PyMYB10promoter regions(A:À1206bp toÀ892bp,B:À926bp toÀ622bp,C:À587bp toÀ277bp,D:À322bp toZs-fruitlet,fruitlet peel of‘Zaosu’pear;ZsR-fruitlet,fruitlet peel of‘Zaosu Red’pear;ZsR(GS)-peel,green-striped peelred-striped peel of‘Zaosu Red’collected in2012;ZsR(FR)-peel,fully red peel of‘Zaosu Red’collected in2012.(For interpretation the reader is referred to the web version of this article.)ation differences were observed in the À1206bp to À892bp and the À587bp to À277bp promoter regions of PyMYB10(Fig.7),indicating that DNA methylation in several regions of the promoter will cause a change of PyMYB10transcriptional activity,and lead to the pigmentation change in pear.DNA methylation appears to be a common epigenetic mechanism to regulate anthocyanin synthesis in members of the Rosaceae.4.ConclusionsDifferences in anthocyanin levels among ‘Zaosu’and its bud sports,as well as the different fruit colour phenotypes (fully red and striped),are caused by differences in the transcript levels of PyMYB10,which encodes the MYB transcription factor that regu-lates PpUFGT2expression.Transcript levels of PyMYB10were asso-ciated with the DNA methylation level in its promoter,especially in the À1206bp to À892bp and the À587bp to À277bp regions.Higher methylation levels were correlated to lower levels of PyMYB10transcription.The results of this study show that the red sport of ‘Zaosu’pear and its red-striped pigmentation pattern are associated with demethylation of the PyMYB10promoter.5.Experimental 5.1.Plant materialAll the materials used in this study were collected from a com-mercial orchard in Weinan City,Shaanxi Province,China.The young leaves and fruitlets of ‘Zaosu’pear and its bud sport ‘Zaosu Red’,and young leaves of ‘Green Zaosu Red’were harvested in May 2011.In July 2011,mature fruit of ‘Zaosu’and ‘Zaosu Red’were collected.In July 2012,mature fruit of ‘Zaosu Red’were re-harvested,and two coloured phenotypes (fully red and striped)were collected.Since stripes cannot be separated into fully red and fully green tissues,these samples are more correctly classified as ‘‘red-stripe enriched’’or ‘‘green-stripe enriched’’.In all cases,leaves and peels were instantly frozen in liq.N 2,and stored at –80°C until extraction of anthocyanin,DNA,and RNA.5.2.Extraction and measurement of total anthocyaninThe total anthocyanin concentration was measured using a pH differential method and is shown as mg cyanidin-3-galactoside (1)per 100g fresh tissue (Dussi et al.,1995).Fruit tissue (1g)was mixed with MeOH containing 0.01%HCl and then centrifuged at 18,514g for 20min at 4°C.The absorbance of a 100l l extract was measured using a DU800spectrophotometer (Beckman Coul-ter,Fullerton,CA,USA)at 510nm and 700nm in buffers with pH 1.0and 4.5.The total anthocyanin content was calculated using the equation A =[(A 510ÀA 700)pH 1.0À(A 510ÀA 700)pH 4.5]with a molar extinction coefficient of cyanidin-3-galactoside (1)of 3.02Â104.5.3.DNA isolation,RNA extraction and cDNA synthesisDNA was isolated using the CTAB method (Doyle,1987).Total RNA was extracted using a modified CTAB method (Zhang et al.,2012).The concentration of total RNA was measured after diges-tion of genomic DNA by DNase I.First-strand cDNA was synthe-sized from 4l g DNA-free RNA using a Revert Aid™First-Strand cDNA Synthesis Kit (Fermentas,Glen Burnie,MD,USA).The cDNA was diluted ten-fold and 2l l was used as the template for gene cloning and real-time quantitative PCR analyses.5.4.Real-time quantitative PCR (Q-PCR)Each Q-PCR reaction mixture contained SYBR Premix Ex Taq™(10.0l l,Takara,Ohtsu,Japan),each primer (0.4l l,10l M),2l l cDNA,and RNase-free H 2O (7.2l l)in a total volume of 20l l.The reaction,performed on a LightCycler 1.5instrument (Roche,Ger-many),started with a preliminary step of 95°C for 30s followed by 40cycles of 95°C for 5s and 60°C for 20s.A template-free con-trol for each primer pair was included for each run.The Q-PCR primers (Supplementary Table 1)were designed using primer 3software (/cgi-bin/primer3/pri-mer3_www.cgi ).All Q-PCR reactions were normalized using the Ct value corresponding to the Pyrus actin gene (PyActin ,JN684184).Each sample was measured three times.5.5.Hierarchical clustering analysisAnthocyanin levels,and genes whose expression showed statis-tical changes were grouped using a two-way hierarchical cluster-ing method using the TIGR MeV v.3.0.1software package (Saeed et al.,2003).Pearson’s distance and average linkage clustering were used for data aggregation.5.6.Cloning of coding and promoter regions of PpUFGT2and PyMYB10Primers for PCR amplification of the coding region of PpUFGT2were designed based on the complete cDNA sequence of PpUFGT2(GenBank accession number JN870846),and those for amplifica-tion of PyMYB10were as described in a previous study on pears (Feng et al.,2010).The primers used to amplify the PpMYB10pro-moter region were based on the sequence supplied by Plant &Food Research,New Zealand.The promoter region of PpUFGT2was iso-lated using a Genome Walking Kit (Clontech,Palo Alto,CA,USA)according to the manufacturer’s ing the promoter sequence obtained for PpUFGT2,primers were redesigned to com-pare sequences among different mutants.All the primers are listed in Supplementary Table 1.PCR products were analysed on 1.0%agarose gels,and for each reaction product,a single fragment was recovered from gels and purified using a DNA purification kit (Takara).The fragment was then ligated into the plasmid pMD18-T vector,transformed into Escherichia coli DH5a compe-tent cells (Takara),and then sequenced (Sangong,Shanghai,China).5.7.Yeast One-hybrid (Y1H)AssayThe Y1H assays were performed using the Matchmaker Gold Yeast One-Hybrid System Kit (Clontech)according to the manufac-turer’s protocols.The fragments of the promoter of PpUFGT2and the whole coding region of PyMYB10were ligated into pAbAi and pGADT7vectors,respectively,to generate the pAbAi-bait plasmid and the construct AD-PyMYB10,respectively.The primers used to clone the PpUFGT2promoter and the PyMYB10coding region are listed in Supplementary Table 1.The pAbAi vector ligated to the PpUFGT2promoter was linearized and transformed into the yeast strain Y1HGold.Transformants were selected on plates con-taining a selective synthetic dextrose medium lacking uracil.The AD-PyMYB10constructs were transformed into strain Y1HGold harbouring pAbAi-bait and screened on SD/-Leu/AbA plates.All transformations and screenings were performed three times.5.8.Analysis of DNA methylation in PyMYB10promoter region Genomic DNA (0.5l g)was treated with sodium bisulfite using an EZ DNA Methylation-Gold kit (D5005,Zymo Research,Orange,CA,USA)according to the manufacturer’s instructions.PCR prod-ucts amplified from the bisulfite-modified DNA were purified andM.Qian et al./Phytochemistry xxx (2014)xxx–xxx7。