1 running title: corresponding author: tianhong li, ph.d. subject

A Role for PacMYBA in ABA-Regulated AnthocyaninBiosynthesis in Red-Colored Sweet Cherry cv. Hong Deng(Prunus avium L.)Xinjie Shen1, Kai Zhao2, Linlin Liu1, Kaichun Zhang3, Huazhao Yuan1, Xiong Liao1, Qi Wang1,Xinwei Guo1, Fang Li1 and Tianhong Li1,*1Department of Pomology, Key Laboratory of Stress Physiology and Molecular Biology for Tree Fruits of Beijing, College of Agriculture andBiotechnology, China Agricultural University, Beijing, 100193, PR China2College of Horticulture and Landscape, Yunnan Agricultural University, Kunming, 650201, PR China3Institution of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Science, Beijing, 100093, PR China*Corresponding author: E-mail, [email protected]; Fax, +86-01062733957(Received September 13, 2013; Accepted January 13, 2014)

The MYB transcription factors and plant hormone ABA havebeen suggested to play a role in fruit anthocyanin biosyn-thesis, but supporting genetic evidence has been lacking insweet cherry. The present study describes the first functionalcharacterization of an R2R3-MYB transcription factor,PacMYBA, from red-colored sweet cherry cv. Hong Deng(Prunus avium L.). Transient promoter assays demonstratedthat PacMYBA physically interacted with several anthocya-nin-related basic helix–loop–helix (bHLH) transcription fac-tors to activate the promoters of PacDFR, PacANS andPacUFGT, which are thought to be involved in anthocyaninbiosynthesis. Furthermore, the immature seeds of transgenicArabidopsis plants overexpressing PacMYBA exhibited ec-topic pigmentation. Silencing of PacMYBA, using aTobacco rattle virus (TRV)-induced gene silencing technique,resulted in sweet cherry fruit that lacked red pigment. ABAtreatment significantly induced anthocyanin accumulation,while treatment with the ABA biosynthesis inhibitor nordi-hydroguaiaretic acid (NDGA) blocked anthocyanin produc-tion. PacMYBA expression peaked after 2 h of pre-incubationin ABA and was 15.2-fold higher than that of sweet cherriestreated with NDGA. The colorless phenotype was alsoobserved in the fruits silenced in PacNCED1, which encodesa key enzyme in the ABA biosynthesis pathway. The en-dogenous ABA content as well as the transcript levels ofsix structural genes and PacMYBA in PacNCED1-RNAi(RNA interference) fruit were significantly lower than inthe TRV vector control fruit. These results suggest thatPacMYBA plays an important role in ABA-regulated antho-cyanin biosynthesis and ABA is a signal molecule that pro-motes red-colored sweet cherry fruit accumulatinganthocyanin.

Keywords: Abscisic acid (ABA) � Anthocyanin � Promoter �

R2R3-MYB transcription factor � Sweet cherry � Virus-induced gene silencing (VIGS).

Abbreviations: ABRE, ABA-responsive element; ANS, antho-cyanidin synthase; bHLH, basic helix–loop–helix; CaMV,Cauliflower mosaic virus; CHS, chalcone synthase; CHI, chal-cone isomerase; CDS, coding sequence; DAFB, days after fullbloom; DFR, dihydroflavonol 4-reductase; F3H, flavanone3-hydroxylase; GFP, green fluorescent protein; LUC, luciferase;NCED, 9-cis-epoxycarotenoid dioxygenase; NDGA, nordihy-droguaiaretic acid; PAL, phenylalanine ammonia lyase; qRT-PCR, real-time quantitative PCR; RT–PCR, reverse transcrip-tion–PCR; RACE, rapid amplification of cDNA ends; RNAi,RNA interference; REN, Renilla; TF, transcription factor;TRV, Tobacco rattle virus; TIS, transcription initiation site;UFGT, UDP-glucose:flavonoid 3-O-glucosyltranferase; UTR,untranslated region; VIGS, virus-induced gene silencing.

Introduction

Anthocyanins are the major water-soluble pigments in higherplants and are responsible for the blue, purple and red colors ofmany fruits and vegetables (Mano et al. 2007, Yuan et al. 2009,Yamagishi et al. 2010, Zifkin et al. 2012). These pigments belongto a diverse group of ubiquitous secondary metabolites knownas flavonoids (Winkel-Shirley 2001), and are believed to have avariety of health-promoting benefits, such as providing protec-tion against oxidative stress, certain cancers, cardiovascular dis-ease and age-related degenerative diseases (Butelli et al. 2008).Anthocyanin compounds also play an important reproductiverole, functioning as attractants in numerous plant or animalinteractions (Allan 2008). The anthocyanin biosynthetic path-way is part of the flavonoid pathway, which is a branch of thephenylpropanoid pathway (Winkel-Shirley 2001). Most of thegenes involved in anthocyanin biosynthesis and regulation wereisolated and characterized in model plants (Grotewold et al.2006, Allan et al. 2008). These genes are involved in boththe early step of dihydroflavonol biosynthesis, including

Plant Cell Physiol. 55(5): 862–880 (2014) doi:10.1093/pcp/pcu013, available online at www.pcp.oxfordjournals.org! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

862 Plant Cell Physiol. 55(5): 862–880 (2014) doi:10.1093/pcp/pcu013 ! The Author 2014.

Regu

larP

aper

Downloaded from https://academic.oup.com/pcp/article-abstract/55/5/862/1810071by gueston 13 February 2018

phenylalanine ammonia lyase (PAL), chalcone synthase (CHS),chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H),as well as in the successive reactions in anthocyanin production,including dihydroflavonol 4-reductase (DFR), anthocyanidinsynthase (ANS) and UDP-glucose:flavonoid 3-O-glucosyltran-ferase (UFGT) (Takos et al. 2006). Recently, the transcriptionfactors (TFs) that regulate the expression of the structural genesinvolved in anthocyanin biosynthesis have been reported inseveral species (Allan et al. 2008, Laitinen et al. 2008). In horti-cultural crops, the R2R3-MYB TFs are known to be importantregulators of anthocyanin biosynthesis. For instance, VvMybA,MdMYB10, GmMYB10, MrMYB1 and PcMYB10 regulate genesinvolved in the anthocyanin biosynthetic pathway in Vitis vini-fera (grapevine), Malus domestica (apple), Garcinia mangostana(mangosteen), Myrica rubra (Chinese bayberry) and Pyrus spp.(pear), respectively (Kobayashi et al. 2002, Espley et al. 2007,Palapol et al. 2009, Niu et al. 2010, Li et al. 2012). The R2R3-MYBTFs have been shown to interact closely with basic helix–loop–helix (bHLH) TFs (Nesi et al. 2001, Zimmermann et al. 2004,Tohge et al. 2005, Hichri et al. 2011). Examples of functionalpartnerships between color-related MYBs and bHLHs includethe Antirrhinum Ros1 and Del bHLHs, Zea mays ZmC1 MYBand ZmB bHLH, apple MdMYB10 and MdbHLH3, and gentianGtMYB3 and GtbHLH1 (Winkel-Shirley 2001, Espley et al. 2007,Allan et al. 2008, Nakatsuka et al. 2008).

Sweet cherry (P. avium L.) is an economically important horti-cultural crop cultivated in temperate regions around the world,with a production in 2011 grossing an estimated US$2 billionworldwide (http://www.fao.org). The accumulation of anthocya-nin pigments in sweet cherry fruit is an important determinantof fruit quality. Recent research showed that cyanidin-3-O-ruti-noside is the main anthocyanin in red-colored cherry, and cher-ries with red-colored fruits have more antioxidant activity thando bicolored cherry cultivars (Liu et al. 2011). As a typical non-climacteric fruit, sweet cherry exhibits a biphasic growth pattern(double sigmoid growth curve) and accumulates anthocyaninmainly in the second growth phase, during a short period (ap-proximately 11 d from the beginning of turning red). In compari-son with climacteric fruits, which are strongly dependent onethylene production for ripening, sweet cherry ripening doesnot require increased ethylene biosynthesis (Gong et al. 2002).In contrast, ABA plays an important role in the ripening of non-climacteric fruits, such as strawberry and sweet cherry (Sethaet al. 2005, Ren et al. 2010, Jia et al. 2011). Although the tran-scriptional regulation of anthocyanin biosynthesis has been stu-died extensively in several horticultural crops, little is knownabout the transcriptional regulation and molecular physiologyof anthocyanin biosynthesis in red-colored sweet cherry (Winkel-Shirley 2001, Espley et al. 2007, Palapol et al. 2009, Niu et al. 2010,Hichri et al. 2010, Li et al. 2012).

The present study describes the identification and func-tional characterization of the sweet cherry R2R3-MYB protein,PacMYBA. This TF was found to contain the conserved R2R3-MYB domain signature and to localize to the nucleus of theplant cell. Overexpression of PacMYBA in Arabidopsis plants

induced ectopic pigmentation in immature seeds. Moreover,PacMYBA physically interacted and cooperated with severalbHLH TFs to regulate the expression of anthocyanin pathwaygenes. We also established a new Tobacco rattle virus (TRV)-induced gene silencing technique, which provides an opportun-ity to regulate gene expression levels in sweet cherry fruit. Thisstrategy was then used to alter the expression of PacMYBA,resulting in sweet cherry fruit that lacked red pigment.Consistent with these results, the expression of PacMYBA waspositively associated with anthocyanin accumulation duringred color development in sweet cherries. We further foundthat ABA significantly induced anthocyanin synthesis, whilethe ABA biosynthesis inhibitor NDGA (nordihydroguaiareticacid) strongly suppressed it in the yellow stage of sweetcherry fruit development. Additionally, all six structural genesof the anthocyanin pathway were significantly up-regulatedupon ABA treatment compared with samples subjected toNDGA treatment. Likewise, PacMYBA expression was up-regu-lated by ABA, but down-regulated by NDGA treatment.Silencing of the PacNCED1 gene controlling ABA levels whichpotentially modulates ABA signaling also resulted in sweetcherry fruit that lacked red pigment. The endogenous ABAcontent as well as the transcript levels of six structural genesand PacMYBA in PacNCED1-RNAi (RNA interference) fruit weresignificantly lower than in the TRV vector control fruit. Theseresults suggest that PacMYBA and ABA are important regula-tors of anthocyanin biosynthesis in red-colored sweet cherries.

Results

Physiological and morphological changes duringred-colored sweet cherry fruit development

Based on the fruit skin color of sweet cherry cv. Hong Deng, wedefined eight visual developmental stages: small green (SG),mid green (MG), big green (BG), degreening (DG), yellow(YW), initial red (IR), full red (FR) and dark red (DR) at about7, 12, 18, 24, 27, 31, 37 and 42 d after anthesis, respectively(Fig. 1A). We observed that ‘Hong Deng’ sweet cherry fruitshad two periods of rapid development under our field condi-tions (from about 46 d after anthesis to ripeness), and exhibiteddistinct physiological and morphological changes, includingchanges in fruit color, size and weight, as well as in levels ofChl, anthocyanin and soluble sugars (Fig. 1). Notably, the levelsof Chl declined continually after the SG stage (Fig. 1C), whereasthe anthocyanin content increased rapidly after the YW stage(Fig. 1D). Levels of soluble sugars (glucose, fructose and sorb-itol), particularly glucose, showed two periods of rapid increaseconcomitant with the DG and FR stages (Fig. 1E). No sucrosewas detected in the sweet cherry fruits of ‘Hong Deng’.

Isolation and sequence analysis of PacMYBA

To isolate and characterize the sweet cherry MYB TF that playsa role in anthocyanin biosynthesis, we designed two degenerate

863Plant Cell Physiol. 55(5): 862–880 (2014) doi:10.1093/pcp/pcu013 ! The Author 2014.

Regulation of anthocyanin synthesis in sweet cherry


http://www.fao.org

Fig. 1 Physiological and morphological changes in red-colored sweet cherry cv. ‘Hong Deng’ fruit during development. Cherry developmentalwas divided into the following eight stages: small green (SG), mid green (MG), big green (BG), degreening (DG), yellow (YW), initial red (IR), fullred (FR) and dark red (DR). (A) Changes in fruit color and size. (B) Changes in fruit weight. (C) Changes in Chl content. (D) Changes inanthocyanin content. (E) Changes in soluble sugar content (Sor, sorbitol; Fru, fructose; Glu, glucose). Error bars represent the SE of threereplicates.


X. Shen et al.


primers based on the R2R3 domain. We obtained a single pu-tative R2R3-MYB gene product, which we named PacMYBA,using RACE (rapid amplification of cDNA ends) PCR. The1,282 bp amplification product was the full-length cDNA ofPacMYBA. The predicted protein of PacMYBA contained 224amino acids, with an isoelectric point of 8.51, and a calculatedmolecular mass of 26 kDa. Alignment of the predicted proteinsequence of PacMYBA with that of other anthocyanin-relatedMYB TFs in the region of the R2R3 domain indicated a highdegree of similarity (Fig. 2A). Compared with apple MdMYB10,PacMYBA shared 84% amino acid identity in the R2R3 DNA-binding domain and 68% identity over the whole protein. Thelevel of amino acid identity with anthocyanin-related R2R3-MYBs (whole protein sequences) from bayberry (MrMYB1),grape (VvMYBA1) and pear (PyMYB10) was 47, 44 and 67%,respectively. A bootstrapped phylogenic tree generated usingMEGA version 5.0 showed that PacMYBA was closely related toMdMYB10 (Fig. 2B). Moreover, a signature motif specificallyrequired for the interaction between MYB and bHLH proteins(Grotewold et al. 2000, Zimmermann et al. 2004) was also foundin the R3 domain of PacMYBA. These results suggest thatPacMYBA might have the same function as MYBs, which areknown to be important regulators of anthocyanin biosynthesis.

The genomic sequence of PacMYBA was subsequently PCR-amplified from genomic DNA using gene-specific primers.Sequence analysis revealed that the PacMYBA clone, with amolecular size of about 3.1 kb, included the approximate-ly1.5 kb upstream region of the start codon. Alignment andcomparison with the corresponding genomic and cDNA se-quences revealed that the gene consisted of three exons andtwo introns (Fig. 2C). The first intron (311 bp) resided in the R2domain, between amino acids 40 and 41, and the second intron(599 bp) in the R3 domain, between amino acids 82 and 83.

Motif analysis of the 1.5 kb region upstream of the startcodon, which is predicted to correspond to the promoter, re-vealed 11 types of cis-acting regulatory elements (Table 1).Interestingly, two ABRE (ABA-responsive element) motifswith the core sequences TACGTG and CCTACGTGGC, respect-ively, were present that were previously shown to be involved inABA responsiveness in Arabidopsis (Simpson et al. 2003).Further, a TATA box at –28 bp from the transcription initiationsite of PacMYBA and an MBS motif, which is a binding motif ofplant R2R3-MYB proteins involved in drought inducibility, werefound in this upstream region.

Subcellular localization, transcriptional activationactivity, and tissue-specific expression ofPacMYBA

To analyze the subcellular localization of PacMYBA, we con-structed a vector harboring a fusion of PacMYBA with greenfluorescent protein (GFP) (PacMYBA-GFP). This plasmid wasintroduced into Arabidopsis thaliana protoplasts, and thefusion protein, which was visualized by fluorescence micros-copy, localized to the nucleus (Supplementary Fig. S1A).

The nuclear localization of PacMYBA was consistent with itspredicted function as a TF.

To test the transactivation ability of PacMYBA, we used ayeast system based on a yeast strain that harbored dual reportergenes, His3 and LacZ, both controlled by the GAL4 upstreamactivation sequence. All transformants containing pBD-PacMYBA, pGAL4 (positive control) and pBD (negative con-trol) grew well on the SD/–Trp medium (Supplementary Fig.S1Ba). However, yeast cells containing the pBD or pBD-PacMYBA plasmids did not grow on the SD/–Trp-Hismedium (Supplementary Fig. S1Bb). In contrast, yeast cellsharboring the pGAL4 plasmid survived on the SD/–Trp-Hismedium and stained blue in a b-galactosidase assay(Supplementary Fig. S1Bc). Therefore, PacMYBA might nothave transcriptional activation ability.

Expression patterns of PacMYBA in different tissues andorgans of red-colored sweet cherry cv. Hong Deng were exam-ined under normal conditions (Supplementary Fig. S1C).PacMYBA was constitutively expressed in almost all organsand tissues examined, including young leaves, flowers, carpo-podia, phloem and mature fruits. PacMYBA expression wasgreatest in the skin of mature fruits, followed by pulp, flowers,carpopodia, phloem and young leaves, suggesting thatPacMYBA functions mainly in fruit.

Anthocyanin accumulation and the expression ofanthocyanin biosynthetic genes during fruitripening

The pattern of anthocyanin accumulation was analyzed during alleight developmental stages (from SG to DR) of red-colored sweetcherry ripening. No anthocyanin accumulated before the YWstage. The concentration increased to 19.43mg g�1 FW at the IRstage, and continued to rise during subsequent ripening, finallyreaching 580.67mg g�1 FW in the DR stage (Fig. 1D). The expres-sion of six anthocyanin biosynthetic genes (PacCHS, PacCHI,PacF3H, PacDFR, PacANS and PacUFGT) and of PacMYBA ateach developmental stage is shown in Fig. 3. The early genes(PacCHS, PacCHI and PacF3H) were expressed at high levelsduring the early stages (SG, MG and BG), declined during veraison(i.e. the onset of ripening; DG, YW and IR), and increased again inthe last two stages (FR and DR) (Fig. 3A–C). In accordance withthe pattern of anthocyanin accumulation during ripening, the lategenes (PacDFR, PacANS and PacUFGT) and PacMYBA were ex-pressed at low levels during the early developmental stages (SG,MG and BG), and expression rapidly increased during veraison(DG, YW and IR) and remained at high levels through to the finaldevelopmental stage (Fig. 3D–G). These results suggest that thetranscript levels of the late genes are closely correlated withanthocyanin accumulation during fruit development.

Isolation and analysis of promoters of PacDFR,PacANS and PacUFGT

PCR-based genomic walking was used to isolate the promoterregions of PacDFR (KF974775), PacANS (KF974776) and




http://pcp.oxfordjournals.org/lookup/suppl/doi:10.1093/pcp/pcu013/-/DC1






Fig. 2 Phylogenetic relationship between the deduced amino acid sequence of PacMYBA and anthocyanin-related MYBs from other species, andgenomic sequence analysis of PacMYBA. (A) Protein sequence alignment of PacMYBA with known anthocyanin MYB regulators from otherspecies. The R2R3-binding domain is underlined. Gray boxes with an arrow indicate specific residues that form the motif implicated in the bHLHcofactor interaction in Arabidopsis (Espley et al., 2007). Motif 6 was identified by Stracke et al. (2001) in the C-terminal domain of Arabidopsis


X. Shen et al.

(continued)


PacUFGT (KF974777), of sizes 712, 1,386 and 734 bp, respect-ively. The regulatory regions of all sequences were analyzedusing PLACE and the PlantCare database. Several putative cis-regulatory elements involved in plant development were iden-tified in these promoters. Two ABA-responsive elements (ABREand CACGTG) were found in the PacDFR promoter [+219 and+196 from the transcription initiation site (TIS)] and one ABA-responsive element (GCCGCGTGG) was found in the PacUFGTpromoter (+13 from the TIS). Several MYB-binding sites, suchas MYBCORE (CNGTTR) and MYB-PLANT (MACCWAMC),were present in all of the promoters analyzed.

PacMYBA activates the promoters of flavonoidpathway genes, which are required foranthocyanin synthesis, in transientlytransformed tobacco leaves

To identify the structural genes of the flavonoid pathway thatare activated by PacMYBA, we conducted transient expressionexperiments using tobacco leaves and the dual luciferase assay

system. Promoters of the flavonoid pathway genes PacDFR,PacANS and PacUFGT were introduced into the pGreenII0800-LUC vector (Hellens et al. 2005) so that activationwould be detected as an increase in LUC activity. Two R2R3-MYB TF genes, AtPAP1 and MdMYB10, which regulate antho-cyanin biosynthesis in Arabidopsis and apple (Zimmermannet al. 2004, Espley et al. 2007), were selected for this analysisin addition to PacMYBA. All TFs were driven by the Cauliflowermosaic virus (CaMV) 35S promoter and were co-transformedwith bHLH class putative anthocyanin biosynthesis regulatorsfrom Arabidopsis and apple, namely AtbHLH2, AtbHLH42 andMdbHLH33. PacMYBA significantly increased the promoteractivity of PacDFR when it was co-transformed with bHLH TFs(Fig. 4A). The highest activity was observed when PacMYBAwas co-infiltrated with AtbHLH42, although MdbHLH33 couldalso significantly activate PacDFR. PacMYBA also activatedthe PacANS promoter, with an approximate 13.8-fold induc-tion compared with the negative control (Fig. 4B). The antho-cyanin-specific promoter of PacUFGT was also significantly

Fig. 2 Continuedanthocyanin-related MYBs. The accession numbers of these proteins, or translated products, in the GenBank database are as follows: AtPAP1

(CAB09230), AtPAP2 (NP176831), CaA (CAE75745), VvMYB1 (AB242302), OsC1 (HQ379703), PhAN2 (AAF66727), IbMYB1 (AB258984), PacMYBA

(KF974774), MaMYB (EU305682), GhMYB10 (CAD87010), MrMYB1 (GQ340767), AmROSEA1 (ABB83826), OgMYB1 (EF570115), LeANT1

(AAQ55181), PmMBF1 (AAA82943), MdMYB10 (EU518249) and ZmC1 (AAA33482). (B) Phylogeny of PacMYBA and MYB proteins from other species

involved in the regulation of anthocyanin biosynthesis. The tree was constructed using MEGA (version 5.0). A minimum evolutionary phylogeny test and

1,000 bootstrap replicates were chosen for the analysis. The scale bar represents 0.2 substitutions per site. (C) Structure of the PacMYBA genomic

sequence. The transcription initiation site (TIS) and exons are indicated by boxes. The introns, promoter and the 30 UTR are indicated by lines. The TIS is

shown as a triangular gray box. The R2 and R3 repeats that constitute the MYB domain are shown as solid black and shaded boxes, respectively. Numbers

refer to position relative to the transcription initiation site of PacMYBA.

Table 1 Cis-acting elements potentially associated with the PacMYBA gene

Motif Strand Distancefrom the TIS

Sequence Function

ABRE + 524 TACGTG Cis-acting element involved in ABA responsiveness

+ 902 CCTACGTGGC

CCGTCC-box + 1201 CCGTCC Cis-acting regulatory element related to meristem-specific activation

MRE + 1103 AACCTAA MYB-binding site involved in light responsiveness

P-box + 12 CCTTTTG Gibberellin-responsive element

MBS + 36 TAACTG MYB-binding site involved in drought inducibility

G-box + 299 GCCACGTGGAA Cis-acting regulatory element involved in light responsiveness

+ 300 CACATGG

+ 524 TACGTG

+ 1,190 CACGAC

– 524 CACGTA

– 545 GCCACGTGGA

Skn-1_motif + 73 GTCAT Cis-acting regulatory element required for endosperm expression

GARE-motif – 204 TAACAAR Gibberellin-responsive element

TGACG-motif + –151 TGACG Cis-acting regulatory element involved in methyljasmonate responsiveness

+ –192 TGACG

CAT-box – 231 GCCACT Cis-acting regulatory element related to meristem expression

TIS, transcription initiation site.




induced by PacMYBA (approximately 8.3-fold compared withthe negative control) (Fig. 4C). AtPAP1 and MdMYB10 wereselected as positive controls, as they were previously shown toactivate the Arabidopsis DFR promoter when co-transformedwith AtbHLH42 and MdbHLH33, respectively (Zimmermannet al. 2004, Espley et al. 2007). The apple MdMYB8 (a MYB

that clusters into a subgroup unrelated to anthocyanin-promot-ing MYBs) was selected as a negative control with low activityagainst the promoter of PacDFR, PacANS and PacUFGT. Ourresults suggest that the activation of promoters of anthocyaninpathway genes was specific to MYBs belonging to the anthocya-nin-associated clade.

Fig. 3 Expression profile of anthocyanin biosynthetic genes and PacMYBA during sweet cherry fruit development. Real-time PCR was used toanalyze PacCHS (A), PacCHI (B), PacF3H (C), PacDFR (D), PacANS (E), PacUFGT (F) and PacMYBA (G) expression patterns. The relative expressionof target genes was calculated using the formula 2–��ct. Each point represents the mean value of three independent experiments performed intriplicate ± SE, with each triplicate derived from mRNA samples independently isolated from five different trees.


X. Shen et al.


Functional testing of PacMYBA in transgenicplants

The function of PacMYBA was tested in a heterologous sys-tem by introducing PacMYBA cDNA under the transcriptionalcontrol of the CaMV 35S promoter into Arabidopsis plants.

A T2 line containing a single T-DNA insertion was chosenfor subsequent analyses. Our reverse transcription–PCR(RT–PCR) results suggested that PacMYBA was only expressedin the transgenic plants (Fig. 5A). Under a stereomicroscope,the immature seeds of transgenic plants displayed red

Fig. 4 PacMYBA interacts with various bHLH transcription factors to activate promoters of anthocyanin pathway genes. The bHLH transcriptionfactors and promoter fragments used for transfection of tobacco leaves are indicated. (A) PacDFR promoter. (B) PacANS promoter. (C) PacUFGTpromoter. The dual luciferase assay shows the promoter activity of the anthocyanin pathway genes expressed as a ratio of firefly luciferase (LUC)to 35S Renilla luciferase (REN) activity, where an increase in activity equates to an increase in LUC relative to REN. Error bars are the SE for sixreplicate reactions.




pigmentation <10 d after anthesis (Fig. 5B), whereas the seedsof wild-type plants (ecotype Columbia) had no pigmentation(Fig. 5C). These data are consistent with the results of Takoset al. (2006) and Feng et al. (2010), indicating that overexpres-sion of PacMYBA could induce anthocyanin synthesis in imma-ture Arabidopsis seeds. These data show that PacMYBA plays arole in anthocyanin biosynthesis.

Silencing of PacMYBA suppresses anthocyaninaccumulation in red-colored sweet cherry fruit

Previous studies reported that TRV-mediated VIGS (virus-induced gene silencing) is a powerful tool for studyingtomato and strawberry fruit development and ripening (Fuet al. 2005, Jia et al. 2011). However, considering that the hostrange of TRV is largely restricted to herbaceous plants, it wasunclear whether TRV could infect sweet cherry fruits. Therefore,we attempted to apply this technique to sweet cherry fruit.

To confirm further whether PacMYBA is involved in regulat-ing anthocyanin biosynthesis in red-colored sweet cherry fruit,we generated PacMYBA-RNAi fruits using 24 DAFB (days afterfull bloom) BG fruits (Fig. 6A), and control fruits were infiltratedwith TRV alone. Three weeks after the inoculation, the controlfruits turned fully red (Fig. 6B), whereas the inoculated sectionsof the RNAi fruits exhibited white and light green tissues (Fig.6C). The white and light green tissues indicate the absence ofanthocyanin, and this phenomenon was accompanied by a de-crease in the level of PacMYBA transcript (Fig. 6D, E). The tran-script levels of PacDFR, PacANS and PacUFGT were significantlydown-regulated in the RNAi sections compared with the controlfruit. In contrast, the transcript levels of PacCHS, PacCHI andPacF3H were almost the same as those of the control fruits(Fig. 6F). These data support the idea that PacMYBA positively

regulates red-colored sweet cherry fruit accumulating anthocya-nin, and PacMYBA might affect anthocyanin biosynthesis viaactivating the transcript levels of flavonoid pathway genessuch as PacDFR, PacANS and PacUFGT.

To confirm that the TRV vector can infect sweet cherryfruits, two pairs of TRV-specific primers were designed (seeprimer information in the Materials and Methods). The PCRproducts were detected in 4-week-old PacMYBA-TRV-infil-trated and TRV vector fruits, but were not detected in fruitsinfiltrated with Agrobacterium alone (Fig. 6G). These resultsdemonstrated that the TRV vector could indeed infect sweetcherry fruits.

The effects of ABA on coloration and on theexpression of PacMYBA and anthocyaninbiosynthetic genes

Sweet cherry fruit (P. avium L.) is classified as a non-climactericfruit, and therefore does not require increased ethylene biosyn-thesis to ripen (Ren et al. 2010). In contrast, ABA is associatedwith fruit maturation processes, such as sugar accumulationand softening, in sweet cherry (Ren et al. 2010). Red-coloredsweet cherry fruit undergoes rapid color development duringripening. Under our field conditions, fruit coloring changedfrom YW to IR in 6 d. We next examined whether treatmentof YW stage fruits with ABA and NDGA altered the expressionof PacMYBA and of anthocyanin biosynthetic genes, andwhether fruit color was affected.

Fruit color and the concentration of anthocyanins after ABAand NDGA treatments are shown in Fig. 7A and B. No antho-cyanins accumulated in the 2 h period of the in vivo incubationassays [a system in which berry tissues were incubated in vivo inABA- or NDGA-containing medium, essentially according to

Fig. 5 Functional testing of PacMYBA in transgenic plants. (A) PacMYBA transcription levels in wild-type and transgenic plants as determined bysemi-quantitative RT–PCR. AtACTIN2 was used as the internal control. (B) Photographs of immature seeds (<10 d after anthesis) from trans-genic Arabidopsis heterologously expressing PacMYBA. (C) Photographs of immature seeds in the siliques of untransformed plants. Photographsin (B) and (C) are representative of one of three separate lines of the T2 generation, and similar phenotypes were observed for all lines. Eachcolumn represents the mean of three replicates. Error bars on each column represent the SE of three replicates. Bars = 0.1 cm.


X. Shen et al.


the technique of Peng et al. (2003), as detailed in the Materialsand Methods]. However, during our 4 d experiments, ABA sig-nificantly induced (up to 132.37 mg g�1 FW) and NDGAstrongly suppressed (no anthocyanins detectable) anthocyaninaccumulation (Fig. 7A, B). The ABA content of fruits was alsoquantified in ABA-treated, control and NDGA-treated fruits(Fig. 7C). In the 2 h in vivo incubation assay, the ABA contentof ABA-treated fruits was 1.78-fold higher than that of thecontrol and 4.43-fold higher than that of NDGA-treatedfruits. After 4 d of treatment, the ABA content of ABA-treated

fruits was 2.59-fold higher than that of the control and 5.2-foldhigher than that of NDGA-treated fruits. These results suggestthat ABA plays an important role in anthocyanin biosynthesisin red-colored sweet cherry cv. Hong Deng. Moreover, ABAmight initiate anthocyanin biosynthesis in red-colored sweetcherry during veraison.

In the present study, 30mmol l�1 ABA was shown to inducePacMYBA expression in cherry fruits after 0.5 h of in vivo incu-bation. The maximum transcript level of PacMYBA (3.79-foldgreater than the control) was attained after 2 h of in vivo

Fig. 6 Silencing of PacMYBA inhibits the accumulation of anthocyanin in sweet cherry fruit. (A) BG fruits, attached to sweet cherry plants, at 24DAFB were inoculated with Agrobacterium containing TRV alone (control fruit) or TRV carrying a PacNCED1 fragment (RNAi fruit). (B)Phenotypes of the control fruit 2 weeks after inoculation. (C) Phenotypes of the PacMYBA-RNAi fruit 2 weeks after inoculation. (D)Anthocyanin contents in control and RNAi fruit. (E) Semi-quantitative RT–PCR and real-time PCR analysis (lower panel) of the PacMYBAtranscription levels in the control and RNAi fruit. Actin mRNA was used as an internal control. (F) The mRNA expression levels of anthocyaninpathway genes in control and RNAi fruit, in which the PacMYBA transcript was down-regulated by 65%. Actin mRNA was used as an internalcontrol. (G) Analysis of the transcripts of the 560 bp TRV1 and 300 bp TRV2 fragments by RT–PCR in fruits inoculated with Agrobacterium alone(lane 1), control fruit (lane 2) and RNAi fruits (lane 3). Error bars on each column represent the SE of three replicates. Bars = 1.0 cm.




Fig. 7 The effects of ABA and NGDA on fruit coloration, ABA content, expression of PacMYBA and anthocyanin biosynthesis genes. (A) Changesin fruit color of ‘Hong Seng’ treated with ABA and NDGA. (B) Changes in anthocyanin concentration in the berries of ‘Hong Deng’ treated withABA and NDGA. (C) ABA content in berries treated with ABA and NDGA. (D) The expression of PacMYBA in response to ABA and NDGAtreatment. (E) Expression of anthocyanin biosynthesis genes in the berries of ‘Hong Deng’ incubated in ABA (30 mmol l�1) or NDGA


X. Shen et al.

(continued)


incubation. The maximum transcript level was maintained at acomparable level for 2 h and then gradually declined. In the ex-periments involving the ABA inhibitor NDGA, the transcriptlevel of PacMYBA was significantly down-regulated (3.94-foldlower than the control) after 2 h of in vivo incubation. Thislow level of PacMYBA transcript was maintained for 6 h andwas then up-regulated, but remained lower than the levelsobserved in the control or ABA treatment at comparable timepoints. In the 4 d treatments, the transcript level of PacMYBA inplants exposed to ABA was 57.23-fold higher than that of plantsexposed to NDGA (Fig. 7D). The expression of six anthocyaninbiosynthetic genes in response to ABA is shown in Fig. 7E, F. Allsix genes were significantly up-regulated after a 2 h in vivo incu-bation in ABA and were strongly down-regulated by NDGA. Thetranscript levels of PacCHI, PacF3H and PacUFGT were 15.32-,12.21- and 82.43-fold greater in ABA-treated samples than inthe control and were strongly suppressed by NDGA (Fig. 7E).The expression patterns of these six genes after 4 d of ABA orNDGA treatment were similar to those in the 2 h treatment(Fig. 7F). Taken together, these results reveal that PacMYBAis up-regulated by ABA and is suppressed by NDGA, indicatingthat this gene might play an important role in the response toABA.

Down-regulation of a 9-cis-epoxycarotenoiddioxygenase (PacNCED1) suppresses anthocyaninaccumulation in red-colored sweet cherry fruit

To silence PacNCED1 in sweet cherry fruit, a mixture ofAgrobacterium strain GV3101 cultures containing pTRV1 andpTRV2 carrying a 579 bp fragment of PacNCED1 (pTRV2-PaNCED1) was syringe-infiltrated at a ratio of 1 : 1 into 24DAFB BG fruits (Fig. 8A), and control fruits were infiltratedwith TRV alone. Three weeks after infiltration, control fruitsturned fully red (both peel and fresh) (Fig. 8B). In contrast,the RNAi fruits exhibited both normal red tissue and TRV-infiltrated transgenic light green or white tissue in one fruit(Fig. 8C). The light green and white tissues indicate the absenceof anthocyanin and this phenomenon was accompanied by adecrease in the level of PacNCED1 transcript (Fig. 8D, E). TheABA content in RNAi sections of fruit was 1.51-fold lower thanin the control fruit, and the IAA content was 1.97-fold higher(Fig. 8F, G). Moreover, the transcript levels of anthocyaninbiosynthetic genes and PacMYBA were also significantlydown-regulated in the RNAi sections compared with the con-trol fruit (Fig. 8H). These results confirm that ABA plays im-portant roles in anthocyanin accumulation in red-coloredsweet cherry fruit.

Discussion

PacMYBA encodes an R2R3-MYB transcriptionfactor that modulates the flavonoid pathway

R2R3-MYB TFs have been shown to interact with bHLH proteinsto regulate flavonoid biosynthesis in various horticultural crops,such as apple, grape, mangosteen and bayberry (Kobayashi et al.2002, Espley et al. 2007, Palapol et al. 2009, Niu et al. 2010).However, it has hitherto not been determined whether R2R3-MYB TFs play roles in anthocyanin biosynthesis in sweet cherry.The present work characterized PacMYBA, the first sweet cherryR2R3-MYB protein shown to regulate the flavonoid pathway inthe red sweet cherry cultivar ‘Hong Deng’. Conserved regions ofthe R2R3-MYB domain were present in PacMYBA, and the se-quence of this protein was similar to that of other R2R3-MYBsinvolved in the regulation of the flavonoid pathway (Fig. 2).PacMYBA was also found to contain the [D/E]Lx2[R/K]x3Lx6Lx3R motif in the R3 domain, which specifies the inter-actions with R-like bHLH proteins (Zimmermann et al. 2004). AKPRPR[S/T] motif, which was defined by Stracke et al. (2001) asmotif 6 in AtPAP1, was found in the C-terminus of PacMYBA.This motif is conserved in many R2R3-MYB TFs that regulate theflavonoid pathway, such as Capsicum annuum A (CaA),GhMYB10 and MrMYB1, and is modified to [K/R] Pxxx [K/T][F/Y] in AmROSEA1, LeANT1 and MdMYB1. The genomic struc-ture of PacMYBA also revealed an orthologous relationship withR2R3-MYB genes, such as PhAN2, NtAn2 and PyMYB10(Quattrocchio et al. 1999, Feng et al. 2010, Pattanaik et al.2010). PacMYBA contains two introns and three exons, but nosignal peptide. The two introns reside in the R2 and R3 domains,respectively (Fig. 2C). This exon/intron and R2R3 domain distri-bution of PacMYBA was conserved in several anthocyanin-related R2R3-MYB genes, such as the A locus of petunia(Borovsky et al. 2004), NtAn2 and PyMYB10. Multiple cis-actingregulator elements were identified in the promoter of PacMYBA,and these elements are possibly involved in the plant’s responseto gibberellic acid, ABA, methyl jasmonate and drought (Table1). Phylogenetic analysis showed that PacMYBA was homolo-gous to AmROSEA1, MdMYB1, MrMYB1 and other anthocya-nin-related MYBs (Schwinn et al. 2006, Allan et al. 2008, Niu et al.2010). These results indicate that PacMYBA is a novel member ofthe MYB anthocyanin activator family that regulates anthocya-nin biosynthesis. PacMYBA might interact with bHLH TFs andform transcriptional complexes that regulate anthocyanin bio-synthesis in red-colored sweet cherry. However, to our know-ledge, no sweet cherry MYBs or bHLHs required for anthocyaninbiosynthesis have been isolated and characterized to date, and so

Fig. 7 Continued(150 mmol l�1) for 2 h. Tissues incubated in 200 mmol l�1 mannitol were used as the control, and the corresponding expression levels were assigned a

value of 1. (F) Expression of anthocyanin biosynthesis genes in berries of 2-year-old shoots. Shoots with fruits and leaves were cut at 27 DAFB, and the

shoots were incubated at 24�C for 4 d under a 16 h photoperiod in 1 mmol l�1 ABA solution, 150 mmol l�1 NDGA solution or distilled water (untreated

control). Tissues immersed in incubation medium containing equilibrium buffer and an equal volume of distilled water were used as a control. Error bars

represent the SE of three replicates. Bars = 1.0 cm.




their contribution to the regulation of this process remains to bedetermined.

PacMYBA is a positive regulator of red-coloredsweet cherry fruit accumulating anthocyanin

In recent years, much progress has been made toward under-standing the molecular mechanism of R2R3-MYB TFs

regulating anthocyanin biosynthesis in several horticulturalcrops (Espley et al. 2007, Laitinen et al. 2008, Palapol et al.2009, Feng et al. 2010, Niu et al. 2010, Wang et al. 2013). Inthis study, we confirmed that PacMYBA could function as ananthocyanin regulator by introducing the coding region of thecDNA into Arabidopsis plants. The immature seeds of trans-genic Arabidopsis plants overexpressing PacMYBA exhibitedectopic pigmentation (Fig. 5B). To provide further molecular

Fig. 8 Down-regulation of PacNCED1 expression inhibits the accumulation of anthocyanin in sweet cherry fruit. (A) BG fruits, attached to sweetcherry plants, at 24 DAFB were inoculated with Agrobacterium containing TRV alone (control fruit) or TRV carrying a PacNCED1 fragment (RNAifruit). (B) Phenotypes of the control fruit 2 weeks after inoculation. (C) Phenotypes of the PacNCED1-RNAi fruit 2 weeks after inoculation. (D)Semi-quantitative RT–PCR and real-time PCR analysis (lower panel) of the PacNCED1 transcription levels in the control and RNAi fruit. ActinmRNA was used as an internal control. (E) Anthocyanin contents in control and RNAi fruit. (F) ABA content in control and RNAi fruit. (G) IAAcontent in control and RNAi fruit. (H) The mRNA expression levels of anthocyanin pathway genes and PacMYBA in control and RNAi fruit, inwhich the PacNCED1 transcript was down-regulated by 65%. Actin mRNA was used as an internal control. Error bars on each column representthe SE of three replicates. Bars = 1.0 cm.


X. Shen et al.


evidence that PacMYBA could indeed play a role as an antho-cyanin biosynthetic activator in sweet cherry fruit, we silencedthe PacMYBA gene during the fruit development. Silencing wasperformed using VIGS, a method successfully used to silencegenes in fruits such as tomato, strawberry and pear (Fu et al.2005, Chai et al. 2011, Jia et al. 2013, Wang et al. 2013). However,it was hitherto unknown whether this powerful system couldbe used on sweet cherry fruit. In the present study, we success-fully silenced PacMYBA using VIGS, and thereby inhibitedanthocyanin synthesis during sweet cherry fruit development(Fig. 6C–E). The transcript levels of PacDFR, PacANS andPacUFGT were significantly down-regulated in the PacMYBA-RNAi fruit (Fig. 6F), indicating that PacMYBA may affect antho-cyanin biosynthesis via regulating the expression of PacDFR,PacANS and PacUFGT. In previous studies, it has been con-firmed that several R2R3-MYB TFs might be specific regulatorsfor DFR or UFGT transcripts such as VvMYBA1, MdMYB1 andMdMYB10 (Kobayashi et al. 2002, Takos et al. 2006, Espley et al.2007). Taken together, this study has provided physiologicaland molecular evidence to demonstrate that PacMYBA is apositive regulator of anthocyanin biosynthesis in red-coloredsweet cherry fruit. Future studies should focus on identifyingmore anthocyanin-related R2R3-MYB TFs and elucidating theirprecise function in regulating anthocyanin biosynthesis.

PacMYBA regulates the anthocyanin biosyntheticpathway by interacting with different bHLHpartners

The b-galactosidase activity assay revealed that PacMYBAmight have no transcriptional activation activity (Supplemen-tary Fig. S1B). This result suggests that PacMYBA may need tointeract with other anthocyanin-related TFs (such as bHLH fac-tors) to control the expression of the flavonoid pathway struc-tural genes in sweet cherry fruit (Zimmermann et al. 2004,Nakatsuka et al. 2008, Hichri et al. 2011). Analysis of the proteinsequence of PacMYBA revealed a signature motif in the R2R3-MYB-binding domain. The motif has been confirmed to bespecific for the interaction between MYB and bHLH proteinsin maize and Arabidopsis (Grotewold et al. 2000, Zimmermannet al. 2004). In this study, we used a dual luciferase reportersystem to test whether PacMYBA could regulate the anthocya-nin biosynthesis genes in the presence and absence of a bHLHpartner in transformed tobacco leaves (Espley et al. 2007, Espleyet al. 2009). Our transient assays, in which PacMYBA was co-expressed with various bHLH proteins, showed that the pro-moters of the flavonoid pathway genes, PacDFR, PacANS andPacUFGT, were significantly induced (11- to 72-fold) (Fig. 4).The highest transactivation of these promoters with PacMYBAwas dependent on AtbHLH42, which regulates proanthocyani-din biosynthesis in Arabidopsis (Gonzalez et al. 2008). Similarresults were obtained for the bayberry R2R3-MYB proteinMrMYB1, which is involved in the regulation of anthocyaninbiosynthesis (Niu et al. 2010). Apple MdbHLH33 also showedhigh levels of transactivation with PacMYBA against the

promoter of PacDFR. Co-expression of the apple R2R3-MYBTF MdMYB10 with MdbHLH33 also significantly increasedthe transactivation of the AtDFR promoter compared withthe control (Espley et al. 2007). PacMYBA showed the highestlevel of sequence similarity to MdMYB10, which has been con-firmed to regulate anthocyanin biosynthesis in apple (Espleyet al. 2007). Interestingly, the activities of PacDFR, PacANS andPacUFGT gene promoters regulated by PacMYBA/AtbHLH42were different, as our data showed. PacMYBA can physicallyinteract with AtbHLH42 to activate these promoters, especiallythe promoters of PacDFR and PacANS. However, the enhancedactivity of the PacUFGT gene promoter was remarkably weakerthan that of the other two promoters. In grape, VvMYBA1 is aspecific regulator of VvUFGT transcripts (Kobayashi et al. 2002).We suppose that the ability of R2R3-MYB TFs to activate thepromoter of anthocyanin biosynthetic genes is different be-tween plant species. In apple, the expression of MdMYB1 iscorrelated well with MdUFGT, but a transient expressionassay showed that the combination of MdMYB1 and a bHLHTF enhanced the promoter of MdDFR rather than that ofMdUFGT (Takos et al. 2006). These results indicate that atranscriptional complex that includes PacMYBA and a bHLHfactor controls the anthocyanin biosynthetic pathway in sweetcherry.

ABA regulates anthocyanin biosynthesis inred-colored sweet cherry

Sweet cherry is defined as non-climacteric because it does notexhibit a peak in respiration and ethylene production duringripening (Ren et al. 2010). In contrast, ABA levels graduallyincreased concomitant with a decline in IAA during veraisonand the subsequent ripening stages (Supplementary Fig. S2A,B). It has been hypothesized that the ratio of ABA to IAA con-stitutes a signal that triggers fruit ripening above a certainthreshold (Perkins-Veazie 1995, Brady et al. 2003, Jia et al.2011). The onset of anthocyanin accumulation in berries is animportant indicator of ripening, and ABA plays an importantrole in this process (Jiang and Joyce 2003, Sun et al. 2010,Castellarin et al. 2011, Jia et al. 2011). However, supportingmolecular evidence that links anthocyanin accumulation withABA has been lacking to date in red-colored sweet cherry. Inthis study, we provide evidence that ABA plays an importantrole in anthocyanin biosynthesis in red-colored sweet cherryfruits. We demonstrate that exogenously applied ABA pro-motes anthocyanin accumulation, whereas NDGA, whichrepresses ABA levels, prevents anthocyanin from accumulatingin the yellow stage berries (Fig. 7A). Most importantly, weprovide genetic evidence that the transcript levels of six antho-cyanin biosynthetic pathway genes are significantly up-regu-lated in berries subjected to ABA treatment, whereas they arestrongly suppressed upon treatment with the ABA inhibitor(Fig. 7E, F). These data suggest that ABA promotes anthocyaninaccumulation in red-colored sweet cherry. Based on all of theseresults, we hypothesize that the increase in ABA content during








veraison is the key signal that triggers anthocyanin biosynthesisin red-colored sweet cherry.

To provide further molecular evidence that ABA plays animportant role in regulating anthocyanin biosynthesis in red-colored sweet cherry, we down-regulated ABA levels in sweetcherry fruits by silencing a gene (PacNCED1) that encodes a keyenzyme in the ABA biosynthesis pathway (Ren et al. 2010). Weconfirmed that endogenous ABA levels could be reduced bysilencing PacNCED1 (Fig. 8D, F), and blocked anthocyanin bio-synthesis (Fig. 8C, E). In PacNCED1-RNAi fruit, the transcriptlevels of six anthocyanin biosynthetic pathway genes andPacMYBA are significantly lower than in the TRV vector controlfruit (Fig. 8H). Interestingly, the IAA content in the PacNCED1-RNAi fruit is significantly higher than in the control fruit(Fig. 8G). Previous studies demonstrated that ABA is involvedin the IAA signal transduction pathway (Brady et al. 2003, Seoand Park 2009, Seo et al. 2009). We thus hypothesized thatABA regulates IAA synthesis in sweet cherry fruits. The datapresented here indeed suggest that ABA plays an importantrole in regulating anthocyanin biosynthesis in sweet cherryfruits.

ABA regulates PacMYBA expression

In some horticultural crops, light is the main factor that regu-lates the expression of anthocyanin-related R2R3-MYB TFgenes, such as MdMYB1, MrMYB1 and PyMYB10 (Takos et al.2006, Feng et al. 2010, Niu et al. 2010). However, in other horti-cultural crops, some of these TFs, such as GmMYB10 andVvMYBA1, appear to be less affected by light and are regulatedby plant hormones (Palapol et al. 2009, He et al. 2010). In red-colored sweet cherry cv. Hong Deng, bagging treatment did notsuppress or delay anthocyanin accumulation or affect tran-script levels of PacMYBA during berry development (data notshown). In the present study, we deternined the effect on theYW stage of red-colored sweet cherry fruits by in vivo incuba-tion assays and field treatments with ABA and NDGA. NDGAclearly reduced red coloration, ABA biosynthesis and anthocya-nin content, whereas ABA strongly induced anthocyanin bio-synthesis (Fig. 7A). Furthermore, we found that NDGAapplication down-regulated the expression of PacMYBA,which then affected the transcription of anthocyanin biosyn-thetic genes. In PacNCED1-RNAi fruit, the transcript level ofPacMYBA was also significantly lower than in control fruit(Fig. 8H). In addition, we identified two ABA-response elem-ents (ABRE and ACGTG) in the PacMYBA promoter by PLACEand PlantCare database analysis (Simpson et al. 2003,Nakashima et al. 2006). In total, these results indicate thatABA production and PacMYBA expression work closely to-gether to control anthocyanin biosynthesis in red-coloredsweet cherry pigmentation. Moreover, ABA might directly regu-late PacMYBA expression at the transcriptional level. Futurework should also focus on deciphering how plant hormonesand environmental factors affect the expression of R2R3-MYBTFs and anthocyanin pathway genes in red-colored sweet

cherry. Knowledge of the functions and behavior of suchmajor genes is valuable both for managing existing cultivarsand for developing new ones.

Materials and Methods

Plant material and treatments

Fruits of P. avium L. cv. Hong Deng were collected from adulttrees (12 years old) during the 2011 season. Trees were grown atthe Beijing Institute of Forestry and Pomology under field con-ditions. Fruits at different developmental and maturation stageswere periodically harvested from April to May. Fruits and othertissues were cut into small pieces and immediately frozen inliquid nitrogen and stored at –80�C for subsequent analysis.

Two-year-old shoots with fruits and leaves were cut at 28DAFB [yellow (YW) stage], just before turning red, and theshoots were placed under a 16 h light/8 h dark regimen for4 d at 24�C in 1 mM ABA and 150 mM NDGA solution. Theuntreated control contained only distilled water. At 4 d afterthe initiation of treatment, ABA and anthocyanin concentra-tions were analyzed, and the fruits were stored at –80�C beforeRNA extraction.

RNA isolation and detection of the TRV vector

Total RNA was extracted from 1 g of fresh sweet cherry tissuesusing the improved hot borate method (Wan and Wilkins1994). After removing genomic DNA using DNase I(TAKARA), the concentration of RNA was determined, andfirst-strand cDNA was synthesized using M-MLV (TAKARA),in accordance with the manufacturer’s instructions.Oligo(dT)18 was used as the primer. Random primers wereused to reverse transcribe the first-strand cDNA of inoculatedsweet cherry fruit to detect the TRV vector. The method andprimers for detecting of the TRV vector were as described byChai et al. (2011).

Isolation of PacMYBA transcription factor andanthocyanin biosynthesis genes

PacMYBA was cloned using ‘Hong Deng’ cDNA and degenerateprimers designed based on the consensus derived from DNAsequences of the R2R3 DNA-binding domain in diverse species.One cDNA species encoding R2R3-MYB domains was obtained.The 50- and 30-untranslated regions (UTRs) of the sequencewere cloned using a SMARTERTM RACE cDNA AmplificationKit (Clontech). The full-length cDNA, named PacMYBA, wasisolated using gene-specific primers based on the 50 and 30

UTRs. The genomic sequence of PacMYBA was cloned usingprimers F, 50-TGAAATAACTAGCAGGCACAA-30 and R, 50-GATGAAAACAAAGCCAGTGAG-30. PCR conditions were 3 minat 94�C, 35 cycles of 30 s at 94 �C, 30 s at 55�C and 2 min at72�C, with a final extension of 72�C for 10 min. The full-lengthcDNAs of anthocyanin biosynthesis genes (except for PacCHS)were isolated using the same method as described for the


X. Shen et al.


isolation of PacMYBA. All primers were designed usingPrimerPremier 5.0 and are listed in Supplementary Table S1.

Promoter isolation and analysis of PacMYBA,PacDFR, PacANS and PacUFGT

Genomic DNA was isolated from young leaves of ‘Hong Deng’using a Multisource Genomic DNA Miniprep Kit (AXYGEN).The promoter region of PacMYBA, PacDFR, PacANS andPacUFGT was isolated using a Genomic Walking Kit(TAKARA). The nested primers used for genomic walking arelisted in Supplementary Table S1. PCR products wereanalyzed on 1% agarose gels, and single fragments were re-covered from gels and purified using a Gel Midi PurificationKit (TIANGEN).

Sequence analysis

MEGA version 5.0 (Tamura et al. 2011) was used for the phylo-genetic and molecular evolutionary analysis. The PacMYBApromoter was analyzed for cis-acting elements using PLACE(http://www.dna.affrc.go.jp/PLACE/signalscan.htm/) and thePlant-CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Sequence alignments wereassembled using ClustalW (Chenna, 2003), and aligned se-quences were edited using BioEdit.

Localization of PacMYBA–green fluorescentprotein (GFP) fusion proteins

The entire coding region of PacMYBA was amplified using thegene-specific primer pair: 50-GAATTC ATGGAGGGCTATAACTTGGGTGT-30 (EcoRI site underlined) and 50-GTCGACT GTCCTTCTGAACATTGGTACACTG-30 (SalI site underlined). ThePCR product was subcloned into the pE3025-GFP vector togenerate pE3025-PacMYBA-GFP, which contained aPacMYBA-GFP fusion construct under the control of theCaMV 35S promoter, as well as a Tobacco etch virus (TEV)enhancer. The construct was confirmed by sequencing andused for polyethylene glycol (PEG)-mediated transient trans-formation of A. thaliana protoplasts as described by Hichret al. (2010). GFP fluorescence was captured with a NIKONECLIPSE TE2000-E fluorescence microscope after incubationfor 16 h at 22�C.

Analysis of PacMYBA transcriptional activationactivity in yeast

The entire coding region of PacMYBA was amplified with thegene-specific primer pair: 50-GAATTC ATGGAGGGCTATAACTTGGGTGT-30 (EcoRI site underlined) and 50-GTCGACT GTCCTTCTGAACATTGGTACACTG-30 (SalI site underlined). ThePCR product was fused in-frame to the DNA-binding domainvector pBD-GAL4, a yeast expression vector with the promoterand terminator of ADH1, to generate a GAL4 DNA-BD-PacMYBA fusion plasmid, named pBD-PacMYBA. GAL4 is atranscription factor involved in the expression of galactose-induced genes. YRG-2 is a yeast strain that carries the His3

and LacZ reporter genes. This strain cannot grow on SDplates without histidine, which cannot induce LacZ (b-galacto-sidase) activity. The pBD (negative control), pGAL4 (positivecontrol) and pBD-PacMYBA plasmids were introduced intoYRG-2 yeast using PEG-mediated transformation. The trans-formed yeast cells were then streaked on SD/–Trp–His (lackingboth tryptophan and histidine) or SD/–Trp (lacking tryptohan)plates. The plates were incubated at 30�C for 3 d and thensubjected to a b-Gal assay to test the transactivation abilityof PacMYBA.

Expression analysis of PacMYBA and anthocyaninstructural genes

The real-time quantitative PCR (qRT-PCR) and semi-quantita-tive RT–PCR primers for genes are given in SupplementaryTable S2. For qRT-PCR, gene expression levels were determinedusing an Applied Biosystems 7500 Real-Time PCR System, trip-licate quantitative assays were performed on each cDNA dilu-tion using UltraSYBR Mixture (CWBIO, Beijing, China), and themeans and corresponding SEs were calculated. Forty PCR cycleswere performed according to the following temperaturescheme: 94�C for 10 s and 60�C for 31 s. The relative quantifi-cation value for PacMYBA was calculated using the 2–��Ct

method (Livak and Schmittgen 2011), and the sweet cherryactin1 gene was used as an internal control. Each time pointrepresents three biological replicates, each consisting of fourtechnical replicates.

The in vivo incubation of berry tissues in mediacontaining ABA or NDGA

The tissues of sweet cherry berries were incubated in vivo withABA or NDGA essentially as described by Peng et al. (2003) withslight modifications. The equilibrium buffer contained50 mmol l�1 MES-Tris (pH 5.5), 1 mmol l�1 ascorbic acid,5 mmol l�1 CaCl2, 1 mmol l�1 MgCl2 and 200 mmol l�1 manni-tol. At 27 DAFB (just before turning red), the sweet cherryberries were sliced into small discs (0.1 cm thick), and thediscs were immediately immersed in equilibration buffer for30 min. A 30 g aliquot of equilibrated tissues was then placedin a 200 ml Erlenmeyer flask containing 90 ml of incubationbuffer that was composed of equilibrium buffer with30mmol l�1 ABA. In experiments that evaluated the effects ofthe NDGA inhibitor, the equilibrated tissues (30 g) were pre-incubated in 90 ml of incubation medium that consisted ofequilibration buffer with 150mmol l�1 NDGA. Tissuesimmersed in incubation medium containing equilibriumbuffer and an equal volume of distilled water were used as acontrol. A pressure of about 60 kPa for 5 min was used for theinfiltration of ABA and NDGA, and then flasks containing theinfiltrated tissues were gently shaken at 25�C for 0.5, 1, 2, 4, 8 or16 h. Then, the incubation medium was removed and the tis-sues were washed three times with double-distilled water,frozen in liquid nitrogen, and stored at –80�C until use. Eachtreatment was repeated in triplicate.






http://www.dna.affrc.go.jp/PLACE/signalscan.htm/

http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

http://bioinformatics.psb.ugent.be/webtools/plantcare/html/



Dual luciferase assay of transiently transformedtobacco leaves

A transient dual luciferase assay was performed as previouslyreported (Hellens et al. 2005). The full-length coding sequence(CDS) of PacMYBA from ‘Hong Deng’ was amplified with thesense primer 50-GAATTCATGGAGGGCTATAACTTGGGTGT-30 and antisense primer 50-GTCGACCTAGTCCTTCTGAACATTGGTACACTG-30, and the fragment was ligated into thepMD18-T Simple vector (TAKARA). The authenticity of thesequence was confirmed by sequencing. The CDS was releasedthrough digestion with EcoRI and SalI ligated into pGreenII002962-SK (Hellens et al. 2005) pre-digested with the same enzymes.The CDS in the final plant expression vector 35S:PacMYBA wasunder the control of a CaMV 35S promoter. Meanwhile, thepromoters of PacDFR, PacANS and PacUFGT, modified to con-tain SalI and BamHI sites at the 50 and 30 ends, were cloned intothe pGreenII0800-LUC vector (Hellens et al. 2005), such that thepromoter was cloned as a transcriptional fusion with the fireflyluciferase (LUC) gene. Thus, TFs that bind to the promoter andincrease the rate of transcription could be identified by an in-crease in luminescence. In the same construct, a luciferase genefrom Renilla (REN), under the control of a 35S promoter, pro-vided an estimate of the extent of transient REN expression.Other pGreenII0029 62-SK vectors harboring AtbHLH2,AtbHLH42, MdbHLH33, MdMYB10 or MdMYB8 were con-structed as previously reported (Grotewold et al. 2000, Espleyet al. 2007, Palapol et al. 2009). All of these plant expressionvectors were introduced into Agrobacterium strain GV3101(MP90) and cultured as described in Espley et al. (2007).

Nicotiana tabacum ‘Samsun’ plants were grown undergreenhouse conditions until at least six leaves were availablefor infiltration with Agrobacterium. Infiltration, transient ex-pression analysis and determination of LUC and REN activitieswere conducted as reported in Espley et al. (2007). Transientexpression was assayed 3 d after infiltration, and the relativeluciferase activities were calculated as the ratio of firefly toRenilla luciferase activity to indicate whether interactions be-tween the MYB proteins (with or without a bHLH) and thepromoters of PacDFR, PacANS and PacUFGT occurred. Alltransfection experiments were performed in triplicate andeach set of promoter experiments was repeated, with similarrelative ratios to the respective control.

Generation of PacMYBA transgenic Arabidopsisplants

The full-length CDS of PacMYBA was ligated into the binaryvector PCB302-3, and used to transform Agrobacterium strainGV3101 (MP90). Arabidopsis plants, ecotype Columbia, weretransformed with Agrobacterium by the floral dip method(Steven and Andrew 1998). T1 transgenic plants were grownon soil at 20�C in a growth chamber with a 16 h daylength andselected by resistance to Basta. Seeds of individual self-fertilizedT2 lines were collected and single-copy insertion lines were se-lected based on a Mendelian segregation ratio of Basta

resistance. RT–PCR was also used to identify transgenicplants. All primers used in this work are listed inSupplementary Table S2.

Construction of the viral vector andagroinoculation

The pTRV1 and pTRV2 VIGS vectors (described by Liu et al.2002) were kindly provided by Dr. Yu-Le Liu. A 495 bp cDNAfragment of PacMYBA was amplified using the primers 50-GAATTCGGTGTGAGAAAAGGAGCTTGG-30 (sense) and 50-GAGCTCGGTGATGTTTGTGATGGCGTA-30 (antisense). A 579 bpcDNA fragment of PaNCED1 was amplified using the primers50-GAATTCCCCGATTGCTTCTGCTTCCAT-30 (sense) and 50-GGTACCGGCCTGCTTCACCAAGTCCTT-30 (antisense). Theamplified fragments were inserted into the pMD19-T vector(TAKARA), digested with EcoRI/SacI or EcoRI/KpnI, and theninserted into the EcoRI–KpnI or EcoRI–KpnI restriction site ofthe pTRV2 viral vector, respectively. The Agrobacterium tume-faciens strain GV3101 containing pTRV1, pTRV2 and pTRV2derivative pTRV2-PaMYBA495 or pTRV2-PaNCED1579 was usedfor RNAi. Agroinoculation and syringe inoculation were per-formed as described by Fu et al. (2005).

Determination of anthocyanin, solouble sugar,Chl, ABA and IAA contents

Anthocyanin content in the berries of red-colored sweet cherrycv. Hong Deng was quantified as described by Niu et al. (2010).The total anthocyanin content was expressed as cyanidinequivalents (molar extinction = 34.300). Measurement ofsoluble sugars was performed as described by Jia et al. (2011).The ABA and IAA contents were measured using anenzyme-linked immunosorbent assay (ELISA) as described bySun et al. (2010). All measurements were performed intriplicate.

GenBank accession numbers

Sequence data from this article has been deposited in GenBankdata libraries under accession numbers: JF748833 (PacCHS),JF740091 (PacCHI), JF740092 (PacF3H), KF974775 (PacDFR),KF974776 (PacANS), KF974777 (PacUFGT), KF974774(PacMYBA), NM_180280 (AtACTIN2) and FJ560908 (PacACT1).

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by the Peoples’s Republic of ChinaNational Natural Science Foundation Project [grant No.31171938]; the Peoples’s Republic of China Special Fund forAgro-scientific Research in the Public Interest [grant No.201003021].


X. Shen et al.




Acknowledgments

We would like to thank Professor Andrew C. Allan(HortResearch, Mt Albert Research Centre, Auckland, NewZealand) for dual luciferase assay vectors, Professor Yule Liu(School of Life Science, Tsinghua University, Beijing, China)for pTRV vectors, Professor Tao Wang (College of BiologicalSciences, China Agricultural University, Beijing, China) for thetransactivation analysis system, and Dr. Bingbing Li (College ofAgriculture and Biotechnology, China Agricultural University,Beijing, China) for her continuous support. We also thankDr. Kathleen Farquharson for English revision.

Disclosures

The authors have no conflicts of interest to declare.

References

Allan, A.C., Hellens, R.P. and Laing, W.A. (2008) MYB transcriptionfactors that colour our fruit. Trends Plant Sci. 13: 99–102.

Borovsky, Y., Oren-Shamir, M., Ovadia, R., De Jong, W. and Paran, I.(2004) The A locus that controls anthocyanin accumulation inpepper encodes a MYB transcription factor homologous toAnthocyanin2 of Petunia. Theor. Appl. Genet. 109: 23–29.

Brady, S.M., Sarkar, S.F., Bonetta, D. and McCourt, P. (2003) TheABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farne-sylation and is involved in auxin signaling and lateral root develop-ment in Arabidopsis. Plant J. 34: 67–75.

Butelli, E., Titta, L., Giorgio, M., Mock, H.P., Matros, A., Peterek, S. et al.(2008) Enrichment of tomato fruit with health-promoting antho-cyanins by expression of select transcription factors. Nat. Biotechnol.26: 1301–1308.

Castellarin, S.D., Gambetta, G.A., Wada, H., Schackel, K.A. andMatthews, M.A. (2011) Fruit ripening in Vitis vinifera: spatiotem-poral relationships among turgor, sugar accumulation, and antho-cyanin biosynthesis. J. Exp. Bot. 62: 4345–4354.

Chai, Y.M., Jia, H.F., Li, C.L., Dong, Q.H. and Shen, Y.Y. (2011) FaPYR1 isinvolved in strawberry fruit ripening. J. Exp. Bot. 62: 5079–5089.

Chenna, R. (2003) Multiple sequence alignment with the Clustal seriesof programs. Nucleic Acids Res. 31: 3497–3500.

Espley, R.V., Brendolise, C., Chagne, D., Kutty-Amma, S., Green, S.,Volz, R. et al. (2009) Multiple repeats of a promoter segmentcauses transcription factor autoregulation in red apples. Plant Cell21: 168–183.

Espley, R.V., Hellens, R.P., Putterill, J., Stevenson, D.E., Kutty-Amma, S.and Allan, A.C. (2007) Red colouration in apple fruit is due to theactivity of the MYB transcription factor, MdMYB10. Plant J. 49:414–427.

Feng, S., Wang, Y., Yang, S., Xu, Y. and Chen, X. (2010) Anthocyaninbiosynthesis in pears is regulated by a R2R3-MYB transcriptionfactor PyMYB10. Planta 232: 245–255.

Fu, D.Q., Zhu, B.Z., Zhu, H.L., Jiang, W.B. and Luo, Y.B. (2005) Virus-induced gene silencing in tomato fruit. Plant J. 43: 299–308.

Gong, Y.P., Fan, X.T. and Mattheis, J.P. (2002) Response of ‘Bing’ and‘Rainier’ sweet cherries to ethylene and 1-methylcyclopropene.J. Amer. Soc. Hortic. Sci. 127: 831–835.

Gonzalez, A., Zhao, M., Leavitt, J.M. and Lloyd, A.M. (2008) Regulationof the anthocyanin biosynthetic pathway by the TTG1/bHLH/Mybtranscriptional complex in Arabidopsis seedlings. Plant J. 53:814–827.

Grotewold, E., Sainz, M.B., Tagliani, L., Hernandez, J.M., Bowen, B. andChandler, V.L. (2000) Identification of the residues in the Mybdomain of maize C1 that specify the interaction with the bHLHcofactor R. Proc. Natl Acad. Sci. USA 97: 13579–13584.

Grotewold, E. (2006) The genetics and biochemistry of floral pigments.Annu. Rev. Plant Biol. 57: 761–780.

He, F., Mu, L., Yan, G.L., Liang, N.N., Pan, Q.H., Wang, J. et al. (2010)Biosynthesis of anthocyanins and their regulation in colored grapes.Molecules 15: 9057–9091.

Hellens, R.P., Allan, A.C., Friel, E.N., Bolitho, K., Grafton, K.,Templeton, M.D. et al. (2005) Transient expression vectors for func-tional genomics, quantification of promoter activity and RNA silen-cing in plants. Plant Methods 1: 13.

Hichri, I., Heppel, S.C., Pillet, J., Leon, C., Czemmel, S., Delrot, S. et al.(2010) The basic helix–loop–helix transcription factor MYC1 isinvolved in the regulation of the flavonoid biosynthesis pathwayin grapevine. Mol. Plant 3: 509–523.

Hichri, I., Barrieu, F., Bogs, J., Kappel, C., Delrot, S. and Lauvergeat, V.(2011) Recent advances in the transcriptional regulation of theflavonoid biosynthetic pathway. J. Exp. Bot. 62: 2465–2483.

Jia, H.F., Chai, Y.M., Li, C.L., Lu, D., Luo, J.J. and Qin, L. (2011) Abscisicacid plays an important role in the regulation of strawberry fruitripening. Plant Physiol. 157: 188–199.

Jia, H.F., Lu, D., Sun, J.H., Li, C.L., Xing, Y., Qin, L. et al. (2013) Type 2Cprotein phosphatase ABI1 is a negative regulator of strawberry fruitripening. J. Exp. Bot. 64: 1677–1687.

Jiang, Y.M. and Joyce, D.C. (2003) ABA effects on ethylene production,PAL activity, anthocyanin and phenolic contents of strawberry fruit.Plant Growth Regul. 39: 171–174.

Kobayashi, S., Ishimaru, M., Hiraoka, K. and Honda, C. (2002) Myb-related genes of the kyoho grape (Vitis labruscana) regulate antho-cyanin biosynthesis. Planta 215: 924–933.

Laitinen, R.A., Ainasoja, M., Broholm, S.K., Teeri, T.H. and Elomaa, P.(2008) Identification of target genes for a MYB-type anthocyaninregulator in Gerbera hybrida. J. Exp. Bot. 59: 3691–3703.

Li, L., Ban, Z.J., Li, X.H., Wu, M.Y., Wang, A.L., Jiang, Y.Q. et al. (2012)Differential expression of anthocyanin biosynthetic genes and tran-scription factor PcMYB10 in pears (Pyrus communis L.). PLoS One 7:e46070.

Liu, Y., Liu, X.Y., Zhong, F., Tian, R.R., Zhang, K.C., Zhang, X.M. et al.(2011) Comparative study of phenolic compounds and antioxidantactivity in different species of cherries. J. Food Sci. 76: C633–C638.

Liu, Y.L., Schiff, M. and Dinesh-Kumar, S.P. (2002) Virus-induced genesilencing in tomato. Plant J. 31: 777–786.

Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expres-sion data using real-time quantitative PCR and the 2–��CT Method.Methods 25: 402–408.

Mano, H., Ogasawara, F., Sato, K., Higo, H. and Minobe, Y. (2007)Isolation of a regulatory gene of anthocyanin biosynthesis in tuber-ous roots of purple-fleshed sweet potato. Plant Physiol. 143:1252–1268.

Nakashima, K., Fujita, Y., Katsura, K., Maruyama, K., Narusaka, Y.,Seki, M. et al. (2006) Transcriptional regulation of ABI3- andABA-responsive genes including RD29B and RD29A in seeds, ger-minating embryos, and seedlings of Arabidopsis. Plant Mol. Biol. 60:51–68.




Nakatsuka, T., Haruta, K.S., Pitaksutheepong, C., Abe, Y., Kakizaki, Y.,Yamamoto, K. et al. (2008) Identification and characterization ofR2R3-MYB and bHLH transcription factors regulating anthocyaninbiosynthesis in gentian flowers. Plant Cell Physiol. 49: 1818–1829.

Nesi, N., Jond, C., Debeaujon, I., Caboche, M. and Lepiniec, L. (2001)The Arabidopsis TT2 gene encodes an R2R3-MYB domain proteinthat acts as a key determinant for proanthocyanidin accumulationin developing seed. Plant Cell 13: 2099–2114.

Niu, S.S., Xu, C.J., Zhang, W.S., Zhang, B., Li, X., Wang, K.L. et al. (2010)Coordinated regulation of anthocyanin biosynthesis in Chinese bay-berry (Myrica rubra) fruit by a R2R3-MYB transcription factor.Planta 231: 887–899.

Palapol, Y., Ketsa, S., Wang, L.K., Ferguson, I.B. and Allan, A.C. (2009)A MYB transcription factor regulates anthocyanin biosynthesis inmangosteen (Garcinia mangostana L.) fruit during ripening. Planta229: 1323–1334.

Pattanaik, S., Kong, Q., Zaitlin, D., Werkman, J.R., Xie, C.H., Patra, B.et al. (2010) Isolation and functional characterization of a floraltissue-specific R2R3-MYB regulator from tobacco. Planta 231:1061–1076.

Peng, Y.B., Lu, Y.F. and Zhang, D.P. (2003) Abscisic acid activatesATPase in developing apple fruit especially in fruit phloem cells.Plant Cell Environ. 26: 1329–1342.

Perkins-Veazie, P. (1995) Growth and ripening of strawberry fruit.Hortic. Rev. 17: 267–297.

Quattrocchio, F., Wing, J., van der Woude, K., Souer, E., de Vetten, N.,Mol, J. et al. (1999) Molecular analysis of the anthocyanin 2 gene ofpetunia and its role in the evolution of flower color. Plant Cell 11:1433–1444.

Ren, J., Sun, L., Wu, J.F., Zhao, S.L., Wang, C.L., Wang, Y.P. et al. (2010)Cloning and expression analysis of cDNAs for ABA 80-hydroxylaseduring sweet cherry fruit maturation and under stress conditions.J. Plant Physiol. 167: 1486–1493.

Schwinn, K., Venail, J., Shang, Y., Mackay, S., Alm, V., Butelli, E. et al.(2006) A small family of MYB-regulatory genes controls floral pig-mentation intensity and patterning in the genus Antirrhinum. PlantCell 18: 831–851.

Seo, P.J. and Park, C.M. (2009) Auxin homeostasis during lateral rootdevelopment under drought condition. Plant Signal. Behav. 4:1002–1004.

Seo, P.J., Xiang, F.N., Qiao, M., Park, J.Y., Lee, Y.N., Kim, S.G. et al. (2009)The MYB96 Transcription factor mediates abscisic acid signalingduring drought stress response in Arabidopsis. Plant Physiol. 151:275–289.

Setha, S., Kondo, S., Hirai, N. and Ohigashi, H. (2005) Quantification ofABA and its metabolites in sweet cherries using deuterium-labeledinternal standards. Plant Growth Regul. 45: 183–188.

Simpson, S.D., Nakashima, K., Narusaka, Y., Seki, M., Shinozaki, K. andShinozaki, K.Y. (2003) Two different novel cis-acting elements of

erd1, a clpA homologous Arabidopsis gene function in induction bydehydration stress and dark-induced senescence. Plant J. 33:259–270.

Steven, J.C. and Andrew, F.B. (1998) Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana.Plant J. 16: 735–743.

Stracke, R., Werber, M. and Weisshaar, B. (2001) The R2R3-MYBgene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 4:447–456.

Sun, L., Zhang, M., Ren, J., Qi, J.X., Zhang, G.J. and Leng, P. (2010)Reciprocity between abscisic acid and ethylene at the onset ofberry ripening and after harvest. BMC Plant Biol. 10: 257.

Takos, A.M., Jaffe, F.W., Jacob, S.R., Bogs, J., Robinson, S.P. andWalker, A.R. (2006) Light-induced expression of a MYB gene regu-lates anthocyanin biosynthesis in red apples. Plant Physiol. 142:1216–1232.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. andKumar, S. (2011) MEGA5: molecular evolutionary genetics analysisusing maximum likelihood, evolutionary distance, and maximumparsimony methods. Mol. Biol. Evol. 28: 2731–2739.

Tohge, T., Nishiyama, Y., Hirai, M.Y., Yano, M., Nakajima, J.,Awazuhara, M. et al. (2005) Functional genomics by integratedanalysis of metabolome and transcriptome of Arabidopsis plantsover-expressing an MYB transcription factor. Plant J. 42: 218–235.

Wan, C.Y. and Wilkins, T.A. (1994) A modified hot borate methodsignificantly enhances the yield of high-quality RNA from cotton(Gossypium hirsutum L.). Anal. Biochem. 223: 7–12.

Wang, Z.G., Meng, D., Wang, A.D., Li, T.L., Jiang, S.L., Cong, P.H. et al.(2013) The Methylation of the PcMYB10 promoter is associatedwith green-skinned sport in max red bartlett pear. Plant Physiol.162: 885–896.

Winkel-Shirley, B. (2001) Flavonoid biosynthesis. a colorful model forgenetics, biochemistry, cell biology, and biotechnology. PlantPhysiol. 126: 485–493.

Yamagishi, M., Shimoyamada, Y., Nakatsuka, T. and Masuda, K. (2010)Two R2R3-MYB genes, homologs of Petunia AN2, regulate antho-cyanin biosyntheses in flower Tepals, tepal spots and leaves ofasiatic hybrid lily. Plant Cell Physiol. 51: 463–474.

Yuan, Y.X., Chiu, L.W. and L, L. (2009) Transcriptional regulation ofanthocyanin biosynthesis in red cabbage. Planta. 230: 1141–1153.

Zifkin, M., Jin, A., Ozga, J.A., Zaharia, L.I., Schernthaner, J.P., Gesell, A.et al. (2012) Gene expression and metabolite profiling of developinghighbush blueberry fruit indicates transcriptional regulation of fla-vonoid metabolism and activation of abscisic acid metabolism.Plant Physiol. 158: 200–224.

Zimmermann, I.M., Heim, M.A., Weisshaar, B. and Uhrig, J.F. (2004)Comprehensive identification of Arabidopsis thaliana MYB tran-scription factors interacting with R/B-like BHLH proteins. Plant J.40: 22–34.


X. Shen et al.


1 running title: corresponding author: tianhong li, ph.d. subject

Documents