A HORT1 Retrotransposon Insertion in the PeMYB11Promoter Causes Harlequin/Black Flowers inPhalaenopsis Orchids1[OPEN]

Chia-Chi Hsu,a,2 Ching-Jen Su,a Mei-Fen Jeng,b Wen-Huei Chen,b and Hong-Hwa Chena,b,3,4

aDepartment of Life Sciences, National Cheng Kung University, Tainan 701, TaiwanbOrchid Research and Development Center, National Cheng Kung University, Tainan 701, Taiwan

ORCID ID: 0000-0002-7323-7434 (H.-H.C.).

The harlequin/black flowers in Phalaenopsis orchids contain dark purple spots and various pigmentation patterns, whichappeared as a new color in 1996. We analyzed this phenotype by microscopy, HPLC, gene functional characterization,genome structure analysis, and transient overexpression system to obtain a better understanding of the black color formationin Phalaenopsis orchids. Most mesophyll cells of harlequin flowers showed extremely high accumulation of anthocyanins as wellas a high expression of Phalaenopsis equestris MYB11 (PeMYB11) as the major regulatory R2R3-MYB transcription factor forregulating the production of the black color. In addition, we analyzed the expression of basic helix-loop-helix factors, WD40repeat proteins, and MYB27- and MYBx-like repressors for their association with the spot pattern formation. To understand thehigh expression of PeMYB11 in harlequin flowers, we isolated the promoter sequences of PeMYB11 from red and harlequinflowers. A retrotransposon, named Harlequin Orchid RetroTransposon 1 (HORT1), was identified and inserted in the upstreamregulatory region of PeMYB11. The insertion resulted in strong expression of PeMYB11 and thus extremely high accumulation ofanthocyanins in the harlequin flowers of the Phalaenopsis Yushan Little Pearl variety. A dual luciferase assay showed that theinsertion of HORT1 enhanced PeMYB11 expression by at least 2-fold compared with plants not carrying the insertion.Furthermore, the presence of HORT1 explains the high mutation rates resulting in many variations of pigmentationpatterning in harlequin flowers of Phalaenopsis orchids.

The harlequin/black flowers in Phalaenopsis orchids,resembling a clown face with painted black spots,appeared as a new color in 1996 (Chen et al., 2004).Harlequin flowers contain dark-purple spots withhighly accumulated anthocyanins and form variouspigmentation patterns on the flowers (SupplementalFig. S1).Harlequin flowers originated from the discovery of

a somaclonal mutant of Phalaenopsis Golden Peoker’Brother’ (Phalaenopsis Misty Green 3 Phalaenopsis LiuTuen-Shen) that has white or yellow flowers with redspots (Supplemental Fig. S2; Chen et al., 2004). Onesomaclonal mutant was named Phalaenopsis Golden

Peoker ’Ever-spring’ and contained purple spots inwhite flowers (Supplemental Fig. S2; Chen et al., 2004).On further tissue culture of Phalaenopsis Golden Peoker’Ever-spring’, three different phenotypes resulted, withsimilar ratios, including Phalaenopsis Golden Peoker’Brother’, Phalaenopsis Golden Peoker ’Ever-spring’,and a new color pattern with large, fused, dark-purplespots, named Phalaenopsis Golden Peoker ’BL’(Supplemental Fig. S2; Chen et al., 2004). Therefore,Phalaenopsis Golden Peoker ’Ever-spring’ and Phalae-nopsis Golden Peoker ’BL’ have harlequin flowers withnear-black color, various pigmentation patterning, andhigh mutation rates, thereby contributing to a new agein harlequin/black flowers in the Phalaenopsis breedinghistory.Anthocyanins are a group of flavonoid compounds

found typically in red, purple, and blue in flowers aswell as fruits (Winkel-Shirley, 2001). The biosyntheticpathway for anthocyanins has been one of the mostcomprehensively studied secondary metabolisms inplants (Grotewold, 2006). All the biosynthetic andregulatory genes involved in the anthocyanin pathwayhave been cloned and characterized from petunia(Petunia sp.), snapdragon (Antirrhinum sp.), and otherplant species (Broun, 2005; Dixon et al., 2005; Koes et al.,2005; Grotewold, 2006). R2R3-MYB and basic helix-loop-helix (bHLH) transcription factors, as well asWD40 repeat (WDR) proteins are the three major

1This work was supported by the Ministry of Science andTechnology, Taiwan (MOST) (grant no. MOST 106-2811-B-006-057-).

2Present address: No. 89, Wen-Hsien Road, Nantou City, NantouCounty 54041, Taiwan

3Author for contact: hhchen@mail.ncku.edu.tw.4Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Hong-Hwa Chen (hhchen@mail.ncku.edu.tw).

C.-C.H. designed the research; C.-C.H. and C.-J.S. performedthe research; C.-C.H. and M.-F.J. analyzed the data; C.-C.H., M.-F.J.,W.-H.C., and H.-H.C. wrote the article.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.00205

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protein families that form regulatory complexes foractivating anthocyanin accumulation (Koes et al., 2005;Feller et al., 2011; Hichri et al., 2011; Petroni and Tonelli,2011). R2R3-MYB transcription factors are key compo-nents that provide the specificity for the downstreamgenes and cause tissue-specific anthocyanin accumu-lation (Borevitz et al., 2000; Zhang et al., 2003). ThebHLH transcription factors are essential for the activityof the R2R3-MYB partner by stabilizing the proteincomplex or promoting its transcription (Hernandezet al., 2004). WDR proteins can physically interactwith theMYB and bHLH factors to control anthocyaninbiosynthesis (Zhang et al., 2003).

Recently, the identification of repressor proteins thatinhibit anthocyanin biosynthetic genes has modifiedthe regulatory mechanism of color pigmentation(Aharoni et al., 2001; Dubos et al., 2008; Matsui et al.,2008). Two differential classes of MYB repressors havebeen identified: R2R3-MYB and R3-MYB repressorswith two and one repeat(s) of the MYB domain region,respectively. Among them, FaMYB1 from strawberry(Fragaria 3 ananassa) and PhMYB27 from petuniaare R2R3-MYB repressors containing Ethylene-responsive element binding factor-associated Amphi-philic Repressionmotifs at their C terminus that repressdownstream gene transcription (Aharoni et al., 2001;Lin-Wang et al., 2010; Albert et al., 2011; Kagale andRozwadowski, 2011; Salvatierra et al., 2013). AtMYBL2from Arabidopsis (Arabidopsis thaliana) is an R3-MYBrepressor and contains a repression motif (TLLLFR) atthe C terminus for its repressive activity (Dubos et al.,2008;Matsui et al., 2008).PhMYBx frompetunia is anR3-MYB repressor without a repressive motif and has anamino acid signature for binding to bHLH partners;therefore, PhMYBx is thought to perform its repressivefunction by competing for bHLH partners with R2R3-MYB activators (Koes et al., 2005; Zhang et al., 2009).Moreover, a network formed by these transcriptionalactivators and repressors is thought to regulate the an-thocyanin accumulation and pigmentation patterns(Albert et al., 2014), although more evidence is needed.

The black color in flowers or fruits has been studiedin a few plants, such as blood oranges (Citrus sinensis;Butelli et al., 2012), purple cauliflower (Brassica oleraceavar botrytis; Chiu et al., 2010), and purple sweet potato(Ipomoea batatas; Mano et al., 2007). In blood oranges, aCopia-like retrotransposon inserted in the upstreamregulatory sequences of a R2R3-MYB transcriptionfactor gene, Ruby, results in extreme anthocyanin ac-cumulation in the fruit (Butelli et al., 2012). A similarsituation was found in purple cauliflower, with aHarbinger DNA transposon inserted in the regulatoryregion of Purple (Pr), which encodes a R2R3-MYBtranscription factor and causes the up-regulation of Prand a purple color in curds (Chiu et al., 2010). In sweetpotato, IbMYB1 is predominantly expressed in thepurple flesh of tuberous roots (Mano et al., 2007).In addition, ectopic expression of a MYB or bHLHtranscription factor can produce the dark-purple colorformation in transgenic plants such as Deep Purple

(MYB) and Leaf Color (bHLH) from petunia (Albertet al., 2009, 2011). Therefore, high expression of theregulatory transcription factors involved in the antho-cyanin biosynthesis pathway may result in the blackfruits and flowers in plants. However, the molecularmechanism for harlequin/black flower formation inPhalaenopsis still needs to be verified.

In Phalaenopsis, three R2R3-MYB transcription factors(Phalaenopsis equestris MYB2 [PeMYB2], PeMYB11, andPeMYB12) have been identified and verified for their rolesin regulating distinct pigmentation patterning in flowers(Hsu et al., 2015). Here, we assessed the expression pro-files of these three PeMYBs to identify the one that is up-regulated in harlequin flowers of Phalaenopsis YushanLittle Pearl, a fourth-generation offspring of PhalaenopsisGolden Peoker that contains a large dark-purple spot onwhite flowers. We also analyzed other regulatory factors,including bHLH factors, WDR protein, R3-MYB, andR2R3-MYB repressors, for their roles in the production ofthe black color. Then we analyzed the upstream regu-latory sequences of these PeMYBs to investigate whyPeMYB11 is up-regulated in harlequin flowers. Finally,we identified a retrotransposon, Harlequin Orchid Retro-Transposon 1 (HORT1), inserted in the promoter se-quence of PeMYB11 in Phalaenopsis Yushan Little Pearl,which resulted in high expression of PeMYB11 and ex-treme anthocyanin accumulation in harlequin flowers.

RESULTS

Harlequin Flowers of Phalaenopsis Orchids Result fromHigh Accumulation of Anthocyanin

To confirm the previous report showing that theharlequin flowers of Phalaenopsis orchids resulted froma high accumulation of anthocyanin but not new an-thocyanin compounds (Kuo and Wu, 2011), weused HPLC analysis of hydrolyzed anthocyaninfrom the flowers of three Phalaenopsis cultivars, white-flower Phalaenopsis Sogo Yukidian ’V3,’ red-flowerPhalaenopsis Red Shoe ’OX1408’, and black-flowerPhalaenopsis Yushan Little Pearl (Fig. 1, A–C). Onlycyanidins were detected in these cultivars, and thedifference between the three cultivars was in the totalamount of cyanidins present in the flowers, with a10-fold increase in Phalaenopsis Yushan Little Pearlas compared with Phalaenopsis Red Shoe ’OX1408’(Fig. 1D). Hence, the extreme accumulation of cyanidin-derived anthocyanins may explain the black colorformation in Phalaenopsis flowers.

The distribution of anthocyanin production in celllayers was reported to differ between the three pig-mentation patterns: the full-red pigmentation with an-thocyanin in subepidermal cells, red spots containinganthocyanin in epidermal cells, and venation patternwith anthocyanin in the region from subepidermal cellsto the xylem (Hsu et al., 2015).

To examine the location of the increased accumula-tion of anthocyanins in the harlequin flowers, crosssections of the red-flower Phalaenopsis Red Shoe

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’OX1408’ and harlequin-flower Phalaenopsis YushanLittle Pearl were observed by microscopy. The red cellscontaining anthocyanins were present in the subepi-dermal cells of both adaxial and abaxial sides in Pha-laenopsis Red Shoe ’OX1408’ (Fig. 1E) but they werepresent in the epidermal cells to most mesophyll celllayers in Phalaenopsis Yushan Little Pearl (Fig. 1F),which suggests that many more mesophyll cells pro-duce anthocyanins to cause the abundant anthocyaninaccumulation and black color formation.

PeMYB11 Is Related to Harlequin Flower Formationin Phalaenopsis

To identify the structural and regulatory genes in-volved in harlequin flower formation in Phalaenopsis,

the flowers of Phalaenopsis Yushan Little Pearl wereseparated into purple spots and white area of theflowers (Fig. 1A) and analyzed for the expression pro-files of anthocyanin-related genes, including threestructural genes, PeF3H5, PeDFR1, and PeANS3, as wellas three regulatory genes, PeMYB2, PeMYB11, andPeMYB12, which have been studied for their regulatoryrole in the anthocyanin biosynthesis pathway (Hsuet al., 2015). High transcript levels of PeF3H5,PeDFR1, and PeANS3 were detected in the purple butnot white part (Fig. 2A), which suggests that a regula-tory gene is responsible for the transactivation of thesethree structural genes in the purple spots. OnlyPeMYB11 had strong expression in purple parts, withno expression of PeMYB2 and PeMYB12 in purple andwhite parts of this flower (Fig. 2B). In addition, a 33.9-fold increase in PeMYB11 expression was detected in

Figure 1. A–C, Phenotypes of Phalaenopsis Sogo Yukidian ‘V39 (A), PhalaenopsisOX Red Shoe ‘OX1408’ (B), and PhalaenopsisYushan Little Pearl (C). Bars5 1 cm. C, The “Purple” and “White” parts were used for gene expression analysis. D, Anthocyanincontents of black flowers of Phalaenopsis Yushan Little Pearl. Anthocyanin contents were compared between Phalaenopsis SogoYukidian ‘V39, Phalaenopsis OX Red Shoe ‘OX1408’, and Phalaenopsis Yushan Little Pearl. Cyanidin at 200 ng was used as astandard to calculate the anthocyanin quantity present in flowers (shown in parentheses). E and F, This is the cross section of theflower petals of Phalaenopsis OX Red Shoe ‘OX1408’ (E) and Phalaenopsis Yushan Little Pearl (F). Bars 5 0.1 mm.

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the purple parts of Phalaenopsis Yushan Little Pearlcompared with the red flower of Phalaenopsis RedShoe ’OX1408’, whereas the structural genesPeF3H5, PeDFR1, and PeANS3 showed 2.1-, 2.6-, and17.7-fold increased expression, respectively (Fig. 2,A and B). In addition, a pair of degenerated primerstargeting the conserved R2R3 domain of MYBtranscription factors was used for amplifying otherpossible factors, and all the amplified sequenceswere PeMYB11. Thus, PeMYB11 was the major reg-ulatory R2R3-MYB transcription factor for the har-lequin flowers in Phalaenopsis, which is consistentwith our previous results that PeMYB11 is respon-sible for the spot pigmentation patterning (Hsuet al., 2015).

To verify whether the sequence of PeMYB11 differedbetween the harlequin flowers of Phalaenopsis YushanLittle Pearl and the other flower colors, we cloned andsequenced the cDNA (complementaryDNA) sequencesof PeMYB11 from Phalaenopsis Red Shoe ’OX1408’ andPhalaenopsis Yushan Little Pearl and named themPeMYB11 and PeMYB11_Pur, respectively. The aminoacid sequences of PeMYB11 and PeMYB11_Pur showed95% identity and 96% similarity. Four amino acidschanges were present in the R2R3 domain, with Tyrchanged to Cys (Y28C), Ala to Thr (A31T), Lys to Met(K42M), and Arg to Gly (R49G; Supplemental Fig. S3).The transcriptional functions of PeMYB11 andPeMYB11_Pur were analyzed in the following experi-ment with the subtitle of “Transient overexpression

Figure 2. Expression profiles of PeMYBs and other regulatory genes in Phalaenopsis Yushan Little Pearl. The relative mRNA a-bundance of geneswas normalized to the expression of PeAct4 and presented asmean6 sd of three technical replicates and threebiological samples performed independently. A and B, Numbers above the boxes indicate the fold of increase for gene expressionin Phalaenopsis Yushan Little Pearl as compared with that of Phalaenopsis Red Shoe ’OX1408,’ which was denoted as 1x. C–L,Expression of purple andwhite parts of harlequin flowers of Phalaenopsis Yushan Little Pearl. Low expression levels were denotedas 1x and used to calculate the fold changes in expression.

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approach confirms the in vivo functions of regulatorygenes in Phalaenopsis flowers.”To confirm that PeMYB11 is the major regulatory

gene responsible for the harlequin phenotype, we usedvirus-induced gene silencing (VIGS) to investigate thein planta function of PeMYB11 in harlequin flowers ofPhalaenopsis OX Red Eagle ‘OX1412’ (Fig. 3A) becauseof its dark-red color in entire flowers and ease in dis-tinguishing the silencing effects on color changes.Phalaenopsis OX Red Eagle ‘OX1412’ highly expressedPeMYB11 but very low PeMYB2 and PeMYB12 ex-pression (Fig. 3B). PeMYB11-silencing resulted in theloss of anthocyanin content with flower colors fadingfrom dark-purple to pink (Fig. 3D). In contrast,PeMYB2-silenced flowers contained a few white areas,with no obvious differences between PeMYB12-silenced and mock flowers (Fig. 3, A, C, and E), whichsuggests that the dark-red color is produced by con-tinuous spot pigmentation patterning. Therefore,PeMYB11 was responsible for the high anthocyaninaccumulation of spot pigmentation patterning in har-lequin flowers of Phalaenopsis orchids.

Other Regulatory Factors Related to Harlequin FlowerFormation in Phalaenopsis

The pigmentation patterns are regulated by aconserved anthocyanin-regulating transcriptionfactor complex consisting of an MYB, bHLH, andWDR-containing protein (the MBW complex; Felleret al., 2011). We cloned three PebHLHs, PebHLH1 toPebHLH3, and one PeWDR1 (Supplemental Table S1) toanalyze their association with PeMYBs in Phalaenopsis.In addition, several putative MYB repressors wereisolated from the orchid transcriptome databaseOrchidBase (Tsai et al., 2013). Phylogenetic analysis

showed that PeMYB4 to PeMYB8 (Hsu et al., 2015)were similar to the R2R3-MYB MYB27-like repres-sors, and PeMYBx06243, PeMYBx07630, andPeMYBx30334 grouped with R3-MYB MYBx-like re-pressors (Supplemental Fig. S4). Reverse transcriptionquantitative PCR (RT-qPCR) was used to verify theexpression profiles of these genes in PhalaenopsisYushan Little Pearl. The expression of PebHLH1 washigher (4.1-fold) in purple than white parts, whereasPebHLH2, PebHLH3, and PeWDR1 were expressedslightly higher (1.8- to 2.0-fold) in white than in purpleparts (Fig. 2, C–F). In addition, only PeMYBx06243could be amplified from flowers of PhalaenopsisYushan Little Pearl, but no expression of PeMYBx07630and PeMYBx30334 was detected. PeMYBx06243 washighly expressed (96.9-fold) in the purple comparedwith the white part (Fig. 2G), even thoughPeMYBx06243 was predicted as a repressor of antho-cyanin production. Moreover, all R2R3-MYB repres-sors, PeMYB4 to PeMYB8, except for PeMYB7, showedslightly higher expression profiles, with a 1.8- to 3.7-fold increase in the white compared with the purplepart of Phalaenopsis Yushan Little Pearl flowers, andPeMYB8 showed the most expression (Fig. 2, H–L). Thefunctions of these regulators with PeMYB11 for antho-cyanin accumulation and pigmentation patterningwere further investigated by a transient overexpressionapproach in Phalaenopsis orchids.

Transient Overexpression Confirms the In Vivo Functionsof Regulatory Genes in Phalaenopsis Flowers

A transient overexpression protocol was developedto investigate the in vivo functions of the regulatorygenes in activating the accumulation of anthocyaninand floral scents in Phalaenopsis flowers (Hsu et al.,

Figure 3. Virus-induced gene silencing(A, C, D, and E) and gene expressionprofiles (B) of three PeMYBs in Phalae-nopsis OX Red Eagle ‘OX1412.’ Theflowers from mock (A), single-silencedPeMYB2 (C), PeMYB11 (D), andPeMYB12 (E) plants are shown. B, Datawere repeated twice for VIGS experi-ments. Bar 5 1 cm.

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2015; Chuang et al., 2018). Agrobacterium tumefacienscontaining various regulatory genes were infiltratedinto white petals of Phalaenopsis Sogo Yukidian ’V39alone or with various bHLH transcription factorsand/or MYB27- and MYBx-like repressors. Over-expression of PeMYB11_Purwith PebHLH2 conferred apink color in white petals (Fig. 4E) and a 6.67-fold in-crease in anthocyanin content as compared with over-expression of PeMYB11_Pur with GUS (Fig. 4G).However, overexpression of PeMYB11 with all threePebHLHs and PeMYB11_Purwith PebHLH1 or PebHLH3conferred no obvious pink color (Fig. 4, A–D, F).Therefore, PeMYB11_Pur from Phalaenopsis YushanLittle Pearl contained higher transactivation activitiesthan PeMYB11 from Phalaenopsis OX Red Shoe

’OX1408’when interacting with PebHLH2. Intriguingly,the different amino acids between PeMYB11_Pur andPeMYB11 were not located in the conserved motif([D/E]Lx2[R/K]x3Lx6Lx3R) for interacting with abHLH transcription factor (Zimmermann et al., 2004).From the molecular modeling analysis, the substitutionof Tyr-28 in PeMYB11 to Cys-28 in PeMYB11_Purwould result in the formation of a disulfide bond be-tween residues Cys-28 and Cys-53 (Supplemental Fig.S5). Cys-49 is specific to plant R2R3 MYB and can forma disulfide bond with Cys-53 to regulate the DNAbinding capacity of the plant MYB by modulating thedimerization capability (Pireyre and Burow, 2015). Inour case, Cys-28 may exert the function of Cys-49 inother plants to form a disulfide bond with Cys-53 and

Figure 4. Transient overexpression assay of PeMYB11 and PeMYB11_Purwith various PebHLHs in white-flowered PhalaenopsisSogo Yukidian ’V39. A–F, Transient overexpression of PeMYB11 (A–C) or PeMYB11_Pur (D–F) was performedwith GUS in the leftpetal or various PebHLHs in the right petal of a single flower. GUS was recruited as a negative control for no correlation toPeMYBs on anthocyanin accumulation. The regions enclosed by blue dotted lines were transfected by Agrobacterium and cut forquantitative anthocyanin content analysis. G, Quantitative anthocyanin content in transient overexpression of PeMYB11 andPeMYB11_Purwith various PebHLHs in Phalaenopsis Sogo Yukidian ’V39. Data are presented asmean6 SD from three plants andtwo transient overexpression assays. Numbers above the boxes indicate the changed anthocyanin contents in PeMYB11 withPebHLHs as compared with that in PeMYB11 with GUS, which was denoted as 1x. FW indicates fresh weight.

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thus regulates the DNA binding capacity ofPeMYB11_Pur by modulating the dimerization capabil-ity. However, the effects of the changed amino acids inPeMYB11_Pur for the transactivation activities needfurther investigation.In addition, we used overexpression of PeMYB2 or

PeMYB12 with the three PebHLHs to verify the associ-ation between MYB and bHLH transcription factors.Overexpression of PeMYB2 with PebHLH1, PebHLH2,and PebHLH3 conferred a similar red color as PeMYB2

with GUS in white petals of Phalaenopsis SogoYukidian ’V39 (Supplemental Fig. S6, A–C). Also, theanthocyanin content was slightly increased (1.34- to1.46-fold) with PeMYB2 with PebHLH1, PebHLH2, andPebHLH3 as compared with PeMYB2 with GUS(Supplemental Fig. S6G). Overexpression of PeMYB12with both PebHLH1 and PebHLH2 conferred aslightly pink color in white petals of PhalaenopsisSogo Yukidian ’V39 (Supplemental Fig. S6, D–F),and the anthocyanin content was increased (8.21- and

Figure 5. Transient overexpression assay of PeMYB2with various repressors in white-flowered Phalaenopsis Sogo Yukidian ’V39. A–F,Transient overexpression of PeMYB2 was performed with GUS in the left petal and various repressors in the right petal of one flower.GUS was used as a negative control for no correlation to PeMYBs on anthocyanin accumulation. The regions enclosed by blue dottedlines were transfected by Agrobacterium and cut for quantitative anthocyanin content analysis. G, Quantitative anthocyanin content intransient overexpression of PeMYB2with various repressors in Phalaenopsis Sogo Yukidian ’V39. Data are presented asmean6 sd fromthree plants and two transient overexpression assays. Numbers above the boxes indicate the changed anthocyanin contents in PeMYB2with repressors as compared to that with GUS, which was denoted as 1x. FW indicates fresh weight.

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10.36-fold) upon overexpression of PeMYB12 withPebHLH1 and PebHLH2 as compared with PeMYB12with GUS (Supplemental Fig. S6H). All these resultssuggest that PebHLH1 and PebHLH2 improve PeMYBfunctions in anthocyanin accumulation in Phalaenopsisflowers, whereas PeMYB11 preferred PebHLH2, andPeMYB12 worked with both PebHLH1 and PebHLH2.

To assess the repressive functions of the predictedPeMYB repressors, including fiveMYB27-like PeMYB4,PeMYB5, PeMYB6, PeMYB7, PeMYB8 and one MYBx-like PeMYBx06243 in Phalaenopsis Sogo Yukidian ’V39,PeMYB2 was chosen for its high activity for anthocya-nin accumulation (Supplemental Fig. S6; Hsu et al.,2015); therefore, the repressive effects of these repres-sors were more sensitive and reliable than usingPeMYB11 or PeMYB12. Overexpression of PeMYB2with all PeMYB repressors resulted in a 32% to 80%reduction in anthocyanin content as compared withPeMYB2 and GUS (Fig. 5). In addition, PeMYBx06243caused a striking reduction of anthocyanin con-tent (88%) with overexpression of PeMYB11_Purand PebHLH2 (Fig. 6, B and D) as compared with

overexpression of PeMYB12 with PebHLH2 (26% re-duction) and PeMYB2 with PebHLH2 (40% reduction;Fig. 6, C and D). Therefore, all these PeMYB repressorsshowed the repressive effects on anthocyanin accu-mulation activated by PeMYB2, PeMYB11, or PeMYB12.The repressive effects of these repressors may be simplybinding the same sites and preventing effective activa-tion by PeMYB activators. Overall, these results showthat the bHLH transcription factors, WDR proteins, andMYB27- and MYBx-like repressors regulate the redcolor formation with all three MYB transcription fac-tors, PeMYB2, PeMYB11, and PeMYB12, in Phalaenopsisflowers, although PeMYB11 was the only transcriptionfactor responsible for the high anthocyanin accumula-tion in harlequin flowers of Phalaenopsis.

A New Retrotransposon, PeHORT, Inserted in theUpstream Regulatory Sequence of PeMYB11

To identify why PeMYB11 was highly expressed inthe purple part of harlequin flowers of Phalaenopsis

Figure 6. Transient overexpression assay of three PeMYBs, and PebHLH2 with PeMYBx repressors in white flowers of Phalae-nopsis Sogo Yukidian ’V39. A–C, Transient overexpression of PeMYB2 (A), PeMYB11_Pur (B), and PeMYB12 (C) with PebHLH2was performedwithGUS in the left petal or PeMYBx in the right petal of a single flower. GUSwas used as a negative control for nocorrelation to PeMYBs on anthocyanin accumulation. The regions enclosed by blue dotted lines were transfected by Agro-bacterium and cut for quantitative anthocyanin content analysis. D, Quantitative anthocyanin content in transient overexpressionof PeMYBs, and PebHLH2 with PeMYBx repressors in Phalaenopsis Sogo Yukidian ’V39. Data are presented as mean 6 sd fromthree plants and two transient overexpression assays. Numbers above the boxes indicate the changed anthocyanin contents inPeMYB2 with repressors as compared with that with GUS, which was denoted as 1x. FW indicates fresh weight.

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Yushan Little Pearl, we isolated a 2.5-kb upstreamregulatory sequence of PeMYB11 from OrchidBase 3.0,which contains the whole genome sequence ofP. equestris (Cai et al., 2015). The 2.5-kb upstream reg-ulatory sequence of PeMYB11 was used to designprimers to clone these promoter sequences fromP. equestris, Phalaenopsis OX Red Shoe ‘OX1408,’ andPhalaenopsis Yushan Little Pearl. Interestingly, thewhite part of Phalaenopsis Yushan Little Pearl had a5.3-kb fragment, and the purple parts had 5.3-kb and2.8-kb fragments. Both the 2.1- and 2.3-kb fragmentswere amplified from P. equestris and Phalaenopsis OXRed Shoe ‘OX1408’, respectively (Fig. 7A). After se-quencing the 5.3- and 2.8-kb sequences, a new retro-transposon was identified and named Harlequin OrchidRetroTransposon 1 (HORT1), which was similar to theGypsy-like retrotransposons present in other plants,such as mulberry (Morus notabilis) and wild strawberry(Fragaria vesca) in Repbase, the most commonly useddatabase of repetitive DNA elements (Bao et al., 2015),although no functional characterization has yet beenreported. The full-length sequence of HORT1 is about

3 kb and contains a 1.6-kb coding sequence with 500-bplong terminal repeats (LTRs) at both ends. Therefore,the 5.3- and 2.8-kb fragments from PhalaenopsisYushan Little Pearl contained 3 kb full length and500-bp LTRs of HORT1, respectively, with the reversedirection inserted in the 1.5-kb upstream regulatorysequence of PeMYB11 (Fig. 7B).In addition, two somaclonal plants of another har-

lequin cultivar, Phalaenopsis Ever-spring Prince‘Plum’ with different pigmentation patterning, wererecruited to confirm the presence of HORT1 in harle-quin flowers (Supplemental Fig. S7, A and B). The 5.3-kb fragment was amplified from both harlequinplants with a dark-red flower or with a mosaic dark-red flower, whereas a 2.3-kb sequence was amplifiedonly from the mosaic dark-red flower, that was simi-lar to the fragment from Phalaenopsis OX RedShoe ‘OX1408’ (Supplemental Fig. S7C). These resultssuggest that the 5.3-kb fragment containing theinsertion of HORT1 in the PeMYB11 promoter is acommon phenomenon for harlequin flowers ofPhalaenopsis orchids.

Figure 7. A and B, Cloning of the upstreamregulatory sequences of PeMYB11 fromP. equestris, Phalaenopsis Red Shoe’OX1408,’ and the purple andwhite parts ofblack flowers of Phalaenopsis Yushan LittlePearl. B, Arrowheads indicate the primersused to construct the promoter sequencesto drive firefly luciferase for the dual lucif-erase assay. C, Dual luciferase assay ofpromoters of PeMYB11 from P. equestris(Pe), Phalaenopsis OX Red Shoe ‘OX1408’(OX1408), and the 2.8- and 5.3-kb se-quences from Phalaenopsis Yushan LittlePearl. Data are presented as means 6 sdfrom three plants, and the Agrobacteriuminfiltrations were repeated three timesindependently.

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To investigate whether the HORT1 sequence affectedthe high expression of PeMYB11 in harlequin flowers,we used a dual luciferase assay. We cloned the 2.3-kbpromoter sequences of PeMYB11 from P. equestris andPhalaenopsis OX Red Shoe ‘OX1408’ as well as the 2.8-and 5.3-kb fragments of Phalaenopsis Yushan LittlePearl into the upstream region of the firefly luciferasegene to analyze the promoter activities in red-flowerPhalaenopsis OX Red Shoe ‘OX1408’. The promotersequences of PeMYB11 from P. equestris and Phalae-nopsis OX Red Shoe ‘OX1408’ showed similar tran-scriptional activities for driving luciferase (Fig. 7C),whereas the 2.8-kb promoter sequence of PeMYB11from Phalaenopsis Yushan Little Pearl containing oneLTR of HORT1 showed a 2-fold increase in transcrip-tional activities. In contrast, the 5.3-kb PeMYB11 pro-moter with full-lengthHORT1, including two LTRs andone coding region, caused only a 0.37-fold increase intranscriptional activities (Fig. 7C), which may resultfrom low transgenic efficiency with a large DNA frag-ment and thus low transcriptional activities. The pro-moter fragments of PeMYB11 harboring only one LTRof HORT1 showed enhanced transactivation activities,such as in the purple part of flowers. This result sug-gests that the insertion of HORT1 in the upstream reg-ulatory sequences of PeMYB11 increased thetranscriptional level of PeMYB11 in planta and resultedin the high expression of PeMYB11 in harlequin flowersof Phalaenopsis Yushan Little Pearl.

DISCUSSION

Harlequin/Black Flowers of Phalaenopsis Orchids

A few plants have dark-purple to black colors inflowers and fruits, which are supposed to catch con-sumers’ eyes. These contain a high content of antho-cyanins with high antioxidant activity (Jayaprakashaand Patil, 2007; Kelebek et al., 2008). Blood orangescontain the activities to reduce oxidative stress (Boninaet al., 2002), protect DNA against oxidative damage(Guarnieri et al., 2007), and reduce cardiovascular riskfactors (de Pascual-Teresa et al., 2010; Paredes-Lópezet al., 2010). Purple cauliflowers have an eye-catchingpurple color, with potent nutritional and health-promoting effects (Chiu et al., 2010). Here, the mar-ketable effects of the harlequin flowers in Phalaenopsisare important as a new color and breeding parentsfor harlequin flowers. The breeding for harlequinflowers started in 1996, and the hybrid number hasreached 3,614 as compared with the total 35,129 hy-brids registered in the Royal Horticultural Society(OrchidWiz, 2018). These harlequin hybrids alsocontain various pigmentation patterns, and evennovel patterns have been found in new hybrids.Therefore, the harlequin flowers are importantbreeding targets in Phalaenopsis and are used as amodel system for studying the molecular mechanismof floral pigmentation patterns.

We analyzed the phenotype of the special color ofharlequin flowers by microscopy, HPLC, gene func-tional characterization, genome structure analysis, andtransient overexpression system to obtain an overallunderstanding of this phenotype in Phalaenopsis or-chids. Most mesophyll cells of harlequin flowers pro-duced extremely high accumulation of anthocyanins aswell as PeMYB11 expression as the major regulatoryR2R3-MYB transcription factor, accompanied by bHLHfactors, WDR proteins, and MYB27- and MYBx-likerepressors to regulate the black color formation.Finally, we identified a retrotransposon, HORT1,inserted in the upstream regulatory sequences ofPeMYB11 that resulted in the high expression ofPeMYB11 and led to highly accumulated anthocyaninsin the harlequin flowers of Phalaenopsis Yushan LittlePearl. Moreover, the presence of the retrotransposonHORT1 is concomitant with the high mutation ratesand thus the multiple pigmentation patterns in harle-quin flowers of Phalaenopsis orchids.

Harlequin Flowers of Phalaenopsis Result from MesophyllCells with High Accumulation of Anthocyanin

The distribution of anthocyanin production affectscolor formation and is regulated by individual genes ofthe R2R3-MYB family. In apple (Malus x domestica),MdMYB1/MdMYBA expression is correlated withanthocyanin accumulation in the red skin of the fruit(Takos et al., 2006; Ban et al., 2007). MdMYB10 is spe-cifically expressed in type 1 red-flesh apple, such as‘Red Field,’ with red pigmentation in the fruit core,cortex, and foliage (Espley et al., 2007), whereasMdMYB110 is responsible for the type 2 red-flesh apple,such as ‘Sangrado,’with its red cortex, white fruit core,and green foliage (Chagné et al., 2013). In Phalaenopsis,the distribution of anthocyanin production in cell layersdiffers between three pigmentation patterns: full-redpigmentation with anthocyanin in the subepidermalcells, red spots containing anthocyanin in epidermalcells, and venation pattern with anthocyanin in the re-gion from subepidermal cells to the xylem. This distri-bution is regulated by three distinct R2R3-MYBtranscription factors, PeMYB2, PeMYB11, and PeMYB12,respectively (Hsu et al., 2015).

Here, we showed that harlequin flowers resultedfrom the high accumulation of anthocyanins that werepresent from both the adaxial and abaxial epidermis tomost mesophyll cells. The highly accumulated antho-cyanins in harlequin flowers resulted from the highamount of anthocyanins produced in epidermal cellsand also most mesophyll cells, which suggests thatHORT1 enhanced the expression of PeMYB11 and alsohad spatial effects on ectopic expression of PeMYB11 inmesophyll cells of flowers.

The anthocyanin content in harlequin flowers ofPhalaenopsis Yushan Little Pearl was increased 10-foldas compared with the red-flower Phalaenopsis RedShoe ’OX1408’, which was coincident with a 33.9-fold

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increase in PeMYB11 transcripts and 2.1- to 17.7-foldincrease in PeF3H5, PeDFR1, and PeANS3 transcripts.However, the insertion of one LTR of HORT1 in thepromoter sequence of PeMYB11 resulted in a 2-foldincrease in transcriptional activities as compared withthe promoter sequence of PeMYB11 alone on a dualluciferase assay. The added mesophyll cell layers withaccumulated anthocyanin production were importantfor the black color in Phalaenopsis.

High Expression of MYB Transcription Factors Is Relatedto Black Color Formation

In blood orange, the massive anthocyanin produc-tion in fruit resulted from a Copia-like retrotransposoninserted in the upstream regulatory region of a R2R3-MYB transcription factor, Ruby, to cause high expres-sion (Butelli et al., 2012). Also in purple cauliflower, theupstream regulatory sequence of a R2R3-MYB tran-scription factor, Purple (Pr), was inserted by aHarbingerDNA transposon and caused up-regulation of Pr andthe purple color formation in curds (Chiu et al., 2010). Inthis study, we screened the expression profiles of genesincluding structural genes, R2R3-MYB activators,bHLH factors, and WDR proteins, as well as R3-MYBand R2R3-MYB repressors to identify the major regu-lator of the black color formation in Phalaenopsis. TheR2R3-MYB PeMYB11 was identified by the retro-transposonHORT1 inserted in the upstream regulatoryregion to highly up-regulate the expression ofPeMYB11. In addition, the changed amino acids ofPeMYB11 from Phalaenopsis OX Red Shoe ’OX1408’ toPhalaenopsis Yushan Little Pearl enhanced its tran-scriptional activities when coexpressed with PebHLH2.

MYB and Other Regulatory Factors Are Related to theBlack Spot Pattern

The spatial and temporal patterning of anthocyaninsis mostly determined by the regulation of the R2R3-MYB factors, whereby each individual factor controlsdistinct patterns with shared bHLH and WDR factors(Schwinn et al., 2006; Albert et al., 2011; Davies et al.,2012; Hsu et al., 2015). The spot pattern has beenstudied in several plants, showing differential expres-sion ofDihydroflavonol 4-reductase2 (Dfr2) for red-purplespots of Clarkia gracilis (Martins et al., 2013), light-induced Lilium hybrid MYB6 (LhMYB6) for red spotsin pink flowers of Asiatic hybrid lily (Lilium spp.)‘Montreux’ (Yamagishi et al., 2010), LhMYB12-Lat forthe splatter-type spots in the Asiatic hybrid lily ‘Latvia’(Yamagishi et al., 2014), and PeMYB11 in Phalaenopsis(Hsu et al., 2015). Moreover, a network formed by thesetranscriptional activators and repressors is thought toregulate anthocyanin accumulation and pigmentationpatterns (Albert et al., 2014). In noninductive condi-tions, R2R3-MYB repressors are expressed and coop-erate with constitutively expressed bHLH factors and

WDR protein to form repressive MBW complexes andinhibit anthocyanin production (Albert et al., 2014). Ininductive conditions, the R2R3-MYB activators areexpressed and form the active MBW complex withbHLH factors and WDR protein to activate anthocya-nin biosynthesis, and the R2R3-MYB and R3-MYB re-pressors were also present for feedback inhibition(Albert et al., 2014). The R3-MYB repressors and WDRproteins are small proteins and capable of intercellularmovement that may relate to the pigmentation pat-terning of red spots inwhite flowers (Albert et al., 2014).In this study, we confirmed that the dark-purple

spots of Phalaenopsis Yushan Little Pearl were regu-lated by the high expression of PeMYB11, and we fur-ther analyzed the effects of the anthocyanin-relatedactivators and repressors for the spot pattern. PeMYB11was responsible for the spot pattern formation, and theinsertion of HORT1 in the upstream regulatory regionof PeMYB11 enhanced its function and resulted in theblack color. In addition, bHLH factors, especiallyPebHLH2, cooperated with PeMYB11 for its transcrip-tional activities, whereas R2R3-MYB repressors wereexpressed higher in the white than purple part (Fig. 2,H–L) and may be slightly related to the “black spot”pattern formation. Moreover, the R3-MYB MYBx-likePeMYBx06243 could inhibit the transcriptional activi-ties of R2R3-MYB activators, although the expression ofPeMYBx06243 was much higher in the purple thanwhite part of Phalaenopsis Yushan Little Pearl. AMYBx-like repressor was found capable of intercellu-lar movement and may contribute to pigmentationpatterning (Albert et al., 2014); therefore, thisPeMYBx06243 protein could be small enough to crossfrom the purple to white part for its repressive func-tion and result in the black-spot pattern. However, thisneeds further investigation.

Transposable Elements Were Related to the Color Changesin Plants

Transposable elements (TEs) related to color changesin plants have been widely reported. TEs affect thegenetic diversity in plants via insertion, excision, andchromosomal rearrangements (Kidwell and Lisch,2002). The mutations with TE insertion and excisionin gene coding regions may disrupt the protein func-tions or change enzymatic activities (Nordborg andWalbot, 1995), whereas the upstream regulatory se-quences with TE insertions may stop, increase, orchange the tissue-specific gene expression profiles(Wicker et al., 2016). For example, most of the colormutants in Japanese morning glory (Ipomoea purpurea)have TE insertions and result in loss-of-function or so-matically unstable genes (Clegg and Durbin, 2003).Various TE types have been involved, including theclass-I short interspersed elements as well as class-IICACTA elements, hAT elements, miniature inverted re-peat transposable elements, and Mutator-like elements(Clegg and Durbin, 2003; Park et al., 2007). In contrast,

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the enhancers or promoters within the TEs may causeupregulated expression in the inserted genes when theTEs are inserted in the upstream regulatory sequencesof color-related genes. Similarly, a Copia-like retro-transposon, Tcs1, and a Harbinger DNA transposoninserted in the upstream regulatory regions ofRuby andPr genes in blood orange and purple cauliflower, re-spectively, caused the up-regulated accumulation ofanthocyanin (Chiu et al., 2010; Butelli et al., 2012). TheLTR of Tcs1 provides a new transcription start site forRuby expression and low temperature-dependent an-thocyanin accumulation in blood oranges (Butelli et al.,2012). However, although Tcs1 is located in the up-stream regulatory regions of Ruby with the same ori-entation in most accessions of blood oranges, oneaccession called Jingxian from China contained a simi-lar retrotransposon, Tcs2, inserted with the oppositeorientation of the Ruby gene (Butelli et al., 2012). Theexpression of Ruby in Jingxian suggests that Tcs2 pro-vides an upstream activating sequence for the expres-sion of Ruby and the production of anthocyanin (Butelliet al., 2012).

In this study, we found a HORT retrotransposoninserted in the upstream regulatory region of PeMYB11with the reverse direction. The retrotransposon mayplay a role as an enhancer to up-regulate the expressionof PeMYB11 and also extend the spatial expression ofPeMYB11 from adaxial epidermal cells to most meso-phyll cell layers, thereby resulting in harlequin flowersof Phalaenopsis orchids.

Previous reports showed a high mutation rate withmicropropagation of Phalaenopsis Golden Peoker ’Ever-spring’: 40% of plants were Phalaenopsis GoldenPeoker ’Brother’, 30% remained as PhalaenopsisGolden Peoker ’Ever-spring’, and 30% were Phalae-nopsis Golden Peoker ’BL’ (Chen et al., 2004).Moreover, harlequin flowers produced various pig-mentation patterns with dark-purple spots on theirflowers, whereas the color patterns for harlequinflowers are still changeable and new patterns emergewith further breeding. Here, we found a HORT ret-rotransposon inserted in the upstream regulatorysequences of PeMYB11 that explained the black colorformation of harlequin flowers and also the highmutation rates and changeable pigmentation pat-terns. Therefore, the harlequin flowers in Phalae-nopsis provide an excellent system for investigatingthe regulation of anthocyanin accumulation, flowerpigmentation patterning, and transposable elementactivation.

MATERIALS AND METHODS

Plant materials

Phalaenopsis Yushan Little Pearl contains a big dark-purple spot on whiteflowers so that it is easy to separate the white and dark-purple parts of theflower for analyzing the gene expression profiles. Phalaenopsis OX Red Shoe‘OX1408’ contains red flowers with various floral pigmentation patterning andwas used for gene expression profile analysis. The white-flower Phalaenopsis

Sogo Yukidian ’V39 was used for transient gene overexpression analysis, be-cause the white sepals/petals were beneficial for detecting anthocyanin accu-mulation. Phalaenopsis OXRed Eagle ‘OX1412’with black flowers was used forVIGS analysis, because the all-black flowers made it easy to distinguish thesilencing effects on color changes. Two somalclonal variants of PhalaenopsisEver-spring Prince ’Plum’ were also recruited for examining the presence ofHORT1 in the promoter of PeMYB11. Phalaenopsis equestriswas a native speciesand was used for cloning the promoter sequences of PeMYB11, because itswhole genome sequence is available (Cai et al., 2015). All plants were purchasedfrom the Taiwan Sugar Corp. and OX Orchid Farm and grown in the green-house at National Cheng Kung University under natural light and controlledtemperature from 23°C to 27°C.

Isolation of Plant RNA and RT-qPCR

For RNAextraction, both sepals andpetals in 1-cm to 2.5-cmfloral budswerecollected. Each sample was immersed in liquid nitrogen and stored at 280°C.Total RNA was extracted by the guanidium thiocyanate method (O’Neill et al.,1993), treated with RNase-Free DNase I (New England Biolabs) to removeresidual DNA, and reverse transcribed to cDNA by use of SuperScript III(Invitrogen). Primer pairs for each gene within the gene-specific regions weredesigned and are listed in Supplemental Table S2. For qPCR, the cDNA tem-plate was mixed with SYBR Green PCRMaster Mix (Applied Biosystems) in anABI Prism 7000 Sequence Detection System (Applied Biosystems). Each samplewas analyzed in triplicate. Reactions involved incubation at 95°C for 10min andthermocycling for 40 cycles (95°C for 15 s and 60°C for 1 min). After amplifi-cation, melting curve analysis was used to verify amplicon specificity andprimer dimer formation. The housekeeping gene PeActin4 (AY134752) wasused for normalization (Chen et al., 2005). Data are presented as mean 6sd of three technical replicates, and three biological samples performedindependently.

VIGS of PeMYBs

VIGS of PeMYB2, PeMYB11, and PeMYB12 was performed in PhalaenopsisOX Red Eagle ‘OX1412’ containing a black color in whole flowers, with thesame constructs and protocol from our previous study (Hsu et al., 2015). Thesequences located downstream from the MYB-R2R3 region to the stop codonwere selected, with 435-, 328-, and 311-bp fragments for PeMYB2, PeMYB11,and PeMYB12, respectively (Hsu et al., 2015). Mock-treated plants were injectedwith an empty plasmid of Cymbidium mosaic virus with a Gateway systemvector as a negative control to confirm that any flower color changes were notcaused by the viral infection. Each treatment involved five plants and was re-peated for two VIGS experiments independently.

Transient Assay by Overexpressing PeMYBs viaAgrobacterium Infiltration

For the transient overexpression assay, the modified binary vectorp1304NhXb (Hsu et al., 2015) was used for overexpression of GUS, PeMYBs,PebHLHs, PeWDR1, and PeMYBx06243 in white flowers of Phalaenopsis SogoYukidian ’V39. These genes were amplified, digested with XhoI, and ligated top1304NhXb to produce the overexpression vectors of these genes driven by theduplicated Cauliflower mosaic virus (CaMV) 35S promoter. The recombinantoverexpression vectors were transformed into Agrobacterium tumefaciensEHA105 by electroproration. The vector-containing A. tumefacienswas culturedovernight at 28°C. After centrifugation, bacterial cell pellets were resuspendedby adding 500 mL Murashige and Skoog medium containing 100 mm aceto-syringone to an optical density value of 600 nm as 0.8 to 1 and allowed to standat room temperature for 0.5 h without shaking before infiltration. The sus-pensions were injected into the basal regions of sepals/petals of flowers ofPhalaenopsis Sogo Yukidian ’V39. A. tumefaciens-infiltrated plants were incu-bated at 25°C in an incubator with a 10-h-light/14-h-dark photoperiod for 5 d.After being photographed, the flowers were detached and stored for antho-cyanin content determination. The transient assay involved five plants in eachexperiment with three experiments repeated independently.

Determination of Anthocyanin Content

Anthocyanin contentwasquantifiedwith theHPLCapproach. Sampleswerecollected at 4dafter transient overexpression assayviaA. tumefaciens infiltration.

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The ground powder was extracted with methanol containing 1% (v/v) HCl at4°C for 20 h and centrifuged at 10,000 Xg for 20min at 4°C. The supernatant wasdried with a vacuum concentrator (SCILOGEX IV-ROEV); then 2N HCI wasadded and sampleswere stored for 1 h at 100°C to hydrolyze the glycosyl groupfrom anthocyanins. The hydrolyzed anthocyanins were recovered by passingthrough the solid-phase extraction column (SUPERLCO, DSC-18 SPE) andeluted with methanol containing 1% (v/v) HCI that can be stored and analyzedby HPLC. Two solvents were used in this study, including solvent A (formicacid: water 5 1:99, % [v/v]) and solvent C (methanol 5 100, % [v/v]). HPLCseparation (Hitachi d-7000, l-7100, L7200, and L7420) was performed with a250 3 4.6-mm column of C18 (Thermo Hypersil BDS C18) with A and C sol-vents applied at a flow rate of 1.0 ml/min for anthocyanin compound extrac-tion. The observation wavelength of the UV detector was set at 530 nm. Thestandard of cyanidin (Sigma-Aldrich cyanidin chloride . 95%) was used inHPLC. Data are presented as mean6 sd from three plants, and overexpressionexperiments were repeated for two transient assays.

Molecular Modeling of PeMYB11 and PeMYB11_Pur

The amino acid sequences of PeMYB11 and PeMYB11_Pur were submittedfor automatic modeling at the SWISS‐MODEL server (Guex and Peitsch, 1997;Schwede et al., 2003). The crystal structure of 3zqc was used as a template. Thecoordinate files were depicted as a 3‐D structure using UCSF Chimera program(Pettersen et al., 2004).

Isolation of the Upstream Promoter Sequences of PeMYB11

Genomic DNA was extracted from young flower buds by the cetyl-triammonium bromide method (Hsu et al., 2014). The 2.5-kb upstream pro-moter sequences of PeMYB11 were isolated by PCR amplification with theprimers designed from the whole-genome sequence of P. equestris (Cai et al.,2015). The amplified bands were recovered from gels with use of the Gel DNAFragment Extraction Kit (Geneaid), and cloned into the pGEM-T Easy Vector(Promega). We randomly selected 10 to 12 colonies for sequencing. The pro-moter sequences were compared with all known DNA sequences with use ofthe default settings of BLASTN from National Center for Biotechnology In-formation (www.ncbi.nlm.nih.gov).

Quantitative Dual Luciferase Assay

The 2-kb promoter sequences of PeMYB11 from P. equestris, PhalaenopsisOX Red Shoe ‘OX1408’, and Phalaenopsis Yushan Little Pearl were ligated intothe upstream region of the firefly (Photinus pyralis) luciferase gene to analyze thepromoter activities in red-flower Phalaenopsis OX Red Shoe ‘OX1408’. TheCaMV 35S promoter driven Renilla luciferase gene was an internal control tonormalize infiltration efficiency. At 28 h after Agrobacterium infiltration, eachsample was ground, and then 1X Passive Luciferase buffer (Promega) wasadded. Luciferase activity was measured by use of the dual-luciferase reporterassay system (Promega) with a Lumat LB 9507 Luminometer (Berthold Tech-nologies), a 10-s premeasurement delay, and a 10-s measurement period foreach assay. The relative luciferase activity was calculated as the ratio of firefly toRenilla luciferase activity. For each analysis, three independent buds wereinfiltrated and analyzed, and theAgrobacterium infiltrationswere repeated threetimes independently. Statistical analysis was performed by Student’s t test, andthe differences were considered significant at P # 0.01.

Accession Numbers

The promoter sequences characterized are deposited at the National Centerfor Biotechnology Information site under the accession numbers PeMYB11promoter (MH670728), PeMYB11 promoter from Phalaenopsis Yushan LittlePearl (MH670729), and HORT1 (MH670730).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Harlequin flowers with various pigmentationpatterns.

Supplemental Figure S2. The breeding history for harlequin flowers.

Supplemental Figure S3. Multiple sequence alignment of the amino acidsof PeMYB11 from Phalaenopsis OX Red Shoe ’OX1408’ and Phalaenop-sis Yushan Little Pearl.

Supplemental Figure S4. Phylogenetic tree inferred from the amino acidsequences of R2R3-MYB and R3-MYB repressors.

Supplemental Figure S5. Modeling of the structure of the DNA bindingdomain of PeMYB11 and PeMYB11_Pur.

Supplemental Figure S6. Transient overexpression assay of PeMYB2 andPeMYB12with various PebHLHs in white-flowered of Phalaenopsis SogoYukidian ’V39.

Supplemental Figure S7. The upstream regulatory sequences of PeMYB11in two somaclonal plants of Phalaenopsis Ever-spring Prince ’Plum’witha dark-red flower and a mosaic dark-red flower.

Supplemental Table S1. Factors regulating anthocyanin accumulation.

Supplemental Table S2. Primers used in this study.

Received February 19, 2019; accepted April 29, 2019; published May 14, 2019.

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