1 2 Running head: Harlequin/black flowers of Phalaenopsis · 5/14/2019 · 13 A HORT1...

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1 Running head: Harlequin/black flowers of Phalaenopsis orchids 2 3 Corresponding author: Hong-Hwa Chen 4 Address: 1, University Road, Tainan 701, Taiwan 5 Tel.: 886-6-275-7575 ext. 65521 6 Email: [email protected] 7 8 Research Area: Genes, development and evolution 9 10 11

Plant Physiology Preview. Published on May 14, 2019, as DOI:10.1104/pp.19.00205

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12 A HORT1 retrotransposon insertion in the PeMYB11 13

promoter causes harlequin/black flowers in Phalaenopsis 14 orchids 15

16 Chia-Chi Hsu1, Ching-Jen Su, Mei-Fen Jeng, Wen-Huei Chen, and 17 Hong-Hwa Chen* 18 Department of Life Sciences (C.-C.H., C.-J.S. H.-H.C.) and Orchid Research and 19 Development Center (M.-F.J., W.-H.C., H.-H.C.), National Cheng Kung University, 20 Tainan 21 22 23 One-sentence Summary: Harlequin/black flowers of Phalaenopsis result 24

from the insertion of a retrotransposon in the PeMYB11 25 promoter 26

27 Author Contributions: 28 C.-C.H. designed the research; C.-C.H. and C.-J.S. performed the research; 29 C.-C.H. and M.-F.J. analyzed the data; C.-C.H., M.-F.J., W.-H.C., and 30 H.-H.C. wrote the article. 31 32 Keywords: black color, harlequin flower, HORT1, orchids, PeMYB11, 33

PeMYBx, Phalaenopsis 34 35



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36 This work was supported by the Ministry of Science and Technology, Taiwan (Grant 37 no. MOST 106-2811-B-006-057-). 38 1Current address: 89, Wen-Hsien Road, Nantou City, Nantou County 54041, Taiwan 39 *Corresponding author 40 E-mail: [email protected] 41 Tel: 886-6-2757575 ext. 58111 42 43



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ABSTRACT 44 The harlequin/black flowers in Phalaenopsis orchids contain dark purple spots 45 and various pigmentation patterns, which appeared as a new color in 1996. We 46 analyzed this phenotype by microscopy, high-performance liquid chromatography, 47 gene functional characterization, genome structure analysis, and transient 48 overexpression system to obtain a better understanding of the black color formation in 49 Phalaenopsis orchids. Most mesophyll cells of harlequin flowers showed extremely 50 high accumulation of anthocyanins as well as a high expression of Phalaenopsis 51 equestris MYB11 (PeMYB11) as the major regulatory R2R3-MYB transcription factor 52 for regulating the production of the black color. In addition, we analyzed the 53 expression of basic helix-loop-helix factors, WD40 repeat proteins, and MYB27- and 54 MYBx-like repressors for their association with the spot pattern formation. To 55 understand the high expression of PeMYB11 in harlequin flowers, we isolated the 56 promoter sequences of PeMYB11 from red and harlequin flowers. A retrotransposon, 57 named Harlequin Orchid RetroTransposon 1 (HORT1), was identified and inserted in 58 the upstream regulatory region of PeMYB11. The insertion resulted in strong 59 expression of PeMYB11 and thus extremely high accumulation of anthocyanins in the 60 harlequin flowers of the P. Yushan Little Pearl variety. A dual luciferase assay showed 61 that the insertion of HORT1 enhanced PeMYB11 expression by at least 2-fold 62 compared with plants not carrying the insertion. Furthermore, the presence of HORT1 63 explains the high mutation rates resulting in many variations of pigmentation 64 patterning in harlequin flowers of Phalaenopsis orchids. 65



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INTRODUCTION 66 The harlequin/black flowers in Phalaenopsis orchids, resembling a clown face 67 with painted black spots, appeared as a new color in 1996 (Chen et al., 2004). 68 Harlequin flowers contain dark-purple spots with highly accumulated anthocyanins 69 and form various pigmentation patterns on the flowers (Supplemental Fig. S1). 70 Harlequin flowers originated from the discovery of a somaclonal mutant of P. 71 Golden Peoker 'Brother' (P. Misty Green x P. Liu Tuen-Shen) that has white or yellow 72 flowers with red spots (Supplemental Fig. S2) (Chen et al., 2004). One somaclonal 73 mutant was named P. Golden Peoker 'Ever-spring' and contained purple spots in white 74 flowers (Supplemental Fig. S2) (Chen et al., 2004). On further tissue culture of P. 75 Golden Peoker 'Ever-spring', three different phenotypes resulted, with similar ratios, 76 including P. Golden Peoker 'Brother', P. Golden Peoker 'Ever-spring', and a new color 77 pattern with large, fused, dark-purple spots, named P. Golden Peoker 'BL' 78 (Supplemental Fig. S2) (Chen et al., 2004). Therefore, P. Golden Peoker 'Ever-spring' 79 and P. Golden Peoker 'BL' have harlequin flowers with near-black color, various 80 pigmentation patterning, and high mutation rates, thereby contributing to a new age in 81 harlequin/black flowers in the Phalaenopsis breeding history. 82 Anthocyanins are a group of flavonoid compounds found typically in red, purple, 83 and blue in flowers as well as fruits (Winkel-Shirley, 2001). The biosynthetic pathway 84 for anthocyanins has been one of the most comprehensively studied secondary 85 metabolisms in plants (Grotewold, 2006). All the biosynthetic and regulatory genes 86 involved in the anthocyanin pathway have been cloned and characterized from 87



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petunia (Petunia sp.), snapdragon (Antirrhinum sp.), and other plant species (Broun, 88 2005; Dixon et al., 2005; Koes et al., 2005; Grotewold, 2006). R2R3-MYB and basic 89 helix-loop-helix (bHLH) transcription factors, as well as WD40 repeat (WDR) 90 proteins are the three major protein families that form regulatory complexes for 91 activating anthocyanin accumulation (Koes et al., 2005; Feller et al., 2011; Hichri et 92 al., 2011; Petroni and Tonelli, 2011). R2R3-MYB transcription factors are key 93 components that provide the specificity for the downstream genes and cause 94 tissue-specific anthocyanin accumulation (Borevitz et al., 2000; Zhang et al., 2003). 95 The bHLH transcription factors are essential for the activity of the R2R3-MYB 96 partner by stabilizing the protein complex or promoting its transcription (Hernandez 97 et al., 2004). WDR proteins can physically interact with the MYB and bHLH factors 98 to control anthocyanin biosynthesis (Zhang et al., 2003). 99 Recently, the identification of repressor proteins that inhibit anthocyanin 100 biosynthetic genes has modified the regulatory mechanism of color pigmentation 101 (Aharoni et al., 2001; Dubos et al., 2008; Matsui et al., 2008). Two differential classes 102 of MYB repressors have been identified: R2R3-MYB and R3-MYB repressors with 103 two and one repeat(s) of the MYB domain region, respectively. Among them, 104 FaMYB1 from strawberry (Fragaria x ananassa) and PhMYB27 from petunia are 105 R2R3-MYB repressors containing Ethylene-responsive element binding 106 factor-associated Amphiphilic Repression (EAR) motifs at their C terminus that 107 repress downstream gene transcription (Aharoni et al., 2001; Lin-Wang et al., 2010; 108 Albert et al., 2011; Kagale and Rozwadowski, 2011; Salvatierra et al., 2013). 109



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AtMYBL2 from Arabidopsis thaliana is an R3-MYB repressor and contains a 110 repression motif (TLLLFR) at the C terminus for its repressive activity (Dubos et al., 111 2008; Matsui et al., 2008). PhMYBx from petunia is an R3-MYB repressor without a 112 repressive motif and has an amino acid signature for binding to bHLH partners, so 113 PhMYBx is thought to perform its repressive function by competing for bHLH 114 partners with R2R3-MYB activators (Koes et al., 2005; Zhang et al., 2009). Moreover, 115 a network formed by these transcriptional activators and repressors is thought to 116 regulate the anthocyanin accumulation and pigmentation patterns (Albert et al., 2014), 117 although more evidence is needed. 118 The black color in flowers or fruits has been studied in a few plants, such as 119 blood oranges (Citrus sinensis) (Butelli et al., 2012), purple cauliflower (Brassica 120 oleracea L. var. botrytis) (Chiu et al., 2010), and purple sweet potato (Ipomoea 121 batatas) (Mano et al., 2007). In blood oranges, a Copia-like retrotransposon inserted 122 in the upstream regulatory sequences of a R2R3-MYB transcription factor gene, Ruby, 123 results in extreme anthocyanin accumulation in the fruit (Butelli et al., 2012). A 124 similar situation was found in purple cauliflower, with a Harbinger DNA transposon 125 inserted in the regulatory region of Purple (Pr), which encodes a R2R3-MYB 126 transcription factor and causes the upregulation of Pr and a purple color in curds 127 (Chiu et al., 2010). In sweet potato, IbMYB1 is predominantly expressed in the purple 128 flesh of tuberous roots (Mano et al., 2007). In addition, ectopic expression of a MYB 129 or bHLH transcription factor can produce the dark-purple color formation in 130 transgenic plants such as Deep Purple (MYB) and Leaf Colour (bHLH) from petunia 131



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(Albert et al., 2009; Albert et al., 2011). Therefore, high expression of the regulatory 132 transcription factors involved in the anthocyanin biosynthesis pathway may result in 133 the black fruits and flowers in plants. However, the molecular mechanism for 134 harlequin/black flower formation in Phalaenopsis still needs to be verified. 135 In Phalaenopsis, three R2R3-MYB transcription factors (PeMYB2, PeMYB11, 136 and PeMYB12) have been identified and verified for their roles in regulating distinct 137 pigmentation patterning in flowers (Hsu et al., 2015). Here, we assessed the 138 expression profiles of these three PeMYBs to identify the one that is upregulated in 139 harlequin flowers of P. Yushan Little Pearl, a fourth-generation offspring of P. Golden 140 Peoker that contains a large dark-purple spot on white flowers. We also analyzed other 141 regulatory factors, including bHLH factors, WDR protein, R3-MYB, and R2R3-MYB 142 repressors, for their roles in the production of the black color. Then we analyzed the 143 upstream regulatory sequences of these PeMYBs to investigate why PeMYB11 is 144 upregulated in harlequin flowers. Finally, we identified a retrotransposon, Harlequin 145 Orchid RetroTransposon 1 (HORT1), inserted in the promoter sequence of PeMYB11 146 in P. Yushan Little Pearl, which resulted in high expression of PeMYB11 and extreme 147 anthocyanin accumulation in harlequin flowers. 148 149 150



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RESULTS 151 Harlequin flowers of Phalaenopsis orchids result from high accumulation of 152 anthocyanin 153 To confirm the previous report showing that the harlequin flowers of 154 Phalaenopsis orchids resulted from a high accumulation of anthocyanin but not new 155 anthocyanin compounds (Kuo and Wu, 2011), we used HPLC analysis of hydrolyzed 156 anthocyanin from the flowers of three Phalaenopsis cultivars, white-flower P. Sogo 157 Yukidian 'V3,' red-flower P. Red Shoe 'OX1408', and black-flower P. Yushan Little 158 Pearl (Fig. 1A-C). Only cyanidins were detected in these cultivars, and the difference 159 between the three cultivars was in the total amount of cyanidins present in the flowers, 160 with a 10-fold increase in P. Yushan Little Pearl as compared with P. Red Shoe 161 'OX1408' (Fig. 1D). Hence, the extreme accumulation of cyanidin-derived 162 anthocyanins may explain the black color formation in Phalaenopsis flowers. 163 The distribution of anthocyanin production in cell layers was reported to differ 164 between the three pigmentation patterns: the full-red pigmentation with anthocyanin 165 in subepidermal cells, red spots containing anthocyanin in epidermal cells, and 166 venation pattern with anthocyanin in the region from subepidermal cells to the xylem 167 (Hsu et al., 2015). 168

To examine the location of the increased accumulation of anthocyanins in the 169 harlequin flowers, cross sections of the red-flower P. Red Shoe 'OX1408' and 170 harlequin-flower P. Yushan Little Pearl were observed by microscopy. The red cells 171 containing anthocyanins were present in the subepidermal cells of both adaxial and 172



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abaxial sides in P. Red Shoe 'OX1408' (Fig. 1E) but they were present in the 173 epidermal cells to most mesophyll cell layers in P. Yushan Little Pearl (Fig. 1F), 174 which suggests that many more mesophyll cells produce anthocyanins to cause the 175 abundant anthocyanin accumulation and black color formation. 176 177



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PeMYB11 is related to harlequin flower formation in Phalaenopsis 178 To identify the structural and regulatory genes involved in harlequin flower 179 formation in Phalaenopsis, the flowers of P. Yushan Little Pearl were separated into 180 purple spots and white area of the flowers (Fig. 1A) and analyzed for the expression 181 profiles of anthocyanin-related genes, including three structural genes, PeF3H5, 182 PeDFR1, and PeANS3, as well as three regulatory genes, PeMYB2, PeMYB11, and 183 PeMYB12, which have been studied for their regulatory role in the anthocyanin 184 biosynthesis pathway (Hsu et al., 2015). High transcript levels of PeF3H5, PeDFR1, 185 and PeANS3 were detected in the purple but not white part (Fig. 2A), which suggests 186 that a regulatory gene is responsible for the transactivation of these three structural 187 genes in the purple spots. Only PeMYB11 had strong expression in purple parts, with 188 no expression of PeMYB2 and PeMYB12 in purple and white parts of this flower (Fig. 189 2B). In addition, a 33.9-fold increase in PeMYB11 expression was detected in the 190 purple parts of P. Yushan Little Pearl compared to the red flower of P. Red Shoe 191 'OX1408', while the structural genes PeF3H5, PeDFR1, and PeANS3 showed 2.1-, 192 2.6-, and 17.7-fold increased expression, respectively (Fig. 2A and B). In addition, a 193 pair of degenerated primers targeting the conserved R2R3 domain of MYB 194 transcription factors was used for amplifying other possible factors, and all the 195 amplified sequences were PeMYB11. Thus, PeMYB11 was the major regulatory 196 R2R3-MYB transcription factor for the harlequin flowers in Phalaenopsis, which is 197 consistent with our previous results that PeMYB11 is responsible for the spot 198 pigmentation patterning (Hsu et al., 2015). 199



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To verify whether the sequence of PeMYB11 differed between the harlequin 200 flowers of P. Yushan Little Pearl and the other flower colors, we cloned and 201 sequenced the cDNA sequences of PeMYB11 from P. Red Shoe 'OX1408' and P. 202 Yushan Little Pearl and named them PeMYB11 and PeMYB11_Pur, respectively. The 203 amino acid sequences of PeMYB11 and PeMYB11_Pur showed 95% identity and 204



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96% similarity. Four amino acids changes were present in the R2R3 domain, with Tyr 205 changed to Cys (Y28C), Ala to Thr (A31T), Lys to Met (K42M), and Arg to Gly 206 (R49G) (Supplemental Fig. S3). The transcriptional functions of PeMYB11 and 207 PeMYB11_Pur were analyzed in the following experiment with the subtitle of 208 "Transient overexpression approach confirms the in vivo functions of regulatory genes 209 in Phalaenopsis flowers". 210 To confirm that PeMYB11 is the major regulatory gene responsible for the 211 harlequin phenotype, we used virus-induced gene silencing (VIGS) to investigate the 212 in planta function of PeMYB11 in harlequin flowers of P. OX Red Eagle ‘OX1412’ 213 (Fig. 3A) because of its dark-red color in entire flowers and ease in distinguishing the 214 silencing effects on color changes. P. OX Red Eagle ‘OX1412’ highly expressed 215 PeMYB11 but very low PeMYB2 and PeMYB12 expression (Fig. 3B). 216 PeMYB11-silencing resulted in the loss of anthocyanin content with flower colors 217 fading from dark-purple to pink (Fig.3D). In contrast, PeMYB2-silenced flowers 218 contained a few white areas, with no obvious differences between PeMYB12-silenced 219 and mock flowers (Fig. 3, A, C, and E), which suggests that the dark-red color is 220 produced by continuous spot pigmentation patterning. Therefore, PeMYB11 was 221 responsible for the high anthocyanin accumulation of spot pigmentation patterning in 222 harlequin flowers of Phalaenopsis orchids. 223 224 Other regulatory factors related to harlequin flower formation in Phalaenopsis 225 The pigmentation patterns are regulated by a conserved anthocyanin-regulating 226



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transcription factor complex consisting of an MYB, bHLH, and WDR-containing 227 protein (the MBW complex) (Feller et al., 2011). We cloned three PebHLHs, 228 PebHLH1 to PebHLH3, and one PeWDR (Table 1) to analyze their association with 229 PeMYBs in Phalaenopsis. In addition, several putative MYB repressors were isolated 230 from the orchid transcriptome database OrchidBase (Tsai et al., 2013). Phylogenetic 231 analysis showed that PeMYB4 to PeMYB8 (Hsu et al., 2015) were similar to the 232 R2R3-MYB MYB27-like repressors, and PeMYBx06243, PeMYBx07630, and 233 PeMYBx30334 grouped with R3-MYB MYBx-like repressors (Supplemental Fig. S4). 234 Reverse transcription quantitative RT-qPCR was used to verify the expression profiles 235 of these genes in P. Yushan Little Pearl. The expression of PebHLH1 was higher 236



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(4.1-fold) in purple than white parts, whereas PebHLH2, PebHLH3, and PeWDR1 237 were expressed slightly higher (1.8- to 2.0–fold) in white than in purple parts (Fig. 2, 238 C-F). In addition, only PeMYBx06243 could be amplified from flowers of P. Yushan 239 Little Pearl, but no expression of PeMYBx07630 and PeMYBx30334 was detected. 240 PeMYBx06243 was highly expressed (96.9-fold) in the purple compared to the white 241 part (Fig. 2G), even though PeMYBx06243 was predicted as a repressor of 242 anthocyanin production. Moreover, all R2R3-MYB repressors, PeMYB4 to PeMYB8, 243 except for PeMYB7, showed slightly higher expression profiles, with a 1.8- to 3.7–244 fold increase in the white compared to the purple part of P. Yushan Little Pearl 245 flowers, and PeMYB8 showed the most expression (Fig. 2, H-L). The functions of 246 these regulators with PeMYB11 for anthocyanin accumulation and pigmentation 247 patterning were further investigated by a transient overexpression approach in 248 Phalaenopsis orchids. 249 250 Transient overexpression confirms the in vivo functions of regulatory genes in 251 Phalaenopsis flowers 252 A transient overexpression protocol was developed to investigate the in vivo 253 functions of the regulatory genes in activating the accumulation of anthocyanin and 254 floral scents in Phalaenopsis flowers (Hsu et al., 2015; Chuang et al., 2018). 255 Agrobacterium tumefaciens containing various regulatory genes were infiltrated into 256 white petals of P. Sogo Yukidian 'V3' alone or with various bHLH transcription 257 factors and/or MYB27- and MYBx-like repressors. Overexpression of PeMYB11_Pur 258



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with PebHLH2 conferred a pink color in white petals (Fig. 4E) and a 6.67-fold 259 increase in anthocyanin content as compared with overexpression of PeMYB11_Pur 260 with GUS (Fig. 4G). However, overexpression of PeMYB11 with all three PebHLHs 261 and PeMYB11_Pur with PebHLH1 or PebHLH3 conferred no obvious pink color (Fig. 262



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4, A-D and F). Therefore, PeMYB11_Pur from P. Yushan Little Pearl contained higher 263 transactivation activities than PeMYB11 from P. OX Red Shoe 'OX1408' when 264 interacting with PebHLH2. Intriguingly, the different amino acids between 265 PeMYB11_Pur and PeMYB11 were not located in the conserved motif 266 ([D/E]Lx2[R/K]x3Lx6Lx3R) for interacting with a bHLH transcription factor 267 (Zimmermann et al., 2004). From the molecular modeling analysis, the substitution of 268 Tyr28 in PeMYB11 to Cys28 in PeMYB11_Pur would result in the formation of a 269 disulfide bond between residues Cys28 and Cys53 (Supplemental Fig. S5). Cys49 is 270 specific to plant R2R3 MYB and can form a disulfide bond with Cys53 to regulate the 271 DNA binding capacity of the plant MYB by modulating the dimerization capability 272 (Pireyre and Burow, 2015). In our case, Cys28 may exert the function of Cys49 in 273 other plants to form a disulfide bond with Cys53 and thus regulates the DNA binding 274 capacity of PeMYB11_Pur by modulating the dimerization capability. However, the 275 effects of the changed amino acids in PeMYB11_Pur for the transactivation activities 276 need further investigation. 277 In addition, we used overexpression of PeMYB2 or PeMYB12 with the three 278 PebHLHs to verify the association between MYB and bHLH transcription factors. 279 Overexpression of PeMYB2 with PebHLH1, PebHLH2, and PebHLH3 conferred a 280 similar red color as PeMYB2 with GUS in white petals of P. Sogo Yukidian 'V3' 281 (Supplemental Fig. S6, A-C). Also, the anthocyanin content was slightly increased 282 (1.34- to 1.46-fold) with PeMYB2 with PebHLH1, PebHLH2, and PebHLH3 as 283 compared to PeMYB2 with GUS (Supplemental Fig. S6G). Overexpression of 284



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PeMYB12 with both PebHLH1 and PebHLH2 conferred a slightly pink color in white 285 petals of P. Sogo Yukidian 'V3' (Supplemental Fig. S6, D-F), and the anthocyanin 286 content was increased (8.21- and 10.36-fold) upon overexpression of PeMYB12 with 287 PebHLH1 and PebHLH2 as compared to PeMYB12 with GUS (Supplemental Fig. 288 S6H). All these results suggest that PebHLH1 and PebHLH2 improve PeMYB 289 functions in anthocyanin accumulation in Phalaenopsis flowers, whereas PeMYB11 290 preferred PebHLH2, and PeMYB12 worked with both PebHLH1 and PebHLH2. 291 To assess the repressive functions of the predicted PeMYB repressors, including 292 five MYB27-like PeMYB4~PeMYB8 and one MYBx-like PeMYBx06243 in P. Sogo 293 Yukidian 'V3', PeMYB2 was chosen for its high activity for anthocyanin accumulation 294 (Supplemental Fig. S6) (Hsu et al., 2015), so the repressive effects of these repressors 295 were more sensitive and reliable than using PeMYB11 or PeMYB12. Overexpression 296 of PeMYB2 with all PeMYB repressors resulted in a 32% to 80% reduction in 297 anthocyanin content as compared with PeMYB2 and GUS (Fig. 5). In addition, 298 PeMYBx06243 caused a striking reduction of anthocyanin content (88%) with 299 overexpression of PeMYB11_Pur and PebHLH2 (Fig. 6, B and D) as compared with 300 overexpression of PeMYB12 with PebHLH2 (26% reduction) and PeMYB2 with 301 PebHLH2 (40% reduction) (Fig. 6, C and D). Therefore, all these PeMYB repressors 302 showed the repressive effects on anthocyanin accumulation activated by PeMYB2, 303 PeMYB11, or PeMYB12. The repressive effects of these repressors may be simply 304 binding the same sites and preventing effective activation by PeMYB activators. 305 Overall, these results show that the bHLH transcription factors, WDR proteins, and 306



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MYB27- and MYBx-like repressors regulate the red color formation with all three 307 MYB transcription factors, PeMYB2, PeMYB11 and PeMYB12, in Phalaenopsis 308 flowers, although PeMYB11 was the only transcription factor responsible for the high 309



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anthocyanin accumulation in harlequin flowers of Phalaenopsis. 310 311 A new retrotransposon, PeHORT, inserted in the upstream regulatory sequence 312 of PeMYB11 313 To identify why PeMYB11 was highly expressed in the purple part of harlequin 314 flowers of P. Yushan Little Pearl, we isolated a 2.5-kb upstream regulatory sequence 315



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of PeMYB11 from OrchidBase 3.0, which contains the whole genome sequence of P. 316 equestris (Cai et al., 2015). The 2.5-kb upstream regulatory sequence of PeMYB11 317



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was used to design primers to clone these promoter sequences from P. equestris, P. 318 OX Red Shoe ‘OX1408,’ and P. Yushan Little Pearl. Interestingly, the white part of P. 319 Yushan Little Pearl had a 5.3-kb fragment and the purple parts had 5.3-kb and 2.8-kb 320 fragments. Both the 2.1- and 2.3-kb fragments were amplified from P. equestris and P. 321 OX Red Shoe ‘OX1408’, respectively (Fig. 7A). After sequencing the 5.3- and 2.8-kb 322 sequences, a new retrotransposon was identified and named Harlequin Orchid 323 RetroTransposon 1 (HORT1), which was similar to the Gypsy-like retrotransposons 324 present in other plants, such as mulberry (Morus notabilis) and wild strawberry 325 (Fragaria vesca) in Repbase, the most commonly used database of repetitive DNA 326 elements (Bao et al., 2015), although no functional characterization has yet been 327 reported. The full-length sequence of HORT1 is about 3 kb and contains a 1.6-kb 328 coding sequence with 500-bp long terminal repeats (LTRs) at both ends. Therefore, 329 the 5.3- and 2.8-kb fragments from P. Yushan Little Pearl contained 3 kb full length 330 and 500-bp LTRs of HORT1, respectively, with the reverse direction inserted in the 331 1.5-kb upstream regulatory sequence of PeMYB11 (Fig. 7B). 332 In addition, two somaclonal plants of another harlequin cultivar, P. Ever-spring 333 Prince 'Plum' with different pigmentation patterning were recruited to confirm the 334 presence of HORT1 in harlequin flowers (Supplemental Fig. S7, A and B). The 5.3-kb 335 fragment was amplified from both harlequin plants with a dark-red flower or with a 336 mosaic dark-red flower, whereas a 2.3-kb sequence was amplified only from the 337 mosaic dark-red flower, that was similar to the fragment from P. OX Red Shoe 338 ‘OX1408’ (Supplemental Fig. S7C). These results suggest that the 5.3-kb fragment 339



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containing the insertion of HORT1 in the PeMYB11 promoter is a common 340 phenomenon for harlequin flowers of Phalaenopsis orchids. 341 To investigate whether the HORT1 sequence affected the high expression of 342 PeMYB11 in harlequin flowers, we used a dual luciferase assay. We cloned the 2.3-kb 343 promoter sequences of PeMYB11 from P. equestris and P. OX Red Shoe ‘OX1408’ as 344 well as the 2.8- and 5.3-kb fragments of P. Yushan Little Pearl into the upstream 345 region of the firefly luciferase gene to analyze the promoter activities in red-flower P. 346 OX Red Shoe ‘OX1408’. The promoter sequences of PeMYB11 from P. equestris and 347 P. OX Red Shoe ‘OX1408’ showed similar transcriptional activities for driving 348 luciferase (Fig. 7C), whereas the 2.8-kb promoter sequence of PeMYB11 from P. 349 Yushan Little Pearl containing one LTR of HORT1 showed a 2-fold increase in 350 transcriptional activities. In contrast, the 5.3-kb PeMYB11 promoter with full-length 351 HORT1, including two LTRs and one coding region, caused only a 0.37-fold increase 352 in transcriptional activities (Fig. 7C), which may result from low transgenic efficiency 353 with a large DNA fragment and thus low transcriptional activities. The promoter 354 fragments of PeMYB11 harboring only one LTR of HORT1 showed enhanced 355 transactivation activities, such as in the purple part of flowers. This result suggests 356 that the insertion of HORT1 in the upstream regulatory sequences of PeMYB11 357 increased the transcriptional level of PeMYB11 in planta and resulted in the high 358 expression of PeMYB11 in harlequin flowers of P. Yushan Little Pearl. 359 360 361



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DISCUSSION 362 Harlequin/black flowers of Phalaenopsis orchids 363 A few plants have dark-purple to black colors in flowers and fruits, which are 364 supposed to catch consumers’ eyes. These contain a high content of anthocyanins with 365 high antioxidant activity (Jayaprakasha and Patil, 2007; Kelebek et al., 2008). Blood 366 oranges contain the activities to reduce oxidative stress (Bonina et al., 2002), protect 367 DNA against oxidative damage (Guarnieri et al., 2007), and reduce cardiovascular 368 risk factors (de Pascual-Teresa et al., 2010; Paredes-López et al., 2010). Purple 369 cauliflowers have an eye-catching purple color, with potent nutritional and 370 health-promoting effects (Chiu et al., 2010). Here, the marketable effects of the 371 harlequin flowers in Phalaenopsis are important as a new color and breeding parents 372 for harlequin flowers. The breeding for harlequin flowers started in 1996, and the 373 hybrid number has reached 3,614 as compared with the total 35,129 hybrids registered 374 in the Royal Horticultural Society (OrchidWiz, 2018). These harlequin hybrids also 375 contain various pigmentation patterns, and even novel patterns have been found in 376 new hybrids. Therefore, the harlequin flowers are important breeding targets in 377 Phalaenopsis and are used as a model system for studying the molecular mechanism 378 of floral pigmentation patterns. 379 We analyzed the phenotype of the special color of harlequin flowers by 380 microscopy, HPLC, gene functional characterization, genome structure analysis, and 381 transient overexpression system to obtain an overall understanding of this phenotype 382 in Phalaenopsis orchids. Most mesophyll cells of harlequin flowers produced 383



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extremely high accumulation of anthocyanins as well as PeMYB11 expression as the 384 major regulatory R2R3-MYB transcription factor, accompanied by bHLH factors, 385 WDR proteins, and MYB27- and MYBx-like repressors to regulate the black color 386 formation. Finally, we identified a retrotransposon, HORT1, inserted in the upstream 387 regulatory sequences of PeMYB11 that resulted in the high expression of PeMYB11 388 and led to highly accumulated anthocyanins in the harlequin flowers of P. Yushan 389 Little Pearl. Moreover, the presence of the retrotransposon HORT1 is concomitant 390 with the high mutation rates and thus the multiple pigmentation patterns in harlequin 391 flowers of Phalaenopsis orchids. 392 393 Harlequin flowers of Phalaenopsis result from mesophyll cells with high 394 accumulation of anthocyanin 395 The distribution of anthocyanin production affects color formation and is 396 regulated by individual genes of the R2R3-MYB family. In apple (Malus x domestica), 397 MdMYB1/MdMYBA expression is correlated with anthocyanin accumulation in the red 398 skin of the fruit (Takos et al., 2006; Ban et al., 2007). MdMYB10 is specifically 399 expressed in type 1 red-flesh apple, such as ‘Red Field,’ with red pigmentation in the 400 fruit core, cortex, and foliage (Espley et al., 2007), whereas MdMYB110 is responsible 401 for the type 2 red-flesh apple, such as ‘Sangrado,’ with its red cortex, white fruit core, 402 and green foliage (Chagné et al., 2012). In Phalaenopsis, the distribution of 403 anthocyanin production in cell layers differs between three pigmentation patterns: 404 full-red pigmentation with anthocyanin in the subepidermal cells, red spots containing 405



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anthocyanin in epidermal cells, and venation pattern with anthocyanin in the region 406 from subepidermal cells to the xylem. This distribution is regulated by three distinct 407 R2R3-MYB transcription factors, PeMYB2, PeMYB11, and PeMYB12, respectively 408 (Hsu et al., 2015). 409 Here, we showed that harlequin flowers resulted from the high accumulation of 410 anthocyanins that were present from both the adaxial and abaxial epidermis to most 411 mesophyll cells. The highly accumulated anthocyanins in harlequin flowers resulted 412 from the high amount of anthocyanins produced in epidermal cells and also most 413 mesophyll cells, which suggests that HORT1 enhanced the expression of PeMYB11 414 and also had spatial effects on ectopic expression of PeMYB11 in mesophyll cells of 415 flowers. 416 The anthocyanin content in harlequin flowers of P. Yushan Little Pearl was 417 increased 10-fold as compared with the red-flower P. Red Shoe 'OX1408', which was 418 coincident with a 33.9-fold increase in PeMYB11 transcripts and 2.1- to 17.7-fold 419 increase in PeF3H5, PeDFR1, and PeANS3 transcripts. However, the insertion of one 420 LTR of HORT1 in the promoter sequence of PeMYB11 resulted in a 2-fold increase in 421 transcriptional activities as compared with the promoter sequence of PeMYB11 alone 422 on a dual luciferase assay. The added mesophyll cell layers with accumulated 423 anthocyanin production were important for the black color in Phalaenopsis. 424 425 High expression of MYB transcription factors is related to black color formation 426 In blood orange, the massive anthocyanin production in fruit resulted from a 427



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Copia-like retrotransposon inserted in the upstream regulatory region of a 428 R2R3-MYB transcription factor, Ruby, to cause high expression (Butelli et al., 2012). 429 Also in purple cauliflower, the upstream regulatory sequence of a R2R3-MYB 430 transcription factor, Purple (Pr), was inserted by a Harbinger DNA transposon and 431 caused upregulation of Pr and the purple color formation in curds (Chiu et al., 2010). 432 In this study, we screened the expression profiles of genes including structural genes, 433 R2R3-MYB activators, bHLH factors, WDR proteins, as well as R3-MYB and 434 R2R3-MYB repressors to identify the major regulator of the black color formation in 435 Phalaenopsis. The R2R3-MYB PeMYB11 was identified by the retrotransposon 436 HORT1 inserted in the upstream regulatory region to highly upregulate the expression 437 of PeMYB11. In addition, the changed amino acids of PeMYB11 from P. OX Red 438 Shoe 'OX1408' to P. Yushan Little Pearl enhanced its transcriptional activities when 439 co-expressed with PebHLH2. 440 441 MYB and other regulatory factors are related to the black spot pattern 442 The spatial and temporal patterning of anthocyanins is mostly determined by the 443 regulation of the R2R3-MYB factors, whereby each individual factor controls distinct 444 patterns with shared bHLH and WDR factors (Schwinn et al., 2006; Albert et al., 2011; 445 Davies et al., 2012; Hsu et al., 2015). The spot pattern has been studied in several 446 plants, showing differential expression of Dihydroflavonol 4-reductase2 (Dfr2) for 447 red-purple spots of Clarkia gracilis (Martins et al., 2013), light-induced Lilium hybrid 448 MYB6 (LhMYB6) for red spots in pink flowers of Asiatic hybrid lily (Lilium spp.) 449



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‘Montreux’ (Yamagishi et al., 2010), LhMYB12-Lat for the splatter-type spots in the 450 Asiatic hybrid lily ‘Latvia’ (Yamagishi et al., 2014), and PeMYB11 in Phalaenopsis 451 (Hsu et al., 2015). Moreover, a network formed by these transcriptional activators and 452 repressors is thought to regulate anthocyanin accumulation and pigmentation patterns 453 (Albert et al., 2014). In non-inductive conditions, R2R3-MYB repressors are 454 expressed and cooperate with constitutively expressed bHLH factors and WDR 455 protein to form repressive MBW complexes and inhibit anthocyanin production 456 (Albert et al., 2014). In inductive conditions, the R2R3-MYB activators are expressed 457 and form the active MBW complex with bHLH factors and WDR protein to activate 458 anthocyanin biosynthesis, and the R2R3-MYB and R3-MYB repressors were also 459 present for feedback inhibition (Albert et al., 2014). The R3-MYB repressors and 460 WDR proteins are small proteins and capable of intercellular movement that may 461 relate to the pigmentation patterning of red spots in white flowers (Albert et al., 462 2014). 463 In this study, we confirmed that the dark-purple spots of P. Yushan Little Pearl 464 were regulated by the high expression of PeMYB11 and we further analyzed the 465 effects of the anthocyanin-related activators and repressors for the spot pattern. 466 PeMYB11 was responsible for the spot pattern formation, and the insertion of HORT1 467 in the upstream regulatory region of PeMYB11 enhanced its function and resulted in 468 the black color. In addition, bHLH factors, especially PebHLH2, cooperated with 469 PeMYB11 for its transcriptional activities, whereas R2R3-MYB repressors were 470 expressed higher in the white than purple part (Fig. 2, H-L) and may be slightly 471



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related to the "black spot" pattern formation. Moreover, the R3-MYB MYBx-like 472 PeMYBx06243 could inhibit the transcriptional activities of R2R3-MYB activators, 473 although the expression of PeMYBx06243 was much higher in the purple than white 474 part of P. Yushan Little Pearl. A MYBx-like repressor was found capable of 475 intercellular movement and may contribute to pigmentation patterning (Albert et al., 476 2014), so this PeMYBx06243 protein could be small enough to cross from the purple 477 to white part for its repressive function and result in the black-spot pattern. However, 478 this needs further investigation. 479 480 Transposable elements were related to the color changes in plants 481 Transposable elements (TEs) related to color changes in plants have been widely 482 reported. TEs affect the genetic diversity in plants via insertion, excision, and 483 chromosomal rearrangements (Kidwell and Lisch, 2002). The mutations with TE 484 insertion and excision in gene coding regions may disrupt the protein functions or 485 change enzymatic activities (Nordborg and Walbot, 1995), whereas the upstream 486 regulatory sequences with TE insertions may stop, increase, or change the 487 tissue-specific gene expression profiles (Wicker et al., 2016). For example, most of 488 the color mutants in Japanese morning glory (Ipomoea purpurea) have TE insertions 489 and result in loss-of-function or somatically unstable genes (Clegg and Durbin, 2003). 490 Various TE types have been involved, including the class-I short interspersed 491 elements (SINEs) as well as class-II CACTA elements, hAT elements, miniature 492 inverted repeat transposable elements (MITEs), and Mutator-like elements (MULEs) 493



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(Clegg and Durbin, 2003; Park et al., 2007). In contrast, the enhancers or promoters 494 within the TEs may cause upregulated expression in the inserted genes when the TEs 495 are inserted in the upstream regulatory sequences of color-related genes. Similarly, a 496 Copia-like retrotransposon, Tcs1, and a Harbinger DNA transposon inserted in the 497 upstream regulatory regions of Ruby and Pr genes in blood orange and purple 498 cauliflower, respectively, caused the upregulated accumulation of anthocyanin (Chiu 499 et al., 2010; Butelli et al., 2012). The LTR of Tcs1 provides a new transcription start 500 site for Ruby expression and low temperature-dependent anthocyanin accumulation in 501 blood oranges (Butelli et al., 2012). However, although Tcs1 is located in the 502 upstream regulatory regions of Ruby with the same orientation in most accessions of 503 blood oranges, one accession called Jingxian from China contained a similar 504 retrotransposon, Tcs2, inserted with the opposite orientation of the Ruby gene (Butelli 505 et al., 2012). The expression of Ruby in Jingxian suggests that Tcs2 provides an 506 upstream activating sequence for the expression of Ruby and the production of 507 anthocyanin (Butelli et al., 2012). 508 In this study, we found a HORT retrotransposon inserted in the upstream 509 regulatory region of PeMYB11 with the reverse direction. The retrotransposon may 510 play a role as an enhancer to upregulate the expression of PeMYB11 and also extend 511 the spatial expression of PeMYB11 from adaxial epidermal cells to most mesophyll 512 cell layers, thereby resulting in harlequin flowers of Phalaenopsis orchids. 513 Previous reports showed a high mutation rate with micropropagation of P. 514 Golden Peoker 'Ever-spring': 40% of plants were P. Golden Peoker 'Brother', 30% 515



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remained as P. Golden Peoker 'Ever-spring', and 30% were P. Golden Peoker 'BL' 516 (Chen et al., 2004). Moreover, harlequin flowers produced various pigmentation 517 patterns with dark-purple spots on their flowers, whereas the color patterns for 518 harlequin flowers are still changeable and new patterns emerge with further breeding. 519 Here, we found a HORT retrotransposon inserted in the upstream regulatory 520 sequences of PeMYB11 that explained the black color formation of harlequin flowers 521 and also the high mutation rates and changeable pigmentation patterns. Therefore, the 522 harlequin flowers in Phalaenopsis provide an excellent system for investigating the 523 regulation of anthocyanin accumulation, flower pigmentation patterning, and 524 transposable element activation. 525 526 MATERIALS AND METHODS 527 Plant materials 528 P. Yushan Little Pearl contains a big dark-purple spot on white flowers, so that it 529 is easy to separate the white and dark-purple parts of the flower for analyzing the gene 530 expression profiles. P. OX Red Shoe ‘OX1408’ contains red flowers with various 531 floral pigmentation patterning and was used for gene expression profile analysis. The 532 white-flower P. Sogo Yukidian 'V3' was used for transient gene overexpression 533 analysis, because the white sepals/petals were beneficial for detecting anthocyanin 534 accumulation. P. OX Red Eagle ‘OX1412’ with black flowers was used for 535 virus-induced gene silencing (VIGS) analysis, because the all-black flowers made it 536 easy to distinguish the silencing effects on color changes. Two somalclonal variants of 537



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P. Ever-spring Prince 'Plum' were also recruited for examining the presence of 538 HORT1 in the promoter of PeMYB11. P. equestris was a native species and was used 539 for cloning the promoter sequences of PeMYB11, because its whole genome sequence 540 is available (Cai et al., 2015). All plants were purchased from the Taiwan Sugar 541 Corp. and OX Orchid Farm and grown in the greenhouse at National Cheng Kung 542 University under natural light and controlled temperature from 23°C to 27°C. 543 544 Isolation of plant RNA and RT-qPCR 545 For RNA extraction, both sepals and petals in 1-cm to 2.5-cm floral buds were 546 collected. Each sample was immersed in liquid nitrogen and stored at -80°C. Total 547 RNA was extracted by the guanidium thiocyanate method (O'Neill et al., 1993), 548 treated with RNase-Free DNase I (New England Biolabs) to remove residual DNA, 549 and reverse transcribed to cDNA by use of SuperScript III (Invitrogen). Primer pairs 550 for each gene within the gene-specific regions were designed and are listed in 551 Supplemental Table S2. For qPCR, the cDNA template was mixed with SYBR Green 552 PCR Master Mix (Applied Biosystems) in an ABI Prism 7000 Sequence Detection 553 System (Applied Biosystems). Each sample was analyzed in triplicate. Reactions 554 involved incubation at 95°C for 10 min and thermocycling for 40 cycles (95°C for 15 555 s and 60°C for 1 min). After amplification, melting curve analysis was used to verify 556 amplicon specificity and primer dimer formation. The housekeeping gene PeActin4 557 (AY134752) was used for normalization (Chen et al., 2005). Data are presented as 558 mean±SD of three technical replicates and three biological samples performed 559



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independently. 560 561 VIGS of PeMYBs 562 VIGS of PeMYB2, PeMYB11, and PeMYB12 was performed in P. OX Red Eagle 563 ‘OX1412’ containing a black color in whole flowers, with the same constructs and 564 protocol from our previous study (Hsu et al., 2015). The sequences located 565 downstream from the MYB-R2R3 region to the stop codon were selected, with 435-, 566 328-, and 311-bp fragments for PeMYB2, PeMYB11, and PeMYB12, respectively (Hsu 567 et al., 2015). Mock-treated plants were injected with an empty plasmid of Cymbidium 568 mosaic virus with a Gateway system vector as a negative control to confirm that any 569 flower color changes were not caused by the viral infection. Each treatment involved 570 five plants and was repeated for two VIGS experiments independently. 571 572 Transient assay by overexpressing PeMYBs via Agrobacterium infiltration 573 For the transient overexpression assay, the modified binary vector p1304NhXb 574 (Hsu et al., 2015) was used for overexpression of GUS, PeMYBs, PebHLHs, PeWDR1, 575 and PeMYBx06243 in white flowers of P. Sogo Yukidian 'V3'. These genes were 576 amplified, digested with XhoI, and ligated to p1304NhXb to produce the 577 overexpression vectors of these genes driven by the duplicated cauliflower mosaic 578 virus (CaMV) 35S promoter. The recombinant overexpression vectors were 579 transformed into Agrobacterium tumefaciens EHA105 by electroproration. The 580 vector-containing A. tumefaciens was cultured overnight at 28°C. After centrifugation, 581



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bacterial cell pellets were resuspended by adding 500 µL Murashige and Skoog 582 medium containing 100 mM acetosyringone to an optical density value of 600 nm as 583 0.8~1 and allowed to stand at room temperature for 0.5 h without shaking before 584 infiltration. The suspensions were injected into the basal regions of sepals/petals of 585 flowers of P. Sogo Yukidian 'V3'. A. tumefaciens-infiltrated plants were incubated at 586 25°C in an incubator with a 10-h-light/14-h-dark photoperiod for 5 d. After being 587 photographed, the flowers were detached and stored for anthocyanin content 588 determination. The transient assay involved five plants in each experiment with three 589 experiments repeated independently. 590 591 Determination of anthocyanin content 592 Anthocyanin content was quantified with the high-performance liquid 593 chromatography (HPLC) approach. Samples were collected at 4 days after transient 594 overexpression assay via A. tumefaciens infiltration. The ground powder was 595 extracted with methanol containing 1% (v/v) HCl at 4°C for 20 h and centrifuged at 596 10,000 Xg for 20 min at 4°C. The supernatant was dried with a vacuum concentrator 597 (SCILOGEX IV-ROEV, CT, USA), then 2N HCI was added and samples were stored 598 for 1 hr at 100°C to hydrolyze the glycosyl group from anthocyanins. The hydrolyzed 599 anthocyanins were recovered by passing through the solid-phase extraction column 600 (SUPERLCO, DSC-18 SPE, Bellefonte, PA, USA) and eluted with methanol 601 containing 1% (v/v) HCI that can be stored and analyzed by HPLC. Two solvents 602 were used in this study, including solvent A (formic acid: water = 1:99, % V/V) and 603



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solvent C (methanol = 100, %V). HPLC separation (Hitachi D-7000, L-7100, L7200 604 and L7420, Japan) was performed with a 250 x 4.6-mm column of C18 (Thermo 605 Hypersil BDS C18, USA) with A and C solvents applied at a flow rate of 1.0 ml/min 606 for anthocyanin compound extraction. The observation wavelength of the UV detector 607 was set at 530 nm. The standard of cyanidin (SIGMA-ALDRICH cyanidin chloride 608 >95 %, St. Louis, MO, USA) was used in HPLC. Data are presented as mean±SD 609 from three plants and overexpression experiments were repeated for two transient 610 assays. 611 612 Molecular modeling of PeMYB11 and PeMYB11_Pur 613 The amino acid sequences of PeMYB11 and PeMYB11_Pur were submitted for 614 automatic modeling at the SWISS‐MODEL server (Guex and Peitsch, 1997; Schwede 615 et al., 2003). The crystal structure of 3zqc was used as a template. The coordinate files 616 were depicted as a 3‐D structure using UCSF Chimera program (Pettersen et al., 617 2004). 618 619 Isolation of the upstream promoter sequences of PeMYB11 620 Genomic DNA was extracted from young flower buds by the cetyltriammonium 621 bromide (CTAB) method (Hsu et al., 2014). The 2.5-kb upstream promoter sequences 622 of PeMYB11 were isolated by PCR amplification with the primers designed from the 623 whole-genome sequence of P. equestris (Cai et al., 2015). The amplified bands were 624 recovered from gels with use of the Gel DNA Fragment Extraction Kit (Geneaid, New 625



36

Taipei City, Taiwan), and cloned into the pGEM-T Easy Vector (Promega, Madison, 626 WI, USA). We randomly selected 10 to 12 colonies for sequencing. The promoter 627 sequences were compared to all known DNA sequences with use of the default 628 settings of BLASTN from NCBI (www.ncbi.nlm.nih.gov). 629 630 Quantitative dual luciferase assay 631 The 2-kb promoter sequences of PeMYB11 from P. equestris, P. OX Red Shoe 632 ‘OX1408’, and P. Yushan Little Pearl were ligated into the upstream region of the 633 firefly (Photinus pyralis) luciferase gene to analyze the promoter activities in 634 red-flower P. OX Red Shoe ‘OX1408’. The CaMV 35S promoter driven Renilla 635 luciferase gene was an internal control to normalize infiltration efficiency. At 28 hours 636 after Agrobacterium infiltration, each sample was ground, and then 1X Passive 637 Luciferase Buffer (Promega, Madison, WI, USA) was added. Luciferase activity was 638 measured by use of the dual-luciferase reporter assay system (Promega) with a Lumat 639 LB 9507 Luminometer (Berthold Technologies, Bad Wildbad, Germany), a 10-sec 640 pre-measurement delay and a 10-sec measurement period for each assay. The relative 641 luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity. 642 For each analysis, three independent buds were infiltrated and analyzed, and the 643 Agrobacterium infiltrations were repeated three times independently. Statistical 644 analysis was performed by Student’s t-test, and the differences were considered 645 significant at p≦0.01. 646 647



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Accession Numbers 648 The promoter sequences characterized are deposited at the NCBI site under the 649 accession numbers: PeMYB11 promoter (MH670728), PeMYB11 promoter from P. 650 Yushan Little Pearl (MH670729), and HORT1 (MH670730). 651 652 Supplemental Data 653 Supplemental Figure S1. Harlequin flowers with various pigmentation patterns. 654 Supplemental Figure S2. The breeding history for harlequin flowers. 655 Supplemental Figure S3. Multiple sequence alignment of the amino acids of 656 PeMYB11 from P. OX Red Shoe 'OX1408' and P. Yushan Little Pearl. 657 Supplemental Figure S4. Phylogenetic tree inferred from the amino acid sequences 658 of R2R3-MYB and R3-MYB repressors. 659 Supplemental Figure S5. Modeling of the structure of the DNA binding domain of 660 PeMYB11 and PeMYB11_Pur. 661 Supplemental Figure S6. Transient overexpression assay of PeMYB2 and PeMYB12 662 with various PebHLHs in white-flowered of P. Sogo Yukidian 'V3'. 663 Supplemental Figure S7. The upstream regulatory sequences of PeMYB11 in two 664 somaclonal plants of P. Ever-spring Prince 'Plum' with a dark-red flower and a mosaic 665 dark-red flower. 666 Supplemental Table S1. Factors regulating anthocyanin accumulation. 667 Supplemental Table S2. Primers used in this study. 668 669



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ACKNOWLEDGMENT 670 This work was supported by the Ministry of Science and Technology, Taiwan (Grant 671 no. MOST 106-2811-B-006-057-). 672 673 FIGURE LEGENDS 674 Figure 1. (A-C) Phenotypes of Phalaenopsis Sogo Yukidian ‘V3’ (A), P. OX Red 675 Shoe ‘OX1408’ (B), and P. Yushan Little Pearl (C). Bars = 1 cm. (C) The “Purple” 676 and “White” parts were used for gene expression analysis. (D) Anthocyanin contents 677 of black flowers of P. Yushan Little Pearl. Anthocyanin contents were compared 678 between P. Sogo Yukidian ‘V3’, P. OX Red Shoe ‘OX1408’, and P. Yushan Little 679 Pearl. Cyanidin at 200 ng was used as a standard to calculate the anthocyanin quantity 680 present in flowers (shown in parentheses). (E-F) This is the cross section of the flower 681 petals of P. OX Red Shoe ‘OX1408’ (E) and P. Yushan Little Pearl (F). Bars = 0.1 682 mm. 683 684 Figure 2. Expression profiles of PeMYBs and other regulatory genes in P. Yushan 685 Little Pearl. The relative mRNA abundance of genes was normalized to the expression 686 of PeAct4 and presented as mean±SD of three technical replicates and three biological 687 samples performed independently. (A, B) Numbers above the boxes indicate the fold 688 of increase for gene expression in P. Yushan Little Pearl as compared to that of P. Red 689 Shoe 'OX1408,' which was denoted as 1x. (C-L) Expression of purple and white parts 690 of harlequin flowers of P. Yushan Little Pearl. Low expression levels were denoted as 691 1x and used to calculate the fold changes in expression. 692 693 Figure 3. Virus-induced gene silencing (A, C, D, and E) and gene expression profiles 694 (B) of three PeMYBs in P. OX Red Eagle ‘OX1412.’ The flowers from mock (A); 695 single-silenced PeMYB2 (C), PeMYB11 (D), and PeMYB12 (E) plants. (B) Data were 696 repeated twice for VIGS experiments. Bar = 1 cm. 697 698 Figure 4. Transient overexpression assay of PeMYB11 and PeMYB11_Pur with 699 various PebHLHs in white-flowered P. Sogo Yukidian 'V3'. (A-F) Transient 700



39

overexpression of PeMYB11 (A-C) or PeMYB11_Pur (D-F) was performed with GUS 701 in the left petal or various PebHLHs in the right petal of a single flower. GUS was 702 recruited as a negative control for no correlation to PeMYBs on anthocyanin 703 accumulation. The regions enclosed by blue dotted lines were transfected by 704 Agrobacterium and cut for quantitative anthocyanin content analysis. (G) Quantitative 705 anthocyanin content in transient overexpression of PeMYB11 and PeMYB11_Pur with 706 various PebHLHs in P. Sogo Yukidian 'V3'. Data are presented as mean±SD from 707 three plants and two transient overexpression assays. Numbers above the boxes 708 indicate the changed anthocyanin contents in PeMYB11 with PebHLHs as compared 709 to that in PeMYB11 with GUS, which was denoted as 1x. FW indicates fresh weight. 710 711 Figure 5. Transient overexpression assay of PeMYB2 with various repressors in 712 white-flowered P. Sogo Yukidian 'V3'. (A-F) Transient overexpression of PeMYB2 713 was performed with GUS in the left petal and various repressors in the right petal of 714 one flower. GUS was used as a negative control for no correlation to PeMYBs on 715 anthocyanin accumulation. The regions enclosed by blue dotted lines were transfected 716 by Agrobacterium and cut for quantitative anthocyanin content analysis. (G) 717 Quantitative anthocyanin content in transient overexpression of PeMYB2 with various 718 repressors in P. Sogo Yukidian 'V3'. Data are presented as mean±SD from three plants 719 and two transient overexpression assays. Numbers above the boxes indicate the 720 changed anthocyanin contents in PeMYB2 with repressors as compared to that with 721 GUS, which was denoted as 1x. FW indicates fresh weight. 722 723 Figure 6. Transient overexpression assay of three PeMYBs, and PebHLH2 with 724 PeMYBx repressors in white flowers of P. Sogo Yukidian 'V3'. (A-C) Transient 725 overexpression of PeMYB2 (A), PeMYB11_Pur (B), and PeMYB12 (C) with 726 PebHLH2 was performed with GUS in the left petal or PeMYBx in the right petal of a 727 single flower. GUS was used as a negative control for no correlation to PeMYBs on 728 anthocyanin accumulation. The regions enclosed by blue dotted lines were transfected 729 by Agrobacterium and cut for quantitative anthocyanin content analysis. (D) 730 Quantitative anthocyanin content in transient overexpression of PeMYBs, and 731 PebHLH2 with PeMYBx repressors in P. Sogo Yukidian 'V3'. Data are presented as 732 mean±SD from three plants and two transient overexpression assays. Numbers above 733



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the boxes indicate the changed anthocyanin contents in PeMYB2 with repressors as 734 compared to that with GUS, which was denoted as 1x. FW indicates fresh weight. 735 736 Figure 7. (A, B) Cloning of the upstream regulatory sequences of PeMYB11 from P. 737 equestris, P. Red Shoe 'OX1408,' and the purple and white parts of black flowers of P. 738 Yushan Little Pearl. (B) Arrowheads indicate the primers used to construct the 739 promoter sequences to drive firefly luciferase for the dual luciferase assay. (C) Dual 740 luciferase assay of promoters of PeMYB11 from P. equestris (Pe), P. OX Red Shoe 741 ‘OX1408’ (OX1408), and the 2.8- and 5.3-kb sequences from P. Yushan Little Pearl. 742 Data are presented as means±SD from three plants and the Agrobacterium infiltrations 743 were repeated three times independently. 744 745 746



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Chuang YC, Hung YC, Tsai WC, Chen WH, Chen HH (2018) PbbHLH4 regulates floral monoterpene biosynthesis in Phalaenopsishttps://plantphysiol.orgDownloaded on December 28, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

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https://www.ncbi.nlm.nih.gov/pubmed?term=Albert NW%2C Davies KM%2C Lewis DH%2C Zhang H%2C Montefiori M%2C Brendolise C%2C Boase MR%2C Ngo H%2C Jameson PE%2C Schwinn KE %282014%29 A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots%2E Plant Cell&dopt=abstract

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Schwinn K, Venail J, Shang Y, Mackay S, Alm V, Butelli E, Oyama R, Bailey P, Davies K, Martin C (2006) A small family of MYB-regulatorygenes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell 18: 831-851


Takos AM, Jaffé FW, Jacob SR, Bogs J, Robinson SP, Walker AR (2006) Light-induced expression of a MYB gene regulatesanthocyanin biosynthesis in red apples. Plant Physiol 142: 1216-1232


Tsai WC, Fu CH, Hsiao YY, Huang YM, Chen LJ, Wang M, Liu ZJ, Chen HH (2013) OrchidBase 2.0: comprehensive collection ofOrchidaceae floral transcriptomes. Plant Cell Physiol 54: e7


Wicker T, Yu Y, Haberer G, Mayer KF, Marri PR, Rounsley S, Chen M, Zuccolo A, Panaud O, Wing RA (2016) DNA transposon activity isassociated with increased mutation rates in genes of rice and other grasses. Nat Commun 7: 12790


Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. PlantPhysiol 126: 485-493


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


Yamagishi M, Toda S, Tasaki K (2014) The novel allele of the LhMYB12 gene is involved in splatter‐type spot formation on the flowertepals of Asiatic hybrid lilies (Lilium spp.). New Phytol 201: 1009-1020


Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A (2003) A network of redundant bHLH proteins functions in all TTG1-dependentpathways of Arabidopsis. Development 130: 4859-4869


Zhang W, Ning G, Lv H, Liao L, Bao M (2009) Single MYB-type transcription factor AtCAPRICE: a new efficient tool to engineer theproduction of anthocyanin in tobacco. Biochem Biophys Res Commun 388: 742-747


Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcriptionfactors interacting with R/B‐like BHLH proteins. The Plant Journal 40: 22-34



https://www.ncbi.nlm.nih.gov/pubmed?term=Pettersen EF%2C Goddard TD%2C Huang CC%2C Couch GS%2C Greenblatt DM%2C Meng EC%2C Ferrin TE %282004%29 UCSF Chimera%2Da visualization system for exploratory research and analysis%2E Journal of computational chemistry&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Salvatierra A%2C Pimentel P%2C Moya%2DLe�n MA%2C Herrera R %282013%29 Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene%2E Phytochemistry&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Schwede T%2C Kopp J%2C Guex N%2C Peitsch MC %282003%29 SWISS%2DMODEL%3A an automated protein homology%2Dmodeling server%2E Nucleic acids research&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Schwinn K%2C Venail J%2C Shang Y%2C Mackay S%2C Alm V%2C Butelli E%2C Oyama R%2C Bailey P%2C Davies K%2C Martin C %282006%29 A small family of MYB%2Dregulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum%2E Plant Cell&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Takos AM%2C Jaff� FW%2C Jacob SR%2C Bogs J%2C Robinson SP%2C Walker AR %282006%29 Light%2Dinduced expression of a MYB gene regulates anthocyanin biosynthesis in red apples%2E Plant Physiol&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Tsai WC%2C Fu CH%2C Hsiao YY%2C Huang YM%2C Chen LJ%2C Wang M%2C Liu ZJ%2C Chen HH %282013%29 OrchidBase 2%2E0%3A comprehensive collection of Orchidaceae floral transcriptomes%2E Plant Cell Physiol 54%3A e&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Yamagishi M%2C Shimoyamada Y%2C Nakatsuka T%2C Masuda K %282010%29 Two R2R3%2DMYB genes%2C homologs of Petunia AN2%2C regulate anthocyanin biosyntheses in flower tepals%2C tepal spots and leaves of Asiatic hybrid lily%2E Plant Cell Physiol&dopt=abstract

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1 2 Running head: Harlequin/black flowers of Phalaenopsis · 5/14/2019 · 13 A HORT1...

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