Identification of an R2R3-MYB gene regulating tepal background coloration in Tricyrtis sp.

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Yuta Shinoku, Yusuke Kanemaki, Mai Shibuya, Kakeru Inagawa, Juria Ono, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8898430/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract The liliaceous ornamental plant Tricyrtis sp. produces unique flowers, whose tepals have many random reddish-purple spots on a light purple background. In our previous studies, we performed comprehensive isolation and expression analysis of the anthocyanin biosynthesis-related genes to elucidate the molecular mechanism underlying flower color pattern formation in this plant, and identified the R2R3-MYB transcription factor gene, TrMYB1 , expressed in tepals. In the present study, we carried out detailed expression and functional analyses of TrMYB1 . Shading treatment of flower buds markedly reduced background coloration of the tepals, while there was little effect on spot formation. In addition, TrMYB1 expression level decreased in the tepals of shaded flower buds. Overexpression of TrMYB1 in Tricyrtis sp. resulted in deeper coloration and significantly increased anthocyanin content in tepals. RNAi-mediated knockdown of TrMYB1 significantly suppressed the expression of the anthocyanin biosynthetic enzyme genes, resulting in a significant decrease in anthocyanin contents and marked reduction in background coloration in the tepals, with little effect on spot formation. These results indicate that tepal background coloration and spot formation may be regulated by different molecular mechanisms, and that background coloration is likely induced by light through activation of TrMYB1 . Biological sciences/Biotechnology Biological sciences/Molecular biology Biological sciences/Plant sciences anthocyanin liliaceous ornamental plant RNAi flower color pattern shading Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Flower color and flower color pattern are among the most important traits for ornamental plants. Flower color is determined primarily by the type and concentration of pigments, as well as the combination of multiple pigments. Whereas flower color patterns, such as spots, variegation, stripes, picotee, and gradation, are caused by different patterns of pigment accumulation in tepals 1 . Among flower pigments, anthocyanins, members of flavonoids, are found in various plant species and are responsible for a wide range of colors including orange, red, purple, and blue 2 , 3 . The anthocyanin biosynthetic pathway is broadly conserved among higher plant species and is well understood 3 . In this pathway, eight enzymes, chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavonoid 3’-hydroxylase (F3’H), flavonoid 3’,5’-hydroxylase (F3’5’H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and anthocyanin synthase (ANS) are involved in the biosynthesis of anthocyanidins and flavonols 3 , 4 . The expression of these enzyme genes is known to be mainly regulated by the MBW transcription factor complex consisting of R2R3-myeloblastosis (MYB), basic helix-loop-helix (bHLH), and WD40 repeats (WDR) proteins 5 . These transcription factors and their functions have also been characterized in various higher plant species 3 , 5 , 6 . Anthocyanin biosynthesis is known to be affected by environmental stimuli such as light, temperature, and drought. Among them, light has been extensively studied as a key factor influencing anthocyanin biosynthesis 7 . It has been reported that shading treatments reduce anthocyanin accumulation in tepals or fruit skins in Eustoma grandiflorum 8 , Gerbera hybrida 9 , Malus domestica 10 , Chrysanthemum morifolium 11 , Solanum melongena 12 , and Lilium regale 13 . The molecular mechanisms underlying light-induced anthocyanin biosynthesis have been investigated in several plant species. In Petunia hybrida , both an R2R3-MYB transcription factor PHZ and a bHLH transcription factor AN1 are expressed in response to light and play key roles in regulating anthocyanin biosynthesis in floral and vegetative organs 14 , 15 . In L . regale , light-induced R2R3-MYB factor LhMYB15 is implicated in the regulation of anthocyanin biosynthesis, contributing to characteristic pigmentation pattern formation in tepals 13 . In C . morifolium , light induces the expression of CmMYB9a while suppressing that of CmBBX28 , a B-box protein that negatively regulates anthocyanin biosynthesis, thereby leading to anthocyanin accumulation in petals 16 . Tricyrtis spp. are liliaceous ornamental plants native to Japan. One of the Tricyrtis cultivars, ‘Shinonome’, produces unique flowers with tepals bearing numerous randomly distributed reddish-purple spots on a light purple background. This flower color pattern is clearly different from variegation patterns caused by transposons 17 – 19 and bicolor patterns caused by post-transcriptional gene silencing 20 , 21 . To elucidate the molecular mechanism of flower color pattern formation in Tricyrtis sp. 'Shinonome', we performed comprehensive isolation and expression analysis of the anthocyanin biosynthesis-related genes in this plant 1 , 22 . To date, seven flavonoid biosynthetic enzyme genes, TrCHS (AB478624; GenBank/EMBL/DDBJ databases; the same applies below), TrCHI (AB908277), TrF3H (LC209222), TrF3’H (AB480691), TrDFR (AB830112), TrFLS (LC103181), and TrANS (LC209106), and three transcription factor genes for the MBW complex, TrMYB1 (AB856412), TrbHLH2 (LC223741), and TrWDR (LC223742), have successfully been isolated. The expression of TrMYB1 in tepals is correlated with that of 'late' biosynthetic enzyme genes ( TrF3’H , TrDFR , TrFLS and TrANS ), suggesting that TrMYB1 may contribute to anthocyanin biosynthesis in tepals through regulation of these genes. However, the role of TrMYB1 in flower color pattern formation remains unclear. In the present study, we examined the effects of light on anthocyanin biosynthesis and the expression of TrMYB1 in the tepals of Tricyrtis sp. ‘Shinonome’ by shading treatment of flower buds, and further analyzed the function of TrMYB1 through overexpression and RNAi-mediated knockdown in transgenic plants. Result Shading treatment of flower buds in Tricyrtis sp. ‘Shinonome’ To examine the effects of light on anthocyanin accumulation and the expression of anthocyanin biosynthesis-related genes in tepals of Tricyrtis sp. 'Shinonome', shading treatment of flower buds was carried out. Under non-shaded conditions, background coloration in tepals was observed from S1 flower buds onwards particularly on the abaxial side of the outer tepals (Fig. 1 A). Spot formation was detectable in tepals of the S1 flower buds and expanded over the entire tepals from S2 onwards (Supplementary Fig. S1 ). In S5 flowers, tepals were characterized by numerous randomly distributed reddish-purple spots on a light purple background. On the other hand, under shaded conditions, background coloration in tepals was suppressed in S1–S4 flower buds (Fig. 1 A). In S5 flowers, tepal background coloration was much reduced compared with those under non-shaded conditions, although reddish-purple spots were produced in the same manner as under non-shaded conditions (Fig. 1 A). Under shaded conditions, the relative amounts of total anthocyanins in outer tepals significantly decreased at all stages of flower development compared with those under non-shaded conditions (Fig. 1 B). Real-time RT-PCR analysis for the expression of anthocyanin biosynthesis-related genes in outer tepals of S4 flower buds revealed that shading treatment reduced the expression levels of TrMYB1 , TrbHLH2 , TrF3'H , TrDFR , and TrANS (Fig. 1 C). On the other hand, no apparent differences in the expression level of TrWDR and TrCHS were observed between shaded and non-shaded conditions (Fig. 1 C). In inner tepals, background coloration was inherently faint, making the effect of shading less apparent than in outer tepals (Supplementary Fig. S2). Overexpression of TrMYB1 in Tricyrtis sp. ‘Shinonome’ To assess the effect of TrMYB1 overexpression, TrMYB1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into Tricyrtis sp. 'Shinonome' (Fig. 2 ). Five independent transgenic lines (TrMYB1-OE2, -OE7, -OE8, -OE9, and -OE12) were obtained (Fig. 3 A), among which TrMYB1-OE7 and -OE8 produced flowers (Fig. 3 B). These transgenic lines showed enhanced pigmentation in most floral organs as well as in leaves compared with the wild type (WT) (Figs. 3 A, B). However, no obvious pigmentation was observed in the basal region of the tepals. In contrast, pigmentation was markedly enhanced in naturally pigmented regions of the tepals of wild-type plants (Fig. 3 B). In particular, TrMYB1-OE2, -OE7, -OE8, and -OE9 showed significantly increased anthocyanin accumulation in leaves (Fig. 3 C), and TrMYB1-OE8 showed significantly increased anthocyanin accumulation in outer tepals of S5 flowers (Fig. 3 D). Real-time RT-PCR analysis using leaves showed that the expression levels of TrMYB1 and enzyme genes ( TrCHS , TrCHI , TrDFR , and TrANS ) significantly increased in all five transgenic lines (Fig. 3 E). TrbHLH2 expression also significantly increased in TrMYB1-OE2, -OE7, and -OE8, whereas no significant changes in TrWDR expression levels were observed in any of the five transgenic lines (Fig. 3 E). RNAi-mediated knockdown of TrMYB1 in Tricyrtis sp. ‘Shinonome’ To further examine the function of TrMYB1 , RNAi-mediated knockdown construct was introduced into Tricyrtis sp. 'Shinonome' (Fig. 2 ). Two independent transgenic lines (TrMYB1-RNAi1 and -RNAi2) were obtained and both produced flowers (Fig. 4 A). In S5 flowers of both transgenic lines, background coloration on both the adaxial and abaxial sides of the tepals almost completely disappeared (Fig. 4 A). By contrast, reddish-purple spots were still detectable, although their pigmentation intensity was markedly reduced compared with that in WT flowers (Fig. 4 A). For both transgenic lines, the relative amounts of total anthocyanins in outer tepals of S5 flowers significantly decreased compared with WT (Fig. 4 B). Real-time RT-PCR analysis using outer tepals of S5 flowers showed that the expression levels of TrMYB1 in TrMYB1-RNAi1 and -RNAi2 were reduced to approximately 10% and 20% of that in WT, respectively (Fig. 4 C). The expression levels of enzyme genes ( TrCHS , TrCHI , TrDFR , and TrANS ) and TrbHLH2 were also substantially reduced in TrMYB1-RNAi1 and -RNAi2 (Fig. 4 C). In particular, the expression levels of TrCHI and TrANS showed significant decreases in both transgenic lines (Fig. 4 C). On the other hand, no significant changes in the TrWDR expression levels were observed in both transgenic lines (Fig. 4 C). Discussion To elucidate the molecular mechanism of flower color pattern formation in tepals of Tricyrtis sp., we previously performed comprehensive isolation and expression analyses of the anthocyanin biosynthesis-related genes 1 , 22 . In the present study, as a next step, we investigated the effect of light on anthocyanin biosynthesis in tepals and performed functional analysis of the R2R3-MYB gene TrMYB1 , which may be involved in the regulation of anthocyanin biosynthesis in tepals. Shading treatment of flower buds or inflorescences has been reported to reduce anthocyanin accumulation in tepals of several plant species, such as G . hybrida 9 , C . morifolium 11 , and L . regale 13 . In the present study, shading treatment of flower buds similarly reduced anthocyanin accumulation in tepals at all developmental stages (Figs. 1 A, B). Interestingly, background coloration in the tepals was largely reduced under shading conditions, whereas spot formation was scarcely affected (Fig. 1 A). This differential response suggests that background coloration and spot formation in tepals are regulated by distinct mechanisms, in which background coloration is light dependent, whereas spot formation is largely light independent. Furthermore, the expression level of TrMYB1 decreased under shading conditions (Fig. 1 C), indicating that TrMYB1 may be positively regulated by light. Together, these findings suggest that TrMYB1 plays an important role in light-induced anthocyanin biosynthesis in tepals of Tricyrtis sp. Light-induced R2R3-MYB gene expression and anthocyanin biosynthesis have also been reported in other plant species, including P . hybrida 14 , 15 , L . regale 13 , and C . morifolium 16 , suggesting that similar regulatory mechanisms may be conserved across a wide range of higher plants. In Arabidopsis thaliana , the anthocyanin biosynthesis–related R2R3-MYB transcription factors PAP1 and MYB12 are directly regulated by the light-responsive bZIP transcription factor HY5 23,24 . HY5 directly interacts with the ubiquitin ligase COP1 and is degraded in the nucleus under dark conditions. In contrast, COP1 is inactivated upon light exposure, preventing HY5 degradation and resulting in its stabilization 25 . COP1 also directly regulates the stability of the R2R3-MYB transcription factors PAP1 and PAP2 26 . Together, these light-dependent mechanisms regulate both the expression and protein stability of R2R3-MYB transcription factors, ultimately modulating anthocyanin biosynthesis. Similar regulatory mechanisms of light-induced anthocyanin biosynthesis mediated by COP1 and HY5 have been reported in other plant species, such as M . domestica and S . melongena 12 . Further studies are needed to clarify the molecular mechanism of light-induced TrMYB1 expression in Tricyrtis sp. In our previous studies, heterologous expression of TrMYB1 increased anthocyanin accumulation in transgenic Pelargonium crispum and Kalanchoe blossfeldiana 27 , 28 , suggesting that TrMYB1 functions as a conserved regulator of anthocyanin biosynthesis across species. To further clarify its role in its native genetic background, we generated transgenic Tricyrtis plants overexpressing TrMYB1 . Overexpression of TrMYB1 resulted in a significant increase in anthocyanin accumulation in the leaves (Figs. 3 A, C). The expression levels of anthocyanin biosynthetic enzyme genes ( TrCHS , TrCHI , TrDFR , and TrANS ) also significantly increased in the leaves of these transgenic plants (Fig. 3 E). These results support the involvement of TrMYB1 in anthocyanin biosynthesis through regulation of anthocyanin biosynthetic genes, consistent with our previous finding that TrMYB1 is associated with regulation of late biosynthetic genes 1 . The expression level of TrbHLH2 was also increased in the leaves of these transgenic plants, suggesting that TrMYB1 may regulate TrbHLH2 either directly or indirectly through regulatory feedback mechanisms. Similarly, overexpression of R2R3-MYB genes has been shown to activate the expression of bHLH genes in various plant species 29 – 32 . In addition, overexpression of TrMYB1 resulted in enhanced pigmentation in most floral organs (Fig. 3 B). However, the absence of ectopic pigmentation in the basal region of the tepals in TrMYB1 -overexpressing lines suggests that additional regulatory factors, such as bHLH and WDR transcription factors, may be required for anthocyanin accumulation in this region. By contrast, enhanced pigmentation was observed in regions that are already pigmented in the tepals of WT plants, implying that TrMYB1 alone may be sufficient to enhance anthocyanin biosynthesis in such regions. In transgenic Tricyrtis sp. plants with RNAi-mediated knockdown of TrMYB1 , both anthocyanin accumulation and the expression levels of anthocyanin biosynthetic enzyme genes, as well as TrbHLH2 , decreased in outer tepals (Figs. 4 B, C). Moreover, the flowers of transgenic plants showed a pronounced reduction of background coloration on both the adaxial and abaxial sides of the tepals, whereas reddish-purple spots were still detectable (Fig. 4 A). These results indicate that background coloration and spot formation may be regulated by different mechanisms, and that TrMYB1 may play a central role in the formation of tepal background coloration through regulation of anthocyanin biosynthesis-related genes in Tricyrtis sp. The involvement of multiple R2R3-MYB transcription factors in the regulation of flower color pattern formation has been reported in several plant species. In P . hybrida , four R2R3-MYB genes have been identified as regulators of anthocyanin biosynthesis in tepals. Among them, AN2 and AN4 control pigmentation in the limb and tube of tepals, respectively 33 , 34 . In addition, DPL regulates veining in the petal tube, and PHZ is light inducible and associated with blushing flower buds 15 . In Clarkia gracilis , four R2R3-MYB genes involved in tepal pigmentation have also been identified 35 . Among them, CgMYB1C controls red spot formation, whereas CgMYB12 regulates background coloration in the basal (cup) region. CgMYB6 and CgMYB11 are also suggested to contribute to background coloration. In the present study, spot pigmentation was clearly reduced in the TrMYB1-RNAi lines. It is possible that additional R2R3-MYB transcription factors are involved in the regulation of spot pigmentation and that their activities may have been partly affected by off-target effects of the RNAi construct. Taken together, these findings suggest that multiple R2R3-MYBs, including TrMYB1 , may cooperatively regulate flower color pattern formation in Tricyrtis sp. In the present study, we demonstrated that TrMYB1 is expressed in response to light and regulates tepal background coloration by promoting anthocyanin biosynthesis. The results obtained here are expected to contribute significantly to studies on flower color pattern formation in ornamental plants. However, the molecular mechanism of tepal spot formation in Tricyrtis remains unclear. In particular, the mechanism underlying the formation of randomly distributed spots of various sizes has not yet been reported and represents a highly intriguing biological phenomenon. Therefore, elucidating the molecular basis of spot formation in Tricyrtis sp. may provide an important model system for understanding the general principles of flower color pattern formation, and further advances in this area are strongly anticipated. Materials and methods Plant materials and callus cultures Potted plants of Tricyrtis sp. ‘Shinonome’ were cultivated in a greenhouse without heating. Embryogenic callus cultures used for transformation were induced and maintained as described by Nakano et al. 36 . Shading treatment of flower buds Shading treatment was carried out by covering shoot apices with aluminum foil from the time of bract emergence until anthesis. Outer tepals were collected from samples with and without shading treatment at five different developmental stages: S1, flower buds of 6–10 mm in length; S2, flower buds of 11–15 mm in length; S3, flower buds of 16–20 mm in length; S4, flower buds just before anthesis (over 21 mm in length); and S5, flowers just after anthesis 1 . RNA isolation and cDNA synthesis Total RNA was extracted using RNeasy Mini Kit (QIAGEN, Hilden, Germany) and treated with RNase-Free DNase Set (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. RT-PCR and real-time RT-PCR Reverse transcription-polymerase chain reaction (RT-PCR) was performed using EmeraldAmp® MAX PCR Master Mix (Takara Bio Inc., Shiga, Japan) on the T100 thermal cycler (Bio-Rad, Hercules, CA, USA) according to Otani et al. 1 . Real-time RT-PCR was performed using KOD SYBR® qPCR Mix (Toyobo Co., Ltd., Osaka, Japan) on the MyGo real-time PCR system (IT-IS Life Science Ltd., Dublin, Ireland). The specificity of the amplification products was confirmed by melting curve analysis. Real-time RT-PCR was performed in biological triplicate for each sample. Relative amounts of transcripts were normalized to the actin gene of Tricyrtis sp. ( TrAct2 ; AB196260) and calculated using the 2 −ΔΔCt method 37 . Primer sets used for these analyses are listed in Supplementary Table S1 online. Measurement of total anthocyanins Total anthocyanins were extracted from outer tepals of flowers at different developmental stages and from leaves using the methanol-HCl method according to Rabino and Mancinelli 38 . Absorbance of the extracts was measured at 530 and 657 nm using a spectrophotometer (Eppendorf BioSpectrometer® basic; Eppendorf, Hamburg, Germany). Total anthocyanin contents were calculated with the following formula: (A 530 − 0.25 × A 657 ) × M⁻¹ [M: weight (g) of the plant material used for extraction]. Vector construction and Agrobacterium -mediated transformation Agrobacterium tumefaciens strains EHA101/pIG- TrMYB1 and EHA101/p TrMYB1 -RNAi were used to produce transgenic plants for TrMYB1 overexpression and RNAi-mediated knockdown, respectively. The T-DNA region of pIG- TrMYB1 contained TrMYB1 driven by the CaMV 35S promoter, neomycin phosphotransferase II ( NPTII ) driven by the Nopaline Synthase ( NOS ) promoter, and hygromycin phosphotransferase ( HPT ) driven by the CaMV35S promoter (Fig. 2 ). The p TrMYB1 -RNAi vector was constructed based on the pANDA35HK vector 39 , 40 . The T-DNA region of p TrMYB1 -RNAi contained two inverted repeats of a 22-bp TrMYB1 trigger sequence fragment driven by the CaMV35S promoter, NPTII driven by the NOS promoter, and HPT driven by the CaMV35S promoter (Fig. 2 ). All primers used for vector construction are listed in Supplementary Table S1 online. Transformation of Tricyrtis sp. 'Shinonome' was performed as previously described by Adachi et al. 41 . To confirm the transgenic nature of regenerated plantlets, PCR analyses were performed using primer sets specific to the T-DNA region (Supplementary Table S1 online). Transgenic plantlets were acclimatized and cultivated in a growth room at 25°C under continuous light-emitting diode (LED) lighting (ca. 170 µmol m – 2 s – 1 ). Morphological, biochemical, and molecular characterizations of transgenic plants were performed during the flowering season. Declarations Additional Information Competing financial interests The authors declare no competing financial interests. (A) Representative flower phenotypes at each developmental stage under non-shaded and shaded conditions. ab, abaxial side; ad, adaxial side. Bar = 1 cm. (B) Relative amounts of total anthocyanins in outer tepals at each developmental stage under non-shaded and shaded conditions. Data are shown as means ± standard errors (SE; n = 9). * indicates significant differences at p < 0.001 by Student’s t-test. (C) Expression levels of anthocyanin biosynthesis-related genes in outer tepals at S4 under non-shaded and shaded conditions. TrAct2 was used as an internal control. Cropped gel images are shown, and the corresponding full-length gel images are provided in Supplementary Figure S2. Funding This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 19K06028 and 24K08880) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Author Contribution Y.S. drafted the manuscript. M.N. and M.O. reviewed and edited the manuscript. Y.K. and M.S. performed the gene isolation and characterization, and Y.S., K.I., and J.O. performed the transgenic experiments. 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MYB transcription factors GmMYBA2 and GmMYBR function in a feedback loop to control pigmentation of seed coat in soybean. J. Exp. Bot. 72 , 4401–4418. 10.1093/jxb/erab152 (2021). Spelt, C., Quattrocchio, F., Mol, J. N. M. & Koes, R. anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes. Plant. Cell. 12 , 1619–1631. 10.1105/tpc.12.9.1619 (2000). Xu, W. et al. Regulation of flavonoid biosynthesis involves an unexpected complex transcriptional regulation of TT8 expression, in Arabidopsis. New. Phytol . 198 , 59–70. 10.1111/nph.12142 (2013). Quattrocchio, F., Wing, J. F., Leppen, H., Mol, J. & Koes, R. E. Regulatory Genes Controlling Anthocyanin Pigmentation Are Functionally Conserved among Plant Species and Have Distinct Sets of Target Genes. Plant. Cell. 5 , 1497–1512. 10.1105/tpc.5.11.1497 (1993). Quattrocchio, F., Wing, J. F., Woude, K., Mol, J. N. M. & Koes, R. Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes. Plant. J. 13 , 1497–1512. 10.1046/j.1365-313X.1998.00046.x (1998). Martins, T. R., Jiang, P. & Rausher, M. D. How petals change their spots: cis-regulatory re-wiring in Clarkia (Onagraceae). New. Phytol . 216 , 510–518. 10.1111/nph.14163 (2017). Nakano, M. et al. Somatic embryogenesis and plant regeneration from callus cultures of several species in the genus Tricyrtis . Vitro Cell. Dev. Bio -Plant . 40 , 274–278. 10.1079/IVP2003506 (2004). Livak, K. J. & Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 –∆∆CT Method. Methods 25 , 402–408. 10.1006/meth.2001.1262 (2001). Rabino, I., Mancinelli, A. L. & Light Temperature, and Anthocyanin Production. Plant. Physiol. 81 , 922–924. 10.1104/pp.81.3.922 (1986). Miki, D. & Shimamoto, K. Simple RNAi Vectors for Stable and Transient Suppression of Gene Function in Rice. Plant. Cell. Physiol. 45 , 490–495. 10.1093/pcp/pch048 (2004). Miki, D., Itoh, R. & Shimamoto, K. RNA Silencing of Single and Multiple Members in a Gene Family of Rice. Plant. Physiol. 138 , 1903–1913. 10.1104/pp.105.063933 (2005). Adachi, Y., Mori, S. & Nakano, M. Agrobacterium-mediated Production of Transgenic Plants in Tricyrtis hirta (Liliaceae). Acta Hort . 673 , 415–419. 10.17660/ActaHortic.2005.673.52 (2005). Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.pdf Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 05 Mar, 2026 Reviews received at journal 04 Mar, 2026 Reviews received at journal 03 Mar, 2026 Reviews received at journal 24 Feb, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviewers invited by journal 24 Feb, 2026 Editor assigned by journal 24 Feb, 2026 Editor invited by journal 24 Feb, 2026 Submission checks completed at journal 19 Feb, 2026 First submitted to journal 19 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8898430","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":594535908,"identity":"13a6e8e9-7167-43d4-84ca-a652c0550028","order_by":0,"name":"Yuta Shinoku","email":"","orcid":"","institution":"Niigata University","correspondingAuthor":false,"prefix":"","firstName":"Yuta","middleName":"","lastName":"Shinoku","suffix":""},{"id":594535909,"identity":"b679471d-6c18-44f1-925a-d9719cc49832","order_by":1,"name":"Yusuke Kanemaki","email":"","orcid":"","institution":"Niigata University","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Kanemaki","suffix":""},{"id":594535910,"identity":"5d331978-b6a8-454f-a50a-d2c9969a7f24","order_by":2,"name":"Mai Shibuya","email":"","orcid":"","institution":"Niigata University","correspondingAuthor":false,"prefix":"","firstName":"Mai","middleName":"","lastName":"Shibuya","suffix":""},{"id":594535911,"identity":"b65127a1-98d7-4aff-9b3e-9dc0bb5d7e53","order_by":3,"name":"Kakeru Inagawa","email":"","orcid":"","institution":"Niigata University","correspondingAuthor":false,"prefix":"","firstName":"Kakeru","middleName":"","lastName":"Inagawa","suffix":""},{"id":594535912,"identity":"05000b4f-80d1-4a77-a5a9-b8c7a8949f77","order_by":4,"name":"Juria Ono","email":"","orcid":"","institution":"Niigata University","correspondingAuthor":false,"prefix":"","firstName":"Juria","middleName":"","lastName":"Ono","suffix":""},{"id":594535914,"identity":"99ee6cce-8069-475a-a4d9-2d8c2ad7cb9a","order_by":5,"name":"Masaru Nakano","email":"","orcid":"","institution":"Niigata University","correspondingAuthor":false,"prefix":"","firstName":"Masaru","middleName":"","lastName":"Nakano","suffix":""},{"id":594535916,"identity":"72c85c95-80bc-4b9f-81c1-fafd9668e37b","order_by":6,"name":"Masahiro Otani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYLCChAoIfSABSZAZp3I2kJYzCC0SxGlhbEPwJXCqhAF++eZnDx7Os7Pb3sB78MCDX3Z1/A08Bgw/ahjYzXFokWxjMzdI3JacPOcAX8KBxL5kCYkDPAaMPccYmC0bsGsxOMZgJpG4jTlZgoHH4EBiD7MEw/03Bgy8DQzMBgdwaWH/JpE4px6mpV5CHmTLX7xaeIC2NBy2A2tJ+HFYwgCohRmfLZJtOWUSCceOJ0gwg2xpOC658QBbwWGZYxI4/cLPfHyb5I+aansJ9h7jjz/+VPPLHWDe+PBNjU0yrhCDgcQGUNTBIgjoJIlkAwJa7CHUH4SIHSEto2AUjIJRMGIAAEB9U1eijjApAAAAAElFTkSuQmCC","orcid":"","institution":"Niigata University","correspondingAuthor":true,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Otani","suffix":""}],"badges":[],"createdAt":"2026-02-17 07:09:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8898430/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8898430/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-46254-x","type":"published","date":"2026-03-28T16:11:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":103505758,"identity":"b35dd505-9912-4345-87ce-aaa3c1794130","added_by":"auto","created_at":"2026-02-26 13:32:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":785684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of shading treatment on flower buds in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTricyrtis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative flower phenotypes at each developmental stage under non-shaded and shaded conditions. ab, abaxial side; ad, adaxial side. Bar = 1 cm. (B) Relative amounts of total anthocyanins in outer tepals at each developmental stage under non-shaded and shaded conditions. Data are shown as means ± standard errors (SE; n = 9). * indicates significant differences at \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 by Student’s t-test. (C)\u003cstrong\u003e \u003c/strong\u003eExpression levels of anthocyanin biosynthesis-related genes in outer tepals at S4 under non-shaded and shaded conditions. \u003cem\u003eTrAct2\u003c/em\u003ewas used as an internal control. Cropped gel images are shown, and the corresponding full-length gel images are provided in Supplementary Figure S2.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8898430/v1/2b8de84e7126f2f7b7af7223.jpg"},{"id":103204435,"identity":"473585d4-643f-4409-b40a-0c9cd00345b9","added_by":"auto","created_at":"2026-02-23 06:59:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":263986,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT-DNA regions of the vectors used for transformation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT-DNA regions of the overexpression vector (pIG-\u003cem\u003eTrMYB1\u003c/em\u003e) and the RNAi-mediated knockdown vector (p\u003cem\u003eTrMYB1\u003c/em\u003e-RNAi). \u003cem\u003egus\u003c/em\u003e linker, an internal fragment of the β-glucuronidase (GUS) gene connecting trigger sequences; \u003cem\u003eHPT\u003c/em\u003e, hygromycin phosphotransferase gene driven by the CaMV35S promoter; LB, left border; \u003cem\u003eNPTII\u003c/em\u003e, neomycin phosphotransferase II gene driven by the \u003cem\u003eNOS\u003c/em\u003e promoter; P-35S, CaMV35S promoter; RB, right border; T, \u003cem\u003eNOS\u003c/em\u003e terminator; \u003cem\u003eTrMYB1\u003c/em\u003e, full-length cDNA or trigger sequence of \u003cem\u003eTrMYB1\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8898430/v1/6685c074035fb4c0d9443496.jpg"},{"id":104397655,"identity":"f355107c-0607-47d9-821b-5e8bc686e2ba","added_by":"auto","created_at":"2026-03-11 11:53:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1303827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrMYB1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpression in transgenic plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative leaf phenotypes of WT and transgenic plants. Bar = 2 cm. (B) Phenotypes of flowers of WT and transgenic plants. Bar = 1 cm. ab, abaxial side; ad, adaxial side. (C) Relative amounts of total anthocyanins in leaves of WT and transgenic plants. Data are shown as means ± SE (n = 3). *, ** and *** indicate significant differences compared with WT at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, respectively, by Dunnett's test following one-way ANOVA. ns, not significant. (D) Relative amounts of total anthocyanins in outer tepals of WT and transgenic plants. Data are shown as means ± SE (n = 3). * indicates significant differences compared with WT at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by Dunnett's test following one-way ANOVA. ns, not significant. (E) Relative expression levels of anthocyanin biosynthesis-related genes in leaves of WT and transgenic plants by real-time RT-PCR. Data are shown as means ± SE (n = 3). *, ** and *** indicate significant differences compared with WT at \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, respectively, by Dunnett's test following one-way ANOVA. ns, not significant.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8898430/v1/cdefbed62975d74e3b5d733c.jpg"},{"id":103204437,"identity":"e973bbb7-1211-46c0-8940-79ed2b3377e8","added_by":"auto","created_at":"2026-02-23 06:59:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1057583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of RNAi-mediated knockdown\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eof \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrMYB1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in transgenic plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phenotypes of flowers of WT and transgenic plants. Bar = 1 cm. ab, abaxial side; ad, adaxial side. (B) Relative amounts of total anthocyanins in outer tepals of WT and transgenic plants. Data are shown as means ± SE (n = 3). * and ** indicate significant differences compared with WT at \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, respectively, by Dunnett's test following one-way ANOVA. (C) Relative expression levels of anthocyanin biosynthesis-related genes in outer tepals of WT and transgenic plants by real-time RT-PCR. Data are shown as means ± SE (n = 3). * and ** indicate significant differences compared with WT at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, respectively, by Dunnett's test following one-way ANOVA. ns, not significant.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8898430/v1/580b7bb407ab01019a8e4072.jpg"},{"id":105755476,"identity":"587aeb90-c2e9-4709-ab7a-da4958053dc1","added_by":"auto","created_at":"2026-03-30 16:27:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4302593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8898430/v1/c38e223a-6005-4ddf-a67b-cf21d3b6e988.pdf"},{"id":103204438,"identity":"c08c86e3-3b47-4491-ae33-dceef4326620","added_by":"auto","created_at":"2026-02-23 06:59:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":211156,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8898430/v1/0601832a604fb5f87f47687c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of an R2R3-MYB gene regulating tepal background coloration in Tricyrtis sp.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFlower color and flower color pattern are among the most important traits for ornamental plants. Flower color is determined primarily by the type and concentration of pigments, as well as the combination of multiple pigments. Whereas flower color patterns, such as spots, variegation, stripes, picotee, and gradation, are caused by different patterns of pigment accumulation in tepals\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among flower pigments, anthocyanins, members of flavonoids, are found in various plant species and are responsible for a wide range of colors including orange, red, purple, and blue\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe anthocyanin biosynthetic pathway is broadly conserved among higher plant species and is well understood\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In this pathway, eight enzymes, chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavonoid 3\u0026rsquo;-hydroxylase (F3\u0026rsquo;H), flavonoid 3\u0026rsquo;,5\u0026rsquo;-hydroxylase (F3\u0026rsquo;5\u0026rsquo;H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and anthocyanin synthase (ANS) are involved in the biosynthesis of anthocyanidins and flavonols\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The expression of these enzyme genes is known to be mainly regulated by the MBW transcription factor complex consisting of R2R3-myeloblastosis (MYB), basic helix-loop-helix (bHLH), and WD40 repeats (WDR) proteins\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These transcription factors and their functions have also been characterized in various higher plant species\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnthocyanin biosynthesis is known to be affected by environmental stimuli such as light, temperature, and drought. Among them, light has been extensively studied as a key factor influencing anthocyanin biosynthesis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. It has been reported that shading treatments reduce anthocyanin accumulation in tepals or fruit skins in \u003cem\u003eEustoma grandiflorum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eGerbera hybrida\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eMalus domestica\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eChrysanthemum morifolium\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eSolanum melongena\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eLilium regale\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The molecular mechanisms underlying light-induced anthocyanin biosynthesis have been investigated in several plant species. In \u003cem\u003ePetunia hybrida\u003c/em\u003e, both an R2R3-MYB transcription factor \u003cem\u003ePHZ\u003c/em\u003e and a bHLH transcription factor \u003cem\u003eAN1\u003c/em\u003e are expressed in response to light and play key roles in regulating anthocyanin biosynthesis in floral and vegetative organs\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eregale\u003c/em\u003e, light-induced R2R3-MYB factor \u003cem\u003eLhMYB15\u003c/em\u003e is implicated in the regulation of anthocyanin biosynthesis, contributing to characteristic pigmentation pattern formation in tepals\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eC\u003c/em\u003e. \u003cem\u003emorifolium\u003c/em\u003e, light induces the expression of \u003cem\u003eCmMYB9a\u003c/em\u003e while suppressing that of \u003cem\u003eCmBBX28\u003c/em\u003e, a B-box protein that negatively regulates anthocyanin biosynthesis, thereby leading to anthocyanin accumulation in petals\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTricyrtis\u003c/em\u003e spp. are liliaceous ornamental plants native to Japan. One of the \u003cem\u003eTricyrtis\u003c/em\u003e cultivars, \u0026lsquo;Shinonome\u0026rsquo;, produces unique flowers with tepals bearing numerous randomly distributed reddish-purple spots on a light purple background. This flower color pattern is clearly different from variegation patterns caused by transposons\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and bicolor patterns caused by post-transcriptional gene silencing\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To elucidate the molecular mechanism of flower color pattern formation in \u003cem\u003eTricyrtis\u003c/em\u003e sp. 'Shinonome', we performed comprehensive isolation and expression analysis of the anthocyanin biosynthesis-related genes in this plant\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. To date, seven flavonoid biosynthetic enzyme genes, \u003cem\u003eTrCHS\u003c/em\u003e (AB478624; GenBank/EMBL/DDBJ databases; the same applies below), \u003cem\u003eTrCHI\u003c/em\u003e (AB908277), \u003cem\u003eTrF3H\u003c/em\u003e (LC209222), \u003cem\u003eTrF3\u0026rsquo;H\u003c/em\u003e (AB480691), \u003cem\u003eTrDFR\u003c/em\u003e (AB830112), \u003cem\u003eTrFLS\u003c/em\u003e (LC103181), and \u003cem\u003eTrANS\u003c/em\u003e (LC209106), and three transcription factor genes for the MBW complex, \u003cem\u003eTrMYB1\u003c/em\u003e (AB856412), \u003cem\u003eTrbHLH2\u003c/em\u003e (LC223741), and \u003cem\u003eTrWDR\u003c/em\u003e (LC223742), have successfully been isolated. The expression of \u003cem\u003eTrMYB1\u003c/em\u003e in tepals is correlated with that of 'late' biosynthetic enzyme genes (\u003cem\u003eTrF3\u0026rsquo;H\u003c/em\u003e, \u003cem\u003eTrDFR\u003c/em\u003e, \u003cem\u003eTrFLS\u003c/em\u003e and \u003cem\u003eTrANS\u003c/em\u003e), suggesting that \u003cem\u003eTrMYB1\u003c/em\u003e may contribute to anthocyanin biosynthesis in tepals through regulation of these genes. However, the role of \u003cem\u003eTrMYB1\u003c/em\u003e in flower color pattern formation remains unclear.\u003c/p\u003e \u003cp\u003eIn the present study, we examined the effects of light on anthocyanin biosynthesis and the expression of \u003cem\u003eTrMYB1\u003c/em\u003e in the tepals of \u003cem\u003eTricyrtis\u003c/em\u003e sp. \u0026lsquo;Shinonome\u0026rsquo; by shading treatment of flower buds, and further analyzed the function of \u003cem\u003eTrMYB1\u003c/em\u003e through overexpression and RNAi-mediated knockdown in transgenic plants.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e \u003cb\u003eShading treatment of flower buds in\u003c/b\u003e \u003cb\u003eTricyrtis\u003c/b\u003e \u003cb\u003esp. \u0026lsquo;Shinonome\u0026rsquo;\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo examine the effects of light on anthocyanin accumulation and the expression of anthocyanin biosynthesis-related genes in tepals of \u003cem\u003eTricyrtis\u003c/em\u003e sp. 'Shinonome', shading treatment of flower buds was carried out. Under non-shaded conditions, background coloration in tepals was observed from S1 flower buds onwards particularly on the abaxial side of the outer tepals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Spot formation was detectable in tepals of the S1 flower buds and expanded over the entire tepals from S2 onwards (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In S5 flowers, tepals were characterized by numerous randomly distributed reddish-purple spots on a light purple background. On the other hand, under shaded conditions, background coloration in tepals was suppressed in S1\u0026ndash;S4 flower buds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In S5 flowers, tepal background coloration was much reduced compared with those under non-shaded conditions, although reddish-purple spots were produced in the same manner as under non-shaded conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder shaded conditions, the relative amounts of total anthocyanins in outer tepals significantly decreased at all stages of flower development compared with those under non-shaded conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Real-time RT-PCR analysis for the expression of anthocyanin biosynthesis-related genes in outer tepals of S4 flower buds revealed that shading treatment reduced the expression levels of \u003cem\u003eTrMYB1\u003c/em\u003e, \u003cem\u003eTrbHLH2\u003c/em\u003e, \u003cem\u003eTrF3'H\u003c/em\u003e, \u003cem\u003eTrDFR\u003c/em\u003e, and \u003cem\u003eTrANS\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). On the other hand, no apparent differences in the expression level of \u003cem\u003eTrWDR\u003c/em\u003e and \u003cem\u003eTrCHS\u003c/em\u003e were observed between shaded and non-shaded conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In inner tepals, background coloration was inherently faint, making the effect of shading less apparent than in outer tepals (Supplementary Fig. S2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eTrMYB1\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eTricyrtis\u003c/b\u003e \u003cb\u003esp. \u0026lsquo;Shinonome\u0026rsquo;\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the effect of \u003cem\u003eTrMYB1\u003c/em\u003e overexpression, \u003cem\u003eTrMYB1\u003c/em\u003e under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into \u003cem\u003eTricyrtis\u003c/em\u003e sp. 'Shinonome' (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Five independent transgenic lines (TrMYB1-OE2, -OE7, -OE8, -OE9, and -OE12) were obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), among which TrMYB1-OE7 and -OE8 produced flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These transgenic lines showed enhanced pigmentation in most floral organs as well as in leaves compared with the wild type (WT) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). However, no obvious pigmentation was observed in the basal region of the tepals. In contrast, pigmentation was markedly enhanced in naturally pigmented regions of the tepals of wild-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In particular, TrMYB1-OE2, -OE7, -OE8, and -OE9 showed significantly increased anthocyanin accumulation in leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and TrMYB1-OE8 showed significantly increased anthocyanin accumulation in outer tepals of S5 flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Real-time RT-PCR analysis using leaves showed that the expression levels of \u003cem\u003eTrMYB1\u003c/em\u003e and enzyme genes (\u003cem\u003eTrCHS\u003c/em\u003e, \u003cem\u003eTrCHI\u003c/em\u003e, \u003cem\u003eTrDFR\u003c/em\u003e, and \u003cem\u003eTrANS\u003c/em\u003e) significantly increased in all five transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). \u003cem\u003eTrbHLH2\u003c/em\u003e expression also significantly increased in TrMYB1-OE2, -OE7, and -OE8, whereas no significant changes in \u003cem\u003eTrWDR\u003c/em\u003e expression levels were observed in any of the five transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRNAi-mediated knockdown of\u003c/b\u003e \u003cb\u003eTrMYB1\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eTricyrtis\u003c/b\u003e \u003cb\u003esp. \u0026lsquo;Shinonome\u0026rsquo;\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further examine the function of \u003cem\u003eTrMYB1\u003c/em\u003e, RNAi-mediated knockdown construct was introduced into \u003cem\u003eTricyrtis\u003c/em\u003e sp. 'Shinonome' (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Two independent transgenic lines (TrMYB1-RNAi1 and -RNAi2) were obtained and both produced flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In S5 flowers of both transgenic lines, background coloration on both the adaxial and abaxial sides of the tepals almost completely disappeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). By contrast, reddish-purple spots were still detectable, although their pigmentation intensity was markedly reduced compared with that in WT flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). For both transgenic lines, the relative amounts of total anthocyanins in outer tepals of S5 flowers significantly decreased compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Real-time RT-PCR analysis using outer tepals of S5 flowers showed that the expression levels of \u003cem\u003eTrMYB1\u003c/em\u003e in TrMYB1-RNAi1 and -RNAi2 were reduced to approximately 10% and 20% of that in WT, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The expression levels of enzyme genes (\u003cem\u003eTrCHS\u003c/em\u003e, \u003cem\u003eTrCHI\u003c/em\u003e, \u003cem\u003eTrDFR\u003c/em\u003e, and \u003cem\u003eTrANS\u003c/em\u003e) and \u003cem\u003eTrbHLH2\u003c/em\u003e were also substantially reduced in TrMYB1-RNAi1 and -RNAi2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In particular, the expression levels of \u003cem\u003eTrCHI\u003c/em\u003e and \u003cem\u003eTrANS\u003c/em\u003e showed significant decreases in both transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). On the other hand, no significant changes in the \u003cem\u003eTrWDR\u003c/em\u003e expression levels were observed in both transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo elucidate the molecular mechanism of flower color pattern formation in tepals of \u003cem\u003eTricyrtis\u003c/em\u003e sp., we previously performed comprehensive isolation and expression analyses of the anthocyanin biosynthesis-related genes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In the present study, as a next step, we investigated the effect of light on anthocyanin biosynthesis in tepals and performed functional analysis of the R2R3-MYB gene \u003cem\u003eTrMYB1\u003c/em\u003e, which may be involved in the regulation of anthocyanin biosynthesis in tepals.\u003c/p\u003e \u003cp\u003eShading treatment of flower buds or inflorescences has been reported to reduce anthocyanin accumulation in tepals of several plant species, such as \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehybrida\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003emorifolium\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eregale\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In the present study, shading treatment of flower buds similarly reduced anthocyanin accumulation in tepals at all developmental stages (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Interestingly, background coloration in the tepals was largely reduced under shading conditions, whereas spot formation was scarcely affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This differential response suggests that background coloration and spot formation in tepals are regulated by distinct mechanisms, in which background coloration is light dependent, whereas spot formation is largely light independent. Furthermore, the expression level of \u003cem\u003eTrMYB1\u003c/em\u003e decreased under shading conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating that \u003cem\u003eTrMYB1\u003c/em\u003e may be positively regulated by light. Together, these findings suggest that \u003cem\u003eTrMYB1\u003c/em\u003e plays an important role in light-induced anthocyanin biosynthesis in tepals of \u003cem\u003eTricyrtis\u003c/em\u003e sp. Light-induced R2R3-MYB gene expression and anthocyanin biosynthesis have also been reported in other plant species, including \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ehybrida\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eregale\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003emorifolium\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, suggesting that similar regulatory mechanisms may be conserved across a wide range of higher plants.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the anthocyanin biosynthesis\u0026ndash;related R2R3-MYB transcription factors PAP1 and MYB12 are directly regulated by the light-responsive bZIP transcription factor HY5\u003csup\u003e23,24\u003c/sup\u003e. HY5 directly interacts with the ubiquitin ligase COP1 and is degraded in the nucleus under dark conditions. In contrast, COP1 is inactivated upon light exposure, preventing HY5 degradation and resulting in its stabilization\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. COP1 also directly regulates the stability of the R2R3-MYB transcription factors PAP1 and PAP2\u003csup\u003e26\u003c/sup\u003e. Together, these light-dependent mechanisms regulate both the expression and protein stability of R2R3-MYB transcription factors, ultimately modulating anthocyanin biosynthesis. Similar regulatory mechanisms of light-induced anthocyanin biosynthesis mediated by COP1 and HY5 have been reported in other plant species, such as \u003cem\u003eM\u003c/em\u003e. \u003cem\u003edomestica\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003emelongena\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Further studies are needed to clarify the molecular mechanism of light-induced \u003cem\u003eTrMYB1\u003c/em\u003e expression in \u003cem\u003eTricyrtis\u003c/em\u003e sp.\u003c/p\u003e \u003cp\u003eIn our previous studies, heterologous expression of \u003cem\u003eTrMYB1\u003c/em\u003e increased anthocyanin accumulation in transgenic \u003cem\u003ePelargonium crispum\u003c/em\u003e and \u003cem\u003eKalanchoe blossfeldiana\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, suggesting that \u003cem\u003eTrMYB1\u003c/em\u003e functions as a conserved regulator of anthocyanin biosynthesis across species. To further clarify its role in its native genetic background, we generated transgenic \u003cem\u003eTricyrtis\u003c/em\u003e plants overexpressing \u003cem\u003eTrMYB1\u003c/em\u003e. Overexpression of \u003cem\u003eTrMYB1\u003c/em\u003e resulted in a significant increase in anthocyanin accumulation in the leaves (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C). The expression levels of anthocyanin biosynthetic enzyme genes (\u003cem\u003eTrCHS\u003c/em\u003e, \u003cem\u003eTrCHI\u003c/em\u003e, \u003cem\u003eTrDFR\u003c/em\u003e, and \u003cem\u003eTrANS\u003c/em\u003e) also significantly increased in the leaves of these transgenic plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results support the involvement of \u003cem\u003eTrMYB1\u003c/em\u003e in anthocyanin biosynthesis through regulation of anthocyanin biosynthetic genes, consistent with our previous finding that \u003cem\u003eTrMYB1\u003c/em\u003e is associated with regulation of late biosynthetic genes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The expression level of \u003cem\u003eTrbHLH2\u003c/em\u003e was also increased in the leaves of these transgenic plants, suggesting that \u003cem\u003eTrMYB1\u003c/em\u003e may regulate \u003cem\u003eTrbHLH2\u003c/em\u003e either directly or indirectly through regulatory feedback mechanisms. Similarly, overexpression of R2R3-MYB genes has been shown to activate the expression of bHLH genes in various plant species\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In addition, overexpression of \u003cem\u003eTrMYB1\u003c/em\u003e resulted in enhanced pigmentation in most floral organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, the absence of ectopic pigmentation in the basal region of the tepals in \u003cem\u003eTrMYB1\u003c/em\u003e-overexpressing lines suggests that additional regulatory factors, such as bHLH and WDR transcription factors, may be required for anthocyanin accumulation in this region. By contrast, enhanced pigmentation was observed in regions that are already pigmented in the tepals of WT plants, implying that \u003cem\u003eTrMYB1\u003c/em\u003e alone may be sufficient to enhance anthocyanin biosynthesis in such regions.\u003c/p\u003e \u003cp\u003eIn transgenic \u003cem\u003eTricyrtis\u003c/em\u003e sp. plants with RNAi-mediated knockdown of \u003cem\u003eTrMYB1\u003c/em\u003e, both anthocyanin accumulation and the expression levels of anthocyanin biosynthetic enzyme genes, as well as \u003cem\u003eTrbHLH2\u003c/em\u003e, decreased in outer tepals (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Moreover, the flowers of transgenic plants showed a pronounced reduction of background coloration on both the adaxial and abaxial sides of the tepals, whereas reddish-purple spots were still detectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These results indicate that background coloration and spot formation may be regulated by different mechanisms, and that \u003cem\u003eTrMYB1\u003c/em\u003e may play a central role in the formation of tepal background coloration through regulation of anthocyanin biosynthesis-related genes in \u003cem\u003eTricyrtis\u003c/em\u003e sp. The involvement of multiple R2R3-MYB transcription factors in the regulation of flower color pattern formation has been reported in several plant species. In \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ehybrida\u003c/em\u003e, four R2R3-MYB genes have been identified as regulators of anthocyanin biosynthesis in tepals. Among them, \u003cem\u003eAN2\u003c/em\u003e and \u003cem\u003eAN4\u003c/em\u003e control pigmentation in the limb and tube of tepals, respectively\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In addition, \u003cem\u003eDPL\u003c/em\u003e regulates veining in the petal tube, and \u003cem\u003ePHZ\u003c/em\u003e is light inducible and associated with blushing flower buds\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eClarkia gracilis\u003c/em\u003e, four R2R3-MYB genes involved in tepal pigmentation have also been identified\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Among them, \u003cem\u003eCgMYB1C\u003c/em\u003e controls red spot formation, whereas \u003cem\u003eCgMYB12\u003c/em\u003e regulates background coloration in the basal (cup) region. \u003cem\u003eCgMYB6\u003c/em\u003e and \u003cem\u003eCgMYB11\u003c/em\u003e are also suggested to contribute to background coloration. In the present study, spot pigmentation was clearly reduced in the TrMYB1-RNAi lines. It is possible that additional R2R3-MYB transcription factors are involved in the regulation of spot pigmentation and that their activities may have been partly affected by off-target effects of the RNAi construct. Taken together, these findings suggest that multiple R2R3-MYBs, including \u003cem\u003eTrMYB1\u003c/em\u003e, may cooperatively regulate flower color pattern formation in \u003cem\u003eTricyrtis\u003c/em\u003e sp.\u003c/p\u003e \u003cp\u003eIn the present study, we demonstrated that \u003cem\u003eTrMYB1\u003c/em\u003e is expressed in response to light and regulates tepal background coloration by promoting anthocyanin biosynthesis. The results obtained here are expected to contribute significantly to studies on flower color pattern formation in ornamental plants. However, the molecular mechanism of tepal spot formation in \u003cem\u003eTricyrtis\u003c/em\u003e remains unclear. In particular, the mechanism underlying the formation of randomly distributed spots of various sizes has not yet been reported and represents a highly intriguing biological phenomenon. Therefore, elucidating the molecular basis of spot formation in \u003cem\u003eTricyrtis\u003c/em\u003e sp. may provide an important model system for understanding the general principles of flower color pattern formation, and further advances in this area are strongly anticipated.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and callus cultures\u003c/h2\u003e \u003cp\u003ePotted plants of \u003cem\u003eTricyrtis\u003c/em\u003e sp. \u0026lsquo;Shinonome\u0026rsquo; were cultivated in a greenhouse without heating. Embryogenic callus cultures used for transformation were induced and maintained as described by Nakano et al.\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eShading treatment of flower buds\u003c/h3\u003e\n\u003cp\u003eShading treatment was carried out by covering shoot apices with aluminum foil from the time of bract emergence until anthesis. Outer tepals were collected from samples with and without shading treatment at five different developmental stages: S1, flower buds of 6\u0026ndash;10 mm in length; S2, flower buds of 11\u0026ndash;15 mm in length; S3, flower buds of 16\u0026ndash;20 mm in length; S4, flower buds just before anthesis (over 21 mm in length); and S5, flowers just after anthesis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eRNA isolation and cDNA synthesis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using RNeasy Mini Kit (QIAGEN, Hilden, Germany) and treated with RNase-Free DNase Set (QIAGEN, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. cDNA was synthesized using ReverTra Ace\u0026reg; qPCR RT Master Mix (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRT-PCR and real-time RT-PCR\u003c/h2\u003e \u003cp\u003eReverse transcription-polymerase chain reaction (RT-PCR) was performed using EmeraldAmp\u0026reg; MAX PCR Master Mix (Takara Bio Inc., Shiga, Japan) on the T100 thermal cycler (Bio-Rad, Hercules, CA, USA) according to Otani et al.\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eReal-time RT-PCR was performed using KOD SYBR\u0026reg; qPCR Mix (Toyobo Co., Ltd., Osaka, Japan) on the MyGo real-time PCR system (IT-IS Life Science Ltd., Dublin, Ireland). The specificity of the amplification products was confirmed by melting curve analysis. Real-time RT-PCR was performed in biological triplicate for each sample. Relative amounts of transcripts were normalized to the actin gene of \u003cem\u003eTricyrtis\u003c/em\u003e sp. (\u003cem\u003eTrAct2\u003c/em\u003e; AB196260) and calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrimer sets used for these analyses are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of total anthocyanins\u003c/h3\u003e\n\u003cp\u003eTotal anthocyanins were extracted from outer tepals of flowers at different developmental stages and from leaves using the methanol-HCl method according to Rabino and Mancinelli\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Absorbance of the extracts was measured at 530 and 657 nm using a spectrophotometer (Eppendorf BioSpectrometer\u0026reg; basic; Eppendorf, Hamburg, Germany). Total anthocyanin contents were calculated with the following formula: (A\u003csub\u003e530\u003c/sub\u003e \u0026minus;\u0026thinsp;0.25 \u0026times; A\u003csub\u003e657\u003c/sub\u003e) \u0026times; M⁻\u0026sup1; [M: weight (g) of the plant material used for extraction].\u003c/p\u003e \u003cp\u003e \u003cb\u003eVector construction and\u003c/b\u003e \u003cb\u003eAgrobacterium\u003c/b\u003e\u003cb\u003e-mediated transformation\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strains EHA101/pIG-\u003cem\u003eTrMYB1\u003c/em\u003e and EHA101/p\u003cem\u003eTrMYB1\u003c/em\u003e-RNAi were used to produce transgenic plants for \u003cem\u003eTrMYB1\u003c/em\u003e overexpression and RNAi-mediated knockdown, respectively. The T-DNA region of pIG-\u003cem\u003eTrMYB1\u003c/em\u003e contained \u003cem\u003eTrMYB1\u003c/em\u003e driven by the CaMV 35S promoter, neomycin phosphotransferase II (\u003cem\u003eNPTII\u003c/em\u003e) driven by the Nopaline Synthase (\u003cem\u003eNOS\u003c/em\u003e) promoter, and hygromycin phosphotransferase (\u003cem\u003eHPT\u003c/em\u003e) driven by the CaMV35S promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The p\u003cem\u003eTrMYB1\u003c/em\u003e-RNAi vector was constructed based on the pANDA35HK vector\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The T-DNA region of p\u003cem\u003eTrMYB1\u003c/em\u003e-RNAi contained two inverted repeats of a 22-bp \u003cem\u003eTrMYB1\u003c/em\u003e trigger sequence fragment driven by the CaMV35S promoter, \u003cem\u003eNPTII\u003c/em\u003e driven by the \u003cem\u003eNOS\u003c/em\u003e promoter, and \u003cem\u003eHPT\u003c/em\u003e driven by the CaMV35S promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All primers used for vector construction are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online.\u003c/p\u003e \u003cp\u003eTransformation of \u003cem\u003eTricyrtis\u003c/em\u003e sp. 'Shinonome' was performed as previously described by Adachi et al.\u003csup\u003e41\u003c/sup\u003e. To confirm the transgenic nature of regenerated plantlets, PCR analyses were performed using primer sets specific to the T-DNA region (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online). Transgenic plantlets were acclimatized and cultivated in a growth room at 25\u0026deg;C under continuous light-emitting diode (LED) lighting (ca. 170 \u0026micro;mol m\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e s\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e). Morphological, biochemical, and molecular characterizations of transgenic plants were performed during the flowering season.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e \u003cb\u003eAdditional Information\u003c/b\u003e \u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCompeting financial interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e \u003cp\u003e(A) Representative flower phenotypes at each developmental stage under non-shaded and shaded conditions. ab, abaxial side; ad, adaxial side. Bar =\u0026thinsp;1 cm. (B) Relative amounts of total anthocyanins in outer tepals at each developmental stage under non-shaded and shaded conditions. Data are shown as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors (SE; n\u0026thinsp;=\u0026thinsp;9). * indicates significant differences at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 by Student\u0026rsquo;s t-test. (C) Expression levels of anthocyanin biosynthesis-related genes in outer tepals at S4 under non-shaded and shaded conditions. \u003cem\u003eTrAct2\u003c/em\u003e was used as an internal control. Cropped gel images are shown, and the corresponding full-length gel images are provided in Supplementary Figure S2.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported in part by Grants-in-Aid for Scientific Research (Nos. 19K06028 and 24K08880) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.S. drafted the manuscript. M.N. and M.O. reviewed and edited the manuscript. Y.K. and M.S. performed the gene isolation and characterization, and Y.S., K.I., and J.O. performed the transgenic experiments. M.O. and M.N. designed and integrated the project.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOtani, M. et al. Comprehensive isolation and expression analysis of the flavonoid biosynthesis-related genes in \u003cem\u003eTricyrtis\u003c/em\u003e spp. \u003cem\u003eBiol. 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Agrobacterium-mediated Production of Transgenic Plants in \u003cem\u003eTricyrtis hirta\u003c/em\u003e (Liliaceae). \u003cem\u003eActa Hort\u003c/em\u003e. \u003cb\u003e673\u003c/b\u003e, 415\u0026ndash;419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17660/ActaHortic.2005.673.52\u003c/span\u003e\u003cspan address=\"10.17660/ActaHortic.2005.673.52\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"anthocyanin, liliaceous ornamental plant, RNAi, flower color pattern, shading","lastPublishedDoi":"10.21203/rs.3.rs-8898430/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8898430/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe liliaceous ornamental plant \u003cem\u003eTricyrtis\u003c/em\u003e sp. produces unique flowers, whose tepals have many random reddish-purple spots on a light purple background. In our previous studies, we performed comprehensive isolation and expression analysis of the anthocyanin biosynthesis-related genes to elucidate the molecular mechanism underlying flower color pattern formation in this plant, and identified the R2R3-MYB transcription factor gene, \u003cem\u003eTrMYB1\u003c/em\u003e, expressed in tepals. In the present study, we carried out detailed expression and functional analyses of \u003cem\u003eTrMYB1\u003c/em\u003e. Shading treatment of flower buds markedly reduced background coloration of the tepals, while there was little effect on spot formation. In addition, \u003cem\u003eTrMYB1\u003c/em\u003e expression level decreased in the tepals of shaded flower buds. Overexpression of \u003cem\u003eTrMYB1\u003c/em\u003e in \u003cem\u003eTricyrtis\u003c/em\u003e sp. resulted in deeper coloration and significantly increased anthocyanin content in tepals. RNAi-mediated knockdown of \u003cem\u003eTrMYB1\u003c/em\u003e significantly suppressed the expression of the anthocyanin biosynthetic enzyme genes, resulting in a significant decrease in anthocyanin contents and marked reduction in background coloration in the tepals, with little effect on spot formation. These results indicate that tepal background coloration and spot formation may be regulated by different molecular mechanisms, and that background coloration is likely induced by light through activation of \u003cem\u003eTrMYB1\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Identification of an R2R3-MYB gene regulating tepal background coloration in Tricyrtis sp.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 06:59:14","doi":"10.21203/rs.3.rs-8898430/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-05T15:49:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T09:12:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T08:50:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T00:49:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254362872841119773552829101129923797565","date":"2026-02-24T09:44:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204978287229475137283594370966735199565","date":"2026-02-24T06:56:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49544617944427247647193115918329260220","date":"2026-02-24T06:24:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-24T06:07:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-24T06:05:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-24T05:30:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-20T01:05:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-20T01:01:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a684af44-5e3d-46e5-8505-80dc41115e06","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":63270569,"name":"Biological sciences/Biotechnology"},{"id":63270570,"name":"Biological sciences/Molecular biology"},{"id":63270571,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-03-30T16:23:33+00:00","versionOfRecord":{"articleIdentity":"rs-8898430","link":"https://doi.org/10.1038/s41598-026-46254-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-28 16:11:55","publishedOnDateReadable":"March 28th, 2026"},"versionCreatedAt":"2026-02-23 06:59:14","video":"","vorDoi":"10.1038/s41598-026-46254-x","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46254-x","workflowStages":[]},"version":"v1","identity":"rs-8898430","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8898430","identity":"rs-8898430","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}