The combination of DNA methylation and positive regulation of anthocyanin biosynthesis by MYB and bHLH transcription factors contributes to the petal blotch formation in Xibei tree peony

Abstract

Xibei tree peony is a distinctive cultivar group that features red–purple blotches in petals. Interestingly, the pigmentations of blotches and non-blotches are largely independent of one another. The underlying molecular mechanism had attracted lots of attention from investigators, but was still uncertain. Our present work demonstrates the factors that are closely related to blotch formation in Paeonia rockii ‘Shu Sheng Peng Mo’. Non-blotch pigmentation is prevented by the silencing of anthocyanin structural genes, among which PrF3H, PrDFR, and PrANS are the three major genes. We characterized two R2R3-MYBs as the key transcription factors that control the early and late anthocyanin biosynthetic pathways. PrMYBa1, which belongs to MYB subgroup 7 (SG7) was found to activate the early biosynthetic gene (EBG) PrF3H by interacting with SG5 member PrMYBa2 to form an ‘MM’ complex. The SG6 member PrMYBa3 interacts with two SG5 (IIIf) bHLHs to synergistically activate the late biosynthetic genes (LBGs) PrDFR and PrANS, which is essential for anthocyanin accumulation in petal blotches. The comparison of methylation levels of the PrANS and PrF3H promoters between blotch and non-blotch indicated a correlation between hypermethylation and gene silencing. The methylation dynamics of PrANS promoter during flower development revealed a potential early demethylating reaction, which may have contributed to the particular expression of PrANS solely in the blotch area. We suggest that the formation of petal blotch may be highly associated with the cooperation of transcriptional activation and DNA methylation of structural gene promoters.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 32030095). We are particularly grateful to Professor Chunhong Yang (Institute of Botany, Chinese Academy of Sciences) for language polishing. We thank Professor Xiaofeng Cao (Institute of Genetics and Development, Chinese Academy of Sciences) for kindly providing guidance on experimental technique. We also thank the anonymous reviewers and editors for comments that greatly improved the manuscript.

Author contributions

L.S.W. and J.Z. conceived and planned the study. J.Z. performed the research. Y.Z.W. and Q.Y.W. helped conduct some of the experiments, the remaining six authors also contributed in various ways. L.S.W. and J.Z. wrote the manuscript. All authors read and approved the final manuscript.

Data availability

All relevant data can be found within the manuscript or the supplementary materials. All data and material reported in this manuscript are available from the corresponding author upon request.

Conflict of interest statement

The authors declare that they have no competing interests.

References

[1] Li J . Chinese Tree Peony (Xibei, Xinan, Jiangnan Volume). Beijing: Chinese Forestry Publishing House, 2005, 53-144.

[2] Zhou S, Zou XH, Zhou ZQ et al. Multiple species of wild tree peonies gave rise to the ’king of flowers’, Paeonia suffruticosa Andrews . Proc R Soc Lond B Biol Sci. 2014; 281: 20141687.

[3] Cooley AM, Willis JH . Genetic divergence causes parallel evolution of flower color in Chilean Mimulus. New Phytol. 2009; 183: 729-39.

[4] Martins TR, Berg JJ, Blinka S et al. Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae). New Phytol. 2013; 197: 958-69.

[5] Yamagishi M, Toda S, Tasaki K . The novel allele of the LhMYB12 gene is involved in splatter-type spot formation on the flower tepals of Asiatic hybrid lilies (Lilium spp.). New Phytol. 2014; 201: 1009-20.

[6] Hsu C, Chen Y, Tsai W et al. Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis spp. Plant Physiol. 2015; 168: 175-91.

[7] Johnson SD, Midgley JJ . Pollination by monkey beetles (Scarabaeidae: Hopliini): do color and dark centers of flowers influence alighting behavior? Environ Entomol. 2001; 30: 861-8.

[8] Ida TY, Kudo G . Floral color change in Weigela middendorffiana (Caprifoliaceae): reduction of geitonogamous pollination by bumble bees . Am J Bot. 2003; 90: 1751-7.

[9] Thomas MM, Rudall PJ, Ellis AG et al. Development of a complex floral trait: the pollinator-attracting petal spots of the beetle daisy, Gorteria diffusa (Asteraceae). Am J Bot. 2009; 96: 2184- 96.

[10] Bradshaw HD, Schemske DW . Allele substitution at a flower colour locus produces a pollinator shift in monkeyflowers. Nature. 2003; 426: 176-8.

[11] Tanaka Y, Sasaki N, Ohmiya A . Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 2008; 54: 733-49.

[12] Rausher MD, Miller RE, Tiffin P . Patterns of evolutionary rate variation among genes of the anthocyanin biosynthetic pathway. Mol Biol Evol. 1999; 16: 266-74.

[13] Winkel-Shirley B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 2001; 126: 485-93.

[14] Wang LS, Hashimoto F, Shiraishi A et al. Phenetics in tree peony species from China by flower pigment cluster analysis. J Plant Res. 2001; 114: 213-21.

[15] Wang LS, Hashimoto F, Shiraishi A et al. Chemical taxonomy of the Xibei tree peony from China by floral pigmentation. J Plant Res. 2004; 117: 47-55.

[16] Zhang J, Wang L, Shu Q et al. Comparison of anthocyanins in non-blotches and blotches of the petals of Xibei tree peony. Sci Hortic. 2007; 114: 104-11.

[17] Zhou L, Wang Y, Ren L et al. Overexpression of Ps-CHI1, a homologue of the chalcone isomerase gene from tree peony (Paeonia suffruticosa), reduces the intensity of flower pigmentation in transgenic tobacco . Plant Cell Tissue Organ Cult. 2014; 116: 285-95.

[18] Du, Wu, Ji H, J, KX et al. Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia. J Exp Bot. 2015; 66: 6563-77.

[19] Zhang Y, Cheng Y, Xu S et al. Tree peony variegated flowers show a small insertion in the F3′H gene of the acyanic flower parts . BMC Plant Biol. 2020; 20: 211.

[20] Chang Y, Hu T, Zhang W et al. Transcriptome profiling for floral development in reblooming cultivar ’High Noon’ of Paeonia suffruticosa. Sci Data. 2019; 6: 217.

[21] Zhang Y, Cheng Y, Ya H et al. Transcriptome sequencing of purple petal spot region in tree peony reveals differentially expressed anthocyanin structural genes. Front Plant Sci. 2015; 6: 964.

[22] Luo X, Sun D, Wang S et al. Integrating full-length transcriptomics and metabolomics reveals the regulatory mechanisms underlying yellow pigmentation in tree peony (Paeonia suffruticosa Andr.) flowers . Hortic Res. 2021; 8: 15.

[23] Shi Q, Zhou L, Wang Y et al. Transcriptomic analysis of Paeonia delavayi wild population flowers to identify differentially expressed genes involved in purple-red and yellow petal pigmentation . PLoS One. 2015; 10: e0135038.

[24] Spelt C, Quattrocchio F, Mol JN et al. anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes . Plant Cell. 2000; 12: 1619-31.

[25] Koes R, Verweij W, Quattrocchio F . Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 2005; 10: 236-42.

[26] Ding B, Patterson EL, Holalu SV et al. Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns. Curr Biol. 2020; 30: 802-814.e8.

[27] Grotewold E . The genetics and biochemistry of floral pigments. Annu Rev Plant Biol. 2006; 57: 761-80.

[28] Schwinn K, Venail J, Shang Y et al. A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell. 2006; 18: 831-51.

[29] Dubos C, Stracke R, Grotewold E et al. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010; 15: 573-81.

[30] Naing AH, Kim CK . Roles of R2R3-MYB transcription factors in transcriptional regulation of anthocyanin biosynthesis in horticultural plants. Plant Mol Biol. 2018; 98: 1-18.

[31] Ma D, Constabel CP . MYB repressors as regulators of phenylpropanoid metabolism in plants. Trends Plant Sci. 2019; 24: 275-89.

[32] Gu, Zhu, Hao ZY, J, Q et al. A novel R2R3-MYB transcription factor contributes to petal blotch formation by regulating organ-specific expression of PsCHS in tree peony (Paeonia suffruticosa). Plant Cell Physiol. 2018; 3: 599-611.

[33] Luan Y, Tang Y, Wang X et al. Tree peony R2R3-MYB transcription factor PsMYB30 promotes petal blotch formation by activating the transcription of the anthocyanin synthase gene . Plant Cell Physiol. 2022; 63: 1101-16.

[34] Sekhon RS, Chopra S . Progressive loss of DNA methylation releases epigenetic gene silencing from a tandemly repeated maize Myb gene . Genetics. 2009; 181: 81-91.

[35] Jullien PE, Susaki D, Yelagandula R et al. DNA methylation dynamics during sexual reproduction in Arabidopsis thaliana. Curr Biol. 2012; 22: 1825-30.

[36] Wittmeyer, Cui, Chatterjee K, J, D et al. The dominant and poorly penetrant phenotypes of maize Unstable factor for orange1 are caused by DNA methylation changes at a linked transposon . Plant Cell. 2019; 30: 3006-23.

[37] Telias A, Lin-Wang K, Stevenson DE et al. Apple skin patterning is associated with differential expression of MYB10. BMC Plant Biol. 2011; 11: 93.

[38] Wang ZG, Meng D, Wang A et al. The methylation of the PcMYB10 promoter is associated with green-skinned sport in Max Red Bartlett pear . Plant Physiol. 2013; 162: 885-96.

[39] Wang Q, Wang Y, Sun H et al. Transposon-induced methylation of the RsMYB1 promoter disturbs anthocyanin accumulation in red-fleshed radish . J Exp Bot. 2020; 71: 2537-50.

[40] Sicilia A, Scialò E, Puglisi I et al. Anthocyanin biosynthesis and DNA methylation dynamics in sweet orange fruit [ Citrus sinensis L. (Osbeck)] under cold stress . J Agric Food Chem. 2020; 68: 7024-31.

[41] Zhu YC, Zhang B, Allan AC et al. DNA demethylation is involved in the regulation of temperature-dependent anthocyanin accumulation in peach. Plant J. 2020; 102: 965-76.

[42] Liu X, Chuang YN, Chiou CY et al. Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars . Planta. 2012; 236: 401-9.

[43] Deng J, Fu Z, Chen S et al. Proteomic and epigenetic analyses of lotus (Nelumbo nucifera) petals between red and white cultivars . Plant Cell Physiol. 2015; 56: 1546-55.

[44] Wu X, Zhou Y, Yao D et al. DNA methylation of LDOX gene contributes to the floral colour variegation in peach . J Plant Physiol. 2020; 246-247: 153116.

[45] Tang M, Xue W, Li X et al. Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemum . New Phytol. 2022; 236: 1075-88.

[46] Zhang X, Xu Z, Yu X et al. Identification of two novel R2R3-MYB transcription factors, PsMYB114L and PsMYB12L, related to anthocyanin biosynthesis in Paeonia suffruticosa. Int J Mol Sci. 2019; 20: 1055.

[47] Zhang Y, Xu S, Cheng Y et al. Functional identification of PsMYB57 involved in anthocyanin regulation of tree peony. BMC Genet. 2020; 21: 124.

[48] Zhang Y, Xu S, Ma H et al. The R2R3-MYB gene PsMYB58 positively regulates anthocyanin biosynthesis in tree peony flowers . Plant Physiol Biochem. 2021; 164: 279-88.

[49] Qi Y, Zhou L, Han L et al. PsbHLH1, a novel transcription factor involved in regulating anthocyanin biosynthesis in tree peony (Paeonia suffruticosa). Plant Physiol Biochem. 2020; 154: 396-408.

[50] Zimmermann IM, Heim MA, Weisshaar B et al. Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins . Plant J. 2004; 40: 22-34.

[51] Wissenbach, Überlacker, Vogt M, B, F et al. Myb genes from Hordeum vulgare: tissue-specific expression of chimeric Myb promoter/ Gus genes in transgenic tobacco . Plant J. 2010; 4: 411-22.

[52] Holton TA, Cornish EC . Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell. 1995; 7: 1071-83.

[53] Amaranatha RM, Francies RM, Nabi Rasool SK et al. How genes paint flowers and seeds. Int J Curr Res. 2014; 6: 4722-32.

[54] Baudry A, Heim MA, Dubreucq B et al. TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J. 2004; 39: 366-80.

[55] Antonio G, Zhao M, Leavitt JM et al. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings . Plant J. 2008; 53: 814-27.

[56] Petroni K, Tonelli C . Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Sci. 2011; 181: 219-29.

[57] Xu W, Dubos C, Lepiniec L . Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 2015; 20: 176-85.

[58] Nesi N, Debeaujon I, Jond C et al. The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques . Plant Cell. 2000; 12: 1863-78.

[59] Toledo-Ortiz G, Huq E, Quail PH . The Arabidopsis basic/helix-loop-helix transcription factor family . Plant Cell. 2003; 15: 1749-70.

[60] Carretero-Paulet L, Galstyan A, Roig-Villanova I et al. Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae . Plant Physiol. 2010; 153: 1398-412.

[61] Ronchi A, Petroni K, Tonelli C . The reduced expression of endogenous duplications (REED) in the maize R gene family is mediated by DNA methylation . EMBO J. 1995; 14: 5318-28.

[62] Finnegan EJ, Genger RK, Peacock WJ et al. DNA methylation in plants. Annu Rev Plant Physiol Plant Mol Biol. 1998; 49: 223-47.

[63] Martienssen R . Transposons, DNA methylation and gene control. Trends Genet. 1998; 14: 263-4.

[64] Shan X, Li Y, Yang S et al. The spatio-temporal biosynthesis of floral flavonols is controlled by differential phylogenetic MYB regulators in Freesia hybrida. New Phytol. 2020; 228: 1864-79.

[65] Yuan YW, Sagawa JM, Frost L et al. Transcriptional control of floral anthocyanin pigmentation in monkeyflowers (Mimulus). New Phytol. 2014; 204: 1013-27.

[66] Nesi N, Jond C, Debeaujon I et al. The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed . Plant Cell. 2001; 13: 2099-114.

[67] Yoshida K, Ma D, Constabel CP . The MYB182 protein down-regulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes. Plant Physiol. 2015; 167: 693-710.

[68] Davies KM, Albert NW, Schwinn KE . From landing lights to mimicry: the molecular regulation of flower colouration and mechanisms for pigmentation patterning. Funct Plant Biol. 2012; 39: 619-38.

[69] Albert NW, Butelli E, Moss SMA et al. Discrete bHLH transcription factors play functionally overlapping roles in pigmentation patterning in flowers of Antirrhinum majus. New Phytol. 2021; 231: 849-63.

[70] Shang Y, Venail J, Mackay S et al. The molecular basis for venation patterning of pigmentation and its effect on pollinator attraction in flowers of Antirrhinum. New Phytol. 2011; 189: 602-15.

[71] Yang Z, Li Y, Gao F et al. MYB21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana. J Exp Bot. 2020; 71: 4140-58.

[72] Mei X, Wan S, Lin C et al. Integration of metabolome and transcriptome reveals the relationship of benzenoid-phenylpropanoid pigment and aroma in purple tea flowers. Front Plant Sci. 2021; 12: 762330.

[73] Teyssier E, Bernacchia G, Maury S et al. Tissue dependent variations of DNA methylation and endoreduplication levels during tomato fruit development and ripening. Planta. 2008; 228: 391-9.

[74] Candaele J, Demuynck K, Mosoti D et al. Differential methylation during maize leaf growth targets developmentally regulated genes. Plant Physiol. 2014; 164: 1350-64.

[75] Gallusci P, Hodgman C, Teyssier E et al. DNA methylation and chromatin regulation during fleshy fruit development and ripening. Front Plant Sci. 2016; 7: 807.

[76] Zhang B, Tieman DM, Jiao C et al. Chilling-induced tomato flavor loss is associated with altered volatile synthesis and transient changes in DNA methylation. Proc Natl Acad Sci USA. 2016; 113: 12580-5.

[77] Ning G, Xiao X, Lv H et al. Shortening tobacco life cycle accelerates functional gene identification in genomic research. Plant Biol. 2012; 14: 934-43.

[78] Finn RD, Coggill P, Eberhardt RY et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016; 44: D279-85.

[79] The UniProt Consortium . UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021; 49: D480-9.

[80] Altschul SF, Madden TL, Schäffer AA et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25: 3389-402.

[81] Ashburner M, Ball CA, Blake JA et al. Gene ontology: tool for the unification of biology. Nat Genet. 2000; 25: 25-9.

[82] Tatusov RL, Galperin MY, Natale DA et al. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000; 28: 33-6.

[83] Kanehisa M, Goto S, Kawashima S et al. The KEGG resource for deciphering the genome. Nucleic Acids Res. 2004; 32: 277D-80.

[84] Xie C, Mao X, Huang J et al. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 2011; 39: W316-22.

[85] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^−ΔΔCT method . Methods. 2001; 25: 402-8.

[86] Sun X, Feng P, Xu X et al. A chloroplast envelope-bound PHD transcription factor mediates chloroplast signals to the nucleus. Nat Commun. 2011; 2: 477.

[87] Lu S, Ye J, Zhu K et al. A fruit ripening-associated transcription factor CsMADS5 positively regulates carotenoid biosynthesis in citrus. J Exp Bot. 2021; 72: 3028-43.

[88] Hellens RP, Edwards EA, Leyland NR et al. pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation . Plant Mol Biol. 2000; 42: 819-32.

[89] Hellens RP, Allan AC, Friel EN et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods. 2005; 1: 13.

[90] Walter M, Chaban C, Schütze K et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 2004; 40: 428-38.

[91] Verweij W, Spelt C, di Sansebastiano GP et al. An H ⁺ P-ATPase on the tonoplast determines vacuolar pH and flower colour . Nat Cell Biol. 2008; 10: 1456-62.