LlR3MYB-mediated flavonoid biosynthesis confers cold stress tolerance in Lilium lancifolium through the LlDREB-LlCHS2 regulatory cascade

Abstract

Lilies ( Lilium spp.) are globally important ornamental crops which are constrained by their narrow thermal tolerance range. However, tiger lily ( Lilium lancifolium), a wild lily species, exhibits remarkable cold tolerance. Based on our previous findings, we proposed that LlR3MYB, an R3-MYB transcription factor (TF), confers cold tolerance via transcriptional regulation of flavonoid metabolism in tiger lily. Here, we revealed that LlR3MYB represents a unique CPC-type R3-MYB TF exhibiting a bifunctional role in flavonoid metabolism. Specifically, LlR3MYB suppresses anthocyanin biosynthesis while promoting non-anthocyanin flavonoid accumulation (i.e. flavonols, flavones, and chalcones) responding to cold stress. Overexpression of LlR3MYB in tobacco and tiger lily increased total flavonoid content but reduced anthocyanin levels, consistent with the upregulation of early biosynthesis genes (e.g. CHS and FLS) and repression of late biosynthesis genes (e.g. DFR and ANS) in the pathway. In contrast, silencing LlR3MYB in tiger lily reduced total flavonoid production, enhanced anthocyanin accumulation, and compromised cold resistance. Mechanistically, LlR3MYB can directly bind to the AC-I element (ACCTACC) and MBSI motif (CAACGGTT) in the LlCHS2 promoter and activating its transcription, with enhanced activation under low temperature conditions. Mutations of critical residues within the C1/C2 repressor motifs may endow LlR3MYB with this transcriptional activation function. Furthermore, LlDREB can directly bind to the DRE motif (ACCGAC) in the LlR3MYB promoter and activating its transcription in a low-temperature-dependent manner. Our findings uncover a branch-specific regulatory mechanism by which MYB TFs fine-tune flavonoid biosynthesis, highlighting their essential role in plant cold stress responses.

Acknowledgements

This study was supported by the Yuelushan Laboratory Breeding Project (YLS-2025-ZY02051), China National Natural Science Foundation (32102417), Hunan Provincial Natural Science Foundation of China (2024JJ6711), Natural Science Foundation of Guangxi (2025GXNSFBA069050), Key Discipline of the State Forestry Administration ([2016] 21), ‘Double First-Class’ Cultivation Discipline of Hunan Province ([2018] 469), and National Long-term Scientific Research Base of Qingxiushan Landscape Architecture in Nanning, Guangxi.

Author contributions

L.Z., Y.Y., and Y.L. conceived and designed the project. H.B., M.L., and Y.Z. performed experiments. Y.Y., H.B., and Q.Z. analyzed data. R.S., W.X., and S.Z. commented on this work. Y.Y. wrote the article.

Data availability

All data can be found within the manuscript and supporting materials.

Conflicts of interest statement

The authors declare that they have no competing interests.

References

[1] He JP, Yao L, Pecoraro L, et al. Cold stress regulates accumulation of flavonoids and terpenoids in plants by phytohormone, transcription process, functional enzyme, and epigenetics. Crit Rev Biotechnol. 2023; 43: 680-97

[2] Guan YL, Hwarari D, Korboe HM, et al. Low temperature stress-induced perception and molecular signaling pathways in plants. Environ Exp Bot. 2023; 207: 105190

[3] Sharma P, Jha AB, Dubey RS, et al. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot. 2012; 2012: 1-26

[4] Gusta LV, Wisniewski M . Understanding plant cold hardiness: an opinion. Physiol Plant. 2013; 147: 4-14

[5] Swati S, Akhtar AS, Israil AM, et al. Abiotic stress and reactive oxygen species: generation, signaling, and defense mechanisms. Antioxidants. 2021; 10: 277-7

[6] Agati G, Azzarello E, Pollastri S, et al. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 2012; 196: 67-76

[7] Agati G, Brunetti C, Nascimento LBDS, et al. Antioxidants by nature: an ancient feature at the heart of flavonoids’ multifunctionality. New Phytol. 2025; 245: 11-26

[8] Marais JPJ, Deavours B, Dixon RA, et al. The Stereochemistry of Flavonoids. In: Grotewold E, ed. The Science of Flavonoids. Springer New York: New York, NY, 2006, 1-46

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

[10] Shen N, Wang TF, Gan Q, et al. Plant flavonoids: classification, distribution, biosynthesis, and antioxidant activity. Food Chem. 2022; 383: 132531

[11] Wang ZB, Yu QB, Shen WX, et al. Functional study of CHS gene family members in citrus revealed a novel CHS gene affecting the production of flavonoids. BMC Plant Biol. 2018; 18: 189

[12] Deng XB, Bashandy H, Ainasoja M, et al. Functional diversification of duplicated chalcone synthase genes in anthocyanin biosynthesis of Gerbera hybrida. New Phytol. 2014; 201: 1469-83

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

[14] Ferreyra MLF, Rius SP, Casati P . Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front Plant Sci. 2012; 3: 222

[15] Davies KM, Schwinn KE, Deroles SC, et al. Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase. Euphytica. 2003; 131: 259-68

[16] Martens S, Mithöfer A . Flavones and flavone synthases. Phytochemistry. 2005; 66: 2399-407

[17] Xie LM, Wang YN, Tao YT, et al. Genome-wide identification and analysis of anthocyanin synthesis-related R2R3-MYB genes in Fragaria pentaphylla. BMC Genomics. 2024; 25: 952

[18] He GR, Zhang R, Jiang SH, et al. The MYB transcription factor RcMYB1 plays a central role in rose anthocyanin biosynthesis. Hortic Res. 2023; 10: uhad080

[19] Yan HL, Pei XN, Zhang H, et al. MYB-mediated regulation of anthocyanin biosynthesis. Int J Mol Sci. 2021; 22: 3103

[20] Mondal SK, Roy S . Genome-wide sequential, evolutionary, organizational and expression analyses of phenylpropanoid biosynthesis associated MYB domain transcription factors in Arabidopsis. J Biomol Struct Dyn. 2018; 36: 1577-601

[21] Yamagishi M. A novel R2R3-MYB transcription factor regulates light-mediated floral and vegetative anthocyanin pigmentation patterns in Lilium regale. Mol Breed. 2015; 36: 3

[22] Liu JT, Osbourn A, Ma PD . MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol Plant. 2015; 8: 689-708

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

[24] Gonzalez A, Zhao MZ, 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

[25] Pelletier MK, Burbulis IE, Winkel-Shirley B . Disruption of specific flavonoid genes enhances the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings. Plant Mol Biol. 1999; 40: 45-54

[26] Stracke R, Ishihara H, Huep G, et al. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J. 2007; 50: 660-77

[27] Albert NW, Thrimawithana AH, Mcghie TK, et al. Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants. New Phytol. 2018; 218: 554-66

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

[29] Matsui K, Umemura Y, Ohme-Takagi M . AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant J. 2008; 55: 954-67

[30] Nguyen NH, Jeong CY, Kang G-H, et al. MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis. Plant J. 2015; 84: 1192-205

[31] Wang YL, Wang YQ, Song ZQ, et al. Repression of MYBL2 by both microRNA858a and HY5 leads to the activation of anthocyanin biosynthetic pathway in Arabidopsis. Mol Plant. 2016; 9: 1395-405

[32] Kim S, Hwang G, Lee S, et al. High ambient temperature represses anthocyanin biosynthesis through degradation of HY5. Front Plant Sci. 2017; 8: 1787

[33] Xu SJ, Chen RZ, Zhang XQ, et al. The evolutionary tale of lilies: Giant genomes derived from transposon insertions and polyploidization. Innovation. 2024; 5: 100726

[34] Liang YW, Gao Q, Li F, et al. The giant genome of lily provides insights into the hybridization of cultivated lilies. Nat Commun. 2025; 16: 45

[35] Chen Y, Hou XR, Zheng YP, et al. The establishment of a genetic transformation system and the Acquisition of Transgenic Plants of oriental hybrid lily (Lilium L.). Int J Mol Sci. 2023; 24: 782

[36] Yu JP, Xu SJ, Liu XY, et al. Starch degradation and sucrose accumulation of lily bulbs after cold storage. Int J Mol Sci. 2022; 23: 4366

[37] Wang JM, Yang Y, Liu XH, et al. Transcriptome profiling of the cold response and signaling pathways in Lilium lancifolium. BMC Genomics. 2014; 15: 203

[38] Yong YB, Li WQ, Wang JM, et al. Identification of gene co-expression networks involved in cold resistance of Lilium lancifolium. Biol Plant. 2018; 62: 287-98

[39] Yong YB, Zhang Y, Lyu YM . A MYB-related transcription factor from Lilium lancifolium L. (LlMYB3) is involved in anthocyanin biosynthesis pathway and enhances multiple abiotic stress tolerance in Arabidopsis thaliana. Int J Mol Sci. 2019; 20: 3195

[40] Benzie IFF, Devaki M . The ferric reducing/antioxidant power (FRAP) assay for non-enzymatic antioxidant capacity: concepts, procedures, limitations and applications: recent trends and applications. In: Apak R, Capanoglu E, Shahidi F, eds. Measurement of Antioxidant Activity & Capacity. John Wiley & Sons, Ltd: Chichester, UK, 2017, 77-106

[41] Ferrer JL, Jez JM, Bowman ME, et al. Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nat Struct Biol. 1999; 6: 775-84

[42] Zhou H, Wang KL, Wang FR, et al. Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phytol. 2019; 221: 1919-34

[43] Landi M, Tattini M, Gould KS . Multiple functional roles of anthocyanins in plant-environment interactions. Environ Exp Bot. 2015; 119: 4-17

[44] Jaakola L. New insights into the regulation of anthocyanin biosynthesis in fruits. Trends Plant Sci. 2013; 18: 477-83

[45] Suzuki N, Mittler R . Reactive oxygen species and temperature stresses: a delicate balance between signaling and destruction. Physiol Plant. 2006; 126: 45-51

[46] Winkel-Shirley B . Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol. 2002; 5: 218-23

[47] Yang XX, Liu C, Li MD, et al. Integrated transcriptomics and metabolomics analysis reveals key regulatory network that response to cold stress in common bean (Phaseolus vulgaris L.). BMC Plant Biol. 2023; 23: 85

[48] Li YT, Zhang J, Wang SJ, et al. Integrative transcriptomic and metabolomic analyses reveal the flavonoid biosynthesis of Pyrus hopeiensis flowers under cold stress. Hortic Plant J. 2023; 9: 395-413

[49] Agati G, Tattini M . Multiple functional roles of flavonoids in photoprotection. New Phytol. 2010; 186: 786-93

[50] An JP, Wang XF, Zhang XW, et al. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnol J. 2020; 18: 337-53

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

[52] Du H, Wang YB, Xie Y, et al. Genome-wide identification and evolutionary and expression analyses of MYB-related genes in land plants. DNA Res. 2013; 20: 437-48

[53] Wang SC, Chen JG . Regulation of cell fate determination by single-repeat R3 MYB transcription factors in Arabidopsis. Front Plant Sci. 2014; 5: 133

[54] Sakai M, Yamagishi M, Matsuyama K . Repression of anthocyanin biosynthesis by R3-MYB transcription factors in lily (Lilium spp.). Plant Cell Rep. 2019; 38: 609-22

[55] Nemie-Feyissa D, Olafsdottir SM, Heidari B, et al. Nitrogen depletion and small R3-MYB transcription factors affecting anthocyanin accumulation in Arabidopsis leaves. Phytochemistry. 2014; 98: 34-40

[56] Yuan YW, Sagawa JM, Young RC, et al. Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus. Genetics. 2013; 194: 255-63

[57] Nakatsuka T, Yamada E, Saito M, et al. Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers. Plant Cell Rep. 2013; 32: 1925-37

[58] Colanero S, Perata P, Gonzali S . The atroviolacea gene encodes an R3-MYB protein repressing anthocyanin synthesis in tomato plants. Front Plant Sci. 2018; 9: 830

[59] Gates DJ, Olson BJSC, Clemente TE, et al. A novel R3 MYB transcriptional repressor associated with the loss of floral pigmentation in Iochroma. New Phytol. 2018; 217: 1346-56

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

[61] Albert NW, Lewis DH, Zhang HB, et al. Members of an R2R3-MYB transcription factor family in petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. Plant J. 2011; 65: 771-84

[62] Albert NW, Davies KM, Lewis DH, et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell. 2014; 26: 962-80

[63] Lai YS, Shimoyamada Y, Nakayama M, et al. Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily (Lilium spp.). Plant Sci. 2012; 193-194: 136-47

[64] Pollastri S, Tattini M . Flavonols: old compounds for old roles. Ann Bot. 2011; 108: 1225-33

[65] Neugart S, Schreiner M . UVB and UVA as eustressors in horticultural and agricultural crops. Sci Hortic. 2018; 234: 370-81

[66] Agati G, Brunetti C, Fini A, et al. Are flavonoids effective antioxidants in plants? Twenty years of our investigation. Antioxidants. 2020; 9: 1098

[67] Singh P, Arif Y, Bajguz A, et al. The role of quercetin in plants. Plant Physiol Biochem. 2021; 166: 10-9

[68] Rice-Evans CA, Miller NJ, Paganga G . Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med. 1996; 20: 933-56

[69] Williams RJ, Spencer JPE, Rice-Evans C . Flavonoids: antioxidants or signalling molecules? Free Radic Biol Med. 2004; 36: 838-49

[70] Rodrigues JA, Espley RV, Allan AC . Genomic analysis uncovers functional variation in the C-terminus of anthocyanin-activating MYB transcription factors. Hortic Res. 2021; 8: 77

[71] Shen H, He XZ, Poovaiah CR, et al. Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks. New Phytol. 2012; 193: 121-36

[72] Kagale S, Rozwadowski K . EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression. Epigenetics. 2011; 6: 141- 6

[73] Yang JT, Liu Y, Yan HY, et al. PlantEAR: functional analysis platform for plant EAR motif-containing proteins. Front Genet. 2018; 9: 590

[74] Chow V, Kirzinger MW, Kagale S . Lend me your EARs: a systematic review of the broad functions of EAR motif-containing transcriptional repressors in plants. Genes. 2023; 14: 270

[75] Kranz HD, Denekamp M, Greco R, et al. Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J. 1998; 16: 263-76

[76] Cavallini E, Matus JT, Finezzo L, et al. The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine. Plant Physiol. 2015; 167: 1448-70

[77] Bian XH, Li W, Niu CF, et al. A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis. New Phytol. 2020; 225: 268-83

[78] Scharf KD, Berberich T, Ebersberger I, et al. The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim Biophys Acta Gene Regul Mech. 2012; 1819: 104-19

[79] Jun JH, Liu CG, Xiao XR, et al. The transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyandin and anthocyanin pigmentation in Medicago truncatula. Plant Cell. 2015; 27: 2860-79

[80] Xu HF, Wang N, Liu JX, et al. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Mol Biol. 2017; 94: 149-65

[81] Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K . AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta Gene Regul Mech. 2012; 1819: 86-96

[82] Rae L, Lao NT, Kavanagh TA . Regulation of multiple aquaporin genes in Arabidopsis by a pair of recently duplicated DREB transcription factors. Planta. 2011; 234: 429-44

[83] Lin RC, Park H, Wang HY . Role of Arabidopsis RAP2.4 in regulating light- and ethylene-mediated developmental processes and drought stress tolerance. Mol Plant. 2008; 1: 42-57

[84] Shaikhali J, Heiber I, Seidel T, et al. The redox-sensitive transcription factor Rap2.4a controls nuclear expression of 2-Cys peroxiredoxin A and other chloroplast antioxidant enzymes. BMC Plant Biol. 2008; 8: 48

[85] Niskanen EA, Malinen M, Sutinen P, et al. Global SUMOylation on active chromatin is an acute heat stress response restricting transcription. Genome Biol. 2015; 16: 153

[86] Vihervaara A, Mahat DB, Guertin MJ, et al. Transcriptional response to stress is pre-wired by promoter and enhancer architecture. Nat Commun. 2017; 8: 255

[87] Mueller B, Mieczkowski J, Kundu S, et al. Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction. Genes Dev. 2017; 31: 451-62

[88] Lyu XW, Rowley MJ, Corces VG . Architectural proteins and pluripotency factors cooperate to orchestrate the transcriptional response of hESCs to temperature stress. Mol Cell. 2018; 71: 940-955.e7

[89] Himanen SV, Sistonen L . New insights into transcriptional reprogramming during cellular stress. J Cell Sci. 2019; 132: jcs238402

[90] Wang GB, Cao FL, Chang L, et al. Temperature has more effects than soil moisture on biosynthesis of flavonoids in Ginkgo (Ginkgo biloba L.) leaves. New For. 2014; 45: 797-812

[91] Wang LM, Sang WN, Xu RR, et al. Alteration of flesh color and enhancement of bioactive substances via the stimulation of anthocyanin biosynthesis in ‘friar’ plum fruit by low temperature and the removal. Food Chem. 2020; 310: 125862

[92] Guo J, Zhou X, Wang TL, et al. Regulation of flavonoid metabolism in ginkgo leaves in response to different day-night temperature combinations. Plant Physiol Biochem. 2020; 147: 133-40

[93] Shen YX, Sun TT, Pan Q, et al. RrMYB5- and RrMYB10-regulated flavonoid biosynthesis plays a pivotal role in feedback loop responding to wounding and oxidation in Rosa rugosa. Plant Biotechnol J. 2019; 17: 2078-95

[94] Deng J, Che XL, Gu Y, et al. Integrated multi-omics investigation revealed the importance of phenylpropanoid metabolism in the defense response of Lilium regale Wilson to fusarium wilt. Hortic Res. 2024; 11: uhae140

[95] Cox J, Hein MY, Luber CA, et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics. 2014; 13: 2513-26

[96] Liu N, Shen GJ, Xu YX, et al. Extensive inter-plant protein transfer between Cuscuta parasites and their host plants. Mol Plant. 2020; 13: 573-85

[97] Wu JH, Zhu CF, Pang JH, et al. OsLOL1, a C2C2-type zinc finger protein, interacts with OsbZIP58 to promote seed germination through the modulation of gibberellin biosynthesis in Oryza sativa. Plant J. 2014; 80: 1118-30

[98] Li WT, Wang K, Chern M, et al. Sclerenchyma cell thickening through enhanced lignification induced by OsMYB30 prevents fungal penetration of rice leaves. New Phytol. 2020; 226: 1850-63