Molecular insights into TT2-type MYB regulators illuminate the complexity of floral flavonoids biosynthesis in Freesia hybrida

Abstract

Proanthocyanidins (PAs), anthocyanins, and flavonols are key flavonoids that play diverse roles in plant physiology and human health. Despite originating from a shared biosynthetic pathway, the regulatory mechanisms of PA biosynthesis and the cooperative regulation of different kinds of flavonoids remain elusive, particularly in flower tissues or organs. Here, we elucidated the regulatory network governing PA biosynthesis in Freesia hybrida ‘Red River®’ by characterizing four TT2-type MYB transcription factors, designated FhMYBPAs. Phylogenetic analysis, subcellular localization, and transactivation assays predicted their roles as PA-related activators. Pearson correlation analysis revealed significant correlations between FhMYBPAs and PA accumulation in various floral tissues and development stages. Functional studies demonstrated that FhMYBPAs activated PA biosynthesis by directly binding to the promoters of target genes, which can be enhanced by FhTT8L. Additionally, a hierarchical and feedback regulatory model involving FhTTG1, FhMYB27, and FhMYBx was proposed for PA biosynthesis. Furthermore, comparative analysis of flavonoid-related MYB factors involving FhPAP1, FhMYB5, FhMYBF1, and FhMYB21L2 highlighted their roles in regulating PA, anthocyanin, and flavonol biosynthesis, with some exhibiting versatile regulations. Overall, our findings provide insights into the spatio-temporal regulation of flavonoids in flowers and expand our understanding of MYB-mediated transcriptional regulation of specialized metabolites in plants.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32100211, 32272751), the China Postdoctoral Science Foundation (2023 M740580), the Department of Science and Technology of Jilin Province (20220508112RC), Science and Technology Projects of HNTI (KY2022YC0003), and the Fundamental Research Funds for the Central Universities (2412023YQ005, 2412024QD024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

X.S., D.Z., R.G., M.Q., L.Z., J.Z., Y.W., Z.Q., N.Z., G.X., and X.G. performed the experiments and helped in analyzing data. X.S. drafted and revised the manuscript together with X.G. and Y.L. X.G. designed the experiments and discussed with Y.L. and L.W. All authors have participated in this research and approved the final manuscript.

Data availability

All data supporting the findings of this study are available within the paper and within its Supplemental data published online.

Conflict of interests

We declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Dixon RA, Xie DY, Sharma SB. Proanthocyanidins - a final fron-tier in flavonoid research? New Phytol. 2005;165: 9-28

[2] Sun S, Qi X, Zhang Z. et al. A structural variation in the promoter of the leucoanthocyanidin reductase gene AaLAR1 enhances freezing tolerance by modulating proanthocyanidin accumu-lation in kiwifruit (Actinidia arguta). Plant Cell Environ. 2024;47: 4048-66

[3] Yu K, Song Y, Lin J. et al. The complexities of proanthocyani-din biosynthesis and its regulation in plants. Plant Commun. 2023;4:100498

[4] Rauf A, Imran M, Abu-Izneid T. et al. Proanthocyanidins: a comprehensive review. Biomed Pharmacother. 2019;116:108999

[5] Qi, Chu M, Yu X. et al. Anthocyanins and proanthocyanidins: chemical structures, food sources, bioactivities, and product development. Food Rev Int. 2023;39: 4581-609

[6] Jonker A, Yu P. The occurrence, biosynthesis, and molecular structure of proanthocyanidins and their effects on legume forage protein precipitation, digestion and absorption in the ruminant digestive tract. Int J Mol Sci. 2017;18:1105

[7] Cai Y, Liang Y, Shi H. et al. Creating yellow seed Camelina sativa with enhanced oil accumulation by CRISPR-mediated disruption of Transparent Testa 8. Plant Biotechnol J. 2024;22: 2773-84

[8] He F, Pan Q, Shi Y. et al. Biosynthesis and genetic regulation of proanthocyanidins in plants. Molecules. 2008;13: 2674-703

[9] Yu D, Huang T, Tian B. et al. Advances in biosynthesis and biolog-ical functions of proanthocyanidins in horticultural plants. Food Secur. 2020;9:1774

[10] Wei X, Ju Y, Ma T. et al. New perspectives on the biosynthesis, transportation, astringency perception and detection methods of grape proanthocyanidins. Crit Rev Food Sci Nutr. 2021;61: 2372-98

[11] Mora J, Pott DM, Osorio S. et al. Regulation of plant tannin synthesis in crop species. Front Genet. 2022;13:870976

[12] Gu Z, Zhou X, Li S. et al. The HD-ZIP IV transcription factor GLABRA2 acts as an activator for proanthocyanidin biosynthesis in Medicago truncatula seed coat. Plant J. 2024;119: 2303-15

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

[14] Verdier J, Zhao J, Torres-Jerez I. et al. MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula. Proc Natl Acad Sci USA. 2012;109: 1766-71

[15] Liu C, Jun JH, Dixon RA. MYB5 and MYB14 play pivotal roles in seed coat polymer biosynthesis in Medicago truncatula. Plant Physiol. 2014;165: 1424-39

[16] Lepiniec L, Debeaujon I, Routaboul JM. et al. Genetics and bio-chemistry of seed flavonoids. Annu Rev Plant Biol. 2006;57: 405-30

[17] Shen Y, Sun T, 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

[18] Jiao T, Huang Y, Wu YL. et al. Functional diversity of subgroup 5 R2R3-MYBs promoting proanthocyanidin biosynthesis and their key residues and motifs in tea plant. Hortic Res. 2023;10:uhad135

[19] Paolocci F, Robbins MP, Passeri V. et al. The strawberry transcrip-tion factor FaMYB1 inhibits the biosynthesis of proanthocyani-dins in Lotus corniculatus leaves. JExp Bot. 2011;62: 1189-200

[20] 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

[21] Zhou H, Lin-Wang K, Wang F. 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

[22] Rao LJM, Yada H, Ono H. et al. Occurrence of antioxidant and radical scavenging proanthocyanidins from the Indian minor spice nagkesar (Mammea longifolia planch and triana syn). Bioorg Med Chem. 2004;12: 31-6

[23] Liu X, Zhang H, Liu Z. et al. PbrMYB186 activation of PbrF3H increased flavonol biosynthesis and promoted pollen tube growth in Pyrus. Mol Hortic. 2024;4: 30-0

[24] Zhu Q, Sui S, Lei X. et al. Ectopic expression of the coleus R2R3 MYB-type proanthocyanidin regulator gene SsMYB3 alters the flower color in transgenic tobacco. PLoS One. 2015;10:e0139392

[25] Yuan Y-W, Rebocho AB, Sagawa JM. et al. Competition between anthocyanin and flavonol biosynthesis produces spatial pattern variation of floral pigments between Mimulus species. Proc Natl Acad Sci USA. 2016;113: 2448-53

[26] Li C, Qiu J, Huang S. et al. AaMYB3 interacts with AabHLH1 to reg-ulate proanthocyanidin accumulation in Anthurium andraeanum (Hort.)-another strategy to modulate pigmentation. Hortic Res. 2019;6:14

[27] 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. JExp Bot. 2020;71: 4140-58

[28] Bao T, Kimani S, Li Y. et al. Allelic variation of terpene synthases drives terpene diversity in the wild species of the Freesia genus. Plant Physiol. 2023;192: 2419-35

[29] Li Y, Shan X, Gao R. et al. Two IIIf clade-bHLHs from Freesia hybrida play divergent roles in flavonoid biosynthesis and trichome formation when ectopically expressed in Arabidopsis. Sci Rep. 2016;6:30514

[30] Li Y, Shan X, Zhou L. et al. The R2R3-MYB factor FhMYB5 from Freesia hybrida contributes to the regulation of anthocyanin and proanthocyanidin in biosynthesis. Front Plant Sci. 2019;9:1935

[31] Li Y, Shan X, Tong L. et al. The conserved and particular roles of the R2R3-MYB regulator FhPAP1 from Freesia hybrida in flower anthocyanin biosynthesis. Plant Cell Physiol. 2020;61: 1365-80

[32] 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

[33] Shan X, Li Y, Yang S. et al. A functional homologue of Arabidopsis TTG1 from Freesia interacts with bHLH proteins to regulate anthocyanin and proanthocyanidin biosynthesis in both Freesia hybrida and Arabidopsis thaliana. Plant Physiol Biochem. 2019;141: 60-72

[34] Li Y, Shan X, Gao R. et al. MYB repressors and MBW activation complex collaborate to fine-tune flower coloration in Freesia hybrida. Commun Biol. 2020;3:396

[35] Li SF, Milliken ON, Pham H. et al. The Arabidopsis MYB5 transcrip-tion factor regulates mucilage synthesis, seed coat development, and trichome morphogenesis. Plant Cell. 2009;21: 72-89

[36] Grotewold E, Sainz MB, Tagliani L. et al. Identification of the residues in the Myb domain of maize C1 that specify the inter-action with the bHLH cofactor R. Proc Natl Acad Sci USA. 2000;97: 13579-84

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

[38] Schaart JG, Dubos C, Romero de la Fuente I. et al. Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (Fra-garia x ananassa) fruits. New Phytol. 2013;197: 454-67

[39] Ahmad S, Yuan C, Cong T. et al. Transcriptome and chemical analyses identify candidate genes associated with flower color shift in a natural mutant of Chrysanthemum × morifolium. Ornam Plant Res. 2022;2: 1-12

[40] Yoshida K, Iwasaka R, Kaneko T. et al. Functional differentiation of Lotus japonicus TT2s, R2R3-MYB transcription factors compris-ing a multigene family. Plant Cell Physiol. 2008;49: 157-69

[41] Mellway RD, Tran LT, Prouse MB. et al. The wound-, pathogen-, and ultraviolet B-responsive MYB134 gene encodes an R2R3 MYB transcription factor that regulates proanthocyanidin synthesis in poplar. Plant Physiol. 2009;150: 924-41

[42] Hancock KR, Collette V, Fraser K. et al. Expression of the R2R3-MYB transcription factor TaMYB14 from Trifolium arvense acti-vates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa. Plant Physiol. 2012;159: 1204-20

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

[44] Gonzalez A, Mendenhall J, Huo Y. et al. TTG1 complex MYBs, MYB5 and TT2, control outer seed coat differentiation. Dev Biol. 2009;325: 412-21

[45] Deluc L, Bogs J, Walker AR. et al. The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries. Plant Physiol. 2008;147: 2041-53

[46] Bogs J, Jaffe FW, Takos AM. et al. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol. 2007;143: 1347-61

[47] Akagi T, Ikegami A, Tsujimoto T. et al. DkMyb4 is a MYB tran-scription factor involved in proanthocyanidin biosynthesis in persimmon fruit. Plant Physiol. 2009;151: 2028-45

[48] James AM, Ma D, Mellway R. et al. Poplar MYB115 and MYB134 transcription factors regulate proanthocyanidin synthesis and structure. Plant Physiol. 2017;174: 154-71

[49] Jiang X, Huang K, Zheng G. et al. CsMYB5a and CsMYB5e from Camellia sinensis differentially regulate anthocyanin and proan-thocyanidin biosynthesis. Plant Sci. 2018;270: 209-20

[50] Panchy N, Lehti-Shiu M, Shiu S-H. Evolution of gene duplication in plants. Plant Physiol. 2016;171: 2294-316

[51] Tsai C-J, Harding SA, Tschaplinski TJ. et al. Genome-wide analy-sis of the structural genes regulating defense phenylpropanoid metabolism in Populus. New Phytol. 2006;172: 47-62

[52] Terrier N, Torregrosa L, Ageorges À. et al. Ectopic expres-sion of VvMYBPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway. Plant Physiol. 2009;149: 1028-41

[53] 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

[54] Huang Y-F, Vialet S, Guiraud JL. et al. A negative MYB regulator of proanthocyanidin accumulation, identified through expres-sion quantitative locus mapping in the grape berry. New Phytol. 2014;201: 795-809

[55] Gao R, Han T, Xun H. et al. MYB transcription factors GmMYBA2 and GmMYBR function in a feedback loop to control pigmenta-tion of seed coat in soybean. JExp Bot. 2021;72: 4401-18

[56] Gao R, Li Y, Wang Y. et al. Unraveling the regulatory network of flower coloration in soybean: insights into roles of GmMYBA3 and GmMYBR1. Crop J. 2024;12: 443-55

[57] Jaakola L, Määttä K, Pirttilä AM. et al. Expression of genes involved in anthocyanin biosynthesis in relation to antho-cyanin, proanthocyanidin, and flavonol levels during bilberry fruit development. Plant Physiol. 2002;130: 729-39

[58] Hichri I, Heppel SC, Pillet J. et al. The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine. Mol Plant. 2010;3: 509-23

[59] Li Y, Bao T, Zhang J. et al. The coordinated interaction or regula-tion between floral pigments and volatile organic compounds. Hortic Plant J. 2024 (In Press).

[60] Zhang C, Dai Z, Ferrier T. et al. MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries. Plant Cell. 2023;35: 4238-65

[61] Cao Y, Zhang R, Xing M. et al. Synergistic actions of three MYB transcription factors underpins the high accumulation of myricetin in Morella rubra. Plant J. 2023;115: 577-94

[62] Zhang L, Wang L, Fang Y. et al. Phosphorylated transcription fac-tor PuHB40 mediates ROS-dependent anthocyanin biosynthesis in pear exposed to high light. Plant Cell. 2024;36: 3562-83

[63] Luan Y, Tao J, Zhao D. Synergistic actions of 3 MYB transcription factors underpin blotch formation in tree peony. Plant Physiol. 2024;196: 1869-86

[64] Uwagaki Y, Matsuda E, Komaki M. et al. Agrobacterium-mediated transformation and regeneration of Freesia x hybrida. Plant Biotechnol. 2015;32: 165-8

[65] Shan X, Li Y, Zhou L. et al. Efficient isolation of protoplasts from freesia callus and its application in transient expression assays. Plant Cell Tissue and Organ Cult. 2019;138: 529-41

[66] Haas BJ, Papanicolaou A, Yassour M. et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity plat-form for reference generation and analysis. Nat Protoc. 2013;8: 1494-512

[67] Chen C, Wu Y, Li J. et al. TBtools-II: a "one for all, all for one" bioinformatics platform for biological big-data mining. Mol Plant. 2023;16: 1733-42

[68] Yoo S-D, Cho Y-H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007;2: 1565-72

[69] 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

[70] Sparkes IA, Runions J, Kearns A. et al. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc. 2006;1: 2019-25