Integrating full-length transcriptomics and metabolomics reveals the regulatory mechanisms underlying yellow pigmentation in tree peony (Paeonia suffruticosa Andr.) flowers

Abstract

Tree peony (Paeonia suffruticosa Andr.) is a popular ornamental plant in China due to its showy and colorful flowers. However, yellow-colored flowers are rare in both wild species and domesticated cultivars. The molecular mechanisms underlying yellow pigmentation remain poorly understood. Here, petal tissues of two tree peony cultivars, “High Noon” (yellow flowers) and “Roufurong” (purple–red flowers), were sampled at five developmental stages (S1–S5) from early flower buds to full blooms. Five petal color indices (brightness, redness, yellowness, chroma, and hue angle) and the contents of ten different flavonoids were determined. Compared to “Roufurong,” which accumulated abundant anthocyanins at S3–S5, the yellow-colored “High Noon” displayed relatively higher contents of tetrahydroxychalcone (THC), flavones, and flavonols but no anthocyanin production. The contents of THC, flavones, and flavonols in “High Noon” peaked at S3 and dropped gradually as the flower bloomed, consistent with the color index patterns. Furthermore, RNA-seq analyses at S3 showed that structural genes such as PsC4Hs, PsDFRs, and PsUFGTs in the flavonoid biosynthesis pathway were downregulated in “High Noon,” whereas most PsFLSs, PsF3Hs, and PsF3’Hs were upregulated. Five transcription factor (TF) genes related to flavonoid biosynthesis were also upregulated in “High Noon.” One of these TFs, PsMYB111, was overexpressed in tobacco, which led to increased flavonols but decreased anthocyanins. Dual-luciferase assays further confirmed that PsMYB111 upregulated PsFLS. These results improve our understanding of yellow pigmentation in tree peony and provide a guide for future molecular-assisted breeding experiments in tree peony with novel flower colors.

References

[1] Li, J. J. Chinese Tree Peony and Herbaceous Peony (China Forestry Publishing House, 1999).

[2] Gu, Z. Y. 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. 0, 1-13 (2018).

[3] Yang, Y. et al. Germplasm resources and genetic breeding of Paeonia: a systematic review. Hortic. Res. 7, 107 (2020).

[4] Han, K., Wang, E. Q. & Liang, C. A. Utilization status and prospect of wild yellow peony in peony breeding. Northwest Hortic. 05, 70-71 (2018).

[5] Zhou, L., Wang, Y. & Peng, Z. H. Advances in study on formation mechanism and genetic engineering of yellow flowers. Silvae Sin. 45, 111-119 (2009).

[6] Huang, J. X., Wang, L. S., Li, X. M. & Lu, Y. Q. Advances in molecular basis and evolution of floral color variation. Chin. J. Bot. 23, 321-333 (2006).

[7] Hosoki, T., Hamada, M., Kando, T., Moriwaki, R. & Inaba, K. Comparative study of anthocyanin in tree peony flowers. J. Jpn Soc. Hort. Sci. 60, 395-403 (1991).

[8] Wang, L. S. et al. Analysis of petal anthocyanins to investigate flower coloration of Zhongyuan (Chinese) and Daikon Island (Japanese) tree peony cultivars. J. Plant Res. 114, 33-43 (2001).

[9] Wang, L. S. et al. Chemical taxonomy of the xibei tree peony from China by floral pigmentation. J. Plant Res. 117, 47-55 (2004).

[10] Zhang, J. et al. Comparison of anthocyanins in non-blotches and blotches of the petals of xibei tree peony. Sci. Hortic. 114, 104-111 (2007).

[11] Li, C. H. et al. Flavonoid composition and anioxidant activity of tree peony (Paeonia section Moutan) yellow flowers. J. Agric. Food Chem. 57, 8496-8503 (2009).

[12] Fan, J., Zhu, W., Kang, H., Ma, H. & Tao, G. Flavonoid constituents and antioxidant capacity in flowers of different zhongyuan tree penoy cultivars. J. Funct. Foods 4, 147-157 (2012).

[13] Wang, S. L. et al. Composition of peony petal fatty acids and flavonoids and their effect on Caenorhabditis elegans lifespan. Plant Physiol. Biochem. 155, 1-12 (2020).

[14] Shi, Q. Q. 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 10, 1-20 (2015a).

[15] Koes, R., Verweij, W. & Quanttrocchio, F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 10, 236-242 (2005).

[16] Tanaka, Y. & Ohmiya, A. Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways. Curr. Opin. Biotechnol. 19, 190-197 (2008).

[17] Hichri, I. et al. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J. Exp. Bot. 62, 2465-2483 (2011).

[18] Spelt, C., Quattrocchio, F., Mol, J. & Koes, R. ANTHOCYANIN1 of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanisms. Plant Cell 14, 2121-2135 (2002).

[19] Zhou, H. et al. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J. 82, 105-121 (2015).

[20] Li, C. et al. PyWRKY26 and PybHLH3 cotargeted the PyMYB114 promoter to regulate anthocyanin biosynthesis and transport in red-skinned pears. Hortic. Res. 7, 37 (2020).

[21] Gou, J. Y., Felippes, F. F., Liu, C. J., Weigel, D. & Wang, J. W. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 23, 1512-1522 (2011).

[22] Zhou, L., Wang, Y. & Peng, Z. H. Molecular characterization and expression analysis of chalcone synthase gene during flower development in tree peony (Paeonia suffruticosa). Afr. J. Biotechnol. 10, 1275-1284 (2011).

[23] Zhou, L., Wang, Y. & Peng, Z. H. Cloning and expression analysis of dihydroflavonol 4-reductase gene PsDFR1 from tree peony (Paeonia suffruticosa). J. Plant Physiol. 47, 885-892 (2011b).

[24] Zhou, L. et al. Overexpression of Ps-CHI1, a homologue of the chalcone isomerase gene from tree peony (Paeonia suffructicosa), reduces the intensity of flower pigmentation in transgenic tobacco. Plant Cell Tiss. Org. 16, 285-295 (2014).

[25] Shi, Q. Q., Zhou, L., Li, K. & Wang, Y. Isolation and expression of PlbHLH3 transcription factor genes in Paeonia lutea. For. Res. 28, 488-496 (2015b).

[26] Shi, Q. Q., Zhou, L., Wang, Y., Zhai, L. J. & Li, L. Isolation and expression of PlWDR3 and PlWDR18 transcription factor genes in Paeonia lutea. J. Northwest Forestry Univ. 32, 97-103 (2017).

[27] Shi, L. Y., Chen, X., Chen, W., Zheng, Y. H. & Yang, Z. F. Comparative transcriptomic analysis of white and red Chinese bayberry (Myrica rubra) fruits reveals flavonoid biosynthesis regulation. Sci. Hortic. 235, 9-20 (2018).

[28] Gao, L. X., Yang, H. X., Liu, H. F., Yang, J. & Hu, Y. H. Extensive transcriptome changes underlying the flower color intensity variation in Paeonia ostii. Front. Plant Sci. 6, 1205 (2016).

[29] Luo, J. R., Shi, Q. Q., Niu, L. X. & Zhang, Y. L. Transcriptomic analysis of leaf in tree peony reveals differentially expressed pigments genes. Molecules 22, e324 (2017).

[30] Guo, S. et al. Complete chloroplast genome sequence and phylogenetic analysis of Paeonia ostii. Molecules 23, e246 (2018).

[31] Zhang, C. et al. Transcriptomic analysis of cut tree peony with glucose supply using the RNA-Seq technique. Plant Cell Rep. 33, 111-129 (2014).

[32] Chaisson, M. J. P. et al. Resolving the complexity of the human genome using single-molecule sequencing. Nature 517, 608-611 (2014).

[33] Chen, X. et al. The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development. Cell 158, 1187-1198 (2014).

[34] Huddleston, J. et al. Reconstructing complex regions of genomes using long-read sequencing technology. Genome Res. 24, 688-696 (2014).

[35] Hackl, T., Hedrich, R., Schultz, J. & Forster, F. Proovread: large-scale high-accuracy pacbio correction through iterative short read consensus. Bioinformatics 30, 3004-3011 (2014).

[36] Wang, X. C. et al. Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis. Plant J. 101, 3 (2020).

[37] Dubos, C. et al. MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573-581 (2010).

[38] Czemmel, S. et al. The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol. 151, 1513-1530 (2009).

[39] Zhong, C. M. et al. The R2R3-MYB transcription factor GhMYB1a regulates flavonol and anthocyanin accumulation in Gerbera hybrida. Hortic. Res. 7, 78 (2020).

[40] Feller, A., Hernandez, J. M. & Grotewold, E. An ACT-like domain participates in the dimerization of several plant basic-helix-loop-helix transcription factors. J. Biol. Chem. 281, 28964-28974 (2006).

[41] Guo, L. P. et al. Transcriptome and chemical analysis reveal putative genes involved in flower color change in Paeonia ‘Coral Sunset’. Plant Physiol. Biochem. 138, 130-139 (2019).

[42] Yang, Q., Yuan, H. H. & Sun, X. B. Preliminary studies on the changes of flower color during the flowering period in two tree peony cultivars. Acta Hortic. Sin. 42, 930-938 (2015).

[43] Zhao, D. Q., Tao, J., Han, C. X. & Ge, J. T. Flower color diversity revealed by differential expression of flavonoid biosynthetic genes and flavonoid accumulation in herbaceous peony (Paeonia lactiflora Pall.). Mol. Biol. Rep. 39, 11263-11275 (2012).

[44] Lou, Q. et al. Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth. J. Exp. Bot. 65, 3157-3164 (2014).

[45] Zhao, D. Q. et al. Transcriptome sequencing of a chimaera reveals coordinated expression of anthocyanin biosynthetic genes mediating yellow formation in herbaceous peony (Paeonia lactiflora Pall.). BMC Genomics 15, 689-706 (2014).

[46] Davies, K. M. et al. Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-Reductase. Euphytica 131, 259-268 (2003).

[47] Duan, Y. B. et al. Genotypic variation of flavonols and antioxidant capacity in broccoli. Food Chem. 338, 127997 (2021).

[48] Zhang, Y. Y. et al. Comparative transcriptomics provides insight into floral color polymorphism in a Pleione limprichtii orchid population. Int. J. Mol. Sci. 21, e247 (2020).

[49] Zhao, D. Q., Wei, M. R., Shi, M., Hao, Z. J. & Tao, J. Identification and comparative profiling of miRNAs in herbaceous peony (Paeonia lactiflora Pall.) with red/yellow bicoloured flowers. Sci. Rep. 7, 44926 (2017).

[50] Stracke, R. et al. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J. 50, 660-677 (2007).

[51] Nakatsuka, T. et al. Isolation and characterization of GtMYBP3 and GtMYBP4, orthologues of R2R3-MYB transcription factors that regulate early flavonoid biosynthesis, in gentian flowers. J. Exp. Bot. 63, 6505-6517 (2012).

[52] Blanco, E. et al. Isolation and characterization of the flavonol regulator CcMYB12 from the globe artichoke [Cynara cardunculus var. scolymus (L.) Fiori]. Front. Plant Sci. 9, 941 (2018).

[53] Huang, W. et al. A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional Chinese medicinal plant, Epimedium sagittatum. Front. Plant Sci. 7, 1089 (2016).

[54] Pandey, A. et al. AtMYB12 expression in tomato leads to large scale differential modulation in transcriptome and flavonoid content in leaf and fruit tissues. Sci. Rep. 5, 12412 (2015).

[55] Zheng, Y. et al. iTAK: a program for genome-wide prediction and classification of plant transcription factors, transcriptional regulators, and protein kinases. Mol. Plant 9, 1667-1670 (2016).

[56] Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, 222-230 (2014).

[57] Koonin, E. V. et al. A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes. Genome Biol. 5, R7 (2004).

[58] Huerta-Cepas, J. et al. eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. 44, 286-293 (2016).

[59] Bairoch, A. & Boeckmann, B. The SWISS-PROT protein sequence data bank. Nucleic Acids Res. 19, 2247-2249 (1991).

[60] Kanehisa, M., Goto, S., Kawashima, S., Okuno, Y. & Hattori, M. The KEGG resource for deciphering the genome. Nucleic Acids Res. 32, 277-280 (2004).

[61] Ashburner, M. et al. Gene Ontology: tool for the unification of biology. Nat. Genet. 25, 25-29 (2000).

[62] Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, e357 (2012).

[63] Robinson, M. D., Mccarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Biogeoences 26, 139-140 (2010).

[64] Leng, N. et al. EBSeq-HMM: a Bayesian approach for identifying gene-expression changes in ordered RNA-seq experiments. Bioinformatics 31, 2614-2622 (2015).

[65] Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Goseq: gene ontology testing for RNA-seq datasets. ResearchGate 28, e573 (2012).

[66] Xie, C. et al. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 39, 316-322 (2011).

[67] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method . Methods 25, 402-408 (2001).

[68] Horsch, R. et al. A simple and general method for transferring genes into plants. Science 227, 1229-1232 (1985).

[69] Intelmann, D., Jaros, D. & Rohm, H. Identification of colour optima of commercial tomato catsup. Eur. Food Res. Technol. 22, 662-666 (2005).