Transcriptome analysis and metabolic profiling reveal the key role of carotenoids in the petal coloration of Liriodendron tulipifera

Abstract

Liriodendron tulipifera, also known as tuliptree, is a popular ornamental horticultural plant with extraordinary tulip-shaped flowers characterized by an orange band near their base. The mechanisms underlying petal band-specific pigmentation during L. tulipifera flower development are unclear. Here, we combined nontargeted and targeted metabolomics and transcriptomics to identify a pathway cascade leading to carotenoid biosynthesis that is specifically activated in the petal band. The comparative analysis of carotenoid metabolites between L. tulipifera and Liriodendron hybrids indicates that γ-carotene, a rare carotene in plants, is the most likely orange pigment responsible for the coloration of the petal band. Phenotypic and transcriptomic analyses of developing petals reveal that the band area is first predefined by the loss of green color. Later, the band is maintained by locally activating and repressing carotenoid and chlorophyll biosynthesis genes, respectively. Two rate-limiting genes of carotene biosynthesis, carotenoid isomerase (CRTISO) and epsilon lycopene cyclase (ε-LCY), encode the core enzymes responsible for petal band-specific orange pigmentation in L. tulipifera. In particular, a putative additional ε-LCY copy specific to L. tulipifera may contribute to the distinct petal coloration pattern, compared with L. chinense. Taken together, our work provides a first glimpse of the metabolome and transcriptome dynamics in tuliptree flower coloration and provides a valuable resource for flower breeding or metabolic engineering as well as for understanding flower evolution in an early woody angiosperm.

References

[1] Bradshaw, H. D. & Schemske, D. W. Allele substitution at a flower colour locus produces a pollinator shift in monkeyflowers. Nature 426, 176-178 (2003).

[2] Hopkins, R. & Rausher, M. D. Pollinator-mediated selection on flower color allele drives reinforcement. Science 335, 1090-1092 (2012).

[3] Mol, J., Grotewold, E. & Koes, R. How genes paint flowers and seeds. Trends Plant Sci. 3, 212-217 (1998).

[4] Albert, N. W., Davies, K. M. & Schwinn, K. E. Gene regulation networks generate diverse pigmentation patterns in plants. Plant Signal. Behav. 9, e29526 (2014).

[5] Clement, J. S. & Mabry, T. J. Pigment evolution in the Caryophyllales: a systematic overview. Bot. Acta 109, 360-367 (1996).

[6] Tanaka, Y., Sasaki, N. & Ohmiya, A. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 54, 733-749 (2008).

[7] 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).

[8] Xu, W. J., Dubos, C. & Lepiniec, L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 20, 176-185 (2015).

[9] Yuan, H., Zhang, J. X., Nageswaran, D. & Li, L. Carotenoid metabolism and regulation in horticultural crops. Hortic. Res.-England 2, https://doi.org/10.1038/hortres.2015.36 (2015).

[10] Sun, T. H. et al. Carotenoid metabolism in plants: the role of plastids. Mol. Plant 11, 58-74 (2018).

[11] Stanley, L. & Yuan, Y. W. Transcriptional regulation of carotenoid biosynthesis in plants: so many regulators, so little consensus. Front. Plant Sci. 10, https://doi.org/10.3389/fpls.2019.01017 (2019).

[12] Frank, H. A. & Brudvig, G. W. Redox functions of carotenoids in photosynthesis. Biochemistry 43, 8607-8615 (2004).

[13] Nambara, E. & Marion-Poll, A. Abscisic acid biosynthesis and catabolism. Annu. Rev. Plant Biol. 56, 165-185 (2005).

[14] Al-Babili, S. & Bouwmeester, H. J. Strigolactones, a novel carotenoid-derived plant hormone. Annu. Rev. Plant Biol. 66, 161-186 (2015).

[15] Eggersdorfer, M. & Wyss, A. Carotenoids in human nutrition and health. Arch. Biochem. Biophys. 652, 18-26 (2018).

[16] Kato, M. et al. Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in citrus fruit. Plant Physiol. 134, 824-837 (2004).

[17] Wang, W. J., Liu, G. S., Niu, H. X., Timko, M. P. & Zhang, H. B. The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90). J. Exp. Bot. 65, 2147-2160 (2014).

[18] Sagawa, J. M. et al. An R2R3-MYB transcription factor regulates carotenoid pigmentation in Mimulus lewisii flowers. N. Phytol. 209, 1049-1057 (2016).

[19] Meng, Y. et al. The MYB activator WHITE PETAL1 associates with MtTT8 and MtWD40-1 to regulate carotenoid-derived flower pigmentation in Medicago truncatula. Plant Cell. https://doi.org/10.1105/tpc.19.00480 (2019).

[20] Parks, C. R., Miller, N. G., Wendel, J. F. & Mcdougal, K. M. Genetic-divergence within the genus Liriodendron (Magnoliaceae). Ann. Missouri Bot. Gard. 70, 658-666 (1983).

[21] Parks, C. R. & Wendel, J. F. Molecular divergence between Asian and North-American species of Liriodendron (Magnoliaceae) with implications for interpretation of fossil floras. Am. J. Bot. 77, 1243-1256 (1990).

[22] Santamour, F. S. & Meyer, F. G. The two tuliptrees. Am. Horticulturist 50, 87-89 (1971).

[23] Santamour, F. S. Interspecific hybrids in Liriodendron and their chemical verification. For. Sci. 18, 233-236 (1972).

[24] Beck, D. E. & Della-Bianca, L. Yellow-Poplar: Characteristics and Management, Agriculture Handbook Vol. 583, 92 (US Department of Agriculture, Washington, 1981).

[25] Santamour, F. S. & McArdle, A. J. Cultivar checklist for Liquidambar and Liriodendron. J. Arboric. 10, 309-312 (1984).

[26] Demuth, P. & Santamour, F. Carotenoid flower pigments in Liriodendron and Magnolia. Bull. Torre. Bot. Club 105, 65-66 (1978).

[27] Mu, J. H., Stains, J. P. & Kao, T. H. Characterization of a pollen-expressed gene encoding a putative pectin esterase of Petunia inflata. Plant Mol. Biol. 25, 539-544 (1994).

[28] Birecka, H., Chaskes, M. J. & Goldstein, J. Peroxidase and senescence. J. Exp. Bot. 30, 565-573 (1979).

[29] Levin, M. et al. The mid-developmental transition and the evolution of animal body plans. Nature 531, 637 (2016).

[30] Cazzonelli, C. I., Roberts, A. C., Carmody, M. E. & Pogson, B. J. Transcriptional control of SET DOMAIN GROUP 8 and CAROTENOID ISOMERASE during Arabidopsis development. Mol. Plant 3, 174-191 (2010).

[31] Soltis, D. E. & Soltis, P. S. Nuclear genomes of two magnoliids. Nat. Plants 5, 6-7 (2019).

[32] Chen, J. et al. Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation. Nat. Plants 5, 18-25 (2019).

[33] Chaw, S. M. et al. Stout camphor tree genome fills gaps in understanding of flowering plant genome evolution. Nat. Plants 5, 63-73 (2019).

[34] Mravec, J. et al. Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature 459, 1136-U1127 (2009).

[35] Tanaka, A. et al. Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a. Proc. Natl Acad. Sci. USA 95, 12719-12723 (1998).

[36] Ilag, L. L., Kumar, A. M. & Soll, D. Light regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis. Plant Cell 6, 265-275 (1994).

[37] Tottey, S. et al. Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide. Proc. Natl Acad. Sci. USA 100, 16119-16124 (2003).

[38] Mok, D. W. S. & Mok, M. C. Cytokinin metabolism and action. Annu. Rev. Plant Phys. 52, 89-118 (2001).

[39] Kobayashi, K. et al. Regulation of root greening by light and auxin/cytokinin signaling in Arabidopsis. Plant Cell 24, 1081-1095 (2012).

[40] Smits, B. L. & Mitchell, H. L. A rich source of γ-carotene. Science 113, 296-297 (1951).

[41] Zhao, C., Hanada, A., Yamaguchi, S., Kamiya, Y. & Beers, E. P. The Arabidopsis Myb genes MYR1 and MYR2 are redundant negative regulators of flowering time under decreased light intensity. Plant J. 66, 502-515 (2011).

[42] Klepikova, A. V., Kasianov, A. S., Gerasimov, E. S., Logacheva, M. D. & Penin, A. A. A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling. Plant J. 88, 1058-1070 (2016).

[43] Obulareddy, N., Panchal, S. & Melotto, M. Guard cell purification and RNA isolation suitable for high-throughput transcriptional analysis of cell-type responses to biotic stresses. Mol. Plant Microbe Interact. 26, 844-849 (2013).

[44] Masierowska, M. L. Floral nectaries and nectar production in brown mustard (Brassica juncea) and white mustard (Sinapis alba) (Brassicaceae). Plant Syst. Evol. 238, 97-107 (2003).

[45] Zhou, Y. W. et al. Floral nectary morphology and proteomic analysis of nectar of Liriodendron tulipifera Linn. Front. Plant Sci. 7, https://doi.org/10.3389/fpls.2016.00826 (2016).

[46] Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644-652 (2011).

[47] Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417-419 (2017).

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

[49] Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284-287 (2012).

[50] Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, https://doi.org/10.1186/1471-2105-9-559 (2008).

[51] Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498-2504 (2003).

[52] Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312-1313 (2014).

[53] Nisar, N., Li, L., Lu, S., Khin, N. C. & Pogson, B. J. Carotenoid Metabolism in Plants. Mol. Plant 8, 68-82 (2015).