The biochemical and molecular investigation of flower color and scent sheds lights on further genetic modification of ornamental traits in Clivia miniata

Abstract

Clivia miniata is renowned for its evergreen and strap-like leaves, whereas its floral color and scent are lacking diversity. Here, anthocyanin, volatile terpene, and carotenoid metabolisms were integrally investigated in C. miniata flowers. The results showed that pelargonidins and lutein might cooperate to confer orange or yellow color to C. miniata flowers, but only a trace amount of (+)-limonene was detected. The expression levels of CmF3^′ H and CmDFR appeared to be responsible for the ratio of cyanidin and pelargonidin derivatives in C. miniata, and the low expression of CmF3^′ H was responsible for the lack of cyanidins in flowers. Moreover, the CmF3^′ H promoter could not be activated by CmMYBAs, suggesting that it was controlled by novel regulators. Only two CmTPSs were functional, with CmTPS2 responsible for (+)-limonene synthesis, contributing to the monotonous flower volatile terpenes of C. miniata. CmCCD1a and CmCCD1b were able to cleave carotenoids at the 5,6 (5^′,6^′), and 9,10 (9^′,10^′) positions to generate volatile apocarotenoids, whereas the substrates found in low-quantities or specific subcellular localizations of CmCCD1s might constrain volatile apocarotenoid release. Consequently, activating F3^′ H and introducing novel F3^′ 5^′ H or versatile TPS may be effective ways to modify the floral color and scent, respectively. Alternatively, modifying the carotenoid flux or CCD1 localization might affect floral color and scent simultaneously. Taking these results together, the present study provides a preliminary deciphering of the genetic constraints underlying flower color and scent development, and proposes possible schemes for further genetic modification of ornamental traits in C. miniata and other plants.

References

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

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

[3] Zhao DQ, Tao J . Recent advances on the development and regulation of flower color in ornamental plants. Front Plant Sci. 2015; 6: 261.

[4] Liu Y, Qian J, Li J et al. Hydroxylation decoration patterns of flavonoids in horticultural crops: chemistry, bioactivity and biosynthesis. Hortic Res 2022; 9: uhab068.

[5] Hermanns AS, Zhou XS, Xu Q et al. Carotenoid pigment accumulation in horticultural plants. Hortic Plant J. 2020; 6: 343-60.

[6] Rosenkranz M, Chen YY, Zhu PY et al. Volatile terpenes-mediators of plant-to-plant communication. Plant J. 2021; 108: 617-31.

[7] Dudareva N, Pichersky E, Gershenzon J . Biochemistry of plant volatiles. Plant Physiol. 2004; 135: 1893-902.

[8] Du F, Wang T, Fan JM et al. Volatile composition and classification of Lilium flower aroma types and identification, polymorphisms, and alternative splicing of their monoterpene synthase genes. Hortic Res. 2019; 6: 110.

[9] Serra S . Recent advances in the synthesis of carotenoid-derived flavours and fragrances. Molecules. 2015; 20: 12817-40.

[10] Han YJ, Wang H, Wang X et al. Mechanism of floral scent production in Osmanthus fragrans and the production and regulation of its key floral constituents, β-ionone and linalool . Hortic Res. 2019; 6: 106.

[11] Byers KJRP, Vela JP, Peng F et al. Floral volatile alleles can contribute to pollinator-mediated reproductive isolation in monkeyflowers (Mimulus) . Plant J. 2014; 80: 1031-42.

[12] Schiestl F, Johnson S . Pollinator-mediated evolution of floral signals. Trends Ecol Evol 2013; 28: 307-15.

[13] Dellinger AS . Pollination syndromes in the 21(st) century: where do we stand and where may we go? New Phytol. 2020; 228: 1193-213.

[14] Cheynier V, Comte G, Davies K et al. Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiol Biochem. 2013; 72: 1-20.

[15] Ambawat S, Sharma P, Yadav N et al. MYB transcription factor genes as regulators for plant responses: an overview. Physiol Mol Biol Plants. 2013; 19: 307-21.

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

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

[18] 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.

[19] Li YQ, 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.

[20] Wheeler LC, Walker JF, Ng J et al. Transcription factors evolve faster than their structural gene targets in the flavonoid pigment pathway. Mol Biol Evol. 2022; 39: msac044.

[21] 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.

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

[23] Vranová E, Coman Schmid D, Gruissem W . Network analysis of the MVA and MEP pathways for isoprenoid synthesis. Annu Rev Plant Biol. 2013; 64: 665-700.

[24] Suzuki M, Kamide Y, Nagata N et al. Loss of function of 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels . Plant J. 2004; 37: 750-61.

[25] Huang Y, Xie FJ, Cao X et al. Research progress in biosynthesis and regulation of plant terpenoids. Biotechnol Biotechnol Eq. 2022; 35: 1800-9.

[26] Bao TT, Shadrack K, Yang S et al. Functional characterization of terpene synthases accounting for the volatilized-terpene heterogeneity in Lathyrus odoratus cultivar flowers. Plant Cell Physiol. 2020; 61: 1733-49.

[27] Gao FZ, Liu B, Li M et al. Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida . J Exp Bot. 2018; 69: 4249-65.

[28] Zhou F, Pichersky E . The complete functional characterisation of the terpene synthase family in tomato. New Phytol. 2020; 226: 1341-60.

[29] Yang S, Wang N, Kimani S et al. Characterization of terpene synthase variation in flowers of wild Aquilegia species from northeastern Asia. Hortic Res. 2022; 9: uhab020.

[30] Sun TH, Rao S, Zhou XS et al. Plant carotenoids: recent advances and future perspectives. Mol Hortic. 2022; 2: 3.

[31] Nisar N, Li L, Lu S et al. Carotenoid metabolism in plants. Mol Plant. 2015; 8: 68-82.

[32] Shi JN, Cao C, Xu JY et al. Research advances on biosynthesis, regulation, and biological activities of apocarotenoid aroma in horticultural plants. J Chem 2020; 2020: 1-11.

[33] Hou X, Rivers J, León P et al. Synthesis and function of apocarotenoid signals in plants. Trends Plant Sci. 2016; 21: 792-803.

[34] Tan BC, Schwartz SH, Zeevaart JA et al. Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA. 1997; 94: 12235-40.

[35] Tan BC, Joseph LM, Deng WT et al. Molecular characterization of the Arabidopsis 9- cis epoxycarotenoid dioxygenase gene family . Plant J 2003; 35: 44-56.

[36] Frusciante S, Diretto G, Bruno M et al. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis. Proc Natl Acad Sci USA 2014; 111: 12246-51.

[37] Zhong YT, Pan X, Wang R et al. ZmCCD10a encodes a distinct type of carotenoid cleavage dioxygenase and enhances plant tolerance to low phosphate . Plant Physiol. 2020; 184: 374-92.

[38] Ahrazem O, Gómez-Gómez L, Rodrigo M et al. Carotenoid cleavage oxygenases from microbes and photosynthetic organisms: features and functions. Int J Mol Sci. 2016; 17: 1781.

[39] Kiepiel I, Johnson SD . Shift from bird to butterfly pollination in Clivia (Amaryllidaceae) . Am J Bot. 2014; 101: 190-200.

[40] Musara C, Aladejana EB, Aladejana AE . Clivia miniata (Lindl.) Bosse, (Amaryllidaceae): botany, medicinal uses, phytochemistry and pharmacological properties . J Appl Pharm Sci 2021; 11: 12-8.

[41] Boutigny AL, Dohin N, Pornin D et al. Overview and detectability of the genetic modifications in ornamental plants. Hortic Res. 2020; 7: 11.

[42] Sannikova VY . Genetic engineering as a way to obtain ornamental plants with a changed flower color. Plant Biotechnol Breed. 2020; 3: 40-5.

[43] Noman A, Aqeel M, Deng J et al. Biotechnological advancements for improving floral attributes in ornamental plants. Front Plant Sci. 2017; 8: 530.

[44] Tiwari SB, Hagen G, Guilfoyle T . The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell. 2003; 15: 533-43.

[45] Tiwari SB, Hagen G, Guilfoyle TJ . Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell. 2004; 16: 533-43.

[46] Gao RF, Han T, Xun H et al. MYB transcription factors GmMYBA2 and GmMYBR function in a feedback loop to control pigmentation of seed coat in soybean. J Exp Bot. 2021; 72: 4401-18.

[47] Shan XT, 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.

[48] Achilonu C, Mathabatha M . Elucidating variable traits of flower pigments in clivian plants’ species. Vegetos. 2017; 30: 9-23.

[49] Conrad F. Molecular systematics, biogeography and dating of the tribe Haemantheae (Amaryllidaceae) and the phylogeography of Clivia . Ph.D. Thesis, Department of Botany, University of Cape Town; 2008.

[50] Yang Y, Li B, Feng C et al. Chemical mechanism of flower color microvariation in Paeonia with yellow flowers. Hortic Plant J 2020; 6: 179-90.

[51] Mekapogu M, Vasamsetti BMK, Kwon OK et al. Anthocyanins in floral colors: biosynthesis and regulation in chrysanthemum flowers. Int J Mol Sci 2020; 21: 6537.

[52] Meng J, Gao Y, Han M et al. In vitro anthocyanin induction and metabolite analysis in Malus spectabilis leaves under low nitrogen conditions. Hortic Plant J 2020; 6: 248-92.

[53] Fu Z, Shang H, Jiang H et al. Systematic identification of the light-quality responding anthocyanin synthesis-related transcripts in Petunia petals . Hortic Plant J 2020; 6: 428-38.

[54] Rausher MD . Evolutionary transitions in floral color. Int J Plant Sci 2008; 169: 7-21.

[55] Li Y, Liu X, Cai X et al. Dihydroflavonol 4-reductase genes from Freesia hybrida play important and partially overlapping roles in the biosynthesis of flavonoids. Front Plant Sci 2017; 8: 428.

[56] Xue X, Cronk QCB . The molecular basis for an ancient colour mutant in sweet pea (Lathyrus odoratus) . Can J Plant Sci 2017; 98: 591-600.

[57] Luo P, Ning G, Wang Z et al. Disequilibrium of flavonol synthase and dihydroflavonol-4-reductase expression associated tightly to white vs. red color flower formation in plants. Front Plant Sci. 2016; 6: 1257.

[58] Li H, Tian J, Yao YY et al. Identification of leucoanthocyanidin reductase and anthocyanidin reductase genes involved in proanthocyanidin biosynthesis in Malus crabapple plants. Plant Physiol Biochem 2019; 139: 141-51.

[59] Bogs J, Downey MO, Harvey JS et al. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol. 2005; 139: 652-63.

[60] Davik J, Aaby K, Buti M et al. Major-effect candidate genes identified in cultivated strawberry (Fragaria × ananassa Duch.) for ellagic acid deoxyhexoside and pelargonidin-3- O-malonylglucoside biosynthesis, key polyphenolic compounds . Hortic Res. 2020; 7: 125.

[61] Saigo T, Wang T, Watanabe M et al. Diversity of anthocyanin and proanthocyanin biosynthesis in land plants. Curr Opin Plant Biol 2020; 55: 93-9.

[62] Aneklaphakij C, Saigo T, Watanabe M et al. Diversity of chemical structures and biosynthesis of polyphenols in nut-bearing species. Front Plant Sci. 2021; 12: 642581-1.

[63] Lui ACW, Lam PY, Chan KH et al. Convergent recruitment of 5^′-hydroxylase activities by CYP75B flavonoid B-ring hydroxylases for tricin biosynthesis in Medicago legumes. New Phytol. 2020; 228: 269-84.

[64] 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.

[65] Peng Y, Lin-Wang K, Cooney JM et al. Differential regulation of the anthocyanin profile in purple kiwifruit (Actinidia species) . Hortic Res. 2019; 6: 3.

[66] Guo H, Lackus ND, KÖllner TG et al. Evolution of a novel and adaptive floral scent in wild tobacco. Mol Biol Evol. 2020; 37: 1090-9.

[67] Rohloff J, Bones AM . Volatile profiling of Arabidopsis thaliana - putative olfactory compounds in plant communication. Phytochemistry. 2005; 66: 1941-55.

[68] Magnard J, Roccia A, Caissard JC et al. Biosynthesis of monoterpene scent compounds in roses. Science. 2015; 349: 81-3.

[69] Ninkuu V, Zhang L, Yan J et al. Biochemistry of terpenes and recent advances in plant protection. Int J Mol Sci. 2021; 22: 5710.

[70] Pichersky E, Raguso RA . Why do plants produce so many terpenoid compounds? New Phytol. 2018; 220: 692-702.

[71] Chen F, Tholl D, Bohlmann J et al. The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011; 66: 212-29.

[72] Zvi MMB, Shklarman E, Masci T et al. PAP1 transcription factor enhances production of phenylpropanoid and terpenoid scent compounds in rose flowers . New Phytol. 2012; 195: 335-45.

[73] Michael RJ, Ranjan A, Kumar RS et al. Light-regulated expression of terpene synthase gene, AtTPS03, is controlled by the bZIP transcription factor, HY5, in Arabidopsis thaliana . Biochem Biophys Res Commun 2020; 529: 437-43.

[74] Yu ZX, Wang LJ, Zhao B et al. Progressive regulation of sesquiterpene biosynthesis in Arabidopsis and patchouli (Pogostemon cablin) by the miR156-targeted SPL transcription factors. Mol Plant 2014; 8: 98-110.

[75] Gou JY, Felippes F, Liu CJ et al. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 2011; 23: 1512-22.

[76] Yang X, Yue Y, Li H et al. The chromosome-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds of Osmanthus fragrans . Hortic Res. 2018; 5: 72.

[77] Zhang L, Chen F, Zhang X et al. The water lily genome and the early evolution of flowering plants. Nature. 2020; 577: 79-84.

[78] Fang Q, Li Y, Liu B et al. Cloning and functional characterization of a carotenoid cleavage dioxygenase 2 gene in safranal and crocin biosynthesis from Freesia hybrida . Plant Physiol Biochem. 2020; 154: 439-50.

[79] Tu Y, Liu F, Guo DD et al. Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation. BMC Plant Biol. 2016; 16: 132.

[80] Clough SJ, Bent AF . Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . Plant J. 1998; 16: 735-43.

[81] Sainsbury F, Thuenemann EC, Lomonossoff GP . pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J. 2009; 7: 682-93.