Sclareol and linalyl acetate are produced by glandular trichomes through the MEP pathway

Abstract

Sclareol, an antifungal specialized metabolite produced by clary sage, Salvia sclarea, is the starting plant natural molecule used for the hemisynthesis of the perfume ingredient ambroxide. Sclareol is mainly produced in clary sage flower calyces; however, the cellular localization of the sclareol biosynthesis remains unknown. To elucidate the site of sclareol biosynthesis, we analyzed its spatial distribution in the clary sage calyx epidermis using laser desorption/ ionization mass spectrometry imaging (LDI–FTICR-MSI) and investigated the expression profile of sclareol biosynthesis genes in isolated glandular trichomes (GTs). We showed that sclareol specifically accumulates in GTs’ gland cells in which sclareol biosynthesis genes are strongly expressed. We next isolated a glabrous beardless mutant and demonstrate that more than 90% of the sclareol is produced by the large capitate GTs. Feeding experiments, using 1-¹³C-glucose, and specific enzyme inhibitors further revealed that the methylerythritol-phosphate (MEP) biosynthetic pathway is the main source of isopentenyl diphosphate (IPP) precursor used for the biosynthesis of sclareol. Our findings demonstrate that sclareol is an MEP-derived diterpene produced by large capitate GTs in clary sage emphasing the role of GTs as biofactories dedicated to the production of specialized metabolites.

References

[1] Clarke, R. The origin of ambergris. LAJAM 5, 7-21 (2006).

[2] Ohloff, G. The Fragrance of Ambergris. 535-573 (ed E.T. Theimer) (Academic Press, 1982).

[3] Rowland, S. J., Sutton, P. A. & Knowles, T. D. J. The age of ambergris. Nat. Prod. Res. 33, 3134-3142 (2019).

[4] Ohloff, G., Schulte-Elte, K. H. & Müller, B. L. Formation of ambergris odorants from ambrein under simulated natural conditions. Helv. Chim. Acta 60, 2763-2766 (1977).

[5] Barrero, A. F., Altarejos, J., AlvarezManzaneda, E. J., Ramos, J. M. & Salido, S. Synthesis of (+/-)-ambrox from (E)-nerolidol and beta-ionone via allylic alcohol [2,3] sigmatropic rearrangement. J. Org. Chem. 61, 2215-2218 (1996).

[6] Barrero, A. F., Alvarezmanzaneda, E. J., Altarejos, J., Salido, S. & Ramos, J. M. Synthesis of ambrox(R) from (-)-sclareol and (+)-cis-abienol. Tetrahedron 49, 10405-10412 (1993).

[7] Zerbe, P. & Bohlmann, J. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering. Trends Biotechnol. 33, 419-428 (2015).

[8] Foutami, I. J., Mariager, T., Rinnan, R., Barnes, C. J. & Ronsted, N. Hundred fifty years of herbarium collections provide a reliable resource of volatile terpenoid profiles showing strong species effect in four medicinal species of salvia across the mediterranean. Front. Plant Sci. 9, 1877 (2018).

[9] Wagner, S., Pfleger, A., Mandl, M. & Böchzelt, H. Changes in the qualitative and quantitative composition of essential oils of clary sage and roman chamomile during steam distillation in pilot plant scale. Distillation-Advances from Modelling to Application, 141-158 (2012).

[10] Nasermoadeli, S. & Rowshan, V. Comparison of Salvia sclarea L. essential oil components in wild and field population. Int. J. Agric. Crop Sci. 4, 1997-2000 (2013).

[11] Lattoo, S. K., Dhar, R. S., Dhar, A. K., Sharma, P. R. & Agarwal, S. G. Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny in Salvia sclarea. Flavour Fragr. J. 21, 817-821 (2006).

[12] Caniard, A. et al. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture. BMC Plant Biol. 12, 119 (2012).

[13] Kuzma, L. et al. Chemical composition and biological activities of essential oil from Salvia sclarea plants regenerated in vitro. Molecules 14, 1438-1447 (2009).

[14] Sharopov, F. S. & Setzer, W. N. The essential oil of Salvia sclarea L. from Tajikistan. Rec. Nat. Prod. 6, 75-79 (2012).

[15] Caissard, J. C. et al. Extracellular localization of the diterpene sclareol in clary sage (Salvia sclarea L., Lamiaceae). PloS ONE 7, e48253 (2012).

[16] Laville, R. et al. Amphilectane diterpenes from Salvia sclarea: biosynthetic considerations. J. Nat. Prod. 75, 121-126 (2012).

[17] Yang, S. X. et al. One-pot synthesis of (-)-ambrox. Sci. Rep. 6, 32650 (2016).

[18] Lange, B. M. The evolution of plant secretory structures and emergence of terpenoid chemical diversity. Annu. Rev. Plant Biol. 66, 139-159 (2015).

[19] Tissier, A. Plant secretory structures: more than just reaction bags. Curr. Opin. Biotechnol. 49, 73-79 (2018).

[20] Sallaud, C. et al. Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes. Plant J. 72, 1-17 (2012).

[21] Spyropoulou, E. A., Haring, M. A. & Schuurink, R. C. Expression of terpenoids 1, a glandular trichome-specific transcription factor from tomato that activates the terpene synthase 5 promoter. Plant Mol. Biol. 84, 345-357 (2014).

[22] Vining, K. J. et al. Draft genome sequence of Mentha longifolia and development of resources for mint cultivar improvement. Mol. Plant 10, 323-339 (2017).

[23] Werker, E. Function of essential oil-secreting glandular hairs in aromatic plants of the lamiaceae-a review. Flavour Fragr. J. 8, 249-255 (1993).

[24] Werker, E., Ravid, U. & Putievsky, E. Glandular hairs and their secretions in the vegetative and reproductive-organs of salvia-sclarea and salvia-dominica. Isr. J. Bot. 34, 239-252 (1985).

[25] Schmiderer, C., Grassi, P., Novak, J., Weber, M. & Franz, C. Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae). Plant Biol. 10, 433-440 (2008).

[26] Tholl, D. Biosynthesis and biological functions of terpenoids in plants. Adv. Biochem. Eng. Biot. 148, 63-106 (2015).

[27] Schalk, M. et al. Toward a biosynthetic route to sclareol and amber odorants. J. Am. Chem. Soc. 134, 18900-18903 (2012).

[28] Gunnewich, N. et al. A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxycopalyl diphosphate. Phytochemistry 91, 93-99 (2013).

[29] Fowler, D. J., Hamilton, J. T. G., Humphrey, A. J. & O’Hagan, D. Plant terpene biosynthesis. The biosynthesis of linalyl acetate in Mentha citrata. Tetrahedron Lett. 40, 3803-3806 (1999).

[30] Zaks, A., Davidovich-Rikanati, R., Bar, E., Inbar, M. & Lewinsohn, E. Biosynthesis of linalyl acetate and other terpenes in lemon mint (Mentha aquatica var. citrata, Lamiaceae) glandular trichomes. Isr. J. Plant Sci. 56, 233-244 (2008).

[31] Jomaa, H. et al. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285, 1573-1576 (1999).

[32] Lichtenthaler, H. K. Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors. Biochem. Soc. Trans. 28, 785-789 (2000).

[33] Lipko, A. & Swiezewska, E. Isoprenoid generating systems in plants-a handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process. Prog. Lipid Res. 63, 70-92 (2016).

[34] Reed, J. & Osbourn, A. Engineering terpenoid production through transient expression in Nicotiana benthamiana. Plant Cell Rep. 37, 1431-1441 (2018).

[35] Opitz, S., Nes, W. D. & Gershenzon, J. Both methylerythritol phosphate and mevalonate pathways contribute to biosynthesis of each of the major isoprenoid classes in young cotton seedlings. Phytochemistry 98, 110-119 (2014).

[36] Wolwer-Rieck, U., May, B., Lankes, C. & Wust, M. Methylerythritol and mevalonate pathway contributions to biosynthesis of mono-, sesqui-, and diterpenes in glandular trichomes and leaves of Stevia rebaudiana Bertoni. J. Agr. Food Chem. 62, 2428-2435 (2014).

[37] Liao, P., Hemmerlin, A., Bach, T. J. & Chye, M. L. The potential of the mevalonate pathway for enhanced isoprenoid production. Biotechnol. Adv. 34, 697-713 (2016).

[38] Alberts, A. W. et al. Mevinolin-a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme-a reductase and a cholesterol-lowering agent. Proc. Natl Acad. Sci. USA 77, 3957-3961 (1980).

[39] Hall, R. et al. Plant metabolomics: the missing link in functional genomics strategies. Plant Cell 14, 1437-1440 (2002).

[40] Amster, I. J. Fourier transform mass spectrometry. J. Mass Spectrom. 31, 1325-1337 (1996).

[41] Ding, J. & Anderegg, R. J. Specific and nonspecific dimer formation in the electrospray ionization mass spectrometry of oligonucleotides. J. Am. Soc. Mass Spectrom. 6, 159-164 (1995).

[42] Stefansson, M., Sjöberg, P. J. R. & Markides, K. E. Regulation of multimer formation in electrospray mass spectrometry. Anal. Chem. 68, 1792-1797 (1996).

[43] Kamel, A. M., Brown, P. R. & Munson, B. Effects of mobile-phase additives, solution pH, ionization constant, and analyte concentration on the sensitivities and electrospray ionization mass spectra of nucleoside antiviral agents. Anal. Chem. 71, 5481-5492 (1999).

[44] Lange, B. M. et al. Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes. Proc. Natl Acad. Sci. USA 97, 2934-2939 (2000).

[45] Lange, B. M. & Turner, G. W. Terpenoid biosynthesis in trichomes-current status and future opportunities. Plant Biotechnol. J. 11, 2-22 (2013).

[46] Figueiredo, A. C., Barroso, J. G., Pedro, L. G. & Scheffer, J. J. C. Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour Fragr. J. 23, 213-226 (2008).

[47] Keene, C. K. & Wagner, G. J. Direct demonstration of duvatrienediol biosynthesis in glandular heads of tobacco trichomes. Plant Physiol. 79, 1026-1032 (1985).

[48] Tissier, A . Glandular trichomes: what comes after expressed sequence tags? Plant J. 70, 51-68 (2012).

[49] Dong, Y. et al. Sample preparation for mass spectrometry imaging of plant tissues: a review. Front. Plant Sci. 7, 60 (2016).

[50] Thunig, J., Hansen, S. H. & Janfelt, C . Analysis of secondary plant metabolites by indirect desorption electrospray ionization imaging mass spectrometry. Anal. Chem. 83, 3256-3259 (2011).

[51] Rodriguez-Concepcion, M. et al. Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. Plant Cell 16, 144-156 (2004).

[52] Balcke, G. U. et al. Multi-omics of tomato glandular trichomes reveals distinct features of central carbon metabolism supporting high productivity of specialized metabolites. Plant Cell 29, 960-983 (2017).

[53] Yan, T. X. et al. HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua. N. Phytol. 213, 1145-1155 (2017).

[54] Yan, T. X. et al. A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua. N. Phytol. 218, 567-578 (2018).

[55] Shi, P. et al. The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua. N. Phytol. 217, 261-276 (2018).

[56] Vaccaro, M., Malafronte, N., Alfieri, M., De Tommasi, N. & Leone, A. Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots. Plant Cell Tiss. Org. 119, 65-77 (2014).

[57] Xu, H. et al. Analysis of the genome sequence of the medicinal plant Salvia miltiorrhiza. Mol. Plant 9, 949-952 (2016).

[58] Dong, A. X. et al. High-quality assembly of the reference genome for scarlet sage, Salvia splendens, an economically important ornamental plant. Gigascience 17, giy068 (2018).

[59] Amme, S. et al. A proteome approach defines protective functions of tobacco leaf trichomes. Proteomics 5, 2508-2518 (2005).

[60] Chain, F. E., Leyton, P., Paipa, C., Fortuna, M. & Brandan, S. A. FT-IR, FT-Raman, UV-visible, and NMR spectroscopy and vibrational properties of the labdane-type diterpene 13-epi-sclareol. Spectrochim. Acta A 138, 303-313 (2015).