Cytokinin and Ethylene Control Indole Alkaloid Production at the Level of the MEP/Terpenoid Pathway in Catharanthus roseus Suspension Cells

Abstract

The Madagascar periwinkle Catharanthus roseus accumulates a number of terpenoid indole alkaloids, some of which have high therapeutic interest. The biotechnological approach with cells in vitro remains an alternative to the field culture of periwinkle for the production of such compounds. We previously reported that two phytohormones, cytokinin and ethylene, remarkably enhanced the accumulation of alkaloids in periwinkle cell suspensions. In this work, we investigated the effects of these hormones on the regulation of several genes of the indole alkaloid biosynthetic pathway. We show that cytokinin and/or ethylene greatly enhanced the expression of the geraniol 10-hydroxylase gene. When given together, these hormones also increased the expression of three genes belonging to the methyl-erythritol pathway. These results make it possible to consider elements of cytokinin and ethylene signalling pathways as tools for improving terpenoid indole alkaloid production through metabolic engineering.

References

• 1 van der Heijden R, Schripsema J, Verpoorte R. The Catharanthus alkaloids: pharmacognosy and biotechnology.  Curr Med Chem. 2004;  11 607-28
• 2 Contin A, van der Heijden R, Lefeber A W, Verpoorte R. The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in a Catharanthus roseus cell culture.  FEBS Lett. 1998;  434 413-6
• 3 Hong S B, Hughes E H, Shanks J V, San K Y, Gibson S I. Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots.  Biotechnol Prog. 2003;  19 1105-8
• 4 Merillon J M, Doireau P, Guillot A, Chenieux J C, Rideau M. Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media.  Plant Cell Rep. 1986;  5 23-6
• 5 Stafford A, Smith L. Effects of modification of the primary precursor level by selection and feeding on indole alkaloids in Catharanthus roseus . In: Morris P et al, editors Secondary metabolism in plant cell cultures. Cambridge; Cambridge University Press 1986: pp 251-6
• 6 Van der Fits L, Memelink J. ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism.  Science. 2000;  289 295-7
• 7 Whitmer S, van der Heiden R, Verpoorte R. Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus .  J Biotechnol. 2000;  79 193-203
• 8 Schiel O, Witte L, Berlin J. Geraniol-10-hydroxylase activity and its relation to monoterpene indole alkaloid accumulation in cell suspension cultures of Catharanthus roseus .  Z Naturforsch. 1987;  42 1075-81
• 9 Collu G, Unver N, Peltenburg-Looman A M, van der Heijden R, Verpoorte R, Memelink J. Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis.  FEBS Lett. 2001;  508 215-20
• 10 Merillon J M, Ouelhazi L, Doireau P, Chenieux J C, Rideau M. Metabolic changes and alkaloid production in habituated and non-habituated cells of Catharanthus roseus grown in hormone-free medium. Comparing hormone-deprivated non-habituated cells with habituated cells.  J Plant Physiol. 1989;  134 54-60
• 11 Veau B, Courtois M, Oudin A, Chenieux J C, Rideau M, Clastre M. Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus .  Biochim Biophys Acta. 2000;  1517 159-63
• 12 Yahia A, Kevers C, Gaspar T, Chenieux J C, Rideau M, Creche J. Cytokinins and ethylene stimulate indole alkaloïds accumulation in cell suspension cultures of Catharanthus roseus by two distinct mechanisms.  Plant Sci. 1998;  133 9-15
• 13 Decendit A, Petit G, Andreu F, Doireau P, Merillon J M, Rideau M. putative sites of cytokinin action during their enhancing effect on indole alkaloid accumulation in periwinkle cell suspensions.  Plant Cell Rep. 1993;  12 710-2
• 14 Gantet P, Imbault N, Thiersault M, Doireau P. Necessity of a functional octadecanoic pathway for indole alkaloid synthesis by Catharanthus roseus cell suspensions cultured in an auxin-starved medium.  Plant Cell Physiol. 1998;  39 220-5
• 15 Cary A J, Liu W, Howell S H. Cytokinin action is coupled to ethylene in its effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings.  Plant Physiol. 1995;  107 1075-82
• 16 Hass C, Lohrmann J, Albrecht V, Sweere U, Hummel F, Yoo S D, Hwang I, Zhu T, Schafer E, Kudla J, Harter K. The response regulator 2 mediates ethylene signalling and hormone signal integration in Arabidopsis .  EMBO J. 2004;  23 3290-302