Identification of marneral synthase, which is critical for growth and development in Arabidopsis

SUMMARYPlants produce structurally diverse triterpenoids, which are important for their life and survival. Mosttriterpenoidsandsterolsshareacommonbiosyntheticintermediate,2,3-oxidosqualene(OS),whichiscyclizedby 2,3-oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta,we characterized a Arabidopsis mrn1 knock-out mutant displaying round-shaped leaves, late flowering, anddelayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation.Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wildtype or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity andpermeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 genepromoter, was specifically detected in shoot and root apical meristems, which are responsible for primarygrowth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots andsiliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobaccoprotoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneralsynthase is important for the growth and development of Arabidopsis.Keywords: Arabidopsis, growth and development, marneral synthase, marnerol, oxidosqualene cyclase,triterpenols.INTRODUCTIONPlants produce more than 20 000 triterpenoids with chemi-cally diverse structures. During plant growth and develop-ment, triterpenoids are not only essential precursors for cellmembranes and steroid hormones (Nes and Heftmann,1981; Benveniste, 2004; Suzuki et al., 2006) but also playroles in defense against pathogen attack (Phillips et al.,2006) and in protection against environmental stresses(Wang et al., 2010). In addition, plant triterpenoids arewidely used as medicines due to their pharmacologicalproperties, such as anti-cancer, anti-inflammatory, andanti-oxidant activities, and anti-plasmodial activity againsthuman malarial parasites (Waller and Yamasaki, 1996;Benoit-Vical et al., 2003; Oliveira et al., 2005; Lima-Junioret al., 2006; Holanda Pinto et al., 2008; Shai et al., 2008; Au-gustin et al., 2011; Osbourn et al., 2011).Triterpenoids are known to be synthesized via the con-densationoffive-carbonisoprenoidprecursorsderivedfromthe mevalonate (MVA) pathway in plants (McGarvey andCroteau, 1995). Farnesyl diphosphate synthase catalyzes theproduction of farnesyl diphosphate (FPP) from isopentenyldiphosphate and dimethylallyl diphosphate. Squalene syn-thase converts FPP to squalene, which is subsequentlyoxidized into 2,3-oxidosqualene (OS) by squalene epoxi-dase. Finally, the biosynthetic intermediate OS is regio- and