Regulation of simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens

Abstract

The white flowers of N. suaveolens emit a complex bouquet of fragrance volatiles. The dominant compounds are benzenoids (e.g. methyl benzoate, methyl salicylate, benzyl benzoate and benzyl salicylate), monoterpenes (1,8-cineole, limonene, sabinene, E-beta-ocimene, beta-beta-myrcene, alpha- and beta-pinene and alpha-terpineole) and sesquiterpenes (e.g. caryophyllene), which are all emitted at higher levels during the night. Here, we show that the simultaneous nocturnal emission of most monoterpenes is realized by a single floral-specific multi-product enzyme (1,8-cineole synthase, CIN), which synthesizes the monoterpenes of the "cineole cassette". Interestingly, N. suaveolens is the only known taxon of the Suaveolentes section to have a flower emitting "cineole cassette of monoterpenes" which is otherwise typical for the Alatae section. Gene sequence analysis of CIN has revealed the highest similarities to other angiosperm monoterpene synthases from Vitis vinifera, Quercus ilex, Citrus unshiu and C. limon, which cluster in the same branch of the terpene synthase B subfamily. However, based on its synthesized products, N. suaveolens CIN shares similarity with enzymes of the Arabidopsis thaliana root and Salvia officinalis leaf. The N. suaveolens CIN gene is only expressed in the stigma/style tissue and petals. Thin sections of petals present the enzyme primarily in the adaxial and abaxial epidermis; this facilitates the comprehensive emission of volatiles in all spacial directions. The oscillation of monoterpene emission is a consequence of the regulation of the CIN gene by the circadian clock, with oscillations occurring at the level of transcript and protein accumulations and of enzyme activity. Light/dark or dark/light transition signals synchronize the slow-running endogenous clock. Two strategies for synchronized scent emission have been established in N. suaveolens flowers: (i) the synthesis of volatile organic compounds by a multi-product enzyme and (ii) the coordination of biosynthetic pathways by a circadian clock.

Artemisia annuaextracts, artemisinin and 1,8-cineole, prevent fruit infestation by a major, cosmopolitan pest of apples

“…However, on the one hand, cloning and expression of the artemisinin biosynthetic genes in microbes as Saccharomyces cerevisiae and Escherichia coli have already led to large-scale microbial production of some artemisinin precursors such as amorpha-4-,11-diene and artemisinic acid (Zeng et al, 2008). Some progress has been made toward deciphering the synthetic pathways for 1,8-cineole (Roeder et al, 2007). On the other hand, apple genome has been recently sequenced (Velasco et al, 2010) and reconstruction of the complete artemisinin or 1,8-cineole biosynthetic pathway in transgenic apple is only a matter of time.…”

Influence of Green Leaf Herbivory by Manduca sexta on Floral Volatile Emission by Nicotiana suaveolens

“…from flowers of N. suaveolens (Roeder et al, 2007). In vitro, this monoterpene synthase is responsible for the formation of 1,8-cineole, b-myrcene, sabinene, D-limonene, a-terpineol, a-pinene, and (E)-b-ocimene.…”

The α-Terpineol to 1,8-Cineole Cyclization Reaction of Tobacco Terpene Synthases

“…For mutation experiments, Nicotiana forgetiana CIN and Nicotiana suaveolens CIN (Roeder et al, 2007;Fähnrich et al, 2012) were cloned into the pET SUMO expression vector (Invitrogen). Nucleotide changes were generated using the Quick Change Site Directed Mutagenesis Kit (Agilent) according to the manufacturer's recommendation.…”