Trichome Density in Relation to Volatiles Emission and 1,8-Cineole Synthase Gene Expression in Thymus albicans Vegetative and Reproductive Organs.

1,8-Cineole is the main volatile produced by Thymus albicans Hoffmanns. & Link 1,8-cineole chemotype. To understand the contribution of distinct plant organs to the high 1,8-cineole production, trichome morphology and density, as well as emitted volatiles and transcriptional expression of the 1,8-cineole synthase (CIN) gene were determined separately for T. albicans leaves, bracts, calyx, corolla and inflorescences. Scanning electron microscopy (SEM) and stereoscope microscopy observations showed the highest peltate trichome density in leaves and bracts, significantly distinct from calyx and corolla. T. albicans volatiles were collected by solid phase micro extraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC/MS) and by GC for component identification and quantification, respectively. Of the 23 components identified, 1,8-cineole was the dominant volatile (57-93 %) in all T. albicans plant organs. The relative amounts of emitted volatiles clearly separated vegetative from reproductive organs. Gene expression of CIN was assigned to all organs analyzed and was consistent with the relatively high emission of 1,8-cineole in leaves and bracts. Further studies will be required to analyze monoterpenoid biosynthesis by each type of glandular trichome.

Molecular cloning and functional characterization of a monoterpene synthase isolated from the aromatic wild shrub Thymus albicans.

The essential oil of Thymus albicans Hoffmanns. & Link, a native shrub from the Iberian Peninsula, is mainly composed of monoterpenes. In this study, a 1,8-cineole synthase was isolated from the 1,8-cineole chemotype. A partial sequence that lacked the complete plastid transit peptide but contained an extended C-terminal when compared to other related terpene synthases was generated by PCR and Rapid Amplification of cDNA Ends (RACE). The predicted mature polypeptide was 593 amino acids in length and shared 78% and 77% sequence similarity with the homologue 1,8-cineole synthase from Rosmarinus officinalis and Salvia officinalis, respectively. The putative protein possessed the characteristic conserved motifs of plant monoterpene synthases including the RRx8W and DDxxD motifs and phylogenetic analysis indicated that the amplified 1,8-cineole synthase bears greater sequence similarity with other 1,8-cineole synthases from Lamiaceae family relative to the terpene synthases from the genus Thymus. Functional expression of the recombinant protein in Escherichia coli revealed that in the presence of geranyl diphosphate (GPP) 1,8-cineole was the major product but that its production was too low for robust quantification. Other minor conversion products included α-pinene, ß-pinene, sabinene and ß-myrcene suggesting the isolated 1,8-cineole synthase may be a multi-product enzyme. To our knowledge, this is the first report of a functionally characterized monoterpene synthase from Thymus albicans.