Abstract
BackgroundThe biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils.ResultsLeaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories.ConclusionsThe protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.
Highlights
The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study
The application to plants of systems biology technologies, such as metabolomics and transcriptomics, has generally followed a non-targeted approach using bulk material composed of numerous tissue types [1]
Our protocol has the potential to be applied to help elucidate the pathways for essential oil biosynthesis as well as that of other natural products recently found in Eucalyptus secretory cavities [26]
Summary
The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. The specialised secretory structures in which essential oils (mono- and sequiterpenes) are synthesised and stored are ideal candidates for fine scale application of Recent advances in elucidating the pathways for essential oil biosynthesis have come with the application of transcriptomics to isolated glandular trichomes from species such as peppermint [11], sweet basil [12], tobacco [13,14], sweet wormwood [15] and hop [16]. Techniques used to harvest trichomes are mostly modifications of the method of glass bead abrasion originally developed for mint species by Gershenzon and co-authors [18], but non-abrasive techniques for physically removing trichomes have been developed [13,14,16]
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