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Summary• The accumulation of terpenoid oil was examined in the leaves of Eucalyptus polybractea at scales ranging from individual oil glands to the whole plant.• Variations in oil composition and concentration of oil were measured and related to both morphological and physiological parameters.• Within a plant, all glands produced oil of broadly similar composition that was not regulated by leaf age or the position of the gland within the leaf. There were, however, distinct differences between plants, suggesting that composition is controlled primarily at the whole-plant level. Oil concentration, too, was regulated primarily at the whole-plant level and was limited by gland capacity. Gland capacity was linked to leaf area and thickness, the final products of leaf expansion.• Leaf and plant oil composition is determined not by a mosaic of glands specializing in producing a single or a small group of compounds, but rather by glands with remarkably similar capacities for terpenoid biosynthesis, although oil concentration, limited by gland capacity, may be linked to leaf expansion rather than biosynthetic capacity.

Terpene deployment was examined in a population of Eucalyptus polybractea (R.Baker) trees. Eucalyptus polybractea is a terpene-accumulating species, which stores terpenes in oil glands beneath the leaf surface. Using regression analysis, we showed that leaf thickness, measured as leaf mass per area (LMA), influenced terpene content, apparently through regulation of gland dimensions, and thus, gland volume. We also examined how environmental factors affected terpene content through regulation of both LMA, and therefore, storage capacity, and the supply of resources for terpene synthesis. Neither water stress, measured using carbon isotope ratios as an indicator, nor nutrient stress, measured as foliar nitrogen and phosphorus content, accounted for observed variation in either terpene content or LMA. Phenolic content, measured as a possible competing carbon sink, did not account for variation in terpene content, and variation in environmental stresses could not account for differences in growth rate. However, both terpenes and total carbon-based secondary metabolites (terpenes and phenolics) showed positive correlations with growth, suggesting plants gain a growth advantage by deploying greater amounts of secondary metabolites.

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.

Cerebral malaria is a major health problem in the developing world. Widespread resistance to existing drugs by the parasite Plasmodium falciparum has coincided with an increase in mortality, particularly in children. One potential source of new drugs comes from plant natural products. We found that commercially available, pharmaceutical grade eucalyptus oil and its principal component 1,8-cineole inhibited the growth and development of chloroquine-sensitive and chloroquine-resistant P. falciparum. This was true both when the oil was added directly to the parasite cultures and when cultures were exposed to the vapours. The development of the parasite was arrested at the early trophozoite stage, irrespective of when the oil was introduced. We used a new approach where the concentration of monoterpenes actually taken up by the cultures was measured directly using HS-GC. We found that the critical concentration required to inhibit and kill the parasite did not adversely affect the host erythrocytes, placing it in the range suitable for drug development. Given the ready availability and existing quality control of eucalyptus oils, this may represent an economically viable adjunct to current antimalarial therapies. Figure 1. Changes in P. falciparum after exposure to eucalyptus oil vapour (IC 90 ). Cultures were synchronized at the ring stage and observed over 96 h (two life cycles). (a) Control, without eucalyptus oil.(b) Culture exposed to eucalyptus oil for 96 h. (c) Culture exposed to eucalyptus oil for 48 h followed by 48 h recovery. Graphs represent the percentage of parasites at a certain life stage as a proportion of the total number of parasites, measured every 24 h Figure 2. Confocal microscopy images of live P. falciparum cells exposed to eucalyptus oil vapour (IC 90 ). Cells were synchronized at the ring stage and followed over 96 h (two life cycles). Cells were CS(l)YFP/ACP(l)DsRed double transfectants, co-labelled with the nuclear dye Hoechst 33 258. 16 Scale bar = 2 μm. (a) Control culture not exposed to eucalyptus oil, at 30 h: late trophozoite stage. (b) Culture exposed to eucalyptus oil at 30 h: stalled at the early trophozoite stage. (c) Control culture, now at 72 h: mid-schizont stage. (d) Exposed culture, now at 72 h: still stalled at the early trophozoite stage