Phytostabilisation of arsenical gold mine tailings using four Eucalyptus species: growth, arsenic uptake and availability after five years.

Arsenic (As) contamination is a worldwide problem. Where arsenic is highly concentrated and confined within a limited area, such as in many mine tailings facilities, phytostabilisation is an attractive technology for long-term remediation. Important characteristics of a plant to be useful for phytostabilisation include As tolerance and low levels of As accumulation, as well as the ability to limit As availability. Performance needs to be monitored over the long term to ensure an ongoing vegetation community, though this is rarely done. In this study, the suitability of four Eucalyptus species (E. cladocalyx, E. melliodora, E. polybractea, E. viridis) for the phytostabilisation of arsenical, sulphidic gold mine tailings was assessed after five years. All four species accumulated low As concentrations, the highest being recorded in mature leaves, ranging from 0.29 to 5.14 microg g(-1) As. E. polybractea had significantly higher foliar As than the other three species but there was also great variation within the species. Between 5-10 times lower concentrations were recorded in stem samples and no As was detected in young leaf tips. There was also significant variation in the growth of trees upon the site. Eucalyptus cladocalyx grew significantly taller than other species although greater variation was detected within the species than between. The variation in tree heights was not correlated with As concentrations in either stems or leaves. Arsenic availability was determined to depths of 2.2 m and found to be low when compared to total As in the tailings. Importantly, no effect of trees on As availability or soil pH was detected. We conclude that E. cladocalyx, in particular is an ideal candidate for the long-term phytostabilisation of As-contaminated land and mine tailings. The variation detected in both As accumulation and growth is also promising for the selection of desirable traits.

Isolation of intact sub-dermal secretory cavities from Eucalyptus.

BACKGROUND: 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. RESULTS: Leaves 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. CONCLUSIONS: The 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.

Research note: Micropropagation of Eucalyptus polybractea selected for key essential oil traits.

A protocol for the micropropagation of Eucalyptus polybractea R.T. Baker (blue mallee) using axillary bud proliferation from lignotuber-derived explants is described. Three different ages of plants were used as explant sources: glasshouse-grown seedlings, field-grown saplings, and coppice of field-grown mature lignotubers. Explants from each source initiated successfully and no significant difference was observed for shoot proliferation, rooting success or hardening success between explant sources. Leaf oil quantity and quality for hardened clones transplanted to a field plantation were assessed after 3 months of growth. Ramets of all clones contained high quality oil with over 80% 1,8-cineole. For seedling-derived clones, foliar oil concentrations of ramets were higher than those of the ortets from which they were derived. For sapling and mature lignotuber derived clones the opposite was the case. This suggests that ontogenetic and physiological constraints may be influencing yield in the young ramets. The age of the explant source did not appear to influence the success of micropropagation, and as a result older plants (for which key oil traits are known) can be selected as elite plants for multiplying selected genotypes via micropropagation.

The accumulation of terpenoid oils does not incur a growth cost in Eucalyptus polybractea seedlings.

The deployment of secondary metabolites, such as terpenes, as anti-herbivore defences is thought to be costly for plants in terms of primary metabolism. Moreover, it is assumed that the cost of this deployment is modified by resource availability. In this study we examined the impact of terpenoid oil accumulation on the growth of Eucalyptus polybractea R.T.Baker seedlings from four maternal half-sib families, under conditions of sufficient and limiting nitrogen. The foliar oil concentration measured was extremely variable, varying almost 20-fold to a maximum of 13% (w / DW). Oil concentration was higher in plants grown under high nitrogen than in low-nitrogen plants, and it was positively correlated with foliar nitrogen concentration. Oil concentration was related to maternal concentration, although this relationship was weak because of the variation encountered. The composition of oil, dominated by monoterpenes, was also extremely variable, although this variation could not be adequately explained by either nitrogen availability or the seedling parentage. Importantly, we detected no negative correlations between oil concentration and relative growth rate (RGR), net assimilation rate (NAR), or leaf nitrogen productivity (LNP). Rather, under nitrogen limiting conditions, positive correlations were detected between oil concentration and all three indices. We conclude that oil accumulation is associated with factors that promote growth and if there is a cost to oil deployment, it could not be detected using the experimental design employed here.

Terpene deployment in Eucalyptus polybractea; relationships with leaf structure, environmental stresses, and growth.

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.