A molecular perspective on terpene variation in Australian Myrtaceae

The terpenoid-dominated essential oils in Australian Myrtaceae mediate many ecological interactions and are important industrially. Of all the significant essential oil-producing families, Myrtaceae is the only one for which there is no molecular information on terpene biosynthesis. Here we summarise available knowledge on terpene biosynthesis and its relevance to the Myrtaceae to provide a foundation for ecological and genetic studies of chemical diversity. There are several steps in the terpene biosynthesis pathway that have potential for influencing the oil yield, profile and composition of leaf oils in Myrtaceae. The biochemical steps that influence oil yield in Myrtaceae probably occur in the steps of the pathway leading up to the synthesis of the terpene backbone. Qualitative differences in oil profiles are more likely to be due to variation in terpene synthases and terpene-modifying enzymes. Most of the information on molecular variation in terpene biosynthesis is based on the analysis of artificially derived mutants but Australian Myrtaceae can provide examples of the same mechanisms in an ecological context

Differences in gene expression within a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory

BACKGROUND Long-lived trees can accumulate mutations throughout their lifetimes that may influence biotic and abiotic interactions. For example, some Eucalyptus trees display marked variation in herbivore defence within a single canopy. These "mosaic" trees support foliage with distinct chemotypes which are differentially favoured by insect and vertebrate herbivores, resulting in susceptible and resistant branches within a single canopy. These mosaic trees provide a unique opportunity to explore the biosynthesis and genetic regulation of chemical defences in the foliage. The biosynthesis of the principal defence compounds, terpenoid-dominated essential oils, is well understood. However, the regulation of the genes involved and thus the control of phenotypic variation within a single tree canopy remains a mystery. RESULTS We sequenced the transcriptomes of the leaves of the two different chemotypes of a chemically mosaic Eucalyptus melliodora tree using 454 pyrosequencing technology. We used gene set enrichment analysis to identify differentially expressed transcripts and found the proportion of differentially expressed genes in the resistant and susceptible foliage similar to the transcript difference between functionally distinct tissues of the same organism, for example roots and leaves. We also investigated sequence differences in the form of single nucleotide polymorphisms and found 10 nucleotides that were different between the two branches. These are likely true SNPs and several occur in regulatory genes. CONCLUSION We found three lines of evidence that suggest changes to a 'master switch' can result in large scale phenotypic changes: 1. We found differential expression of terpene biosynthetic genes between the two chemotypes that could contribute to chemical variation within this plant. 2. We identified many genes that are differentially expressed between the two chemotypes, including some unique genes in each branch. These genes are involved in a variety of processes within the plant and many could contribute to the regulation of secondary metabolism, thus contributing to the chemical variation. 3. We identified 10 SNPs, some of which occur in regulatory genes that could influence secondary metabolism and thus contribute to chemical variation. Whilst this research is inherently limited by sample size, the patterns we describe could be indicative of other plant genetic mosaics.This work was supported by a Discovery grant from the Australian Research Council to WJF (DP0877063)

Four terpene synthases contribute to the generation of chemotypes in tea tree (Melaleuca alternifolia)

BACKGROUND: Terpene rich leaves are a characteristic of Myrtaceae. There is significant qualitative variation in the terpene profile of plants within a single species, which is observable as "chemotypes". Understanding the molecular basis of chemotypic variation will help explain how such variation is maintained in natural populations as well as allowing focussed breeding for those terpenes sought by industry. The leaves of the medicinal tea tree, Melaleuca alternifolia, are used to produce terpinen-4-ol rich tea tree oil, but there are six naturally occurring chemotypes; three cardinal chemotypes (dominated by terpinen-4-ol, terpinolene and 1,8-cineole, respectively) and three intermediates. It has been predicted that three distinct terpene synthases could be responsible for the maintenance of chemotypic variation in this species. RESULTS: We isolated and characterised the most abundant terpene synthases (TPSs) from the three cardinal chemotypes of M. alternifolia. Functional characterisation of these enzymes shows that they produce the dominant compounds in the foliar terpene profile of all six chemotypes. Using RNA-Seq, we investigated the expression of these and 24 additional putative terpene synthases in young leaves of all six chemotypes of M. alternifolia. CONCLUSIONS: Despite contributing to the variation patterns observed, variation in gene expression of the three TPS genes is not enough to explain all variation for the maintenance of chemotypes. Other candidate terpene synthases as well as other levels of regulation must also be involved. The results of this study provide novel insights into the complexity of terpene biosynthesis in natural populations of a non-model organism.The research was funded by the Australian Research Council (ARC) Discovery Program (DP14101755), the Australian Government Rural Industries Research and Development Corporation (RIRDC) and the Australia Tea Tree Industry Association (ATTIA). Grants from the Go8-DAAD Research Scheme and a Humboldt Research Award from the Alexander von Humboldt Foundation to WJF underpinned this collaborative research. Each of the funding bodies granted the funds based on a research proposal. They had no influence over the experimental design, data analysis or interpretation, or writing the manuscript

Tanulástámogató szolgáltatások a Savaria Egyetemi Központ Könyvtárában

Missing values, n (%) in variables by country; n (%). (DOCX 15 kb

Intensive sampling identifies previously unknown chemotypes, population divergence and biosynthetic connections among terpenoids in Eucalyptus tricarpa

Australian members of the Myrtaceae produce large quantities of ecologically and economically important terpenes and display abundant diversity in both yield and composition of their oils. In a survey of the concentrations of leaf terpenes inEucalyptus t

A Biochemical Interpretation of Terpene Chemotypes in Melaleuca alternifolia

The variation of foliar monoterpenes in the Australian Tea Tree (Melaleuca alternifolia) has been of significant interest both to the essential oil industry as well as to ecologists. The majority of studies on leaf chemistry have been aimed directly towards obtaining oil of higher quality or quantity. In the current study, we aimed to understand how molecular mechanisms contribute to the chemical variability of this species, based on chemical analysis of the leaf oils from a biochemical perspective. Correlations between monoterpenes across the species as well as within chemotypes show strong, persistent patterns, which enable us to establish groups based on possible common biosynthetic origins. We found that three distinct enzymes corresponding to these groups: a sabinene-hydrate synthase, a 1,8-cineole synthase, and a terpinolene synthase may be sufficient to explain all six chemotypes in M. alternifolia

Intensive sampling identifies previously unknown chemotypes, population divergence and biosynthetic connections among terpenoids in 'Eucalyptus tricarpa'

Australian members of the Myrtaceae produce large quantities of ecologically and economically important terpenes and display abundant diversity in both yield and composition of their oils. In a survey of the concentrations of leaf terpenes in 'Eucalyptus tricarpa' (L.A.S. Johnson) L.A.S. Johnson & K.D. Hill, which were previously known from few samples, exceptional variability was found in composition. The aim was to characterize the patterns of variation and covariation among terpene components in this species and to use this information to enhance our understanding of their biosynthesis. There were marked discontinuities in the distributions of numerous compounds, including the overall proportions of mono- and sesquiterpenes, leading us to delineate three distinct chemotypes. Overall, positive covariation predominated, but negative covariation suggested competitive interactions involved in monoterpene synthesis. Two groups of covarying monoterpenes were found, each of which was positively correlated with a group of sesquiterpenes and negatively correlated with the alternate sesquiterpene group. These results imply substantial cross-talk between mono- and sesquiterpene biosynthesis pathways. However, only those compounds hypothesized to share final carbocation intermediates or post-processing steps were strongly positively correlated within chemotypes. This suggests that the broader patterns of covariation among groups of compounds may result from co-regulation of multiple biosynthetic genes, controlling the complex terpene profiles of the chemotypes of 'Eucalyptus'

Mosaic Eucalypt Trees Suggest Genetic Control at a Point That Influences Several Metabolic Pathways

Mosaic trees contain more than one phenotype. The two Eucalyptus mosaic trees studied here (E. melliodora and E. sideroxylon) are predominantly susceptible to insect herbivory, with the leaves on a single large branch on each tree resisting herbivory. W