Amanda Padovan

The Eucalyptus terpene synthase gene family

BackgroundTerpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts.ResultsThe genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus.ConclusionsOur data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

The evolution of foliar terpene diversity in Myrtaceae

Plant terpenes play many roles in natural systems, from altering plant–animal interactions, to altering the local abiotic environment. Additionally, many industries depend on terpenes. For example, commercially used essential oils, including tea tree oil and lavender oil, are a mixture of terpenes. Many species of the family Myrtaceae form a key resource for these industries due to the high concentration of terpenes found predominately in their leaves. The frequency of chemotypic differences within many species and populations can lead to costly errors in industry. Terpene diversity in Myrtaceae is driven by variation in the terpene synthase enzymes, which catalyse the conversion a few common substrates into thousands of terpene structures. We review terpene diversity within and between species of Myrtaceae and relate this to variation in the terpene synthase enzymes to reconstruct the evolution of foliar terpene diversity in Myrtaceae. We found that (1) high inter- and intra-species variation exists in terpene profile and that α-pinene the most likely ancestral foliar terpene, and (2) that high concentration of 1,8-cineole (a compound which is regarded as the signature compound of Myrtaceae) is limited to just four Myrtaceae sub-families. We suggest that the terpene synthase enzymes do not limit terpene diversity in this family and variation in these enzymes suggests a mode of enzymatic evolution that could lead to high 1,8-cineole production. Our analysis highlights the need to standardise methods for collecting and reporting foliar terpene data, and we discuss some methods and issues here. Although there are many gaps in the published data, our large scale analysis using the results of many studies, shows the value of a family wide analysis for understanding both the evolution and industrial potential of terpene-producing plants.

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

BackgroundLong-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.ResultsWe 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.ConclusionWe 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.

The molecular basis of host plant selection in Melaleuca quinquenervia by a successful biological control agent

Melaleuca quinquenervia (Cav) S.T. Blake (broadleaf paperbark) is an Australian tree that has become a serious weed in many places around the world. Two insects Oxyops vitiosa (the melaleuca weevil), and Boreioglycaspis melaleucae (the melaleuca psyllid), which were introduced to Florida as part of a biological control programme, have been very effective in reducing survival and reproduction of this weed. There are two terpene chemotypes of M. quinquenervia; one rich in the sesquiterpene E-nerolidol whereas the other is rich in viridiflorol. Viridiflorol is a strong feeding deterrent for the melaleuca weevil and retards larval development. The larvae therefore avoid the viridiflorol-rich chemotype, in contrast, female melaleuca psyllids prefer to oviposit on these leaves. To identify the molecular basis of these preferences, we isolated and characterised two terpene synthases from the viridiflorol-rich chemotype, both of which utilise farnesyl pyrophosphate and have the same product profile. Chemotypic variation in terpenes in M. quinquenervia is under strong genetic control and the reproductive potential of each chemotype is limited by a different insect. These insects could, therefore, be selective agents for the maintenance of chemotypic variation in M. quinquenervia.

Transcriptome Sequencing of Two Phenotypic Mosaic Eucalyptus Trees Reveals Large Scale Transcriptome Re-Modelling

Phenotypic mosaic trees offer an ideal system for studying differential gene expression. We have investigated two mosaic eucalypt trees from two closely related species (Eucalyptus melliodora and E. sideroxylon), which each support two types of leaves: one part of the canopy is resistant to insect herbivory and the remaining leaves are susceptible. Driving this ecological distinction are differences in plant secondary metabolites. We used these phenotypic mosaics to investigate genome wide patterns of foliar gene expression with the aim of identifying patterns of differential gene expression and the somatic mutation(s) that lead to this phenotypic mosaicism. We sequenced the mRNA pool from leaves of the resistant and susceptible ecotypes from both mosaic eucalypts using the Illumina HiSeq 2000 platform. We found large differences in pathway regulation and gene expression between the ecotypes of each mosaic. The expression of the genes in the MVA and MEP pathways is reflected by variation in leaf chemistry, however this is not the case for the terpene synthases. Apart from the terpene biosynthetic pathway, there are several other metabolic pathways that are differentially regulated between the two ecotypes, suggesting there is much more phenotypic diversity than has been described. Despite the close relationship between the two species, they show large differences in the global patterns of gene and pathway regulation.

Transcriptome analysis of terpene chemotypes of Melaleuca alternifolia across different tissues

Plant chemotypes or chemical polymorphisms are defined by discrete variation in secondary metabolites within a species. This variation can have consequences for ecological interactions or the human use of plants. Understanding the molecular basis of chemotypic variation can help to explain how variation of plant secondary metabolites is controlled. We explored the transcriptomes of the 3 cardinal terpene chemotypes of Melaleuca alternifolia in young leaves, mature leaves, and stem and compared transcript abundance to variation in the constitutive profile of terpenes. Leaves from chemotype 1 plants (dominated by terpinen-4-ol) show a similar pattern of gene expression when compared to chemotype 5 plants (dominated by 1,8-cineole). Only terpene synthases in young leaves were differentially expressed between these chemotypes, supporting the idea that terpenes are mainly synthetized in young tissue. Chemotype 2 plants (dominated by terpinolene) show a greater degree of differential gene expression compared to the other chemotypes, which might be related to the isolation of plant populations that exhibit this chemotype and the possibility that the terpinolene synthase gene in M. alternifolia was derived by introgression from a closely related species, Melaleuca trichostachya. By using multivariate analyses, we were able to associate terpenes with candidate terpene synthases.

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

BackgroundTerpene 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.ResultsWe 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.ConclusionsDespite 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.

Antarctica’s ecological isolation will be broken by storm-driven dispersal and warming

Antarctica has long been considered biologically isolated1. Global warming will make parts of Antarctica more habitable for invasive taxa, yet presumed barriers to dispersal—especially the Southern Ocean’s strong, circumpolar winds, ocean currents and fronts—have been thought to protect the region from non-anthropogenic colonizations from the north1,2. We combine molecular and oceanographic tools to directly test for biological dispersal across the Southern Ocean. Genomic analyses reveal that rafting keystone kelps recently travelled >20,000 km and crossed several ocean-front ‘barriers’ to reach Antarctica from mid-latitude source populations. High-resolution ocean circulation models, incorporating both mesoscale eddies and wave-driven Stokes drift, indicate that such Antarctic incursions are remarkably frequent and rapid. Our results demonstrate that storm-forced surface waves and ocean eddies can dramatically enhance oceanographic connectivity for drift particles in surface layers, and show that Antarctica is not biologically isolated. We infer that Antarctica’s long-standing ecological differences have been the result of environmental extremes that have precluded the establishment of temperate-adapted taxa, but that such taxa nonetheless frequently disperse to the region. Global warming thus has the potential to allow the establishment of diverse new species—including keystone kelps that would drastically alter ecosystem dynamics—even without anthropogenic introductions.Genomic tools and ocean circulation models show that organisms surface-drift across the Southern Ocean frequently. The extreme cold therefore keeps Antarctica biologically isolated, but as the climate warms new species may establish quickly.

Association genetics of essential oil traits in Eucalyptus loxophleba: explaining variation in oil yield

York gum (Eucalyptus loxophleba Benth) is widely planted in semi-arid regions of Australia for the production of Eucalyptus oil, a mixture of terpenes dominated by the monoterpene 1,8-cineole. Increasing oil yield in this species would improve the profitability of this crop and enhance its use in sustainable land management systems in Australia. To this end, we sequenced ten structural genes in the terpene biosynthetic pathway of ~400 individuals of E. loxophleba. Of the 4353 allelic variants identified, 1347 had a minor allele frequency >0.01. These were associated with three key traits of essential oil yield (concentration of 1,8-cineole, α-pinene and total terpenes). Three variants associated with α-pinene, two with 1,8-cineole and eight with total terpenes (13 total). The variants were mostly located in introns of the final three biosynthetic steps of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway (mcs, hds and hdr). Effect size varied from 2.7 to 6.8%, comparable to similar studies in forest trees. The cumulative effect size of the unlinked variants was 34.8% for total terpenes, although this is likely to be a high estimate. These results provide the basis for the development of molecular breeding methods for improving essential oil yield in this industrially important species.

Accuracy of Genomic Prediction for Foliar Terpene Traits in Eucalyptus polybractea

Unlike agricultural crops, most forest species have not had millennia of improvement through phenotypic selection, but can contribute energy and material resources and possibly help alleviate climate change. Yield gains similar to those achieved in agricultural crops over millennia could be made in forestry species with the use of genomic methods in a much shorter time frame. Here we compare various methods of genomic prediction for eight traits related to foliar terpene yield in Eucalyptus polybractea, a tree grown predominantly for the production of Eucalyptus oil. The genomic markers used in this study are derived from shallow whole genome sequencing of a population of 480 trees. We compare the traditional pedigree-based additive best linear unbiased predictors (ABLUP), genomic BLUP (GBLUP), BayesB genomic prediction model, and a form of GBLUP based on weighting markers according to their influence on traits (BLUP|GA). Predictive ability is assessed under varying marker densities of 10,000, 100,000 and 500,000 SNPs. Our results show that BayesB and BLUP|GA perform best across the eight traits. Predictive ability was higher for individual terpene traits, such as foliar α-pinene and 1,8-cineole concentration (0.59 and 0.73, respectively), than aggregate traits such as total foliar oil concentration (0.38). This is likely a function of the trait architecture and markers used. BLUP|GA was the best model for the two biomass related traits, height and 1 year change in height (0.25 and 0.19, respectively). Predictive ability increased with marker density for most traits, but with diminishing returns. The results of this study are a solid foundation for yield improvement of essential oil producing eucalypts. New markets such as biopolymers and terpene-derived biofuels could benefit from rapid yield increases in undomesticated oil-producing species.