The status of genomics and genetics research in the Myrtaceae, a large family of dicotyledonous woody plants, is reviewed with Eucalyptus as the focal genus. The family contains over 5,650 species in 130 to 150 genera, predominantly of neo-tropical and Southern Hemisphere distribution. Several genera are well known for their economic importance worldwide. Myrtaceae are typically diploids with small to intermediate genome size. Microsatellites have been developed for several genera while higher throughput marker systems such as diversity arrays technology and single nucleotide polymorphism are available for Eucalyptus. Molecular data have been fundamental to current perspectives on the phylogeny, phylogeography and taxonomy of the Myrtaceae, while numerous studies of genetic diversity have been carried out particularly as it relates to endangered, rare, fragmented, overharvested or economically important species. Large expressed sequence tag collections for species of Eucalyptus have recently become public to support the annotation of the Eucalyptus grandis genome. Transcriptomics in Eucalyptus has advanced by microarrays and next-generation sequencing focusing on wood development. Linkage maps for Eucalyptus display high synteny across species and have been extensively used to map quantitative trait loci for a number of traits including growth, wood quality, disease and insect resistance. Candidate gene-based association genetics have successfully found marker–trait associations for wood and fiber traits. Genomic selection experiments have demonstrated clear potential to improve the efficiency of breeding programs while freeze-tolerant transgenic Eucalyptus trials have recently been initiated. The recently released E. grandis genome, sequenced to an average coverage of 8×, will open up exceptional opportunities to advance Myrtaceae genetics and genomics research.

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Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

Leaf-cutting ants of the Attini tribe are a major pest of agricultural and forestry productions in the New World. Economic losses caused by these ants were estimated at several million dollars per year. These ants need to live in symbiosis with a basidiomycete fungus. Due to their mutualistic interaction with the symbiotic fungus, management of Attini ants can be done with insecticides or fungicides or both. So far, synthetic pesticides were the main control means, albeit with negative effects on the environment. Very few studies describe alternative methods for the control of leaf-cutting ants such as the use of insecticidal and fungicidal plant extracts. There is therefore a need of knowledge on phytochemicals and plants that could be used as insecticides and fungicides. Here, we review chemicals of plant origin and species with insecticidal and fungicidal activities. We establish a list of plants and phytochemicals that could manage leaf-cutting ants and also other insects, notably insects that use fungus-based agriculture. An exhaustive literature search of 1965 references from 1923 to 2010 was conducted using scientific databases, chemical databases, botanical databases, and books to identify published papers related to insecticidal and fungicidal chemical compounds stemmed from plant species. The major points are the following: (1) 119 and 284 chemicals have been cited in the literature for their insecticidal and fungicidal activities, respectively; (2) 656 and 1,064 plant species have significant insecticidal and fungicidal activities, respectively; (3) 3 main chemical classes were most cited for these activities: alkaloids, phenolics, and terpenoids; (4) 20 interesting chemicals with the both insecticidal and fungicidal activities were found; and (5) 305 plant species containing these chemicals were cited. To conclude, 20 chemicals: caryophyllene oxide, cinnamaldehyde, eugenol, helenalin, linalool, menthone, myristicin, pulegone, thymol, anethole, anisaldehyde, elemicin, isopimpinellin, plumbagin, podophyllotoxin, psoralen, xanthotoxin, anonaine, solamargine, and tomatine; two main plant families, Lamiaceae and Apiaceae; and 17 species of these families were particularly interesting for the management of leaf-cutting ants.

Insecticidal and antifungal chemicals produced by plants: a review

Plant secondary compounds (PSCs), also called secondary metabolites, have high chemical and structural diversity and appear as non-volatile or volatile compounds These compounds may have evolved to have specific physiological and ecological functions in the adaptation of plants to their growth environment PSCs are produced by several metabolic pathways and many PSCs are specific for a few plant genera or families In forest ecosystems, full-grown trees constitute the majority of plant biomass and are thus capable of producing significant amounts of PSCs We summarize older literature and review recent progress in understanding the effects of abiotic and biotic factors on PSC production of forest trees and PSC behavior in forest ecosystems The roles of different PSCs under stress and their important role in protecting plants against abiotic and biotic factors are also discussed There was strong evidence that major climate change factors, CO2 and warming, have contradictory effects on the main PSC groups CO2 increases phenolic compounds in foliage, but limits terpenoids in foliage and emissions Warming decreases phenolic compounds in foliage but increases terpenoids in foliage and emissions Other abiotic stresses have more variable effects PSCs may help trees to adapt to a changing climate and to pressure from current and invasive pests and pathogens Indirect adaptation comes via the effects of PSCs on soil chemistry and nutrient cycling, the formation of cloud condensation nuclei from tree volatiles and by CO2 sequestration into PSCs in the wood of living and dead forest trees

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Climate Change Effects on Secondary Compounds of Forest Trees in the Northern Hemisphere.

Australia has a mostly dry, open, fire-shaped landscape of sclerophyllous and xeromorphic flora dominated by eucalypt and acacia trees, with diverse shrubs from a few families such as Myrtaceae, Proteaceae, and Fabaceae. Using molecular phylogenies to test hypotheses derived from the fossil record, we review the principal forces that transformed the ancestral Gondwanan rainforest through the Cenozoic. Today's vegetation is a mix of ancient radiations that have persisted in Australia through dramatic climate change since before the breakup of Gondwana, and more recent lineages whose ancestors arrived by trans-oceanic dispersal. Signatures in the fossil record of lineage turnover and trait evolutionary change are detected in phylogenies, but often at earlier dates. The Australian biota is a sample of the wider region, with extinction of some taxa and radiation of others (due to chance and opportunity), but biotic and abiotic interactions have resulted in a unique flora and fauna.

How Was the Australian Flora Assembled Over the Last 65 Million Years? A Molecular Phylogenetic Perspective

Terpenoids 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. The 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. Our 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.

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The Eucalyptus terpene synthase gene family

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.

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A molecular perspective on terpene variation in Australian Myrtaceae

http://biology-assets.anu.edu.au/hosted_sites/BillsLab/papers/Keszei%20et%20al%202010%20(fragments).pdf

Functional and evolutionary relationships between terpene synthases from Australian 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.

The evolution of foliar terpene diversity in Myrtaceae

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.

A biochemical interpretation of terpene chemotypes in Melaleuca alternifolia.

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. 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. 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.

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Andras Keszei

Papers

A molecular perspective on terpene variation in Australian Myrtaceae

Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

The evolution of foliar terpene diversity in Myrtaceae

A biochemical interpretation of terpene chemotypes in Melaleuca alternifolia.

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