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

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s12870-017-1107-2) contains supplementary material, which is available to authorized users.

Transcriptome analysis of terpene chemotypes of Melaleuca alternifolia across different tissues

“…Using a targeted transcript assembly for genes involved in terpene biosynthesis, we were able to associate terpene synthases with individual terpenes. Comparing our results to three functionally characterized TPS genes (Padovan et al, ), we find close associations between the characterized cineole synthase and 1,8‐cineole, terpinolene synthase and terpinolene and to some degree between sabinene hydrate synthase and cis‐ and trans‐ sabinene hydrate, sabinene and terpinen‐4‐ol, especially in young leaves (Figure ).…”

“…The sPLS analysis in young leaves showed a strong correlation between the main monoterpene groups dominated by 1,8‐cineole, terpinolene, and cis ‐sabinene hydrate and the genes cineole synthase ( MaCin1 ), terpinolene synthase ( MaTln ), and sabinene hydrate synthase ( MaSaH ), respectively (Figure ). These genes were previously functionally characterized by Padovan and coworkers (Padovan et al, ). MaTPS9 , MaTPS19 , and MaTPS21 also showed a strong correlation with terpenes related to and including 1,8‐cineole, while MaTPS20 correlated with terpinolene and related compounds.…”

“…Targeted or whole genome sequencing of chemotypes 1 and 5 could determine whether the observed chemotypic differences are based on allelic variation between the sabinene hydrate synthase and the cineole synthase . These TPS genes differ by just 68 nucleotides (out of 1,806) and 36 amino acids (out of 563), three of which are within the catalytic pocket of the enzyme (Padovan et al, ), which has been shown to be sufficient to change the product profile of TPS enzymes (Keeling, Weisshaar, Lin, & Bohlmann, ). This is within the observed nucleotide diversity for exons of genes related to plant secondary metabolism in Myrtaceae; 2.6 to 4.7 allelic variants per 100 bp , based on four Eucalyptus species (Külheim, Yeoh, Maintz, Foley, & Moran, ).…”

“…Young leaves showed the highest correlation between terpenes and transcript counts for genes that have already been functionally characterized (Padovan et al, ). Gene expression of sabinene hydrate synthase , terpinolene synthase , and cineole synthase correlated strongly with their major products.…”

“…We used two terpene synthases, sabinene hydrate synthase and cineole synthase which had been isolated from M . alternifolia (Padovan et al, ) as controls. These genes have pairwise nucleotide identities of 96.6% and at the threshold of 0.94, two separate contigs were maintained, one for each gene, while at 0.92, they were merged into a single contig.…”