Abstract
The Australian sexually deceptive orchid, Chiloglottis trapeziformis, employs a unique UV-B-dependent floral volatile, chiloglottone 1, for specific male wasp pollinator attraction. Chiloglottone 1 and related variants (2,5-dialkylcyclohexane-1,3-diones), represent a unique class of specialized metabolites presumed to be the product of cyclization between two fatty acid (FA) precursors. However, the genes involved in the biosynthesis of precursors, intermediates, and transcriptional regulation remains to be discovered. Chiloglottone 1 production occurs in the aggregation of calli (callus) on the labellum under continuous UV-B light. Therefore, deep sequencing, transcriptome assembly, and differential expression (DE) analysis were performed across different tissue types and UV-B treatments. Transcripts expressed in the callus and labellum (∼23,000 transcripts) were highly specialized and enriched for a diversity of known and novel metabolic pathways. DE analysis between chiloglottone-emitting callus versus the remainder of the labellum showed strong coordinated induction of entire FA biosynthesis and β-oxidation pathways including genes encoding Ketoacyl-ACP Synthase, Acyl-CoA Oxidase, and Multifunctional Protein. Phylogenetic analysis revealed potential gene duplicates with tissue-specific differential regulation including two Acyl-ACP Thioesterase B and a Ketoacyl-ACP Synthase genes. UV-B treatment induced the activation of UVR8-mediated signaling and large-scale transcriptome changes in both tissues, however, neither FA biosynthesis/β-oxidation nor other lipid metabolic pathways showed clear indications of concerted DE. Gene co-expression network analysis identified three callus-specific modules enriched with various lipid metabolism categories. These networks also highlight promising candidates involved in the cyclization of chiloglottone 1 intermediates (e.g., Bet v I and dimeric α,β barrel proteins) and orchestrating regulation of precursor pathways (e.g., AP2/ERF) given a strong co-regulation with FA biosynthesis/β-oxidation genes. Possible alternative biosynthetic routes for precursors (e.g., aldehyde dehydrogenases) were also indicated. Our comprehensive study constitutes the first step toward understanding the biosynthetic pathways involved in chiloglottone 1 production in Chiloglottis trapeziformis – supporting the roles of FA metabolism in planta, gene duplication as a potential source of new genes, and co-regulation of novel pathway genes in a tissue-specific manner. This study also provides a new and valuable resource for future discovery and comparative studies in plant specialized metabolism of other orchids and non-model plants.
Highlights
The intriguing pollination strategy of sexual deception is known to be used by several hundred plants species representing more than 22 genera and spanning three plant families (Bohman et al, 2016)
RNA-seq was performed on C. trapeziformis to gain insights into the biochemical and regulatory pathways that are active in flowers (Figure 2A), and to search for clues into chiloglottone 1 biosynthesis
We show that coordinated induction of entire fatty acid (FA) biosynthesis and β-oxidation pathways occurs in chiloglottoneemitting callus
Summary
The intriguing pollination strategy of sexual deception is known to be used by several hundred plants species representing more than 22 genera and spanning three plant families (Bohman et al, 2016). Known examples are dominated by the orchids These orchids sexually lure specific male insect pollinators by ‘semiochemicals’ that mimic the sex pheromone, with pollination often achieved during attempted copulation by the pollinator with the flower (Schiestl et al, 1999; Ayasse et al, 2003; Schiestl, 2003; Franke et al, 2009; Bohman et al, 2014; Phillips et al, 2014). Within Australian sexually deceptive orchids, where several hundred species across multiple genera are pollinated by male thynnine wasps, at least four different floral volatile chemical systems are involved in pollinator attraction: (1) Specific blends of unique cyclohexanediones, called chiloglottones, which are widely employed across Chiloglottis orchids (Schiestl, 2003; Franke et al, 2009; Peakall et al, 2010). Within Australian sexually deceptive orchids, where several hundred species across multiple genera are pollinated by male thynnine wasps, at least four different floral volatile chemical systems are involved in pollinator attraction: (1) Specific blends of unique cyclohexanediones, called chiloglottones, which are widely employed across Chiloglottis orchids (Schiestl, 2003; Franke et al, 2009; Peakall et al, 2010). (2) A specific blend of alkyl- and hydroxymethyl-pyrazines in Drakaea glyptodon (Bohman et al, 2014). (3) Unique (methylthio)phenols in Caladenia crebra (Bohman et al, 2017), and (4) A combination of two biosynthetically unrelated compounds (2-hydroxy-6methylacetophenone and (S)-β-citronellol) in Caladenia plicata (Xu et al, 2017)
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