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. 2016 Oct 25;113(43):12132-12137.
doi: 10.1073/pnas.1610379113. Epub 2016 Oct 7.

Terpene Synthase Genes in Eukaryotes Beyond Plants and Fungi: Occurrence in Social Amoebae

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Free PMC article

Terpene Synthase Genes in Eukaryotes Beyond Plants and Fungi: Occurrence in Social Amoebae

Xinlu Chen et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including β-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations.

Keywords: amoebae; chemical ecology; evolution; terpene synthases; volatiles.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Distribution of terpene synthase (TPS) genes among the major lineages of eukaryotes with sequenced genomes. A total of 168 species (Table S1), which did not include any species from land plants and fungi (Holomycota), were analyzed. The phylogeny of eukaryotes was adapted from Adl et al. (14) and Burki (15) with five supergroups recognized: Opisthokonta, Amoebozoa, Excavata, Archaeplastida, and SAR (stramenopiles + alveolates + Rhizaria). The first number (before the slash) indicates the number of species in certain lineages that were determined to contain TPS genes. The second number (after the slash) indicates the total number of species in that lineage that were analyzed. NA, not analyzed. The “+2” indicates that two additional species from Amoebozoa were identified to contain TPS genes in the nonredundant database at NCBI.
Fig. 2.
Fig. 2.
Phylogenetic reconstruction of newly identified eukaryotic TPSs with known TPSs. The set of known TPSs includes representative TPSs from fungi and bacteria. Also included were the microbial terpene synthase-like proteins identified from the plant Selaginella moellendorffii. The newly identified eukaryotic TPSs include a total of 50 putative full-length TPSs identified from six species (five species from Dictyosteliida and N. gruberi) (Table S2). TPSs are color-coded based on their source. The TPSs from Dictyosteliida form clade I and the TPSs from N. gruberi form clade II.
Fig. S1.
Fig. S1.
Life cycle of the social amoeba D. discoideum. D. discoideum amoebae propagate vegetatively as a unicellular organism when food (naturally bacteria) is abundant. Upon starvation, social amoebae transition into multicellular development. During development, individual cells aggregate and differentiate, forming a multicellular slug that migrates and then forms a fruiting body in a highly coordinated process. The cartoons at the bottom half of the figure depict various stages during multicellular development: streaming, loose aggregate, tipped aggregate, slug, Mexican hat, and fruiting bodies.
Fig. 3.
Fig. 3.
Expression patterns of nine terpene synthase genes in D. discoideum (DdTPS1-9). This analysis was based on published RNAseq data (24), which were obtained at seven time points during a complete developmental program in which individual D. discoideum cells aggregated and differentiated, forming a multicellular slug that migrated and then formed a fruiting body in a highly coordinated process that lasted approximately 24 h. The expression levels of nine DdTPS genes were measured by RPKM (reads per kilobase per million sequenced reads) and then displayed on a log2(RPKM+1) scale in this line plot. The line plot shows the transcript abundance (y axis; log-scale) of nine DdTPS genes. The cartoons depict various stages during multicellular development: vegetative, individual cells (0 h), streaming (8 h), loose aggregate (10 h), tipped aggregate (14 h), slug (16 h), Mexican hat (20 h), and fruiting bodies (24 h).
Fig. 4.
Fig. 4.
Sesquiterpene synthase activity of D. discoideum terpene synthases. Genes were heterologously expressed in E. coli, and crude protein extracts were incubated with the substrate FPP. Enzyme products were collected by using solid-phase microextraction and analyzed by GC/MS. GC traces (Left) and mass spectra of major products (Right) are shown. 1, (E,E)-α-farnesene*; 2, unidentified sesquiterpene hydrocarbon; 3, β-maaliene; 4, aristolene; 5, calarene; 6, unidentified sesquiterpene hydrocarbon; 7, unidentified sesquiterpene hydrocarbon; 8, unidentified sesquiterpene hydrocarbon; 9, (E)-nerolidol*; 10, β-elemene*; 11, (E)-β-farnesene*; 12, unidentified sesquiterpene hydrocarbon; 13, β-barbatene*; 14, unidentified sesquiterpene; cont, contamination. Compounds marked with asterisks (*) were identified by using authentic standards. Each assay was repeated at least three times, and a representative GC chromatogram is shown.
Fig. S2.
Fig. S2.
Monoterpene synthase activity of D. discoideum terpene synthases. Genes were heterologously expressed in E. coli, and crude protein extracts were incubated with the substrate GPP. Enzyme products were collected by using solid-phase microextraction and analyzed by GC/MS. 1, (Z)-β-ocimene; 2, (E)-β-ocimene; 3, allo-ocimene; 4, linalool; 5, β-myrcene; 6, limonene; 7, α-terpineol.
Fig. S3.
Fig. S3.
Diterpene synthase activity of D. discoideum terpene synthases. Genes were heterologously expressed in E. coli, and crude protein extracts were incubated with the substrate GGPP. Enzyme products were extracted with hexane and analyzed by using GC/MS. GC traces (Left) and mass spectra of major products (Right) are shown. I, unidentified diterpene; II, unidentified diterpene; III, unidentified diterpene; IV, unidentified oxygenated diterpene; V, unidentified diterpene.
Fig. 5.
Fig. 5.
D. discoideum emits diverse volatile terpenes during development. The beginning of multicellular development was set as 0 h. Volatiles were collected once every 4 h. Shown are GC/MS chromatograms from one representative replicate. All of the peaks labeled with a number are sesquiterpenes, and the number corresponds to the number of the peak in Fig. 4. 1, (E,E)-α-farnesene*; 2, unidentified sesquiterpene hydrocarbon; 5, calarene; 6, unidentified sesquiterpene hydrocarbon; 7, unidentified sesquiterpene hydrocarbon; 8, unidentified sesquiterpene hydrocarbon; 9, (E)-nerolidol*; 12, unidentified sesquiterpene hydrocarbon; 13, β-barbatene*. Compounds marked with asterisks (*) were identified by using authentic standard. “li” represents the monoterpene linalool, and “di” represents an unidentified diterpene. The letters indicate nonterpene volatiles. “a” represents 2-phenylethanol; “b”, “c”, and “d” represent unidentified compounds.
Fig. S4.
Fig. S4.
Relative abundance of individual volatile terpenes at each time point during the 24 h of multicellular development of D. discoideum. The emission rates were calculated based on the peak area of individual compound in GC chromatograms from three biological replicates with Fig. 5 as one of the replicates.

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