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.terpene synthases | amoebae | volatiles | evolution | chemical ecology

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono-and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg 2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

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Key Message Distinct catalytic features of the Poaceae TPS-a subfamily arose early in grass evolution and the reactions catalyzed have become more complex with time. Abstract The structural diversity of terpenes found in nature is mainly determined by terpene synthases (TPS). TPS enzymes accept ubiquitous prenyl diphosphates as substrates and convert them into the various terpene skeletons by catalyzing a carbocation-driven reaction. Based on their sequence similarity, terpene synthases from land plants can be divided into different subfamilies, TPS-a to TPS-h. In this study, we aimed to understand the evolution and functional diversification of the TPS-a subfamily in the Poaceae (the grass family), a plant family that contains important crops such as maize, wheat, rice, and sorghum. Sequence comparisons showed that aside from one clade shared with other monocot plants, the Poaceae TPS-a subfamily consists of five well-defined clades I-V, the common ancestor of which probably originated very early in the evolution of the grasses. A survey of the TPS literature and the characterization of representative TPS enzymes from clades I-III revealed clade-specific substrate and product specificities. The enzymes in both clade I and II function as sesquiterpene synthases with clade I enzymes catalyzing initial C10-C1 or C11-C1 ring closures and clade II enzymes catalyzing C6-C1 closures. The enzymes of clade III mainly act as monoterpene synthases, forming cyclic and acyclic monoterpenes. The reconstruction and characterization of clade ancestors demonstrated that the differences among clades I-III were already present in their ancestors. However, the ancestors generally catalyzed simpler reactions with less double-bond isomerization and fewer cyclization steps. Overall, our data indicate an early origin of key enzymatic features of TPS-a enzymes in the Poaceae, and the development of more complex reactions over the course of evolution.

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