Xinlu Chen

Terpene synthase genes in eukaryotes beyond plants and fungi: Occurrence in social amoebae

Significance Many living organisms use terpenes for ecological interactions. Terpenes are biosynthesized by terpene synthases (TPSs), but classic TPS genes are known to exist only in plants and fungi among the eukaryotes. In this study, TPS genes were identified in six species of amoebae with five of them being multicellular social amoebae. Amoebal TPSs showed closer relatedness to fungal TPSs than bacterial TPSs. In the social amoeba Dictyostelium discoideum, all nine TPS genes encoded active enzymes and most of their terpene products were released as volatiles in a development-specific manner. This study highlights a wider distribution of TPS genes in eukaryotes than previously thought and opens a door to studying the function and evolution of TPS genes and their products. 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.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

Marchantia polymorpha, like all liverworts, accumulates a large array of terpenes, and this process depends on a unique family of terpene synthases. 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 Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

Microbial-type terpene synthase genes occur widely in nonseed land plants, but not in seed plants.

Significance Terpenoids are ubiquitous products made by land plants with diverse biological functions. Their formation in seed plants is catalyzed by typical plant terpene synthases (TPSs), a well-characterized group of enzymes. In contrast, our knowledge of terpenoid biosynthesis in nonseed plants is very limited. By systematically analyzing the transcriptomes and/or genomes of more than 1000 plant species, we report that microbial terpene synthase-like genes, which are only distantly related to typical plant TPS genes, are widely distributed in nonseed plants, but virtually absent in seed plants. The study provides insights into the evolution of TPS genes in early land plants and opens the door to investigating the diversity and functions of terpenoids in nonseed plants. The vast abundance of terpene natural products in nature is due to enzymes known as terpene synthases (TPSs) that convert acyclic prenyl diphosphate precursors into a multitude of cyclic and acyclic carbon skeletons. Yet the evolution of TPSs is not well understood at higher levels of classification. Microbial TPSs from bacteria and fungi are only distantly related to typical plant TPSs, whereas genes similar to microbial TPS genes have been recently identified in the lycophyte Selaginella moellendorffii. The goal of this study was to investigate the distribution, evolution, and biochemical functions of microbial terpene synthase-like (MTPSL) genes in other plants. By analyzing the transcriptomes of 1,103 plant species ranging from green algae to flowering plants, putative MTPSL genes were identified predominantly from nonseed plants, including liverworts, mosses, hornworts, lycophytes, and monilophytes. Directed searching for MTPSL genes in the sequenced genomes of a wide range of seed plants confirmed their general absence in this group. Among themselves, MTPSL proteins from nonseed plants form four major groups, with two of these more closely related to bacterial TPSs and the other two to fungal TPSs. Two of the four groups contain a canonical aspartate-rich “DDxxD” motif. The third group has a “DDxxxD” motif, and the fourth group has only the first two “DD” conserved in this motif. Upon heterologous expression, representative members from each of the four groups displayed diverse catalytic functions as monoterpene and sesquiterpene synthases, suggesting these are important for terpene formation in nonseed plants.

Studying Plant Secondary Metabolism in the Age of Genomics

Collectively plants produce an enormous diversity of secondary metabolites. In the genomics age, the study of plant secondary metabolite biosynthesis has been transformed by various genomic tools. The field of metabolomics is continuingly adding novelty and complexity to our information on the chemistry of plant secondary metabolism. The availability of whole-genome sequences for an ever-increasing list of plants enables our examination of the genomic basis of secondary metabolite production. By integrating large-scale sequencing/bioinformatics, metabolomics, transcriptomics, proteomics and in vitro biochemistry, functional genomics holds the promise of expediting functional characterization of genes of plant secondary metabolism. Overall, the increasing volume of biochemical knowledge about plant metabolism, together with the genetic and molecular tools generated in recent years, paves the way for rationally designed, more effective genetic engineering of plant secondary metabolism for enhanced plant defense, improved quality and production of valuable chemicals, and many other applications.

Mechanisms of the Diterpene Cyclases β‐Pinacene Synthase from Dictyostelium discoideum and Hydropyrene Synthase from Streptomyces clavuligerus

Two diterpene cyclases, one from the social amoeba Dictyostelium discoideum and the other from the bacterium Streptomyces clavuligerus, with products containing a Z-configured double bond between the original C2 and C3 of geranylgeranyl diphosphate, were extensively investigated for their mechanisms through isotopic labelling experiments. The participation of geranyllinalyl diphosphate, in analogy to the role of linalyl and nerolidyl diphosphate for mono- and sesquiterpene biosynthesis, as an intermediate towards diterpenes with a Z-configured C2=C3 double bond is discussed.

An (E,E)‐α‐farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect‐induced volatiles

Summary Plant terpene synthase genes (TPSs) have roles in diverse biological processes. Here, we report the functional characterization of one member of the soybean TPS gene family, which was designated GmAFS. Recombinant GmAFS produced in Escherichia coli catalysed the formation of a sesquiterpene (E,E)‐α‐farnesene. GmAFS is closely related to (E,E)‐α‐farnesene synthase gene from apple, both phylogenetically and structurally. GmAFS was further investigated for its biological role in defence against nematodes and insects. Soybean cyst nematode (SCN) is the most important pathogen of soybean. The expression of GmAFS in a SCN‐resistant soybean was significantly induced by SCN infection compared with the control, whereas its expression in a SCN‐susceptible soybean was not changed by SCN infection. Transgenic hairy roots overexpressing GmAFS under the control of the CaMV 35S promoter were generated in an SCN‐susceptible soybean line. The transgenic lines showed significantly higher resistance to SCN, which indicates that GmAFS contributes to the resistance of soybean to SCN. In soybean leaves, the expression of GmAFS was found to be induced by Tetranychus urticae (two‐spotted spider mites). Exogenous application of methyl jasmonate to soybean plants also induced the expression of GmAFS in leaves. Using headspace collection combined with gas chromatography–mass spectrometry analysis, soybean plants that were infested with T. urticae were shown to emit a mixture of volatiles with (E,E)‐α‐farnesene as one of the most abundant constituents. In summary, this study showed that GmAFS has defence roles in both below‐ground and above‐ground organs of soybean against nematodes and insects, respectively.

Terpenoid Secondary Metabolites in Bryophytes: Chemical Diversity, Biosynthesis and Biological Functions

Abstract Bryophytes are close extant relatives of the ancestral land plant. As such, they have retained many innovations that had enabled the adaptation of early land plants to the terrestrial environment. One of such important innovations is the elaboration of an enormously diverse array of secondary metabolites. This article reviews current knowledge of terpenoid secondary metabolites, which constitute the largest family of plant metabolites, in bryophytes from three perspectives: chemical diversity, biosynthesis, and biological functions. The diversity of terpenoids in bryophytes, particularly in liverworts, is enormously rich. More than 1600 terpenoids have been reported from this plant group. While many terpenoids are observed in both bryophytes and seed plants, some are unique to bryophytes. It is just the beginning for us to understand the molecular and biochemical basis underlying terpenoid biosynthesis in bryophytes. Compared to seed plants, which have only one type of terpene synthase genes, so-called typical plant terpene synthase genes, bryophytes employ not only typical plant terpene synthase genes but also another class of terpene synthase genes called microbial-terpene synthase-like (MTPSL) genes for terpenoid biosynthesis. Biochemical studies suggest the MTPSLs are largely responsible for the terpenoid diversity in bryophytes, particularly sesquiterpenes and monoterpenes. Existing literature indicates that terpenoids made by bryophytes have important functions in diverse biological and ecological processes, particularly as defenses against biotic and abiotic stresses. The continued development of genomic resources and molecular tool kit for bryophytes will accelerate characterization of terpenoids biosynthesis and their biological functions in this important lineage of plants.

Diversity and Functional Evolution of Terpene Synthases in Dictyostelid Social Amoebae

Dictyostelids, or social amoebae, have a unique life style in forming multicellular fruiting bodies from unicellular amoeboids upon starvation. Recently, dictyostelids were found to contain terpene synthase (TPS) genes, a gene type of secondary metabolism previously known to occur only in plants, fungi and bacteria. Here we report an evolutionary functional study of dictyostelid TPS genes. The number of TPS genes in six species of dictyostelids examined ranges from 1 to 19; and the model species Dictyostelium purpureum contains 12 genes. Using in vitro enzyme assays, the 12 TPS genes from D. purpureum were shown to encode functional enzymes with distinct product profiles. The expression of the 12 TPS genes in D. purpureum is developmentally regulated. During multicellular development, D. purpureum releases a mixture of volatile terpenes dominated by sesquiterpenes that are the in vitro products of a subset of the 12 TPS genes. The quality and quantity of the terpenes released from D. purpureum, however, bear little resemblance to those of D. discoideum, a closely related dictyostelid. Despite these variations, the conserved clade of dictyostelid TPSs, which have an evolutionary distance of more than 600 million years, has the same biochemical function, catalyzing the formation of a sesquiterpene protoillud-7-ene. Taken together, our results indicate that the dynamic evolution of dictyostelid TPS genes includes both purifying selection of an orthologous group and species-specific expansion with functional divergence. Consequently, the terpenes produced by these TPSs most likely have conserved as well as species-adaptive biological functions as chemical languages in dictyostelids.

The rice terpene synthase gene OsTPS19 functions as an (S)-limonene synthase in planta, and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae

Summary Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up‐regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)‐limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)‐limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)‐limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.

Biochemical characterization in Norway spruce (Picea abies) of SABATH methyltransferases that methylate phytohormones

Indole-3-acetic acid (IAA), gibberellins (GAs), salicylic acid (SA) and jasmonic acid (JA) exist in methyl ester forms in plants in addition to their free acid forms. The enzymes that catalyze methylation of these carboxylic acid phytohormones belong to a same protein family, the SABATH methyltransferases. While the genes encoding these enzymes have been isolated from a small number of flowering plants, little is known about their occurrence and evolution in non-flowering plants. Here, we report the systematic characterization of the SABATH family from Norway spruce (Picea abies), a gymnosperm. The Norway spruce genome contains ten SABATH genes (PaSABATH1-10). Full-length cDNA for each of the ten PaSABATH genes was cloned and expressed in Escherichia coli. Recombinant PaSABATHs were tested for activity with IAA, GA, SA, and JA. Among the ten PaSABATHs, five had activity with one or more of the four substrates. PaSABATH1 and PaSABATH2 had the highest activities with IAA and SA, respectively. PaSABATH4, PaSABATH5 and PaSABATH10 all had JA as a preferred substrate but with notable differences in biochemical properties. The structural basis of PaSABATHs in discriminating various phytohormone substrates was inferred based on structural models of the enzyme-substrate complexes. The phylogeny of PaSABATHs with selected SABATHs from other plants implies that the enzymes methylating IAA are conserved in seed plants whereas the enzymes methylating JA and SA have independent evolution in gymnosperms and angiosperms.