Diterpenoids constitute a diverse class of metabolites with critical functions in plant development, defense, and ecological adaptation. Major monocot crops, such as maize () and rice (), deploy diverse blends of specialized diterpenoids as core components of biotic and abiotic stress resilience. Here, we describe the genome-wide identification and functional characterization of stress-related diterpene synthases (diTPSs) in the dedicated bioenergy crop switchgrass (). Mining of the allotetraploid switchgrass genome identified an expansive diTPS family of 31 members, and biochemical analysis of 11 diTPSs revealed a modular metabolic network producing a diverse array of diterpenoid metabolites. In addition to -copalyl diphosphate (CPP) and-kaurene synthases predictably involved in gibberellin biosynthesis, we identified -CPP and-labda-13-en-8-ol diphosphate (LPP) synthases as well as two diTPSs forming (+)-labda-8,13-dienyl diphosphate (8,13-CPP) and cis-trans-clerodienyl diphosphate (CT-CLPP) scaffolds not observed previously in plants. Structure-guided mutagenesis of the (+)-8,13-CPP andCT-CLPP synthases revealed residue substitutions in the active sites that altered product outcome, representing potential neofunctionalization events that occurred during diversification of the switchgrass diTPS family. The conversion of -CPP,-LPP, -CPP, and-CT-CLPP by promiscuous diTPSs further yielded distinct labdane-type diterpene olefins and alcohols. Of these metabolites, the formation of 9β-hydroxy--pimar-15-ene and the expression of the corresponding genes were induced in roots and leaves in response to oxidative stress and ultraviolet irradiation, indicating their possible roles in abiotic stress adaptation. Together, these findings expand the known chemical space of diterpenoid metabolism in monocot crops toward systematically investigating and ultimately improving stress resilience traits in crop species.

SignificanceMany insects release volatile terpenes for chemical communication. However, the biosynthetic origin and evolution of these infochemicals are mostly unknown. We show that the harlequin bug, Murgantia histrionica, a stink bug pest (Hemiptera) of crucifer crops, produces a terpene aggregation pheromone by an enzyme that is unrelated to microbial and plant terpene synthases. M. histrionica terpene synthase activity is highly sex- and tissue-specific and makes a sesquiterpene alcohol, so far unknown in animals, as pheromone precursor. The enzyme evolved from ancestral isoprenyl diphosphate synthases and provides new evidence for de novo biosynthesis of terpenes in hemipteran insects. Knowledge of pheromone biosynthesis in stink bugs may lead to the development of new controls of these pests.

Switchgrass (Panicum virgatum L.), a perennial C4 grass, represents an important species in natural and anthropogenic grasslands of North America. Its resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. However, little is known about the mechanisms of resistance of switchgrass against pathogens and herbivores. Volatile compounds such as terpenes have important activities in plant direct and indirect defense. Here, we show that switchgrass leaves emit blends of monoterpenes and sesquiterpenes upon feeding by the generalist insect herbivore Spodoptera frugiperda (fall armyworm) and in a systemic response to the treatment of roots with defense hormones. Belowground application of methyl jasmonate also induced the release of volatile terpenes from roots. To correlate the emission of terpenes with the expression and activity of their corresponding biosynthetic genes, we identified a gene family of 44 monoterpene and sesquiterpene synthases (mono- and sesqui-TPSs) of the type-a, type-b, type-g, and type-e subfamilies, of which 32 TPSs were found to be functionally active in vitro. The TPS genes are distributed over the K and N subgenomes with clusters occurring on several chromosomes. Synteny analysis revealed syntenic networks for approximately 30–40% of the switchgrass TPS genes in the genomes of Panicum hallii, Setaria italica, and Sorghum bicolor, suggesting shared TPS ancestry in the common progenitor of these grass lineages. Eighteen switchgrass TPS genes were substantially induced upon insect and hormone treatment and the enzymatic products of nine of these genes correlated with compounds of the induced volatile blends. In accordance with the emission of volatiles, TPS gene expression was induced systemically in response to belowground treatment, whereas this response was not observed upon aboveground feeding of S. frugiperda. Our results demonstrate complex above and belowground responses of induced volatile terpene metabolism in switchgrass and provide a framework for more detailed investigations of the function of terpenes in stress resistance in this monocot crop.

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Carrot (Daucus carota L.) is an important root vegetable crop with high nutritional value, characteristic flavor, and benefits to human health. D. carota tissues produce an essential oil that is rich in volatile terpenes and plays a major role in carrot aroma and flavor. Although terpene composition represents a critical quality attribute of carrots, little is known about the biosynthesis of terpenes in this crop. Here, we functionally characterized 19 terpene synthase (TPS) genes in an orange carrot (genotype DH1) and compared tissue-specific expression profiles and in vitro products of their recombinant proteins with volatile terpene profiles from DH1 and four other colored carrot genotypes. In addition to the previously reported (E)-β-caryophyllene synthase (DcTPS01), we biochemically characterized several TPS proteins with direct correlations to major compounds of carrot flavor and aroma including germacrene D (DcTPS7/11), γ-terpinene (DcTPS30) and α-terpinolene (DcTPS03). Random forest analysis of volatiles from colored carrot cultivars identified nine terpenes that were clearly distinct among the cultivars and likely contribute to differences in sensory quality. Correlation of TPS gene expression and terpene metabolite profiles supported the function of DcTPS01 and DcTPS03 in these cultivars. Our findings provide a roadmap for future breeding efforts to enhance carrot flavor and aroma.