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Terpene volatile messengers are widespread among animals. New discoveries have been made about the role and independent evolution of pathways and enzymes in animal terpene biosynthesis, especially in the large group of insects.

Insects use diverse arrays of small molecules such as metabolites of the large class of terpenes for intra-and inter -specific communication and defense. These molecules are synthesized by specialized metabolic pathways; however, the origin of enzymes involved in terpene biosynthesis and their evolution in insect genomes is still poorly understood. We addressed this question by investigating the evolution of isoprenyl diphosphate synthase (IDS)-like genes with terpene synthase (TPS) function in the family of stink bugs (Pentatomidae) within the large order of piercing-sucking Hemipteran insects. Stink bugs include species of global pest status, many of which emit structurally related 15-carbon sesquiterpenes as sex or aggregation pheromones. We provide evidence for the emergence of IDS-type TPS enzymes at the onset of pentatomid evolution over 100 million years ago, coinciding with the evolution of flowering plants. Stink bugs of different geographical origin maintain small IDS -type families with genes of conserved TPS function, which stands in contrast to the diversification of TPS genes in plants. Expanded gene mining and phylogenetic analysis in other hemipteran insects further provides evidence for an ancient emergence of IDS-like genes under presumed selection for terpene-mediated chemical interactions, and this process occurred independently from a similar evolution of IDS-type TPS genes in beetles. Our findings further suggest differences in TPS diversification in insects and plants in conjunction with different modes of gene functionalization in chemical interactions. National Science Foundation; US Department of Agriculture National Institute of Food and Agriculture; [MCB1920914]; [MCB1920922]; [MCB1920925]; [2016-67013-24759] Published version Acknowledgements This work was supported by the National Science Foundation (MCB1920914, MCB1920922, MCB1920925) to AVM, P.O., and D.T., respectively, and the US Department of Agriculture National Institute of Food and Agriculture (2016-67013-24759) to DT, AK, DEG-R, and DCW. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.

Gene functional descriptions, which are typically derived from sequence similarity to experimentally validated genes in a handful of model species, offer a crucial line of evidence when searching for candidate genes that underlie trait variation. Plant responses to environmental cues, including gene expression regulatory variation, represent important resources for understanding gene function and crucial targets for plant improvement through gene editing and other biotechnologies. However, even after years of effort and numerous large-scale functional characterization studies, biological roles of large proportions of protein coding genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, a public and updateable data resource consisting of transcript abundance assays from 2,090 samples derived from 604 tissues or conditions across 18 diverse species. We integrated across these diverse conditions and genotypes by analyzing expression profiles, building gene clusters that exhibited tissue/condition specific expression, and testing for transcriptional modulation in response to environmental queues. For example, we discovered extensive phylogenetically constrained and condition-specific expression profiles across many gene families and genes without any functional annotation. Such conserved expression patterns and other tightly co-expressed gene clusters let us assign expression derived functional descriptions to 64,620 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome-next.jgi.doe.gov), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Soilborne plant diseases are a major constraint to crop production worldwide. Effective and economical management of these diseases is dependent on the ability to accurately detect and diagnose their signs and/or symptoms prior to widespread development in a crop. Sensor-based technologies are promising tools for automated crop disease detection, but research is still needed to optimize and validate methods for the detection of specific plant diseases. The overarching goal of our research is to use the peanut-stem rot plant disease system to identify and evaluate sensor-based technologies that can be utilized for the detection of soilborne plant diseases. Here we summarize the current state of sensor-based technologies for plant disease detection and provide examples from our own research that illustrate the advantages and limitations of different sensor-based methods for detecting soilborne diseases. In addition, the potential to adapt different sensor-based technologies to practical use in the field is discussed.

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)-beta -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), gamma -terpinene (DcTPS30) and alpha -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. US-Israel Binational Agricultural Research and Development FundUS-Israel Binational Science Foundation [IS-4745-14R] We are thankful to Dr. Reuben Peters (Iowa State University) for providing the pDEST15-BjKS construct. This work was supported by grant IS-4745-14R from the US-Israel Binational Agricultural Research and Development Fund (to M.I. and D.T.).

Published version

Stink bugs (Hemiptera: Pentatomidae) are known to release pheromones that belong to the class of terpene compounds. Cyclic 15-carbon sesquiterpenes with a bisabolane type skeleton typically serve as aggregation pheromones in common pentatomid pests such as the southern green stink bug (Nezara viridula) and the brown marmorated stink bug (Halyomorpha halys). This chapter describes recent findings in the de novo biosynthesis of terpene pheromones in stink bugs with a focus on terpene synthase (TPS) enzymes that convert farnesyl diphosphate (FDP), a common intermediate of terpene biosynthesis, to pheromone precursors. The TPS proteins share sequence similarity with trans-isoprenyl diphosphate synthases (IDSs) of the core terpene pathway indicating that insect TPSs have evolved from IDS progenitors. The transition from IDS to TPS function has been described in other insects (e.g. Coleoptera) and gives rise to the discovery of similar enzymes and pathways and the study of their evolutionary emergence in terpene pheromone and semiochemical biosynthesis in different insect lineages.

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.

The belowground parts, roots in vascular plants and rhizoids in bryophytes, play critical roles for adaptations and productivity of land plants. The rhizosphere, which is the narrow region of soil that surrounds roots/rhizoids, is enormously rich in nonplant species. How roots/rhizoids interact with such diverse soil organisms remains largely uncharacterized. Nonetheless, some constituents of root exudates, which are metabolites that are produced by roots and rhizoids and secreted into the rhizosphere, have been demonstrated to impact root-soil organism interactions. Germane to this chapter, there is increasing evidence that volatile organic compounds, a subset of root metabolites, play important roles in mediating diverse types of interactions between roots and other soil organisms. This chapter discusses four types of such belowground interactions: (1) volatile-mediated belowground plant–plant interactions; (2) volatile-mediated belowground plant–insect interactions; (3) root-beneficial microbe interactions mediated by volatiles, and (4) plant defense against microbial pathogens mediated by volatiles.

Summary Soil communities are diverse taxonomically and functionally. This ecosystem experiences highly complex networks of interactions, but may also present functionally independent entities. Plant roots, a metabolically active hotspot in the soil, take an essential part in below-ground interactions. While plants are known to release an extremely high portion of the fixated carbon to the soil, less information is known about the composition and role of C-containing compounds in the rhizosphere, in particular those involved in chemical communication. Specialized metabolites (or secondary metabolites) produced by plants and their associated microbes have a critical role in various biological activities that modulate the behavior of neighboring organisms. Thus, elucidating the chemical composition and function of specialized metabolites in the rhizosphere is a key element in understanding interactions in this below-ground environment. Here, we review key classes of specialized metabolites that occur as mostly non-volatile compounds in root exudates or are emitted as volatile organic compounds (VOCs). The role of these metabolites in below-ground interactions and response to nutrient deficiency, as well as their tissue and cell type-specific biosynthesis and release are discussed in detail.

Chemical communication is ubiquitous. The identification of conserved structural elements in visual and acoustic communication is well established, but comparable information on chemical communication displays (CCDs) is lacking. We assessed the phenotypic integration of CCDs in a meta‐analysis to characterize patterns of covariation in CCDs and identified functional or biosynthetically constrained modules. Poorly integrated plant CCDs (i.e. low covariation between scent compounds) support the notion that plants often utilize one or few key compounds to repel antagonists or to attract pollinators and enemies of herbivores. Animal CCDs (mostly insect pheromones) were usually more integrated than those of plants (i.e. stronger covariation), suggesting that animals communicate via fixed proportions among compounds. Both plant and animal CCDs were composed of modules, which are groups of strongly covarying compounds. Biosynthetic similarity of compounds revealed biosynthetic constraints in the covariation patterns of plant CCDs. We provide a novel perspective on chemical communication and a basis for future investigations on structural properties of CCDs. This will facilitate identifying modules and biosynthetic constraints that may affect the outcome of selection and thus provide a predictive framework for evolutionary trajectories of CCDs in plants and animals., The project was supported by the Graduate School ‘Evolutionary Networks: Organisms, Reactions, Molecules’ (E‐Norm) of the Heinrich‐Heine‐University, Düsseldorf, Germany, the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG JU 2856/1‐1 and DFG JU 2856/2‐2).

Carotenogenesis has been intensively studied in carrot roots, and transcriptional regulation is thought to be the major factor in carotenoid accumulation in these organs. However, little is known about the transcriptional regulation of carotenoid biosynthetic genes concerning carotenoid accumulation during infestation by the obligate parasite Phelipanche aegyptiaca. HPLC analysis revealed a decrease in carotenoid levels of the different carrot cultivars when parasitized by P. aegyptiaca. Besides, we isolated and analyzed P. aegyptiaca tubercles parasitizing the various carrot root cultivars and show that they accumulate different carotenoids compared to those in non-infested carrot roots. Expression analysis of PHYTOENE SYNTHASE (PSY1) and CAROTENOID ISOMERASE (CRTISO) as well as the strigolactone apocarotenoid biosynthetic genes DWARF27 (D27), CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 revealed that their transcript levels showed significant variation in P. aegyptiaca infested carrot roots. After parasite infestation, the expression of these genes was strongly reduced, as were the carotenoid levels and this was more pronounced in the uncommon non-orange varieties. We also analyzed the parasite genes encoding D27, CCD7 and CCD8 and show that they are expressed in tubercles. This raises important questions of whether the parasite produces its carotenoids and apocarotenoids including strigolactones and whether the latter might have a role in tubercle development.

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. Community Science Program grant of Department of Energy Joint Genome Institute [WIP 2568]; Translational Plant Sciences Program at Virginia Tech; Office of Science of the US Department of EnergyUnited States Department of Energy (DOE) [DE-AC02-05CH11231] This work was supported by Community Science Program grant (WIP 2568) of the Department of Energy Joint Genome Institute and funds by the Translational Plant Sciences Program at Virginia Tech. The work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under contract no. DE-AC02-05CH11231.

Despite the phylogenetic distance between plants and insects, these two groups of organisms produce some secondary metabolites in common. Identical structures belonging to chemical classes such as the simple monoterpenes and sesquiterpenes, iridoid monoterpenes, cyanogenic glycosides, benzoic acid derivatives, benzoquinones and naphthoquinones are sometimes found in both plants and insects. In addition, very similar glucohydrolases involved in activating two component defenses, such as glucosinolates and cyanogenic glycosides, occur in both plants and insects. While this trend was first noted many years ago, researchers have long struggled to find convincing explanations for such co‐occurrence. In some cases, identical compounds may be produced by plants to interfere with their function in insects. But, in other cases, plant and insect compounds may simply have parallel functions, probably in defense or attraction, and their co‐occurrence is a coincidence. The biosynthetic origin of such co‐occurring metabolites may be very different in insects as compared to plants. Plants and insects may have different pathways to the same metabolite, or similar sequences of intermediates, but different enzymes. Further knowledge of the ecological roles and biosynthetic pathways of secondary metabolites may shed more light on why plants and insects produce identical substances.

The original version of this article unfortunately contained a mistake. Under the heading "Insects" in "Methods and Materials" the sentence "A colony of N. viridula originated with field collections near Tifton, Georgia, USA" is incorrect.

Carrot is considered one of the leading horticultural crops in the world in terms of its nutritional value, health benefits, and unique flavor based on its high content of carotenoids and volatile aroma compounds. Terpenes such as monoterpenes and sesquiterpenes represent some of the predominant volatile compounds that contribute to carrot aroma and flavor. Variation of terpene composition based on genotypic differences or environmental factors has significant effects on taste perception by consumers and, therefore, is a critical quality attribute for carrot breeders and growers. Surprisingly, little is known about the biosynthesis of volatile terpenes in carrots and the various enzymes involved in their formation. In this chapter, we provide an overview of volatile terpene sampling and terpene diversity in different D. carota genotypes. Facilitated by the recent elucidation of the carrot genome, we further describe and discuss latest findings of the function of genes and enzymes in the terpene synthase family involved in the biosynthesis of carrot terpene volatiles.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (15,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication. US Department of Agriculture National Institute of Food and Agriculture [2016-67013-24759]; Virginia Polytechnic Institute; State University Department of Biochemistry; State University Department of Biological Sciences; Translational Plant Sciences Program We thank Megan V. Herlihy and Jeremy K. Turner for assisting with harlequin bug rearing and volatile collection and Filadelfo Guzman for assisting with GC analyses. This work was supported by US Department of Agriculture National Institute of Food and Agriculture Grant 2016-67013-24759 (to D.T., D.E.G.-R., A.K., T.P.K., C.T., and D.C.W.) and the Virginia Polytechnic Institute and State University Departments of Biochemistry and Biological Sciences and the Translational Plant Sciences Program.

Diterpenoids constitute a diverse class of metabolites with critical functions in plant development, defense, and ecological adaptation. Major monocot crops, such as maize (Zea mays) and rice (Oryza sativa), 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 (Panicum virgatum). 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 ent-copalyl diphosphate (CPP) and ent-kaurene synthases predictably involved in gibberellin biosynthesis, we identified syn-CPP and ent-labda-13-en-8-ol diphosphate (LPP) synthases as well as two diTPSs forming (+)-labda-8,13E-dienyl diphosphate (8,13-CPP) and ent-neo-cis-trans-clerodienyl diphosphate (CT-CLPP) scaffolds not observed previously in plants. Structure-guided mutagenesis of the (+)-8,13-CPP and ent-neo-CT-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 ent-CPP, ent-LPP, syn-CPP, and ent-neo-CT-CLPP by promiscuous diTPSs further yielded distinct labdane-type diterpene olefins and alcohols. Of these metabolites, the formation of 9β-hydroxy-syn-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.

Insects use a wide range of structurally diverse pheromones for intra-specific communication. Compounds in the class of terpenes are emitted as sex, aggregation, alarm, or trail pheromones. Despite the common occurrence of terpene pheromones in different insect lineages, their origin from dietary host plant precursors or de novo biosynthetic pathways often remains unknown. Several stink bugs (Hemiptera: Pentatomidae) release bisabolene-type sesquiterpenes for aggregation and mating. Here we provide evidence for de novo biosynthesis of the sex pheromone trans−/cis-(Z)-α-bisabolene epoxide of the Southern green stink bug, Nezara viridula. We show that an enzyme (NvTPS) related to isoprenyl diphosphate synthases (IDSs) of the core terpene metabolic pathway functions as a terpene synthase (TPS), which converts the general intermediate (E,E)-farnesyl diphosphate (FPP) to the putative pheromone precursor (+)-(S,Z)-α-bisabolene in vitro and in protein lysates. A second identified IDS-type protein (NvFPPS) makes the TPS substrate (E,E)-FPP and functions as a bona fide FPP synthase. NvTPS is highly expressed in male epidermal tissue associated with the cuticle of ventral sternites, which is in agreement with the male specific release of the pheromone from glandular cells in this tissue. Our study supports findings of the function of similar TPS enzymes in the biosynthesis of aggregation pheromones from the pine engraver beetle Ips pini, the striped flea beetle Phyllotreta striolata, and the harlequin bug Murgantia histrionica, and hence provides growing evidence for the evolution of terpene de novo biosynthesis by IDS-type TPS families in insects.

Plant roots secrete a significant portion of their assimilated carbon into the rhizosphere. The putative sugar transporter SWEET2 is highly expressed in Arabidopsis roots. Expression patterns of SWEET2-β-glucuronidase fusions confirmed that SWEET2 accumulates highly in root cells and thus may contribute to sugar secretion, specifically from epidermal cells of the root apex. SWEET2-green fluorescent protein fusions localized to the tonoplast, which engulfs the major sugar storage compartment. Functional analysis of SWEET2 activity in yeast showed low uptake activity for the glucose analog 2-deoxyglucose, consistent with a role in the transport of glucose across the tonoplast. Loss-of-function sweet2 mutants showed reduced tolerance to excess glucose, lower glucose accumulation in leaves, and 15-25% higher glucose-derived carbon efflux from roots, suggesting that SWEET2 has a role in preventing the loss of sugar from root tissue. SWEET2 root expression was induced more than 10-fold during Pythium infection. Importantly, sweet2 mutants were more susceptible to the oomycete, showing impaired growth after infection. We propose that root-expressed vacuolar SWEET2 modulates sugar secretion, possibly by reducing the availability of glucose sequestered in the vacuole, thereby limiting carbon loss to the rhizosphere. Moreover, the reduced availability of sugar in the rhizosphere due to SWEET2 activity contributes to resistance to Pythium.

The harlequin bug, Murgantia histrionica (Hahn), is an agricultural pest in the continental United States, particularly in southern states. Reliable gene sequence data are especially useful to the development of species-specific, environmentally friendly molecular biopesticides and effective biolures for this insect. Here, mRNAs were sampled from whole insects at the 2nd and 4th nymphal instars, as well as sexed adults, and sequenced using Illumina RNA-Seq technology. A global assembly of these data identified 72,540 putative unique transcripts bearing high levels of similarity to transcripts identified in other taxa, with over 99% of conserved single-copy orthologs among insects being detected. Gene ontology and protein family analyses were conducted to explore the functional potential of the harlequin bug’s gene repertoire, and phylogenetic analyses were conducted on gene families germane to xenobiotic detoxification, including glutathione S-transferases, carboxylesterases and cytochrome P450s. Genic content in harlequin bug was compared with that of the closely related invasive pest, the brown marmorated stink bug, Halyomorpha halys (Stål). Quantitative analyses of harlequin bug gene expression levels, experimentally validated using quantitative real-time PCR, identified genes differentially expressed between life stages and/or sexes.

Volatile compounds are usually associated with an appearance/presence in the atmosphere. Recent advances, however, indicated that the soil is a huge reservoir and source of biogenic volatile organic compounds (bVOCs), which are formed from decomposing litter and dead organic material or are synthesized by underground living organism or organs and tissues of plants. This review summarizes the scarce available data on the exchange of VOCs between soil and atmosphere and the features of the soil and particle structure allowing diffusion of volatiles in the soil, which is the prerequisite for biological VOC-based interactions. In fact, soil may function either as a sink or as a source of bVOCs. Soil VOC emissions to the atmosphere are often 1-2 (0-3) orders of magnitude lower than those from aboveground vegetation. Microorganisms and the plant root system are the major sources for bVOCs. The current methodology to detect belowground volatiles is described as well as the metabolic capabilities resulting in the wealth of microbial and root VOC emissions. Furthermore, VOC profiles are discussed as non-destructive fingerprints for the detection of organisms. In the last chapter, belowground volatile-based bi- and multi-trophic interactions between microorganisms, plants and invertebrates in the soil are discussed.

Harlequin bug, Murgantia histrionica (Hahn) (Hemiptera: Pentatomidae), is a piercing-sucking pest of cole crops, causing cosmetic damage in low populations, while heavy pest pressure can kill plants or entire fields. Field studies were conducted to evaluate a trap crop for control of harlequin bug in collard. Field-cage choice tests found that potential trap crop plant species, mustard (Brassica juncea 'Southern Giant Curled'), rapeseed (B. napus 'Athena'), rapini (B. rapa), and arugula (Eruca satica) attracted more harlequin bugs than collard (B. oleracea 'Champion') and a nonbrassica control, bean (Phaseolus vulgaris'Bronco'). Mustard was the most consistently selected by harlequin bug over collard in choice tests, and was found to be an effective trap crop for reducing feeding injury on collard at two experimental sites. Augmentation of the mustard trap crop with a systemic, neonicotinoid insecticide provided no added control of harlequin bug for the 10 wk duration in the spring season.

MicroRNAs (miRNAs) play essential roles in plant and animal development, but the cause and effect of miRNA expression divergence between closely related species and in interspecific hybrids or allopolyploids are unknown. Here, we show differential regulation of a miR163-mediated pathway in allotetraploids and their progenitors, Arabidopsis thaliana and Arabidopsis arenosa. miR163 is a recently evolved miRNA in A. thaliana and highly expressed in A. thaliana, but its expression was undetectable in A. arenosa and repressed in resynthesized allotetraploids. Repression of A. arenosa MIR163 (Aa MIR163) is caused by a weak cis-acting promoter and putative trans-acting repressor(s) present in A. arenosa and allotetraploids. Moreover, ectopic Aa MIR163 precursors were processed more efficiently in A. thaliana than in resynthesized allotetraploids, suggesting a role of posttranscriptional regulation in mature miR163 abundance. Target genes of miR163 encode a family of small molecule methyltransferases involved in secondary metabolite biosynthetic pathways that are inducible by a fungal elicitor, alamethicin. Loss of miR163 or overexpression of miR163 in mir163 mutant plants alters target transcript and secondary metabolite profiles. We suggest that cis- and trans-regulation of miRNA and other genes provides a molecular basis for natural variation of biochemical and metabolic pathways that are important to growth vigor and stress responses in Arabidopsis-related species and allopolyploids.

Summary Some plant terpenes such as sterols and carotenes are part of primary metabolism and found essentially in all plants. However, the majority of the terpenes found in plants are classified as ‘secondary’ compounds, those chemicals whose synthesis has evolved in plants as a result of selection for increased fitness via better adaptation to the local ecological niche of each species. Thousands of such terpenes have been found in the plant kingdom, but each species is capable of synthesizing only a small fraction of this total. In plants, a family of terpene synthases (TPSs) is responsible for the synthesis of the various terpene molecules from two isomeric 5-carbon precursor ‘building blocks’, leading to 5-carbon isoprene, 10-carbon monoterpenes, 15-carbon sesquiterpenes and 20-carbon diterpenes. The bryophyte Physcomitrella patens has a single TPS gene, copalyl synthase/kaurene synthase (CPS/KS), encoding a bifunctional enzyme producing ent-kaurene, which is a precursor of gibberellins. The genome of the lycophyte Selaginella moellendorffii contains 18 TPS genes, and the genomes of some model angiosperms and gymnosperms contain 40–152 TPS genes, not all of them functional and most of the functional ones having lost activity in either the CPS- or KS-type domains. TPS genes are generally divided into seven clades, with some plant lineages having a majority of their TPS genes in one or two clades, indicating lineage-specific expansion of specific types of genes. Evolutionary plasticity is evident in the TPS family, with closely related enzymes differing in their product profiles, subcellular localization, or the in planta substrates they use.

Summary Aromatic l-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5′-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an l-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-l-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant–plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C 20 -precursor ( E , E )-geranyllinalool to the insect-induced C 16 -homoterpene ( E , E )-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for ( E , E )-geranyllinalool and its C 15 -analog ( E )-nerolidol, which is converted to the respective C 11 -homoterpene ( E )-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn -elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivore-induced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase.

Volatile secondary metabolites emitted by plants contribute to plant–plant, plant–fungus, and plant–insect interactions. The C16-homoterpene TMTT (for 4,8,12-trimethyltrideca-1,3,7,11-tetraene) is emitted after herbivore attack by a wide variety of plant species, including Arabidopsis thaliana, and is assumed to play a role in attracting predators or parasitoids of herbivores. TMTT has been suggested to be formed as a degradation product of the diterpene alcohol (E,E)-geranyllinalool. Here, we report the identification of Terpene Synthase 04 (TPS04; At1g61120) as a geranyllinalool synthase (GES). Recombinant TPS04/GES protein expressed in Escherichia coli catalyzes the formation of (E,E)-geranyllinalool from the substrate geranylgeranyl diphosphate. Transgenic Arabidopsis lines carrying T-DNA insertions in the TPS04 locus are deficient in (E,E)-geranyllinalool and TMTT synthesis, a phenotype that can be complemented by expressing the GES gene under the control of a heterologous promoter. GES transcription is upregulated under conditions that induce (E,E)-geranyllinalool and TMTT synthesis, including infestation of plants with larvae of the moth Plutella xylostella and treatment with the fungal peptide alamethicin or the octadecanoid mimic coronalon. Induction requires jasmonic acid but is independent from salicylic acid or ethylene. This study paves the ground to address the contribution of TMTT in ecological interactions and to elucidate the signaling network that regulates TMTT synthesis.

Plants produce an excess of volatile organic compounds, which are important in determining the quality and nutraceutical properties of fruit and root crops, including the taste and aroma of carrots (Daucus carota L.). A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of volatile terpenes in a diverse collection of fresh carrots (D. carota L.). Here, we report on a transcriptome-based identification and functional characterization of two carrot terpene synthases, the sesquiterpene synthase, DcTPS1, and the monoterpene synthase, DcTPS2. Recombinant DcTPS1 protein produces mainly (E)-β-caryophyllene, the predominant sesquiterpene in carrot roots, and α-humulene, while recombinant DcTPS2 functions as a monoterpene synthase with geraniol as the main product. Both genes are differentially transcribed in different cultivars and during carrot root development. Our results suggest a role for DcTPS genes in carrot aroma biosynthesis.

Terpene synthases are the primary enzymes in the formation of low-molecular-weight terpene metabolites. Rapid progress in the biochemical and molecular analysis of terpene synthases has allowed significant investigations of their evolution, structural and mechanistic properties, and regulation. The organization of terpene synthases in large gene families, their characteristic ability to form multiple products, and their spatial and temporal regulation during development and in response to biotic and abiotic factors contribute to the time-variable formation of a diverse group of terpene metabolites. The structural diversity and complexity of terpenes generates an enormous potential for mediating plant-environment interactions. Engineering the activities of terpene synthases provides opportunities for detailed functional evaluations of terpene metabolites in planta.

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.

Summary Emission of methylsalicylate (MeSA), and occasionally of methylbenzoate (MeBA), from Arabidopsis thaliana leaves was detected following the application of some forms of both biotic and abiotic stresses to the plant. Maximal emission of MeSA was observed following alamethicin treatment of leaves. A gene (AtBSMT1) encoding a protein with both benzoic acid (BA) and salicylic acid (SA) carboxyl methyltransferase activities was identified using a biochemical genomics approach. Its ortholog (AlBSMT1) in A. lyrata, a close relative of A. thaliana, was also isolated. The AtBSMT1 protein utilizes SA more efficiently than BA, whereas AlBSMT1 catalyzes the methylation of SA less effectively than that of BA. The AtBSMT1 and AlBSMT1 genes showed expression in leaves under normal growth conditions and were more highly expressed in the flowers. In A. thaliana leaves, the expression of AtBSMT1 was induced by alamethicin, Plutella xylostella herbivory, uprooting, physical wounding, and methyl jasmonate. SA was not an effective inducer. Using a β-glucuronidase (GUS) reporter approach, the promoter activity of AtBSMT1 was localized to the sepals of flowers, and also to leaf trichomes and hydathodes. Upon thrip damage to leaves, AtBSMT1 promoter activity was induced specifically around the lesions.

Arabidopsis is believed to be mostly self-pollinated, although several lines of genetic and morphological evidence indicate that insect-mediated outcrossing occurs with at least a low frequency in wild populations. Here, we show that Arabidopsis flowers emit both monoterpenes and sesquiterpenes, potential olfactory cues for pollinating insects. Of the 32 terpene synthase genes in the Arabidopsis genome, 20 were found to be expressed in flowers, 6 of these exclusively or almost exclusively so. Two terpene synthase genes expressed exclusively in the flowers and one terpene synthase gene expressed almost exclusively in the flowers were characterized and found to encode proteins that catalyze the formation of major floral volatiles. A beta-glucuronidase fusion construct with a promoter of one of these genes demonstrated that gene expression was restricted to the sepals, stigmas, anther filaments, and receptacles, reaching a peak when the stigma was receptive to cross pollen. The observation that Arabidopsis flowers synthesize and emit volatiles raises intriguing questions about the reproductive behavior of Arabidopsis in the wild and allows detailed investigations of floral volatile biosynthesis and its regulation to be performed with this model plant system.

Terpenoids (isoprenoids) represent the largest and most diverse class of chemicals among the myriad compounds produced by plants. Plants employ terpenoid metabolites for a variety of basic functions in growth and development but use the majority of terpenoids for more specialized chemical interactions and protection in the abiotic and biotic environment. Traditionally, plant-based terpenoids have been used by humans in the food, pharmaceutical, and chemical industries, and more recently have been exploited in the development of biofuel products. Genomic resources and emerging tools in synthetic biology facilitate the metabolic engineering of high-value terpenoid products in plants and microbes. Moreover, the ecological importance of terpenoids has gained increased attention to develop strategies for sustainable pest control and abiotic stress protection. Together, these efforts require a continuous growth in knowledge of the complex metabolic and molecular regulatory networks in terpenoid biosynthesis. This chapter gives an overview and highlights recent advances in our understanding of the organization, regulation, and diversification of core and specialized terpenoid metabolic pathways, and addresses the most important functions of volatile and nonvolatile terpenoid specialized metabolites in plants.

© 2015 American Society of Plant Biologists. All rights reserved. Plant-derived volatile compounds such as terpenes exhibit substantial structural variation and serve multiple ecological functions. Despite their structural diversity, volatile terpenes are generally produced from a small number of core 5- to 20-carbon intermediates. Here, we present unexpected plasticity in volatile terpene biosynthesis by showing that irregular homo/norterpenes can arise from different biosynthetic routes in a tissue specific manner. While Arabidopsis thaliana and other angiosperms are known to produce the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) or its C16-analog (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene by the breakdown of sesquiterpene and diterpene tertiary alcohols in aboveground tissues, we demonstrate that Arabidopsis roots biosynthesize DMNT by the degradation of the C30 triterpene diol, arabidiol. The reaction is catalyzed by the Brassicaceae-specific cytochrome P450 monooxygenase CYP705A1 and is transiently induced in a jasmonate-dependent manner by infection with the root-rot pathogen Pythium irregulare. CYP705A1 clusters with the arabidiol synthase gene ABDS, and both genes are coexpressed constitutively in the root stele and meristematic tissue. We further provide in vitro and in vivo evidence for the role of the DMNT biosynthetic pathway in resistance against P. irregulare. Our results show biosynthetic plasticity in DMNT biosynthesis in land plants via the assembly of triterpene gene clusters and present biochemical and genetic evidence for volatile compound formation via triterpene degradation in plants.

Terpene specialized metabolites exhibit multiple functions in plant-environment interactions and plant development. Molecular biologists investigating the biochemistry and molecular function of terpenes need to apply robust but yet sensitive analytical methods optimized and adapted to the structural diversity and often varying concentrations of terpene compounds in plant tissues. Here we present hands-on protocols for sample preparation and GC-MS or LC-MS/MS analysis of selected diterpene and triterpene hydrocarbons or oxygenated derivatives from roots and shoots of Arabidopsis and rice.

In the conifer Abies grandis (grand fir), a secreted oleoresin rich in mono-, sesqui-, and diterpenes serves as a constitutive and induced defense against insects and pathogenic fungi. Geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) synthase, two enzymes which form the principal precursors of the oleoresin mono- and sesquiterpenes, were isolated from the stems of 2-year-old grand fir saplings. These enzymes were partially purified by sequential chromatography on DEAE-Sepharose, Mono-Q, and phenyl-Sepharose to remove competing phosphohydrolase and isopentenyl diphosphate (IPP) isomerase activities. GPP and FPP synthase formed GPP and E,E-FPP, respectively, as the sole products of the enzymatic condensation of IPP and dimethylallyl diphosphate (DMAPP). The properties of both enzymes are broadly similar to those of other prenyltransferases. The apparent native molecular masses are 54 +/- 3 kDa for GPP synthase and 110 +/- 6 kDa fo

Sequence analysis of the 330-kb genome of chlorella virus Paramecium bursaria chlorella virus 1 (PBCV-1) revealed an open reading frame, A237R, that encodes a protein with 34% amino acid identity to homospermidine synthase from Rhodopseudomonas viridis. Expression of the a237r gene product in Escherichia coli established that the recombinant enzyme catalyzes the NAD(+)-dependent formation of homospermidine from two molecules of putrescine. The a237r gene is expressed late in PBCV-1 infection. Both uninfected and PBCV-1-infected chlorella, as well as PBCV-1 virions, contain homospermidine, along with the more common polyamines putrescine, spermidine, and cadaverine. The total number of polyamine molecules per virion ( approximately 539) is too small to significantly neutralize the virus double-stranded DNA (>660,000 nucleotides). Consequently, the biological significance of the homospermidine synthase gene is unknown. However, the gene is widespread among the chlorella viruses. To our knowledge, this is the first report of a virus encoding an enzyme involved in polyamine biosynthesis.

Floral scent is an important trait of ornamental roses that has provided sensual pleasures for humans since antiquity. However, most modern rose cultivars used for cut flowers have little fragrance as a result of breeding preferences for traits such as color and longevity. Restoring scent attributes by breeding or biotechnological means ( 1 ) requires a detailed understanding of the biosynthesis of rose scent. Rose fragrance consists of hundreds of volatile compounds with diverse biosynthetic origins whose amounts vary among the different rose varieties. Genomic approaches over the past 15 years have identified several genes and enzymes involved in rose scent production ( 2 ), but these efforts have not succeeded in elucidating the biosynthetic steps in the formation of geraniol and other monoterpenes that constitute one of the major groups of rose fragrances. On page [81][1] of this issue, Magnard et al. ( 3 ) describe the discovery of an unexpected enzymatic pathway to these scent compounds. [1]: /lookup/doi/10.1126/science.aab0696

The Escherichia coli mutant speE deficient in the gene encoding for spermidine synthase has no absolute requirement for spermidine but shows a retarded growth rate. This growth retardation could be unspecifically restored to the respective wild type level by exogenously supplied polyamines such as spermidine, spermine and homospermidine as well as the diamines putrescine and cadaverine. In comparison to the respective wild type, the mutant shows a two-fold increased level of endogenous putrescine but displays a reduced ability to accumulate the diamines putrescine and cadaverine. The ability to accumulate polyamines is not affected. The deleted spermidine synthase gene of the mutant was substituted by heterologous expression of the hss gene from Rhodopseudomonas viridis encoding homospermidine synthase.