Streamlined screening platforms lead to the discovery of pachysiphine synthase from Tabernanthe iboga.

Plant-specialized metabolism is largely driven by the oxidative tailoring of key chemical scaffolds catalyzed by cytochrome P450 (CYP450s) enzymes. Monoterpene indole alkaloids (MIAs) tabersonine and pseudo-tabersonine, found in the medicinal plant Tabernanthe iboga (commonly known as iboga), are tailored with oxidations, and the enzymes involved remain unknown. Here, we developed a streamlined screening strategy to test the activity of T. iboga CYP450s in Nicotiana benthamiana. Using multigene constructs encoding the biosynthesis of tabersonine and pseudo-tabersonine scaffolds, we aimed to uncover the CYP450s responsible for oxidative transformations in these scaffolds. Our approach identified two T. iboga cytochrome P450 enzymes: pachysiphine synthase (PS) and 16-hydroxy-tabersonine synthase (T16H). These enzymes catalyze an epoxidation and site-specific hydroxylation of tabersonine to produce pachysiphine and 16-OH-tabersonine, respectively. This work provides new insights into the biosynthetic pathways of MIAs and underscores the utility of N. benthamiana and Catharanthus roseus as platforms for the functional characterization of plant enzymes.

Phenylphenalenones and Linear Diarylheptanoid Derivatives Are Biosynthesized via Parallel Routes in Musella lasiocarpa, the Chinese Dwarf Banana.

Here, we use transcriptomic data from seeds of Musella lasiocarpa to identify five enzymes involved in the formation of dihydrocurcuminoids. Characterization of the substrate specificities of the enzymes reveals two distinct dihydrocurcuminoid pathways leading to phenylphenalenones and linear diarylheptanoid derivatives, the major seed metabolites. Furthermore, we demonstrate the stepwise conversion of dihydrobisdemethoxycurcumin to the phenylphenalenone 4'-hydroxylachnanthocarpone by feeding intermediates to M. lasiocarpa root protein extract.

Reinventing metabolic pathways: Independent evolution of benzoxazinoids in flowering plants.

Benzoxazinoids (BXDs) form a class of indole-derived specialized plant metabolites with broad antimicrobial and antifeedant properties. Unlike most specialized metabolites, which are typically lineage-specific, BXDs occur sporadically in a number of distantly related plant orders. This observation suggests that BXD biosynthesis arose independently numerous times in the plant kingdom. However, although decades of research in the grasses have led to the elucidation of the BXD pathway in the monocots, the biosynthesis of BXDs in eudicots is unknown. Here, we used a metabolomic and transcriptomic-guided approach, in combination with pathway reconstitution in Nicotiana benthamiana, to identify and characterize the BXD biosynthetic pathways from both Aphelandra squarrosa and Lamium galeobdolon, two phylogenetically distant eudicot species. We show that BXD biosynthesis in A. squarrosa and L. galeobdolon utilize a dual-function flavin-containing monooxygenase in place of two distinct cytochrome P450s, as is the case in the grasses. In addition, we identified evolutionarily unrelated cytochrome P450s, a 2-oxoglutarate-dependent dioxygenase, a UDP-glucosyltransferase, and a methyltransferase that were also recruited into these BXD biosynthetic pathways. Our findings constitute the discovery of BXD pathways in eudicots. Moreover, the biosynthetic enzymes of these pathways clearly demonstrate that BXDs independently arose in the plant kingdom at least three times. The heterogeneous pool of identified BXD enzymes represents a remarkable example of metabolic plasticity, in which BXDs are synthesized according to a similar chemical logic, but with an entirely different set of metabolic enzymes.

Biosynthesis, herbivore induction, and defensive role of phenylacetaldoxime glucoside.

Aldoximes are well-known metabolic precursors for plant defense compounds such as cyanogenic glycosides, glucosinolates, and volatile nitriles. They are also defenses themselves produced in response to herbivory; however, it is unclear whether aldoximes can be stored over a longer term as defense compounds and how plants protect themselves against the potential autotoxic effects of aldoximes. Here, we show that the Neotropical myrmecophyte tococa (Tococa quadrialata, recently renamed Miconia microphysca) accumulates phenylacetaldoxime glucoside (PAOx-Glc) in response to leaf herbivory. Sequence comparison, transcriptomic analysis, and heterologous expression revealed that 2 cytochrome P450 enzymes, CYP79A206 and CYP79A207, and the UDP-glucosyltransferase UGT85A123 are involved in the formation of PAOx-Glc in tococa. Another P450, CYP71E76, was shown to convert PAOx to the volatile defense compound benzyl cyanide. The formation of PAOx-Glc and PAOx in leaves is a very local response to herbivory but does not appear to be regulated by jasmonic acid signaling. In contrast to PAOx, which was only detectable during herbivory, PAOx-Glc levels remained high for at least 3 d after insect feeding. This, together with the fact that gut protein extracts of 3 insect herbivore species exhibited hydrolytic activity toward PAOx-Glc, suggests that the glucoside is a stable storage form of a defense compound that may provide rapid protection against future herbivory. Moreover, the finding that herbivory or pathogen elicitor treatment also led to the accumulation of PAOx-Glc in 3 other phylogenetically distant plant species suggests that the formation and storage of aldoxime glucosides may represent a widespread plant defense response.

Metabolism of plant-derived toxins from its insect host increases the success of the entomopathogenic fungus Beauveria bassiana.

Beauveria bassiana is a soil fungus that parasitizes a large number of arthropod species, including numerous crop pests, causing white muscardine disease and is therefore used as a biological insecticide. However, some insects, such as the cabbage aphid (Brevicoryne brassicae), defend themselves chemically by sequestering dietary pro-toxins (glucosinolates) from their Brassicales host plants. Glucosinolates are accumulated by cabbage aphids and activated to form toxic isothiocyanates when under attack. While isothiocyanate formation protects aphids against most attackers, B. bassiana is still able to infect the cabbage aphid under natural conditions. We therefore investigated how this fungus is able to circumvent the chemical defense system of the cabbage aphid. Here, we describe how B. bassiana infection activates the cabbage aphid defense system, but the resulting toxins are metabolized by B. bassiana via the mercapturic acid pathway, of which the first step is catalyzed by glutathione-S-transferases of low substrate specificity. This detoxification pathway enhances B. bassiana growth when isothiocyanates are present in natural concentrations, and so appears to be an important factor in fungal parasitization of these chemically defended aphids.

Ancient origin and conserved gene function in terpene pheromone and defense evolution of stink bugs and hemipteran 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.

Biosynthesis of iridoid sex pheromones in aphids.

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Alternative transcript splicing regulates UDP-glucosyltransferase-catalyzed detoxification of DIMBOA in the fall armyworm (Spodoptera frugiperda).

Herbivorous insects often possess the ability to detoxify chemical defenses from their host plants. The fall armyworm (Spodoptera frugiperda), which feeds principally on maize, detoxifies the maize benzoxazinoid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by stereoselective re-glucosylation using a UDP-glucosyltransferase, SfUGT33F28. SfUGT33F28 activity is induced by feeding on a DIMBOA-containing diet, but how this induction is regulated is unknown. In the present work, we describe the alternative splicing of the SfUGT33F28 transcript. Variant transcripts are differentially expressed in response to DIMBOA, and this transcriptional response is mediated by an insect aryl hydrocarbon receptor. These variants have large deletions leading to the production of truncated proteins that have no intrinsic UGT activity with DIMBOA but interact with the full-length enzyme to raise or lower its activity. Therefore, the formation of SfUGT33F28 splice variants induces DIMBOA-conjugating UGT activity when DIMBOA is present in the insect diet and represses activity in the absence of this plant defense compound.

Biosynthesis and antifungal activity of fungus-induced O-methylated flavonoids in maize.

Fungal infection of grasses, including rice (Oryza sativa), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), induces the formation and accumulation of flavonoid phytoalexins. In maize (Zea mays), however, investigators have emphasized benzoxazinoid and terpenoid phytoalexins, and comparatively little is known about flavonoid induction in response to pathogens. Here, we examined fungus-elicited flavonoid metabolism in maize and identified key biosynthetic enzymes involved in the formation of O-methylflavonoids. The predominant end products were identified as two tautomers of a 2-hydroxynaringenin-derived compound termed xilonenin, which significantly inhibited the growth of two maize pathogens, Fusarium graminearum and Fusarium verticillioides. Among the biosynthetic enzymes identified were two O-methyltransferases (OMTs), flavonoid OMT 2 (FOMT2), and FOMT4, which demonstrated distinct regiospecificity on a broad spectrum of flavonoid classes. In addition, a cytochrome P450 monooxygenase (CYP) in the CYP93G subfamily was found to serve as a flavanone 2-hydroxylase providing the substrate for FOMT2-catalyzed formation of xilonenin. In summary, maize produces a diverse blend of O-methylflavonoids with antifungal activity upon attack by a broad range of fungi.

Volatile emission and biosynthesis in endophytic fungi colonizing black poplar leaves.

Plant volatiles play a major role in plant-insect interactions as defense compounds or attractants for insect herbivores. Recent studies have shown that endophytic fungi are also able to produce volatiles and this raises the question of whether these fungal volatiles influence plant-insect interactions. Here, we qualitatively investigated the volatiles released from 13 endophytic fungal species isolated from leaves of mature black poplar (Populus nigra) trees. The volatile blends of these endophytes grown on agar medium consist of typical fungal compounds, including aliphatic alcohols, ketones and esters, the aromatic alcohol 2-phenylethanol and various sesquiterpenes. Some of the compounds were previously reported as constituents of the poplar volatile blend. For one endophyte, a species of Cladosporium, we isolated and characterized two sesquiterpene synthases that can produce a number of mono- and sesquiterpenes like (E)-ß-ocimene and (E)-ß-caryophyllene, compounds that are dominant components of the herbivore-induced volatile bouquet of black poplar trees. As several of the fungus-derived volatiles like 2-phenylethanol, 3-methyl-1-butanol and the sesquiterpene (E)-ß-caryophyllene, are known to play a role in direct and indirect plant defense, the emission of volatiles from endophytic microbial species should be considered in future studies investigating tree-insect interactions.

Identification of a Sulfatase that Detoxifies Glucosinolates in the Phloem-Feeding Insect Bemisia tabaci and Prefers Indolic Glucosinolates.

Cruciferous plants in the order Brassicales defend themselves from herbivory using glucosinolates: sulfur-containing pro-toxic metabolites that are activated by hydrolysis to form compounds, such as isothiocyanates, which are toxic to insects and other organisms. Some herbivores are known to circumvent glucosinolate activation with glucosinolate sulfatases (GSSs), enzymes that convert glucosinolates into inactive desulfoglucosinolates. This strategy is a major glucosinolate detoxification pathway in a phloem-feeding insect, the silverleaf whitefly Bemisia tabaci, a serious agricultural pest of cruciferous vegetables. In this study, we identified and characterized an enzyme responsible for glucosinolate desulfation in the globally distributed B. tabaci species MEAM1. In in vitro assays, this sulfatase showed a clear preference for indolic glucosinolates compared with aliphatic glucosinolates, consistent with the greater representation of desulfated indolic glucosinolates in honeydew. B. tabaci might use this detoxification strategy specifically against indolic glucosinolates since plants may preferentially deploy indolic glucosinolates against phloem-feeding insects. In vivo silencing of the expression of the B. tabaci GSS gene via RNA interference led to lower levels of desulfoglucosinolates in honeydew. Our findings expand the knowledge on the biochemistry of glucosinolate detoxification in phloem-feeding insects and suggest how detoxification pathways might facilitate plant colonization in a generalist herbivore.

Poplar protease inhibitor expression differs in an herbivore specific manner.

BACKGROUND: Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. RESULTS: We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. CONCLUSIONS: We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

Glucosylation prevents plant defense activation in phloem-feeding insects.

The metabolic adaptations by which phloem-feeding insects counteract plant defense compounds are poorly known. Two-component plant defenses, such as glucosinolates, consist of a glucosylated protoxin that is activated by a glycoside hydrolase upon plant damage. Phloem-feeding herbivores are not generally believed to be negatively impacted by two-component defenses due to their slender piercing-sucking mouthparts, which minimize plant damage. However, here we document that glucosinolates are indeed activated during feeding by the whitefly Bemisia tabaci. This phloem feeder was also found to detoxify the majority of the glucosinolates it ingests by the stereoselective addition of glucose moieties, which prevents hydrolytic activation of these defense compounds. Glucosylation of glucosinolates in B. tabaci was accomplished via a transglucosidation mechanism, and two glycoside hydrolase family 13 (GH13) enzymes were shown to catalyze these reactions. This detoxification reaction was also found in a range of other phloem-feeding herbivores.

The reconstruction and biochemical characterization of ancestral genes furnish insights into the evolution of terpene synthase function in the Poaceae.

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. 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.

The Occurrence of Sulfated Salicinoids in Poplar and Their Formation by Sulfotransferase1.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus × canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

The Fall Armyworm Spodoptera frugiperda Utilizes Specific UDP-Glycosyltransferases to Inactivate Maize Defensive Benzoxazinoids.

The relationship between plants and insects is continuously evolving, and many insects rely on biochemical strategies to mitigate the effects of toxic chemicals in their food plants, allowing them to feed on well-defended plants. Spodoptera frugiperda, the fall armyworm (FAW), accepts a number of plants as hosts, and has particular success on plants of the Poaceae family such as maize, despite their benzoxazinoid (BXD) defenses. BXDs stored as inert glucosides are converted into toxic aglucones by plant glucosidases upon herbivory. DIMBOA, the main BXD aglucone released by maize leaves, can be stereoselectively re-glucosylated by UDP-glycosyltransferases (UGTs) in the insect gut, rendering it non-toxic. Here, we identify UGTs involved in BXD detoxification by FAW larvae and examine how RNAi-mediated manipulation of the larval glucosylation capacity toward the major maize BXD, DIMBOA, affects larval growth. Our findings highlight the involvement of members of two major UGT families, UGT33 and UGT40, in the glycosylation of BXDs. Most of the BXD excretion in the frass occurs in the form of glucosylated products. Furthermore, the DIMBOA-associated activity was enriched in the gut tissue, with a single conserved UGT33 enzyme (SfUGT33F28) being dedicated to DIMBOA re-glucosylation in the FAW gut. The knock-down of its encoding gene reduces larval performance in a strain-specific manner. This study thus reveals that a single UGT enzyme is responsible for detoxification of the major maize-defensive BXD in this pest insect.

An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco.

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining, we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(-)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions. We used these lines to test ovipositing moths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but the magnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity.

A terpene synthase-cytochrome P450 cluster in Dictyostelium discoideum produces a novel trisnorsesquiterpene.

Terpenoids are enormously diverse, but our knowledge of their biosynthesis and functions is limited. Here we report on a terpene synthase (DdTPS8)-cytochrome P450 (CYP521A1) gene cluster that produces a novel C12 trisnorsesquiterpene and affects the development of Dictyostelium discoideum. DdTPS8 catalyzes the formation of a sesquiterpene discoidol, which is undetectable from the volatile bouquet of wild type D. discoideum. Interestingly, a DdTPS8 knockout mutant lacks not only discoidol, but also a putative trisnorsesquiterpene. This compound was hypothesized to be derived from discoidol via cytochrome P450 (CYP)-catalyzed oxidative cleavage. CYP521A1, which is clustered with DdTPS8, was identified as a top candidate. Biochemical assays demonstrated that CYP521A1 catalyzes the conversion of discoidol to a novel trisnorsesquiterpene named discodiene. The DdTPS8 knockout mutant exhibited slow progression in development. This study points to the untapped diversity of natural products made by D. discoideum, which may have diverse roles in its development and chemical ecology.

Author Correction: An IDS-Type Sesquiterpene Synthase Produces the Pheromone Precursor (Z)-α-Bisabolene in Nezara viridula.

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.