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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.

Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development., (© 2024. The Author(s).)

Phenylphenalenones (PhPNs), phytoalexins in wild bananas (Musaceae), are known to act against various pathogens. However, the abundance of PhPNs in many Musaceae plants of economic importance is low. Knowledge of the biosynthesis of PhPNs and the application of biosynthetic approaches to improve their yield is vital for fighting banana diseases. However, the processes of PhPN biosynthesis, especially those involved in methylation modification, remain unclear. Musella lasiocarpa is a herbaceous plant belonging to Musaceae, and due to the abundant PhPNs, their biosynthesis in M. lasiocarpa has been the subject of much attention. In this study, we assembled a telomere-to-telomere gapless genome of M. lasiocarpa as the reference, and further integrated transcriptomic and metabolomic data to mine the candidate genes involved in PhPN biosynthesis. To elucidate the diversity of PhPNs in M. lasiocarpa , three screened O -methyltransferases (Ml01G0494, Ml04G2958, and Ml08G0855) by phylogenetic and expressional clues were subjected to in vitro enzymatic assays. The results show that the three were all novel O -methyltransferases involved in the biosynthesis of PhPN phytoalexins, among which Ml08G0855 was proved to function as a multifunctional enzyme targeting multiple hydroxyl groups in PhPN structure. Moreover, we tested the antifungal activity of PhPNs against Fusarium oxysporum and found that the methylated modification of PhPNs enhanced their antifungal activity. These findings provide valuable genetic resources in banana breeding and lay a foundation for improving disease resistance through molecular breeding., Competing Interests: The authors declare that there are no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Larvae of the Salicaceae-adapted Notodontidae have developed a unique mechanism to metabolize the chemical defenses of their Salicaceae host plants. Salicinoids and salicortinoids are enzymatically transformed into salicyloyl, benzoyl and mixed salicyloyl-benzoyl quinates. The source of quinates and benzoates was previously unknown. To elucidate the origin of quinate and benzoate in the metabolic end-products, we fed Cerura vinula caterpillars with 13 C-labelled poplar defense compounds. Caffeoylquinic acids (CQAs), such as chlorogenic acid, neochlorogenic acid and their methyl esters, were identified as the source of quinates in the caterpillar's metabolism. Benzoyl substituents in the quinate end-products were found to originate from compounds such as tremulacin or trichocarpin. Salicaceae-adapted Notodontidae caterpillars have the ability to overcome their host plant's chemical defense by metabolizing CQAs and salicinoids, both abundant defense compounds in Salicacea plants, by a strategy of transformation and recombination. We believe that our study opens up avenues for understanding salicortinoid biotransformation at the enzymatic level., (© 2023. The Author(s).)

Phenylphenalenones (PPs) are phytoalexins protecting banana plants (Musaceae) against various pathogens. However, how plants synthesize PPs is still poorly understood. In this work, we investigated the major secondary metabolites of developing seed coats of Musella lasiocarpa to determine if this species might be a good model system to study the biosynthesis of PPs. We found that PPs are major components of M. lasiocarpa seed coats at middle and late developmental stages. Two previously undescribed PP dimers ( M-4 and M-6 ) and a group of unreported diarylheptanoid (DH) derivatives named musellins A-F ( B-7 , B-9 , B-10 , B-12 , B-14 , and B-15 ) were isolated along with 14 known compounds. Musellin D ( B-12 ) and musellin F ( B-15 ) contain the first reported furo[3,2- c ]pyran ring and represent a previously undescribed carbon skeleton. The chemical structures of all new compounds were characterized by spectroscopic data, including NMR, HRESIMS, and ECD analysis. Plausible biosynthetic pathways for the formation of PPs and DHs are proposed.

Lepidopteran specialist herbivores of the Notodontidae family have adapted to thrive on poplar and willow species (Salicaceae). Previous research showed that Cerura vinula, a member of the Notodontidae family occurring throughout Europe and Asia, uses a unique mechanism to transform salicortinoids, the host plant's defense compounds, into quinic acid-salicylate conjugates. However, how the production of this conjugates relates to the detoxification of salicortinoids and how this transformation proceeds mechanistically have remained unknown. To find the mechanisms, we conducted gut homogenate incubation experiments with C. vinula and re-examined its metabolism by analyzing the constituents of its frass. To estimate the contribution of spontaneous degradation, we examined the chemical stability of salicortinoids and found that salicortinoids were degraded very quickly by midgut homogenates and that spontaneous degradation plays only a marginal role in the metabolism. We learned how salicortinoids are transformed into salicylate after we discovered reductively transformed derivatives, which were revealed to play key roles in the metabolism. Unless they have undergone the process of reduction, salicortinoids produce toxic catechol. We also studied constituents in the frass of the Notodontidae species Cerura erminea, Clostera anachoreta, Furcula furcula, Notodonta ziczac, and Pheosia tremula, and found the same metabolites as those described for C. vinula. We conclude that the process whereby salicortinoids are reductively transformed represents an important adaption of the Notodontidae to their Salicaceae host species., (© 2023. The Author(s).)

Cardiac glycosides are a large class of secondary metabolites found in plants. In the genus Asclepias , cardenolides in milkweed plants have an established role in plant-herbivore and predator-prey interactions, based on their ability to inhibit the membrane-bound Na + /K + -ATPase enzyme. Milkweed seeds are eaten by specialist lygaeid bugs, which are the most cardenolide-tolerant insects known. These insects likely impose natural selection for the repeated derivatisation of cardenolides. A first step in investigating this hypothesis is to conduct a phytochemical profiling of the cardenolides in the seeds. Here, we report the concentrations of 10 purified cardenolides from the seeds of Asclepias curassavica . We report the structures of new compounds: 3- O - β -allopyranosyl coroglaucigenin ( 1 ), 3-[4'- O - β -glucopyranosyl- β -allopyranosyl] coroglaucigenin ( 2 ), 3'- O - β -glucopyranosyl-15- β -hydroxycalotropin ( 3 ), and 3- O - β -glucopyranosyl-12- β -hydroxyl coroglaucigenin ( 4 ), as well as six previously reported cardenolides ( 5 - 10 ). We test the in vitro inhibition of these compounds on the sensitive porcine Na + /K + -ATPase. The least inhibitory compound was also the most abundant in the seeds-4'- O - β -glucopyranosyl frugoside ( 5 ). Gofruside ( 9 ) was the most inhibitory. We found no direct correlation between the number of glycosides/sugar moieties in a cardenolide and its inhibitory effect. Our results enhance the literature on cardenolide diversity and concentration among tissues eaten by insects and provide an opportunity to uncover potential evolutionary relationships between tissue-specific defense expression and insect adaptations in plant-herbivore interactions.

A precursor-directed approach to access N-hydroxyalkyl phenylbenzoisoquinolindiones (PBIQs) has been developed. Incubation of plant material of Xiphidium caeruleum with hydroxylamines of various chain lengths (C 2 , C 4 , C 6 , C 8 , C 10 and C 12 ) resulted in 11 new 5-hydroxy- and 5-methoxy PBIQs with different N-hydroxyalkyl side chain lengths. The antiproliferative effect and the cytotoxicity against HUVEC, K-562, and HeLa cell lines of 26 previously reported PBIQs and the 11 newly synthesized N-hydroxyalkyl PBIQs was determined for the first time. The results revealed that introducing long-chain N-aliphatic amine moieties improved the antiproliferative effect and cytotoxicity of PBIQs when compared to derivatives with N-amino acids as side chains., (© 2022 The Authors. Published by Wiley-VCH GmbH.)

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudoaspidosperma alkaloids pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors.

Deploying toxins in complex mixtures is thought to be advantageous and is observed during antagonistic interactions in nature. Toxin mixtures are widely utilized in medicine and pest control, as they are thought to slow the evolution of detoxification counterresponses in the targeted organisms. Here we show that caterpillars rearrange key constituents of two distinct plant defense pathways to postingestively disable the defensive properties of both pathways. Specifically, phenolic esters of quinic acid, chlorogenic acids (CAs), potent herbivore and ultraviolet (UV) defenses, are reesterified to decorate particular sugars of 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) and prevent their respective anti–herbivore defense functions. This was discovered through the employment of comparative metabolomics of the leaves of Nicotiana attenuata and the frass of this native tobacco’s specialist herbivore, Manduca sexta larvae. Feeding caterpillars on leaves of transgenic plants abrogated in each of the two pathways, separately and together, revealed that one of the fully characterized frass conjugates, caffeoylated HGL-DTG, originated from ingested CA and HGL-DTGs and that both had negative effects on the defensive function of the other compound class, as revealed by rates of larval mass gain. This negative defensive synergy was further explored in 183 N. attenuata natural accessions, which revealed a strong negative covariance between the two defense pathways. Further mapping analyses in a biparental recombinant inbred line (RIL) population imputed quantitative trait loci (QTLs) for the two pathways at distinct genomic locations. The postingestive repurposing of defense metabolism constituents reveals a downside of deploying toxins in mixtures, a downside which plants in nature have evolved to counter.

Pyrethrum pulchrum is a rare Mongolian plant species that has been traditionally used as an ingredient in various remedies. Bioactivity-guided fractionation performed on the methanol extract of its aerial parts led to the isolation of 2 previously undescribed guaianolide-type sesquiterpene lactones, namely 1 β ,10 β -epoxy-8 α -hydroxyguaia-3,11(13)-dien-6,12-olide (1: ) and 1,8,10-trihydroxyguaia-3,11(13)-dien-6,12-olide (2: ), along with the isolation or chromatographic identification of 11 compounds, arglabin (3: ), 3 β -hydroxycostunolide (4: ), isocostic acid (5: ), ( E )-9-(2-thienyl)-6-nonen-8-yn-3-ol (6: ), ( Z )-9-(2-thienyl)-6-nonen-8-yn-3-ol (7: ), N 1 , N 5 , N 10 , N 14 -tetra-p-coumaroyl spermine (8: ), chlorogenic acid (9: ), 3,5-di- O -caffeoylquinic acid (10: ), 3,5-di- O -caffeoylquinic acid methyl ester (11: ), 3,4-di- O -caffeoylquinic acid (12: ), and tryptophan (13: ). Their structures were assigned based on spectroscopic and spectrometric data. The antimicrobial, antiproliferative and cytotoxic activities of selected compounds were evaluated. The new compounds showed weak to moderate antimicrobial activity. Arglabin (3: ), the major sesquiterpene lactone found in the methanol extract of P. pulchrum , exhibited the highest activity against human cancer lines, while compound 1: also possesses significant antiproliferative activity against leukemia cells., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Although much is known about plant traits that function in nonhost resistance against pathogens, little is known about nonhost resistance against herbivores, despite its agricultural importance. Empoasca leafhoppers, serious agricultural pests, identify host plants by eavesdropping on unknown outputs of jasmonate (JA)-mediated signaling. Forward- and reverse-genetics lines of a native tobacco plant were screened in native habitats with native herbivores using high-throughput genomic, transcriptomic, and metabolomic tools to reveal an Empoasca -elicited JA-JAZi module. This module induces an uncharacterized caffeoylputrescine-green leaf volatile compound, catalyzed by a polyphenol oxidase in a Michael addition reaction, which we reconstitute in vitro; engineer in crop plants, where it requires a berberine bridge enzyme-like 2 (BBL2) for its synthesis; and show that it confers resistance to leafhoppers. Natural history-guided forward genetics reveals a conserved nonhost resistance mechanism useful for crop protection.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Phytochemical investigation of the ethanol extract of underground parts of Iris tenuifolia Pall. afforded five new compounds; an unusual macrolide termed moniristenulide ( 1 ), 5-methoxy-6,7-methylenedioxy-4- O -2'-cycloflavan ( 2 ), 5,7,2',3'-tetrahydroxyflavanone ( 3 ), 5-hydroxy-6,7-dimethoxyisoflavone-2'- O - β -d-glucopyranoside ( 9 ), 5,2',3'-dihydroxy-6,7-dimethoxyisoflavone ( 10 ), along with seven known compounds ( 4 - 8 , 11 - 12 ). The structures of all purified compounds were established by analysis of 1D and 2D NMR spectroscopy and HR-ESI-MS. The antimicrobial activity of the compounds 1 - 3 , 5 , 9 , and 10 was investigated using the agar diffusion method against fungi, Gram-positive and Gram-negative bacteria. In consequence, new compound 3 was found to possess the highest antibacterial activity against Enterococcus faecalis VRE and Mycobacterium vaccae . Cell proliferation and cytotoxicity tests were also applied on all isolated compounds and plant crude extract in vitro with the result of potent inhibitory effect against leukemia cells. In particular, the newly discovered isoflavone 10 was active against both of the leukemia cells K-562 and THP-1 while 4 - 6 of the flavanone type compounds were active against only THP-1.

Several medical plants belonging to the genera Passiflora , Viola , and Crataegus accumulate flavonoid C-glycosides, which likely contribute to their efficacy. Information regarding their phase I and II metabolism in the liver are lacking. Thus, in vitro liver metabolism of orientin, isoorientin, schaftoside, isoschaftoside, vitexin, and isovitexin, all of which accumulated in Passiflora incarnata L., was investigated by incubation in subcellular systems with human liver microsomes and human liver S9 fraction. All metabolite profiles were comprehensively characterized using HPLC-DAD and UHPLC-MS/MS analysis. Mono-glycosylic flavones of the luteolin-type orientin and isoorientin showed a broad range of mono-glucuronidated and mono-sulfated metabolites, whereas for mono-glycosylic flavones of the apigenin-type vitexin and isovitexin, only mono-glucuronidates could be detected. For di-glycosylic flavones of the apigenin-type schaftosid and isoschaftosid, no phase I or II metabolites were identified. The main metabolite of isoorientin was isolated using solid-phase extraction and prep. HPLC-DAD and identified as isoorientin-3'- O -α-glucuronide by NMR analysis. A second isolated glucuronide was assigned as isoorientin 4'- O -α-glucuronide. These findings indicate that vitexin and isovitexin are metabolized preferentially by uridine 5'-diphospho glucuronosyltransferases (UGTs) in the liver. As only orientin and isoorientin showed mono-sulfated and mono-glucuronidated metabolites, the dihydroxy group in 3',4'-position may be essential for additional sulfation by sulfotransferases (SULTs) in the liver. The diglycosylic flavones schaftoside and isoschaftoside are likely not accepted as substrates of the used liver enzymes under the chosen conditions.

Gut enzymes can metabolize plant defense compounds and thereby affect the growth and fitness of insect herbivores. Whether these enzymes also influence feeding preference is largely unknown. We studied the metabolization of taraxinic acid β-D-glucopyranosyl ester (TA-G), a sesquiterpene lactone of the common dandelion ( Taraxacum officinale ) that deters its major root herbivore, the common cockchafer larva ( Melolontha melolontha ). We have demonstrated that TA-G is rapidly deglucosylated and conjugated to glutathione in the insect gut. A broad-spectrum M. melolontha β-glucosidase, Mm_bGlc17, is sufficient and necessary for TA-G deglucosylation. Using cross-species RNA interference, we have shown that Mm_bGlc17 reduces TA-G toxicity. Furthermore, Mm_bGlc17 is required for the preference of M. melolontha larvae for TA-G-deficient plants. Thus, herbivore metabolism modulates both the toxicity and deterrence of a plant defense compound. Our work illustrates the multifaceted roles of insect digestive enzymes as mediators of plant-herbivore interactions., Competing Interests: MH, TR, SI, AR, SG, JF, PR, MR, CP, NL, LH, ZB, YM, WH, CR, JG, ME No competing interests declared, (© 2021, Huber et al.)

Two-component plant defenses such as cyanogenic glucosides are produced by many plant species, but phloem-feeding herbivores have long been thought not to activate these defenses due to their mode of feeding, which causes only minimal tissue damage. Here, however, we report that cyanogenic glycoside defenses from cassava (Manihot esculenta), a major staple crop in Africa, are activated during feeding by a pest insect, the whitefly Bemisia tabaci, and the resulting hydrogen cyanide is detoxified by conversion to beta-cyanoalanine. Additionally, B. tabaci was found to utilize two metabolic mechanisms to detoxify cyanogenic glucosides by conversion to non-activatable derivatives. First, the cyanogenic glycoside linamarin was glucosylated 1-4 times in succession in a reaction catalyzed by two B. tabaci glycoside hydrolase family 13 enzymes in vitro utilizing sucrose as a co-substrate. Second, both linamarin and the glucosylated linamarin derivatives were phosphorylated. Both phosphorylation and glucosidation of linamarin render this plant pro-toxin inert to the activating plant enzyme linamarase, and thus these metabolic transformations can be considered pre-emptive detoxification strategies to avoid cyanogenesis.

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins in Papaver nudicaule L. (Iceland poppy) are unique due to their rarity and unparalleled flavoalkaloid structure. Nudicaulins are derived from pelargonidin glycoside and indole, products of the flavonoid and indole/tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, differential gel electrophoresis (DIGE)-based proteome, and ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based metabolome data of P. nudicaule petals with chemical investigations. We identified candidate genes and proteins for all biosynthetic steps as well as some key metabolites across five stages of petal development. Candidate genes of amino acid biosynthesis showed a relatively stable expression throughout petal development, whereas most candidate genes of flavonoid biosynthesis showed increasing expression during development followed by downregulation in the final stage. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were co-expressed with flavonoid biosynthesis genes, and are probably providing free indole. The fusion of indole with pelargonidin glycosides was retraced synthetically and promoted by high precursor concentrations, an excess of indole, and a specific glycosylation pattern of pelargonidin. Thus, nudicaulin biosynthesis combines the enzymatic steps of two different pathways with a spontaneous fusion of indole and pelargonidin glycoside under precisely tuned reaction conditions.

The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase (CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3 . Additionally, the discovery of an O -methyltransferase specific for 6'-hydroxycinchoninone 4 suggested the final step order to be cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.

Some plant species are less susceptible to herbivore infestation than others. The reason for this is often unknown in detail but is very likely due to an efficient composition of secondary plant metabolites. Strikingly, carnivorous plants of the genus Nepenthes show extremely less herbivory both in the field and in green house. In order to identify the basis for the efficient defense against herbivorous insects in Nepenthes, we performed bioassays using larvae of the generalist lepidopteran herbivore, Spodoptera littoralis. Larvae fed with different tissues from Nepenthes x ventrata grew significantly less when feeding on a diet containing leaf tissue compared with pitcher-trap tissue. As dominating metabolite in Nepenthes tissues, we identified a naphthoquinone, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone). When plumbagin was added at different concentrations to the diet of S. littoralis larvae, an EC 50 value for larval growth inhibition was determined with 226.5 µg g -1 diet. To further determine the concentration causing higher larval mortality, sweet potato leaf discs were covered with increasing plumbagin concentrations in no-choice-assays; a higher mortality of the larvae was found beyond 60 µg plumbagin per leaf, corresponding to 750 µg g -1 . Plant-derived insecticides have long been proposed as alternatives for pest management; plumbagin and derivatives might be such promising environmentally friendly candidates.

Olive (Olea europaea) is an important crop in Europe, with high cultural, economic and nutritional significance. Olive oil flavor and quality depend on phenolic secoiridoids, but the biosynthetic pathway of these iridoids remains largely uncharacterized. We discovered two bifunctional cytochrome P450 enzymes, catalyzing the rare oxidative C-C bond cleavage of 7-epi-loganin to produce oleoside methyl ester (OeOMES) and secoxyloganin (OeSXS), both through a ketologanin intermediary. Although these enzymes are homologous to the previously reported Catharanthus roseus secologanin synthase (CrSLS), the substrate and product profiles differ. Biochemical assays provided mechanistic insights into the two-step OeOMES and CrSLS reactions. Model-guided mutations of OeOMES changed the product profile in a predictable manner, revealing insights into the molecular basis for this change in product specificity. Our results suggest that, in contrast to published hypotheses, in planta production of secoxy-iridoids is secologanin-independent. Notably, sequence data of cultivated and wild olives point to a relation between domestication and OeOMES expression. Thus, the discovery of this key biosynthetic gene suggests a link between domestication and secondary metabolism, and could potentially be used as a genetic marker to guide next-generation breeding programs., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Foundation.)

Wood is a habitat for a variety of organisms, including saprophytic fungi and bacteria, playing an important role in wood decomposition. Wood inhabiting fungi release a diversity of volatiles used as signaling compounds to attract or repel other organisms. Here, we show that volatiles of Schizophyllum commune are active against wood-decay fungi and bacteria found in its mycosphere. We identified sesquiterpenes as the biologically active compounds, that inhibit fungal growth and modify bacterial motility. The low number of cultivable wood inhabiting bacteria prompted us to analyze the microbial community in the mycosphere of S. commune using a culture-independent approach. Most bacteria belong to Actinobacteria and Proteobacteria, including Pseudomonadaceae, Sphingomonadaceae, Erwiniaceae, Yersiniaceae and Mariprofundacea as the dominating families. In the fungal community, the phyla of ascomycetes and basidiomycetes were well represented. We propose that fungal volatiles might have an important function in the wood mycosphere and could meditate interactions between microorganisms across domains and within the fungal kingdom., Competing Interests: The authors have declared that no competing interests exist.

Many plant specialized metabolites function in herbivore defense, and abrogating particular steps in their biosynthetic pathways frequently causes autotoxicity. However, the molecular mechanisms underlying their defense and autotoxicity remain unclear. Here, we show that silencing two cytochrome P450s involved in diterpene biosynthesis in the wild tobacco Nicotiana attenuata causes severe autotoxicity symptoms that result from the inhibition of sphingolipid biosynthesis by noncontrolled hydroxylated diterpene derivatives. Moreover, the diterpenes' defensive function is achieved by inhibiting herbivore sphingolipid biosynthesis through postingestive backbone hydroxylation products. Thus, by regulating metabolic modifications, tobacco plants avoid autotoxicity and gain herbivore defense. The postdigestive duet that occurs between plants and their insect herbivores can reflect the plant's solutions to the "toxic waste dump" problem of using potent chemical defenses., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Two novel lathyrane-type diterpenoids, the Euphorbia factors L 2a (1) and L 2b (2), and their stereoisomer Euphorbia factor L 2 (3) were obtained from seeds of Euphorbia lathyris . Both Euphorbia factors L 2a and L 2b possess an unprecedented trans-gem -dimethylcyclopropane as structural feature. Also, the Euphorbia factor L 2a is the first example of a lathyrane diterpenoid with an endocyclic 12( Z )-double bond. The structures of the molecules and their absolute configurations were elucidated by comprehensive spectroscopic analyses, Cu-Kα radiation X-ray diffraction, and comparison with calculated electronic circular dichroism (ECD) data. The Euphorbia factor L 2b exhibited an inhibitory effect against U937 cell line with an IC 50 value of 0.87 μM., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Boswellic acids, and particularly 11-keto-boswellic acids, triterpenoids derived from the genus Boswellia ( Burseraceae ), are known for their anti-inflammatory and potential antitumor efficacy. Although boswellic acids generally occur as α-isomers (oleanane type) and β-isomers (ursane type), 11-keto-boswellic acid (KBA) was found only as the β-isomer, β-KBA. Here, the existence and natural occurrence of the respective α-isomer, 11-keto-α-boswellic acid (α-KBA), is demonstrated for the first time. Initially, α-KBA was synthesized and characterized by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy, and a highly selective, sensitive, and accurate high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed by Design of Experiments (DoE) using a pentafluorophenyl stationary phase. This method allowed the selective quantification of individual 11-keto-boswellic acids and provided evidence for α-KBA in Boswellia spp. oleogum resins. The contents of α-KBA as well as further boswellic acids and the composition of essential oils were used to chemotaxonomically classify 41 Boswellia oleogum resins from 9 different species. Moreover, α-KBA exhibited cytotoxicity against three treatment-resistant triple-negative breast cancer (TNBC) cell lines in vitro and also induced apoptosis in MDA-MB-231 xenografts in vivo. The respective β-isomer and the acetylated form demonstrate higher cytotoxic efficacies against TNBC cells. This provides further insights into the structure-activity relationship of boswellic acids and could support future developments of potential anti-inflammatory and antitumor drugs.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Israni, Wouters, Luck, Seibel, Ahn, Paetz, Reinert, Vogel, Erb, Heckel, Gershenzon and Vassão.)

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.

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes with a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families secoiridoids, coumarins, flavonoids, phenylethanoids, and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations, thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Comparative ascaroside profiling of Caenorhabditis nematodes using HPLC-ESI-(-)-MS/MS precursor ion scanning revealed a class of highly species-specific ascaroside dimers. Their 2- and 4-isomeric, homo- and heterodimeric structures were identified using a combination of HPLC-ESI-(+)-HR-MS/MS spectrometry and high-resolution dqf-COSY NMR spectroscopy. Structure assignments were confirmed by total synthesis of representative examples. Functional characterization using holding assays indicated that males of Caenorhabditis remanei and Caenorhabditis nigoni are exclusively retained by their conspecific ascaroside dimers, demonstrating that dimerization of conserved monomeric building blocks represents a yet undescribed mechanism that generates species-specific signaling molecules in the Caenorhabditis genus.

Triple negative human breast cancer (TNBC) is an aggressive cancer subtype with poor prognosis. Besides the better-known artemisinin, Artemisia annua L. contains numerous active compounds not well-studied yet. High-performance liquid chromatography coupled with diode-array and mass spectrometric detection (HPLC-DAD-MS) was used for the analysis of the most abundant compounds of an Artemisia annua extract exhibiting toxicity to MDA-MB-231 TNBC cells. Artemisinin, 6,7-dimethoxycoumarin, arteannuic acid were not toxic to any of the cancer cell lines tested. The flavonols chrysosplenol d and casticin selectively inhibited the viability of the TNBC cell lines, MDA-MB-231, CAL-51, CAL-148, as well as MCF7, A549, MIA PaCa-2, and PC-3. PC-3 prostate cancer cells exhibiting high basal protein kinase B (AKT) and no ERK1/2 activation were relatively resistant, whereas MDA-MB-231 cells with high basal ERK1/2 and low AKT activity were more sensitive to chrysosplenol d treatment. In vivo, chrysosplenol d and casticin inhibited MDA-MB-231 tumor growth on chick chorioallantoic membranes. Both compounds induced mitochondrial membrane potential loss and apoptosis. Chrysosplenol d activated ERK1/2, but not other kinases tested, increased cytosolic reactive oxygen species (ROS) and induced autophagy in MDA-MB-231 cells. Lysosomal aberrations and toxicity could be antagonized by ERK1/2 inhibition. The Artemisia annua flavonols chrysosplenol d and casticin merit exploration as potential anticancer therapeutics.

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., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Belowground (BG) herbivory can influence aboveground (AG) herbivore performance and food preference via changes in plant chemistry. Most evidence for this phenomenon derives from studies in herbaceous plants but studies in woody plants are scarce. Here we investigated whether and how BG herbivory on black poplar (Populus nigra) trees by Melolontha melolontha larvae influences the feeding preference of Lymantria dispar (gypsy moth) caterpillars. In a food choice assay, caterpillars preferred to feed on leaves from trees that had experienced attack by BG herbivores. Therefore, we investigated the effect of BG herbivory on the phytochemical composition of P. nigra trees alone and in combination with AG feeding by L. dispar caterpillars. BG herbivory did not increase systemic AG tree defences like volatile organic compounds, protease inhibitors and salicinoids. Jasmonates and salicylic acid were also not induced by BG herbivory in leaves but abscisic acid concentrations drastically increased together with proline and few other amino acids. Leaf coating experiments with amino acids suggest that proline might be responsible for the caterpillar feeding preference via presumptive phagostimulatory properties. This study shows that BG herbivory in poplar can modify the feeding preference of AG herbivores via phytochemical changes as a consequence of root-to-shoot signaling., (© 2019 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Three previously undescribed compounds, two prenyleudesmanes ( 1 and 2 ), and one hexanorlanostane ( 3 ), were isolated from the roots of Lonicera macranthoides . Their structures were established based on 1D and 2D nuclear magnetic resonance (NMR) spectra and high-resolution electrospray ionization mass spectral (HR-ESI-MS) data. The absolute configurations of 1 and 3 were determined by X-ray diffraction. To the best of our knowledge, this is the first time that the absolute configuration of a prenyleudesmane with a trans -decalin system and a hexanorlanostane have been unambiguously confirmed by single-crystal X-ray diffraction with Cu Kα radiation. Thecompounds were tested for their antiproliferative activity on the cancer cell lines (HepG2 and HeLa). The compounds 1 - 3 exhibited moderate inhibitory effects against two human cancer cell lines.

Larvae of the leaf beetle Phaedon cochleariae synthesize the iridoid chysomelidial via the mevalonate pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated defensive pathway by the ω-hydroxylation of geraniol to (2E,6E)-2,6-dimethylocta-2,6-diene-1,8-diol (ω-OH-geraniol). Here we identify and characterize the P450 monooxygenase CYP6BH5 as the geraniol hydroxylase using integrated transcriptomics, proteomics and RNA interference (RNAi). In the fat body, 73 cytochrome P450s were identified, and CYP6BH5 was among those that were expressed specifically in fat body. Double stranded RNA mediated knockdown of CYP6BH5 led to a significant reduction of ω-hydroxygeraniol glucoside in the hemolymph and, later, of the chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted geraniol to ω-OH-geraniol. In addition to geraniol, CYP6BH5 also catalyzes hydroxylation of other monoterpenols, such as nerol and citronellol to the corresponding α,ω-dihydroxy compounds., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Artemisia annua L. has gained increasing attention for its anticancer activity. However, beside artemisinin, less is known about the possible bioactive ingredients of Artemisia annua and respective herbal preparations. We hypothesized that, in addition to artemisinin, Artemisia annua preparations might contain multiple ingredients with potential anticancer activity., Methods: MDA-MB-231 triple negative human breast cancer (TNBC) cells along with other treatment resistant, metastatic cancer cell lines were used to investigate in vitro and in vivo the anticancer efficacy of an Artemisia annua extract marketed as a herbal preparation, which contained no detectable artemisinin (limit of detection = 0.2 ng/mg). The extract was characterized by HPLC-DAD and the most abundant compounds were identified by 1 H- and 13 C NMR spectroscopy and quantified by UHPLC-MS/MS. Cell viability and various apoptotic parameters were quantified by flow cytometry. In vitro data were validated in two in vivo cancer models, the chick chorioallantoic membrane (CAM) assay and in orthotopic breast cancer xenografts in nude mice., Results: The Artemisia annua extract, the activity of which could be enhanced by acetonitrile maceration, inhibited the viability of breast (MDA-MB-231 and MCF-7), pancreas (MIA PaCa-2), prostate (PC-3), non-small cell lung cancer (A459) cells, whereas normal mammary epithelial cells, lymphocytes, and PBMC were relatively resistant to extract treatment. Likewise, the extract's most abundant ingredients, chrysosplenol D, arteannuin B, and casticin, but not arteannuic acid or 6,7-dimethoxycoumarin, inhibited the viability of MDA-MB-231 breast cancer cells. The extract induced accumulation of multinucleated cancer cells within 24 h of treatment, increased the number of cells in the S and G 2 /M phases of the cell cycle, followed by loss of mitochondrial membrane potential, caspase 3 activation, and formation of an apoptotic hypodiploid cell population. Further, the extract inhibited cancer cell proliferation, decreased tumor growth, and induced apoptosis in vivo in TNBC MDA-MB-231 xenografts grown on CAM as well as in nude mice., Conclusion: An extract of an artemisinin-deficient Artemisia annua herbal preparation exhibits potent anticancer activity against triple negative human breast cancer. New active ingredients of Artemisia annua extract with potential anticancer activity have been identified., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

A novel class of species-specific modular ascarosides that integrate additional fatty acid building blocks was characterized in the nematode Caenorhabditis remanei using a combination of HPLC-ESI-(-)-MS/MS precursor ion scanning, microreactions, HR-MS/MS, MS n , and NMR techniques. The structure of the dominating component carrying a cyclopropyl fatty acid moiety was established by total synthesis. Biogenesis of this female-produced male attractant depends on cyclopropyl fatty acid synthase ( cfa ), which is expressed in bacteria upon entering their stationary phase.

We investigated the organ-specific phytochemistry of the inflorescences, leaves at different stages of senescence, and roots of Xiphidium caeruleum (Haemodoraceae) and elucidated the structure of six undescribed compounds. Among these, a phenylcarbamoylnaphthoquinone (PCNQ), representing the first member of a class of undescribed phenylphenalenone-derived nitrogenous compounds, was identified and its spontaneous formation elaborated. Starting from phenylbenzoisochromenone glucosides, the reaction cascade proceeds through oxidative decarboxylation and several oxidation steps to an anhydride, which is further converted to a carboxy-phenylnaphthoquinone. In the presence of amino acids, this carboxy-phenylnaphthoquinone readily reacts to PCNQs. Hence, the carboxy-phenylnaphthoquinone was hypothesized to be involved in plant defense because of its reactivity towards amino acids. It was also hypothesized that reduced levels of the corresponding glucosidic phenylbenzoisochromenone precursors in older leaves may foster pathogen-driven senescence., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceA Y51 ) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Nudicaulins are yellow flower pigments accounting for the color of the petals of Papaver nudicaule (Papaveraceae). These glucosidic compounds belong to the small group of indole/flavonoid hybrid alkaloids. Here we describe in vivo and in vitro experiments which substantiate the strongly pH-dependent conversion of pelargonidin glucosides to nudicaulins as the final biosynthetic step of these alkaloids. Furthermore, we report the first synthesis of nudicaulin aglycon derivatives, starting with quercetin and ending up at the biomimetic fusion of a permethylated anthocyanidin with indole. A small library of nudicaulin derivatives with differently substituted indole units was prepared, and the antimicrobial, antiproliferative and cell toxicity data of the new compounds were determined. The synthetic procedure is considered suitable for preparing nudicaulin derivatives which are structurally modified in the indole and/or the polyphenolic part of the molecule and may have optimized pharmacological activities.

The cabbage stem flea beetle ( Psylliodes chrysocephala) is a key pest of oilseed rape in Europe, and is specialized to feed on Brassicaceae plants armed with the glucosinolate-myrosinase defense system. Upon tissue damage, the β-thioglucosidase enzyme myrosinase hydrolyzes glucosinolates (GLS) to form toxic isothiocyanates (ITCs) which deter non-adapted herbivores. Here, we show that P. chrysocephala selectively sequester GLS from their host plants and store these throughout their life cycle. In addition, P. chrysocephala metabolize GLS to desulfo-GLS, which implies the evolution of GLS sulfatase activity in this specialist. To assess whether P. chrysocephala can largely prevent GLS hydrolysis in ingested plant tissue by sequestration and desulfation, we analyzed the metabolic fate of 4-methylsulfinylbutyl (4MSOB) GLS in adults. Surprisingly, intact and desulfo-GLS together accounted for the metabolic fate of only 26% of the total ingested GLS in P. chrysocephala , indicating that most ingested GLS are nevertheless activated by the plant myrosinase. The presence of 4MSOB-ITC and the corresponding nitrile in feces extracts confirmed the activation of ingested GLS, but the detected amounts of unmetabolized ITCs were low. P. chrysocephala partially detoxifies ITCs by conjugation with glutathione via the conserved mercapturic acid pathway. In addition to known products of the mercapturic acid pathway, we identified two previously unknown cyclic metabolites derived from the cysteine-conjugate of 4MSOB-ITC. In summary, the cabbage stem flea beetle avoids ITC formation by specialized strategies, but also relies on and extends the conserved mercapturic acid pathway to prevent toxicity of formed ITCs.

The regulator of G-protein signalling, Thn1, is involved in sexual development through pheromone signalling in the mushroom forming basidiomycete Schizophyllum commune affecting hyphal morphology and mating interactions. Thn1 plays a key role in coordinating sesquiterpene production, pheromone response and sexual development. The gene thn1 is transcriptionally regulated in response to mating with a role in clamp cell development and hydrophobin gene transcription. Further, it negatively regulates cAMP signalling and secondary metabolism. Disruption of thn1 affects dikaryotization by reducing clamp fusion and development with predominant non-fused pseudoclamps. Enhanced protein kinase A (PKA) activities in Δthn1 strains indicate that Thn1 regulates pheromone signalling by de-activating G-protein α subunits, which control cAMP-dependent PKA. The repressed formation of aerial hyphae could be linked to a reduced metabolic activity and to a transcriptional down-regulation of hyd6 and sc3 hydrophobin genes. Thn1 was also shown to be necessary for the biosynthesis of sesquiterpenes and an altered spectrum of sesquiterpenes in Δthn1 is linked to transcriptional up-regulation of biosynthesis genes. Proteome analysis indicated changes in cytoskeletal structure affecting actin localization, linking the major regulator Thn1 to growth and development of S. commune. The results support a role for Thn1 in G-protein signalling connecting development and secondary metabolism., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

High-through-put (HTP) screening for functional arbuscular mycorrhizal fungi (AMF)-associations is challenging because roots must be excavated and colonization evaluated by transcript analysis or microscopy. Here we show that specific leaf-metabolites provide broadly applicable accurate proxies of these associations, suitable for HTP-screens. With a combination of untargeted and targeted metabolomics, we show that shoot accumulations of hydroxy- and carboxyblumenol C-glucosides mirror root AMF-colonization in Nicotiana attenuata plants. Genetic/pharmacologic manipulations indicate that these AMF-indicative foliar blumenols are synthesized and transported from roots to shoots. These blumenol-derived foliar markers, found in many di- and monocotyledonous crop and model plants ( Solanum lycopersicum, Solanum tuberosum, Hordeum vulgare, Triticum aestivum, Medicago truncatula and Brachypodium distachyon ), are not restricted to particular plant-AMF interactions, and are shown to be applicable for field-based QTL mapping of AMF-related genes., Competing Interests: MW European patent application EP 18 15 8922.7, MS Martin Schäfer: European patent application EP 18 15 8922.7, DL Dapeng Li: European patent application EP 18 15 8922.7, RH Rayko Halitschke: European patent application EP 18 15 8922.7, CD, CP, YS, SL, JD, KG, PF, MB, UP No competing interests declared, EM Erica McGale: European patent application EP 18 15 8922.7, SH Sven Heiling: European patent application EP 18 15 8922.7, MH Reviewing editor, eLife, IB Senior editor, eLife; European patent application EP 18 15 8922.7, (© 2018, Wang et al.)

While the characterization of the biosynthetic pathway of monoterpene indole alkaloids (MIAs) in leaves of Catharanthus roseus is now reaching completion, only two enzymes from the root counterpart dedicated to tabersonine metabolism have been identified to date, namely tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT). Albeit the recombinant MAT catalyzes MIA acetylation at low efficiency in vitro, we demonstrated that MAT was inactive when expressed in yeast and in planta, suggesting an alternative function for this enzyme. Therefore, through transcriptomic analysis of periwinkle adventitious roots, several other BAHD acyltransferase candidates were identified based on the correlation of their expression profile with T19H and found to localize in small genomic clusters. Only one, named tabersonine derivative 19-O-acetyltransferase (TAT) was able to acetylate the 19-hydroxytabersonine derivatives from roots, such as minovincinine and hörhammericine, following expression in yeast. Kinetic studies also showed that the recombinant TAT was specific for root MIAs and displayed an up to 200-fold higher catalytic efficiency than MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in Nicotiana benthamiana were in agreement with the prominent role of TAT in acetylation of root-specific MIAs, thereby redefining the molecular determinants of the root MIA biosynthetic pathway. Finally, identification of TAT provided a convenient tool for metabolic engineering of MIAs in yeast enabling efficiently mixing different biosynthetic modules spatially separated in the whole plant. This combinatorial synthesis associating several enzymes from Catharanthus roseus resulted in the conversion of tabersonine in tailor-made MIAs bearing both leaf and root-type decorations., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Salicortin is a phenolic glucoside produced in Salicaceae as a chemical defense against herbivory. The specialist lepidopteran herbivorous larvae of Cerura vinula are able to overcome this defense. We examined the main frass constituents of C. vinula fed on Populus nigra leaves, and identified 11 quinic acid derivatives with benzoate and/or salicylate substitution. We asked whether the compounds are a result of salicortin breakdown and sought answers by carrying out feeding experiments with highly 13 C-enriched salicortin. Using HRMS and NMR analyses, we were able to confirm that salicortin metabolism in C. vinula proceeds through deglucosylation and ester hydrolysis, after which saligenin is oxidatively transformed into salicylic acid and, eventually, conjugated to quinic acid. To the best of our knowledge, this is the first report of a detoxification pathway based on conjugation with quinic acid.

Phenylbenzoisochromenone glucosides (oxa-phenylphenalenone glucosides) occurring in some phenylphenalenone-producing plants of the Haemodoraceae undergo conversion to phenylbenzoisoquinolindiones (aza-phenylphenalenones) in extracts of Xiphidium caeruleum. Precursor-directed biosynthetic experiments were used to generate a series of new phenylbenzoisoquinolindiones from native phenylbenzoisochromenone glucosides and external amines, amino acids, and peptides. Intermediates of the conversion were isolated, incubated with cell-free extracts, and exposed to reactions under oxidative or inert conditions, respectively, to elucidate the entire pathway from phenylbenzoisochromenones to phenylbenzoisoquinolindiones. An intermediate in this pathway, a reactive hydroxylactone/aldehyde, readily binds not only to amines in vitro but may also bind to the N-terminus of biogenic peptides and proteins of herbivores and pathogens in vivo. The deactivation of biogenic amino compounds by N-terminal modification is discussed as the key reaction of a novel phenylphenalenone-based plant defense mechanism. According to these data, the ecological function of phenylphenalenone-type compounds in the Haemodoraceae, subfamily Haemodoroideae, has been substantiated.

The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B 12 , which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B 12 In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as cofactors by a cobamide-containing enzyme are provided. IMPORTANCE Guided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans is reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl- or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme., (Copyright © 2018 American Society for Microbiology.)

Piriformospora indica , an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. P. indica fungal cell wall extract induces cytoplasmic calcium elevation in host plant roots. Here, we show that cellotriose (CT) is an elicitor-active cell wall moiety released by P. indica into the medium. CT induces a mild defense-like response, including the production of reactive oxygen species, changes in membrane potential, and the expression of genes involved in growth regulation and root development. CT-based cytoplasmic calcium elevation in Arabidopsis ( Arabidopsis thaliana ) roots does not require the BAK1 coreceptor or the putative Ca 2+ channels TPC1, GLR3.3, GLR2.4, and GLR2.5 and operates synergistically with the elicitor chitin. We identified an ethyl methanesulfonate-induced mutant ( cytoplasmic calcium elevation mutant ) impaired in the response to CT and various other cellooligomers ( n = 2-7), but not to chitooligomers ( n = 4-8), in roots. The mutant contains a single nucleotide exchange in the gene encoding a poly(A) ribonuclease (AtPARN; At1g55870) that degrades the poly(A) tails of specific mRNAs. The wild-type PARN cDNA, expressed under the control of a 35S promoter, complements the mutant phenotype. Our identification of cellotriose as a novel chemical mediator casts light on the complex P. indica -plant mutualistic relationship., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

(9Z)-Methyl 4-dihydrotrisporate B and (9Z)-methyl trisporate B, pheromones of Zygomycetes fungi, have been synthesized using Stille cross-coupling from previously described cyclohexenone precursors. Conducting the coupling without protection groups allowed for a short and stereospecific synthesis route of the late trisporoids. Stability studies for both the compounds revealed (9Z)-methyl trisporate B to be very unstable against UV irradiation.

Phytochemical investigation of Idesia polycarpa (Salicaceae) resulted in the structure elucidation of nine previously undescribed phenolic natural products along with six known compounds. The compounds are structurally related to salicinoids that are known defense compounds from Salix and Populus species. The I. polycarpa diet was toxic, as shown in feeding experiments with larvae of Lymantria dispar, an herbivorous broadleaf tree generalist insect, and with larvae of Cerura vinula, a specialist adapted to poplar. The survival rate and mass gain of larvae was significantly lower when they fed on I. polycarpa leaves, compared to larvae fed on Populus nigra leaves. Potential reasons for the poor performance of both herbivores on I. polycarpa leaves are discussed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)