[Show abstract][Hide abstract] ABSTRACT: Abutilon theophrasti is one of the most problematic weeds worldwide. Rye mulches, rich in benzoxazinoids, could not suppress Abutilon (Herbiseeds) due to its avoidance of high BOA/BOA detoxification product accumulation and phytotoxic 2-aminophenoxazinone production. Since Abutilon theophrasti seed coats harbour a variety of fungi and bacteria, a participation of those organisms in BOA degradation was assumed. The avoidance of high detoxification product/BOA accumulation in the roots was correlated with the presence of the Zygomycete Actinomucor elegans, which is known to colonize Abutilon seed coats. The microorganism(s) produced plant growth promoting compounds, whereby tryptophan excretion was stimulated by BOA. In Fenton reactions with BOA, tryptophan accelerates the destruction of the allelochemical via hydroxylated BOA molecules, when used in defined ratios. Destruction of BOA via hydroxylated molecules presents a new pathway for BOA degradation without accumulation of 2-aminophenoxazinone. During this process tryptophan is also degraded. Applications of high concentrations of tryptophan led, however, to a higher, channelled synthesis of BOA-6-O-glucoside in the roots, probably to slow down oxygen consuming Fenton reactions. From all possible hydroxylated BOA molecules, BOA-6-OH is the best substrate for Oglucosylation. Incubations in presence of the mono-oxygenase effector 2-bromo-4´-nitroacetophenone revealed that those enzymes are mainly responsible for BOA-6-OH synthesis and only a minor part results from Fenton reactions. The study elucidates the important role of plant-microorganism associations in the survival of allelopathic attacks.
7th World Congress on Allelopathy: “Complex Interactions in a Changing Climate”, International Allelopathy Society., Vigo, Spain; 07/2014
[Show abstract][Hide abstract] ABSTRACT: The increasing resistance level of insect pest species is a major concern to agriculture worldwide. The cotton bollworm, Helicoverpa armigera, is one of the most important pest species due to being highly polyphagous, geographically widespread, and resistant towards many chemical classes of insecticides. We previously described the mechanism of fenvalerate resistance in Australian populations conferred by the chimeric cytochrome P450 monooxygenase CYP337B3, which arose by unequal crossing-over between CYP337B1 and CYP337B2. Here, we show that this mechanism is also present in the cypermethrin-resistant FSD strain from Pakistan. The Pakistani and the Australian CYP337B3 alleles differ by 18 synonymous and three nonsynonymous SNPs and additionally in the length and sequence of the intron. Nevertheless, the activity of both CYP337B3 proteins is comparable. We demonstrate that CYP337B3 is capable of metabolizing cypermethrin (trans- and especially cis-isomers) to the main metabolite 4'-hydroxycypermethrin, which exhibits no intrinsic toxicity towards susceptible larvae. In a bioassay, CYP337B3 confers a 7-fold resistance towards cypermethrin in FSD larvae compared to susceptible larvae from the Australian TWB strain lacking CYP337B3. Linkage analysis shows that presence of CYP337B3 accounts for most of the cypermethrin resistance in the FSD strain; up-regulation of other P450s in FSD plays no detectable role in resistance. The presence or absence of CYP337B3 can be easily detected by a simple PCR screen, providing a powerful tool to rapidly distinguish resistant from susceptible individuals in the field and to determine the geographical distribution of this resistance gene. Our results suggest that CYP337B3 evolved twice independently by unequal crossing-over between CYP337B2 and two different CYP337B1 alleles.
Insect biochemistry and molecular biology. 07/2014;
[Show abstract][Hide abstract] ABSTRACT: Nudicaulins are unique alkaloids responsible for the yellow color of the petals of some papaveraceaous plants. To elucidate the unknown biosynthetic origin of the skeleton, a (13) CO2 -pulse/chase experiment was performed with growing Papaver nudicaule plants. (13) C NMR analysis revealed more than 20 multiple (13) C-enriched isotopologues in nudicaulins from the petals of (13) CO2 -labeled plants. The complex labeling pattern was compared with the isotopologue composition of a kaempferol derivative that was isolated from petals of the same (13) CO2 -labeled plants. The deconvolution of the labeling profiles indicated that the nudicaulin scaffold is assembled from products or intermediates of indole metabolism, the phenylpropanoid pathway, and the polyketide biosynthesis. Naringenin-type compounds and tryptophan/tryptamine are potential substrates for the condensation reaction finally generating the aglycone skeleton of nudicaulins.
[Show abstract][Hide abstract] ABSTRACT: The global yield of bananas-one of the most important food crops-is severely hampered by parasites, such as nematodes, which cause yield losses up to 75%. Plant-nematode interactions of two banana cultivars differing in susceptibility to Radopholus similis were investigated by combining the conventional and spatially resolved analytical techniques (1)H NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging, and Raman microspectroscopy. This innovative combination of analytical techniques was applied to isolate, identify, and locate the banana-specific type of phytoalexins, phenylphenalenones, in the R. similis-caused lesions of the plants. The striking antinematode activity of the phenylphenalenone anigorufone, its ingestion by the nematode, and its subsequent localization in lipid droplets within the nematode is reported. The importance of varying local concentrations of these specialized metabolites in infected plant tissues, their involvement in the plant's defense system, and derived strategies for improving banana resistance are highlighted.
Proceedings of the National Academy of Sciences 12/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.
[Show abstract][Hide abstract] ABSTRACT: Aldoximes are known as floral and vegetative plant volatiles but also as biosynthetic intermediates for other plant defense compounds. While the cytochrome P450 monooxygenases (CYP) from the CYP79 family forming aldoximes as biosynthetic intermediates have been intensively studied, little is known about the enzymology of volatile aldoxime formation. We characterized two P450 enzymes, CYP79D6v3 and CYP79D7v2, which are involved in herbivore-induced aldoxime formation in western balsam poplar (Populus trichocarpa). Heterologous expression in Saccharomyces cerevisiae revealed that both enzymes produce a mixture of different aldoximes. Knockdown lines of CYP79D6/7 in gray poplar (Populus × canescens) exhibited a decreased emission of aldoximes, nitriles, and alcohols, emphasizing that the CYP79s catalyze the first step in the formation of a complex volatile blend. Aldoxime emission was found to be restricted to herbivore-damaged leaves and is closely correlated with CYP79D6 and CYP79D7 gene expression. The semi-volatile phenylacetaldoxime decreased survival and weight gain of gypsy moth (Lymantria dispar) caterpillars, suggesting that aldoximes may be involved in direct defense. The wide distribution of volatile aldoximes throughout the plant kingdom and the presence of CYP79 genes in all sequenced genomes of angiosperms suggest that volatile formation mediated by CYP79s is a general phenomenon in the plant kingdom.
[Show abstract][Hide abstract] ABSTRACT: Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In the present study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes, characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 mRNA are specifically located to leaf epidermis, which also host the next step of vindoline biosynthesis. Comparison of high and low vindoline accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and re-directs the biosynthetic flux towards the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351 that encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: 2,4-Dihydroxy-9-phenyl-1H-phenalen-1-one (4-hydroxyanigorufone, 1), a compound isolated from Anigozanthos flavidus and Monochoria elata, displayed a high radical scavenging capacity in the ORAC assay. A systematic approach was adopted in order to explore the effect of each functional group. H-Atom transfer from the phenolic hydroxyl, a captodative effect from the hydroxy ketone, and the presumed involvement of the phenyl ring in the termination step of the radical reaction were disclosed as relevant features of this type of antioxidant.
[Show abstract][Hide abstract] ABSTRACT: Norway spruce (Picea abies) forests suffer periodic fatal attacks by the bark beetle Ips typographus and its fungal associate, Ceratocystis polonica. P. abies protects itself against fungal and bark beetle invasion by production of terpenoid resins, but it is unclear whether resins or other defenses are effective against the fungus. We investigated stilbenes, a group of phenolic compounds found in P. abies bark with a diaryl-ethene skeleton with known antifungal properties. During C. polonica infection, stilbene biosynthesis was up-regulated as evidenced by elevated transcript levels of stilbene synthase genes. However, stilbene concentrations actually declined during infection and this was due to fungal metabolism. C. polonica converted stilbenes to ring-opened, deglycosylated and dimeric products. Chromatographic separation of C. polonica protein extracts confirmed that these metabolites arose from specific fungal enzyme activities. Comparison of C. polonica strains showed that rapid conversion of host phenolics is associated with higher virulence. C. polonica is so well adapted to its host's chemical defenses that it is even able to use host phenolic compounds as its sole carbon source.
[Show abstract][Hide abstract] ABSTRACT: The intense color of yellow Papaver nudicaule flowers is conferred by the presence of nudicaulins, a group of alkaloids with a unique pentacyclic skeleton composed of an indole ring and a polyphenolic moiety. Petals from eight different Papaveraceae species composed of different color varieties were probed for the presence of nudicaulins. In addition to their occurrence in yellow P. nudicaule flowers, nudicaulins I-VIII were detected and quantified in orange flowers of P. nudicaule, and in yellow and orange Papaver alpinum flowers. Meconopsis cambrica petals showed a divergent nudicaulin spectrum, with compounds having an attached 3-hydroxy-3-methyl-glutaryl group (HMG) instead of a malonyl unit at one of the glucose units. Flavonols and anthocyanins that accompany nudicaulins were identified. The taxonomical significance of the occurrence of nudicaulins is briefly discussed.
[Show abstract][Hide abstract] ABSTRACT: A new class of unique sesterterpenoids, colquhounoids A-C (1-3), were identified from the peltate glandular trichomes of Colquhounia coccinea var. mollis (Lamiaceae) through precise laser-microdissection coupled with UPLC/MS/MS and spectroscopic analyses and X-ray diffraction. Very interestingly, their structural features and defensive function are closely related to leucosceptroid-class sesterterpenoids harbored by the glandular trichomes of another Lamiaceae taxon, Leucosceptrum canum, even though this is morphologically distinct and taxonomically distant.
[Show abstract][Hide abstract] ABSTRACT: 2-Hydroxy-4-phenyl-1H-phenalen-1-one (isoanigorufone, 1), a phytoalexin exclusive of Musaceae, was synthesized starting from 3-(2-hydroxynaphthalen-1-yl)propanenitrile in nine steps in an overall yield of 10%. Hydrolysis of ethyl 3-(2-phenylnaphthalen-1-yl)propanoate obtained from Suzuki–Miyaura coupling between the parent triflate and phenylboronic acid afforded the corresponding propionic acid which, after Friedel–Crafts acylation and bromine-mediated dehydrogenation, was subjected to Yang–Finnegan epoxidation to furnish 1. The preparation of analogs using this procedure is also discussed.
[Show abstract][Hide abstract] ABSTRACT: Larvae of the Chrysomelina species Phaedon cochleariae, Hydrothassa marginella, Phratora vulgatissima, Gastrophysa viridula, Gastrophysa atrocyanea, Gastrophysa cyanea and Gastrophysa polygoni produce the iridoid chrysomelidial (1) to defend themselves against predators. Feeding experiments with a deuterated precursor ([(2) H(5) ]8-hydroxygeraniol 9) and in vitro isotope exchange experiments with defensive secretion in (2) H(2) O revealed differences in the cyclisation of the ultimate precursor 8-oxogeranial (8) to 1, between members of the genus Gastrophysa and all other species. In P. cochleariae, H. marginella and P. vulgatissima 1 is most likely produced by a Rauhut-Currier-type cyclisation via a "transoid dienamine", with loss of a single deuterium atom from C(4) of the precursor. In contrast, members of the genus Gastrophysa cyclise 8 via a "cisoid dienamine" intermediate, with exchange of all three deuterium atoms from the methyl group at C(3). To study whether the different cyclisation modes influence the stereochemistry of 1, the absolute configuration of 1 of the larvae was determined by GC-MS on a chiral column. In accordance with literature (J. Meinwald, T. H. Jones, J. Am. Chem. Soc. 1978, 100, 1883 and N. Shimizu, R. Yakumaru, T. Sakata, S. Shimano, Y. Kuwahara, J. Chem. Ecol. 2012, 38, 29), we found (5S,8S)-chrysomelidial (1) in H. marginella and P. vulgatissima, but P. cochleariae and all investigated members of the genus Gastrophysa synthesise (5R,8R)-chrysomelidial (1).
[Show abstract][Hide abstract] ABSTRACT: This present work describes the application of liquid chromatograpy-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1), alternariol (2), 3'-hydroxyalternariol monomethyl ether (3), and alternariol monomethyl ether (4), were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max) cell cultures as a model. EC(50) values which range from 0.11 (±0.02) to 4.69 (±0.47) μM showed the high cytotoxicity of these compounds.
[Show abstract][Hide abstract] ABSTRACT: The formation of 4-deoxyaurones, which serve as UV nectar guides in Bidens ferulifolia (Jacq.) DC., was established by combination of UV photography, mass spectrometry, and biochemical assays and the key step in aurone formation was studied. The yellow flowering ornamental plant accumulates deoxy type anthochlor pigments (6'-deoxychalcones and the corresponding 4-deoxyaurones) in the basal part of the flower surface whilst the apex contains only yellow carotenoids. For UV sensitive pollinating insects, this appears as a bicoloured floral pattern which can be visualized in situ by specific ammonia staining of the anthochlor pigments. The petal back side, in contrast, shows a faintly UV absorbing centre and UV absorbing rays along the otherwise UV reflecting petal apex. Matrix-free UV laser desorption/ionisation mass spectrometric imaging (LDI-MSI) indicated the presence of 9 anthochlors in the UV absorbing areas. The prevalent pigments were derivatives of okanin and maritimetin. Enzyme preparations from flowers, leaves, stems and roots of B. ferulifolia and from plants, which do not accumulate aurones e.g. Arabidopsis thaliana, were able to convert chalcones to aurones. Thus, aurone formation could be catalyzed by a widespread enzyme and seems to depend mainly on a specific biochemical background, which favours the formation of aurones at the expense of flavonoids. In contrast to 4-hydroxyaurone formation, hydroxylation and oxidative cyclization to the 4-deoxyaurones does not occur in one single step but is catalyzed by two separate enzymes, chalcone 3-hydroxylase and aurone synthase (catechol oxidase reaction). Aurone formation shows an optimum at pH 7.5 or above, which is another striking contrast to 4-hydroxyaurone formation in Antirrhinum majus L. This is the first example of a plant catechol oxidase type enzyme being involved in the flavonoid pathway and in an anabolic reaction in general.
PLoS ONE 01/2013; 8(5):e61766. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25-143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. Δ(9)-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, Δ(9)-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit.
[Show abstract][Hide abstract] ABSTRACT: The Diels-Alder cycloaddition of nitroethylene to some androsta-14,16-dien-17-yl acetates has been studied. The addition occurs stereoselectively, giving predominantly head-to-head-adduct. 14β-Cyanomethyl steroids were obtained via the reductive cleavage reaction of bridged nitro compounds. The structures of the new compounds have been fully characterized by 2D NMR and tandem mass-spectrometry methods.