Promiscuous CYP87A enzyme activity initiates cardenolide biosynthesis in plants.

Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families1,2. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases3-9. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and ß-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.

Biosynthesis of isoxazolin-5-one and 3-nitropropanoic acid containing glucosides in juvenile Chrysomelina.

Stable-isotope-labeled precursors were used to establish the biosynthetic pathway leading from ß-alanine towards isoxazolin-5-one glucoside 1 and its 3-nitropropanoate (3-NPA) ester 2 in Chrysomelina larvae. Both structural elements originate from sequestered plant-derived ß-alanine or from propanoyl-CoA that is derived from the degradation of some essential amino acids, e.g. valine. ß-Alanine is converted into 3-NPA and isoxazolinone 5 by consecutive oxidations of the amino group of ß-Ala. Substituting the diphospho group of α-UDP-glucose with 5 generates the isoxazolin-5-one glucoside 1, which serves in the circulating hemolymph of the larva as a platform for esterification with 3-nitropropanoyl-CoA. The pathway was validated with larvae of Phaedon cochleariae, Chrysomela populi as well as Gastrophysa viridula.

The Green Gut: Chlorophyll Degradation in the Gut of Spodoptera littoralis.

Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous insects. The spectrum of ingested and digested chlorophyll metabolites compares well to the pattern of early chlorophyll-degradation products in senescent plants. Intact chlorophyll is rapidly degraded by proteins in the front- and midgut. Unlike plants, insects convert both chlorophyll a and b into the corresponding catabolites. MALDI-TOF/MS imaging allowed monitoring the distribution of the chlorophyll catabolites along the gut of Spodoptera littoralis larvae. The chlorophyll degradation in the fore- and mid-gut is strongly pH dependent, and requires alkaline conditions. Using LC-MS/MS analysis we identified a lipocalin-type protein in the intestinal fluid of S. littoralis homolog to the chlorophyllide a binding protein from Bombyx mori. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes are covered with the chlorophyllide binding protein tightly bound via its GPI-anchor to the gut membrane. A function in defense against gut microbes is discussed.

Chlorophyll degradation in the gut of generalist and specialist Lepidopteran caterpillars.

Plant feeding herbivores excrete most of the ingested chlorophyll (Chl) as partly degraded derivatives lacking the phytol side chain and the central magnesium ion. An ecological role of digested and degraded Chls in the interactions between insects, their food plant and other insects has been described recently. To gain more information on common degradation patterns in plant-feeding insects, the orals secretions and frass of five Lepidopteran caterpillars covering generalists and specialists, namely Spodoptera littoralis, Spodoptera eridania, Heliothis virescens, Helicoverpa armigera, Manduca sexta, and, for comparison, of the leaf beetle larva Chrysomela lapponica were analyzed for chlorophyll catabolites. The major degradation products were determined as pheohorbide a/b and pyropheophorbide a/b by using LC-MS, LC-NMR, UV, and fluorescence spectrometry. The compounds were not present in fresh leaves of the food plants (Phaseolus lunatus, Nicotiana tabacum). The catabolite spectrum in generalists and specialists was qualitatively similar and could be attributed to the action of gut proteins and the strongly alkaline milieu in the digestive tract. Due to the anaerobic environment of the larval gut, the tetrapyrrole core of the Chl catabolites was not cleaved. Substantial amounts of Chl a/b metabolites were strongly complexed by a protein in the mid-gut.

Bifunctional lipocalin ameliorates murine immune complex-induced acute lung injury.

Molecules that simultaneously inhibit independent or co-dependent proinflammatory pathways may have advantages over conventional monotherapeutics. OmCI is a bifunctional protein derived from blood-feeding ticks that specifically prevents complement (C)-mediated C5 activation and also sequesters leukotriene B4 (LTB4) within an internal binding pocket. Here, we examined the effect of LTB4 binding on OmCI structure and function and investigated the relative importance of C-mediated C5 activation and LTB4 in a mouse model of immune complex-induced acute lung injury (IC-ALI). We describe two crystal structures of bacterially expressed OmCI: one binding a C16 fatty acid and the other binding LTB4 (C20). We show that the C5 and LTB4 binding activities of the molecule are independent of each other and that OmCI is a potent inhibitor of experimental IC-ALI, equally dependent on both C5 inhibition and LTB4 binding for full activity. The data highlight the importance of LTB4 in IC-ALI and activation of C5 by the complement pathway C5 convertase rather than by non-C proteases. The findings suggest that dual inhibition of C5 and LTB4 may be useful for treatment of human immune complex-dependent diseases.

Glucose and glucose esters in the larval secretion of Chrysomela lapponica; selectivity of the glucoside import system from host plant leaves.

Larvae of Chrysomela lapponica (Coleoptera: Chrysomelidae) sequester characteristic O-glucosides from the leaves of their food plants, namely Betula and/or Salix The present study focuses on birch-feeding larvae of C. lapponica from the Altai region in East Kazakhstan. As in other sequestering leaf beetle larvae, the compounds are transported intact via different membrane barriers into the defensive system, followed by glucoside cleavage and subsequent transformations of the plant-derived aglycones. Unlike previous studies with model compounds, we studied the sequestration of phytogenic precursors by analyzing the complex pattern of glucosides present in food plant Betula rotundifolia (39 compounds) and compared this composition with the aglycones present as butyrate esters in the defensive secretion. In addition to the analytic approach, the insect's ability, to transport individual glucosides was tested by using hydrolysis-resistant thioglucoside analogs, applied onto the leaf surface. The test compounds reach the defensive system intact and without intermediate transformation. No significant difference of the transport capacity and selectivity was observed between larvae of birch-feeding population from Kazakhstan, and previous results for larvae of birch-feeding population from the Czech Republic or willow-feeding populations. Overall, the transport of the phytogenic glucosides is highly selective and highly efficient, since only minor compounds of the spectrum of phytogenic glucoside precursors contribute to the limited number of aglycones utilized in the defensive secretion. Interestingly, salicortin 44 and tremulacin 60 were found in the leaves, but no aldehyde or esters of salicylalcohol. Surprisingly, we observed large amounts of free glucose, together with small amounts of 6-O-butyrate esters of glucose (27a/b and 28a/b).

Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle.

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them.

The chlorophyll catabolite, pheophorbide a, confers predation resistance in a larval tortoise beetle shield defense.

Larval insect herbivores feeding externally on leaves are vulnerable to numerous and varied enemies. Larvae of the Neotropical herbivore, Chelymorpha alternans (Chrysomelidae:Cassidinae), possess shields made of cast skins and feces, which can be aimed and waved at attacking enemies. Prior work with C. alternans feeding on Merremia umbellata (Convolvulaceae) showed that shields offered protection from generalist predators, and polar compounds were implicated. This study used a ubiquitous ant predator, Azteca lacrymosa, in field bioassays to determine the chemical constitution of the defense. We confirmed that intact shields do protect larvae and that methanol-water leaching significantly reduced shield effectiveness. Liquid chromatography-mass spectrometry (LC-MS) of the methanolic shield extract revealed two peaks at 20.18 min and 21.97 min, both with a molecular ion at m/z 593.4, and a strong UV absorption around 409 nm, suggesting a porphyrin-type compound. LC-MS analysis of a commercial standard confirmed pheophorbide a (Pha) identity. C. alternans shields contained more than 100 microg Pha per shield. Shields leached with methanol-water did not deter ants. Methanol-water-leached shields enhanced with 3 microg of Pha were more deterrent than larvae with solvent-leached shields, while those with 5 microg additional Pha provided slightly less deterrence than larvae with intact shields. Solvent-leached shields with 10 microg added Pha were comparable to intact shields, even though the Pha concentration was less than 10% of its natural concentration. Our findings are the first to assign an ecological role for a chlorophyll catabolite as a deterrent in an insect defense.

Biosynthesis and accumulation of ergoline alkaloids in a mutualistic association between Ipomoea asarifolia (Convolvulaceae) and a clavicipitalean fungus.

Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role.

Granular chitin in the epidermis of nudibranch molluscs.

Chitin is usually found in stiff extracellular coatings typified by the arthropod exoskeleton, and is not associated with the soft, flexible mollusc skin. Here, we show, however, that chitin in nudibranch gastropods (Opisthobranchia, Mollusca) occurs as intracellular granules that fill the epidermal cells of the skin and the epithelial cells of the stomach. In response to nematocysts fired by tentacles of prey Cnidaria, the epidermal cells of eolid nudibranchs (Aeolidacea) release masses of chitin granules, which then form aggregates with the nematocyst tubules, having the effect of insulating the animal from the deleterious action of the Cnidaria tentacles. Granular chitin, while protecting the animal, does not interfere with the suppleness and flexibility of the skin, in contrast to the stiffness of chitin armor. The specialized epidermis enables nudibranchs to live with and feed on Cnidaria.

Evidence for a radical mechanism of the dechlorination of chlorinated propenes mediated by the tetrachloroethene reductive dehalogenase of Sulfurospirillum muftivorans.

The reductive dehalogenation of chlorinated propenes was studied with the tetrachloroethene reductive dehalogenase purified from Sulfurospirillum multivorans to obtain indications for a radical mechanism of this reaction. When reduced methyl viologen (MV), which is a radical cation, was applied as electron donor for the reduction of different chloropropenes, a significant part of MV could not be rereduced with Ti(III) citrate, indicating that a part of the MV was consumed in a side reaction. Mass spectrometric analysis of assays with MV as electron donor revealed the formation of side products, the masses of which might account for the formation of adducts from a chloropropenyl radical and reduced methyl viologen. With Ti(III) citrate as sole electron donor, 2,3-dichloropropene was reduced and as a side product, 2,5-dichloro-1,5-hexadiene was formed demonstrating that the reductive dechlorination of 2,3-dichloropropene proceeds via a radical reaction mechanism. The results support different dehalogenation mechanisms forthe reductive dechlorination of chloropropenes and halogenated ethenes.

The structure of OMCI, a novel lipocalin inhibitor of the complement system.

The complement (C) system is a potent innate immune defence system against parasites. We have recently characterised and expressed OmCI, a 16 kDa protein derived from the soft tick Ornithodoros moubata that specifically binds C5, thereby preventing C activation. The structure of recombinant OmCI determined at 1.9 A resolution confirms a lipocalin fold and reveals that the protein binds a fatty acid derivative that we have identified by mass spectrometry as ricinoleic acid. We propose that OmCI could sequester one of the fatty acid-derived inflammatory modulators from the host plasma, thereby interfering with the host inflammatory response to the tick bite. Mapping of sequence differences between OmCI and other tick lipocalins with different functions, combined with biochemical investigations of OmCI activity, supports the hypothesis that OmCI acts by preventing interaction with the C5 convertase, rather than by blocking the C5a cleavage site.

Induced resistance enzymes in wild plants-do 'early birds' escape from pathogen attack?

Systemic acquired resistance (SAR) of plants to pathogens is a well-defined phenomenon. The underlying signalling pathways and its application in crop protection are intensively studied. However, most studies are conducted on crop plants or on Arabidopsis as a model plant. The taxonomic distribution of this phenomenon and its dependence on life history are thus largely unknown. We quantified activities of three classes of resistance-related enzymes in 18 plant species to investigate whether plants with varying life histories differ in their investment in disease resistance. Enzyme activities were quantified in untreated plants, and in plants induced with BION, a chemical resistance elicitor. All species showed constitutive activities of chitinase, peroxidase, or glucanase. However, constitutive chitinase activities varied by 30 times, and peroxidase by 50 times, among species. Several species did not respond to the induction treatment, while enzyme activities in other species increased more than threefold after BION application. Plant species differ dramatically in the presence and inducibility of resistance enzymes. This variation could be related to life history: While all resistance enzymes were significantly induced in larger perennial plants that flower during summer, spring geophytes hardly showed inducible resistance. These plants grow in an environment that is characterised by a low-pathogen pressure, and thus may simply 'escape' from infection. Our study presents the first comparative data set on resistance-related enzymes in noncultivated plants. The current view on SAR-narrowed by the concentration on cultivated crops-is not sufficient to understand the ecological and evolutionary relevance of this widespread plant trait.