Plants of the order Ranunculales, especially members of the species Papaver , accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum ) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum , 69 show increased expression in morphinan alkaloid‐containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7‐epi ‐salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo‐keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.

A simple and regiospecific synthesis of 4-alkoxy(amino)-2-trifluoromethyl pyrroles from 5-azido-4-alkoxy(amino)-1,1,1-trifluoro-pent-3-en-2-ones by an aza-Wittig cyclization of aminophosphoranes is described. The structures of the pyrroles and their synthetic intermediates were supported by NMR and HRMS analysis.

Sorghum SbSTS1 was the first example of a stilbene synthase gene in monocots. Previously, we demonstrated that the gene was involved in defense responses. To examine its biochemical function in planta, SbSTS1 was overexpressed in transgenic Arabidopsis. Metabolite analysis revealed that cis -resveratrol glucoside (piceid) accumulated as the major stilbene in the transgenic lines. Using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring mode, up to 580 μg g −1 FW of cis -piceid were detected in 2-week-old plants, which represent a convenient source of the cis -isomers for pharmacological investigations. Our results also suggested the presence of unknown stilbene isomerase activities in Arabidopsis.

Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s-1. The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8α-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.

8-Prenylnaringenin, a flavonoid, is the strongest known phytoestrogen (plant derived estrogen mimic) used in phytomedicinal applications. Starting from xanthohumol a byproduct of hops-extraction, 8-prenylnaringenin can be synthesized via isoxanthohumol. Of various demethylation procedures tested, the best yield (92%) is obtained by treatment with scandium trifluoromethanesulfonate and potassium iodide without any need of protection. The demethylation with AlBr3/collidine and of the TIPS protected isoxanthohumol provides good results too.

Utilizing the multiple multicomponent macrocyclization including bifunctional building blocks (MiB) strategy, a library of nonracemic, nonrepetitive peptoid-containing steroid−biaryl ether hybrid macrocycles was built. Up to 16 new bonds, including those of the macrocyclization, can be formed in one pot simultaneously while introducing varied elements of diversity. Functional diversity is generated primarily by choosing Ugi-reactive functional building blocks, bearing the respective recognition or catalytic motifs. These appear attached to the peptoid backbone of the macrocyclic cavity, similar to side chains of amino acids found in enzyme active sites. Likewise, skeletal diversity is based on the variation of defined bifunctional building blocks which allow the parallel formation of macrocyclic cavities that are highly diverse in shape and size and thus perspectively in function. This straightforward approach is suitable to generate multifunctional macrocycles for applications in catalysis, supramolecular, or biological chemistry.

0

Cultured cells of Eschscholzia californica respond to a yeast glycoprotein elicitor by producing benzophenanthridine alkaloids, which are excreted into the cell wall and the outer medium. These compounds, preferentially sanguinarine, are efficient phytoalexins because of their ability to intercalate double‐stranded DNA (dsDNA), penetrate membranes and inhibit various enzymes containing SH‐groups. Externally added sanguinarine is rapidly taken up by intact cells and converted to dihydrosanguinarine, which is substituted intracellularly according to the biosynthetic route. A 29.5 kDa soluble enzyme that catalyses the reduction of sanguinarine and chelerythrine by either NADPH or NADH has been isolated and purified to homogeneity. Benzophenanthridines that accumulate in the outer medium, mainly 10‐OH‐chelerythrine, chelirubine and macarpine, are converted by the isolated enzyme and by intact cells at much slower rates than sanguinarine. The cellular capacity of uptake and conversion of sanguinarine largely surpasses the rate of alkaloid production. We conclude that the sanguinarine produced by intact cells, after excretion and binding to cell wall elements, is rapidly reabsorbed and reduced to the less toxic dihydrosanguinarine, which then undergoes further biosynthetic reactions. This recycling process would allow the presence of the toxic phytoalexin at the cellular surface without taking the risk of injuring the producing cell.

Toxic effects of both essential and non‐essential heavy metals are well documented in plants. Very little is known, however, about their modes of toxicity, about tolerance mechanisms and the signalling cascades involved in mediating transcriptional responses to toxic metal excess. We analysed transcriptome changes upon Cd2+ and Cu2+ exposure in roots of Arabidopsis thaliana and the Cd2+‐hypertolerant metallophyte Arabidopsis halleri . Particularly, three categories of genes were identified with the help of this comparative approach: (1) common responses, which might indicate stable and functionally relevant changes conserved across plant species; (2) metallophyte‐specific responses as well as transcripts differentially regulated between the two species, representing candidate genes for Cd2+ hypertolerance; and (3) those specifically responsive to Cd2+ and therefore indicative of toxicity mechanisms or potentially involved in signalling cascades. Our data define, for instance, Arabidopsis core responses to Cd2+ and Cu2+. In addition, they suggest that Cd2+ exposure very rapidly results in apparent Zn deficiency, and they show the existence of highly specific Cd2+ responses and distinct signalling cascades. Array results were independently confirmed by real‐time quantitative PCR, thereby further validating cross‐species transcriptome analysis with oligonucleotide microarrays.

Plants respond to mechanical wounding or herbivore attack with a complex scenario of sequential, antagonistic or synergistic action of different signals leading to defense gene expression. Tomato plants were used as a model system since the peptide systemin and the lipid-derived jasmonic acid (JA) were recognized as essential signals in wound-induced gene expression. In this review recent data are discussed with emphasis on wound-signaling in tomato. The following aspects are covered: (i) systemin signaling, (ii) JA biosynthesis and action, (iii) orchestration of various signals such as JA, H2O2, NO, and salicylate, (iv) local and systemic response, and (v) amplification in wound signaling. The common occurrence of JA biosynthesis and systemin generation in the vascular bundles suggest JA as the systemic signal. Grafting experiments with JA-deficient, JA-insensitive and systemin-insensitive mutants strongly support this assumption.