Phytochemistry (v.66, #20)

Author Index (II).

Flavones and flavone synthases by Stefan Martens; Axel Mithöfer (2399-2407).
Flavones represent one of the largest subgroups within the flavonoids. Two independently evolved and mechanistically different enzymes can convert the precursors, flavanones, into flavones. Various biological activities of flavones in plants and in human nutrition and health make them valuable targets for metabolic engineering.Within the secondary metabolite class of flavonoids which consist of more than 9000 known structures, flavones define one of the largest subgroups. Their natural distribution is demonstrated for almost all plant tissues. Various flavone aglyca and their O- or C-glycosides have been described in the literature. The diverse functions of flavones in plants as well as their various roles in the interaction with other organisms offer many potential applications, not only in plant breeding but also in ecology, agriculture and human nutrition and pharmacology. In this context, the antioxidative activity of flavones, their use in cancer prevention and treatment as well as the prevention of coronary heart disease should be emphasized. The therapeutic potential of flavones makes these compounds valuable targets for drug design, including recombinant DNA approaches. The biosynthesis of flavones in plants was found to be catalyzed by two completely different flavone synthase proteins (FNS), a unique feature within the flavonoids. The first, FNS I, a soluble dioxygenase, was only described for members of the Apiaceae family so far. The second, FNS II, a membrane bound cytochrome P450 enzyme, has been found in all other flavone accumulating tissues. This phenomenon is particularly of interest from the evolutionary point of view concerning the flavone biosynthesis and functions in plants. Recently, FNS I and FNS II genes have been cloned from a number of plant species. This now enables detailed biochemical and molecular characterizations and also the development of direct metabolic engineering strategies for modifications of flavone synthesis in plants to improve their nutritional and/or biopharmaceutical value.
Keywords: Flavonoids; Flavones; Flavone synthases; FNS;

Substances isolated from Mandragora species by Lumír O. Hanuš; Tomáš Řezanka; Jaroslav Spížek; Valery M. Dembitsky (2408-2417).
The present state of knowledge in the chemistry of mandragora plant is reviewed. Isolations and identifications of the compounds were done from all parts of this plant. Up-to-date more than 80 substances were identified in different species of the genus Mandragora.The present state of knowledge in the chemistry of mandragora plant is reviewed. Isolations and identifications of the compounds were done from all parts of this plant. Up-to-date more than 80 substances were identified in different species of the genus Mandragora.
Keywords: Mandragora; Mandrake; Solanaceae; Alkaloids; Natural substances;

A β-(1 → 3)-arabinopyranosyltransferase that transfers a single arabinopyranose onto arabino-oligosaccharides in mung bean (Vigna radiate) hypocotyls by Tadashi Ishii; Teruko Konishi; Yuki Ito; Hiroshi Ono; Mayumi Ohnishi-Kameyama; Ikuko Maeda (2418-2425).
A β-(1 → 3)-arabinopyranosyltransferase that transfers a single arabinopyranose onto arabino-oligosaccharides was characterized.Arabinopyranosyltransferase (ArapT) activity that results in the transfer of a single arabinopyranose (Arap) residue from UDP-β-l-arabinopyranose (UDP-Arap) to exogenous (1 → 5)-linked α-l-arabino-oligosaccharides labeled with 2-aminobenzamide (2-AB) at their reducing ends was identified in a particulate preparation obtained from 3-day-old mung bean (Vigna radiate L. Wilezek) hypocotyls. The transferred Ara residue was shown to be β-(1 → 3)-linked to O-3 of the non-reducing terminal Araf residues of the oligosaccharide using nuclear magnetic resonance spectroscopy together with glycosyl composition and glycosyl linkage composition analyses. The 2AB-labeled arabino-octasaccharide was the most effective acceptor substrate analyzed, although arabino-oligosaccharides with a degree of polymerization between 4 and 7 were also acceptor substrates. Maximum ArapT activity was obtained at pH 6.5–7.0, and 20 °C in the presence of 25 mM Mn2+ and 0.5% Triton X-100.
Keywords: Vigna radiate; Arabinogalactan; Arabinan; Arabino-oligosaccharide; Pectin; Rhamnogalacturonon I; β-(1 → 3)-Arabinopyranosyltransferase;

An antimicrobial peptide Ar-AMP from amaranth (Amaranthus retroflexus L.) seeds by Aleksey Lipkin; Veronika Anisimova; Aleksandra Nikonorova; Aleksey Babakov; Eberhardt Krause; Mikhael Bienert; Eugene Grishin; Tsezi Egorov (2426-2431).
A 30-residue antimicrobial peptide Ar-AMP with six cysteine residues was isolated from the seeds of amaranth Amaranthus retroflexus L. In spite of the fact that seeds were collected in 1967 and lost their germination capacity, Ar-AMP retained its biological activities.A 30-residue antimicrobial peptide Ar-AMP was isolated from the seeds of amaranth Amaranthus retroflexus L. essentially by a single step procedure using reversed-phase HPLC, and its in vitro biological activities were studied. The complete amino acid sequence of Ar-AMP was determined by Edman degradation in combination with mass spectrometric methods. In addition, the cDNA encoding Ar-AMP was obtained and sequenced. The cDNA encodes a precursor protein consisting of the N-terminal putative signal sequence of 25 amino acids, a mature peptide of 30 amino acids and a 34-residue long C-terminal region cleaved during post-translational processing. According to sequence similarity the Ar-AMP belongs to the hevein-like family of antimicrobial peptides with six cysteine residues. In spite of the fact that seeds were collected in 1967 and lost their germination capacity, Ar-AMP retained its biological activities. It effectively inhibited the growth of different fungi tested: Fusarium culmorium (Smith) Sacc., Helminthosporium sativum Pammel., King et Bakke, Alternaria consortiale Fr., and Botrytis cinerea Pers., caused morphological changes in Rhizoctonia solani Kühn at micromolar concentrations and protected barley seedlings from H. sativum infection.
Keywords: Amaranthus retroflexus; Amaranth; Weeds; Cysteine-rich peptide; Antimicrobial peptide; Hevein-like peptide; cDNA; Cloning;

Nicotine demethylation in Nicotiana cell suspension cultures: N′-formylnornicotine is not involved by Trixie Ann Bartholomeusz; Ramneek K. Bhogal; Roland Molinié; François-Xavier Felpin; Monique Mathé-Allainmat; Anna-Carolin Meier; Birgit Dräger; Jacques Lebreton; Albrecht Roscher; Richard J. Robins; François Mesnard (2432-2440).
Label from [13C,2H3-methyl]nicotine fed to Nicotiana plumbaginifolia suspension cell cultures is incorporated into cotinine but to a much lesser extent into N′-formylnornicotine. This labelling pattern directly shows that N′-formylnornicotine is not intermediate in the demethylation mechanism, but is formed by the condensation of nornicotine with formaldehyde.Nicotine or nornicotine enriched with stable isotopes in either the N′-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography–mass spectroscopy (GC–MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N′-demethylation product. In addition, six minor metabolites appeared during the course of nicotine metabolism, four of which were identified as cotinine, myosmine, N′-formylnornicotine and N′-carboethoxynornicotine. While cotinine was formed from [13C,2H3-methyl]nicotine without dilution of label, N′-formylnornicotine was labelled at only about 6% of the level of nicotine and N′-carboethoxynornicotine was unlabelled. Feeding with [1′-15N]nornicotine resulted in incorporation without dilution of label into both N′-formylnornicotine and N′-carboethoxynornicotine. This pattern strongly indicates that, while nornicotine and cotinine are derived directly from nicotine, N′-formylnornicotine and N′-carboethoxynornicotine are metabolites of nornicotine. Thus, it is directly demonstrated that N′-formylnornicotine is not an intermediate in nicotine demethylation.
Keywords: Nicotiana plumbaginifolia; Solanaceae; Nicotine; Nornicotine; N′-Formylnornicotine; Demethylation;

Amplified fragment length polymorphism and metabolomic profiles of hairy roots of Psoralea corylifolia L. by Gauri Abhyankar; V.D. Reddy; C.C. Giri; K.V. Rao; V.V.S. Lakshmi; S. Prabhakar; M. Vairamani; B.S. Thippeswamy; P.S. Bhattacharya (2441-2457).
Hairy root cultures of Psoralea corylifolia were developed. AFLP and Metabolomic profiles showed striking variations between the clones. An Isoflavonoid, formononetin and its glycoside were identified for the first time from hairy root cultures of P. corylifolia.A reproducible protocol for establishment of hairy root cultures of Psoralea corylifolia L. was developed using Agrobacterium rhizogenes strain ATCC 15834. The hairy root clones exhibited typical sigmoid growth curves. Genomic and metabolomic profiles of hairy root clones along with that of untransformed control were analysed. Hairy root clones, Ps I and Ps II, showed significant differences in their amplified fragment length polymorphism (AFLP) profiles as compared to that of control, besides exhibiting Ri T-DNA-specific bands. These results amply indicate the stable integration of Ri T-DNA into the genomes of these clones. Further, the variations observed between clones in the AFLP profiles suggest the variable lengths and independent nature of Ri T-DNA integrations into their genomes. An isoflavonoid, formononetin, and its glycoside were present only in the hairy root clones while they were absent in the untransformed control. Variations observed in the metabolite profiles of these clones may be attributed to the random T-DNA integrations and associated changes caused by them in the recipient genomes. GC/MS analyses revealed the production of three and six clone-specific compounds in Ps I and Ps II, respectively, suggesting that the clones are dissimilar in their secondary metabolism. HPLC/UV–MS analyses disclosed substantial increases in the total isoflavonoids produced in Ps I (184%) and Ps II (94%) compared to untransformed control.
Keywords: Psoralea corylifolia; Papilionaceae; Hairy roots; Metabolomics; AFLP; Isoflavonoids; Formononetin;

Metabolic distinction of Ulmus minor xylem tissues after inoculation with Ophiostoma novo-ulmi by Juan A. Martín; Alejandro Solla; Manuel A. Coimbra; Luis Gil (2458-2467).
Changes in the major chemical components of xylem are investigated in susceptible and resistant Ulmus minor trees, after inoculation with Ophiostoma novo-ulmi, using FT-IR spectroscopy and principal component analysis.Dutch elm disease (DED) is the most devastating and widespread disease of elms. The pathogen, Ophiostoma novo-ulmi, spreads systemically causing xylem vessels blocking and cavitation, and ultimately resulting in the development of a wilt syndrome. Twig samples from susceptible and resistant Ulmus minor trees were harvested at 0, 5, 15, 30, 60, and 120 days post-inoculation (dpi) with O. novo-ulmi. Fourier transform-infrared (FT-IR) spectroscopy, in tandem with chemometrics, was used to monitor changes in wood chemistry as consequence of infection. Principal component analysis distinguished between spectra from inoculated and control elms, and from susceptible- and resistant-inoculated elms. By 30 dpi, infected xylem showed reduced relative levels of carbohydrates and enhanced relative levels of phenolic compounds, probably due to the degradation of cell wall polysaccharides by fungal enzymes and the synthesis of host defence compounds. On 15 dpi, samples from resistant-inoculated elms showed higher levels of starch than samples from susceptible-inoculated elms, suggesting that availability of starch reserves could affect the tree’s capacity for defensive responses. The results showed the power of FT-IR spectroscopy for analysing changes in the major components of elm xylem as consequence of infection by DED, and its potential for detecting metabolic profiles related to host resistance.
Keywords: Ulmus minor; Ophiostoma novo-ulmi; Dutch elm disease; Metabolite fingerprinting; FT-IR metabolite profiles; PCA; Wood degradation; Plant resistance;

Four feruloyl amides and two cross-linked feruloyl dimers were isolated from potato common scab lesions and characterized by NMR techniques.Four feruloyl amides, N-trans-feruloyloctopamine (1), N-cis-feruloyloctopamine (2), N-trans-feruloyltyramine (3), N-cis-feruloyltyramine (4), a cross-linked N-trans-feruloyltyramine dimer (5), and a cross-linked N-cis-feruloyltyramine dimer (6) were isolated from potato common scab lesions. The compounds were purified by TLC and characterized by a combination of 1H and 13C NMR spectroscopic techniques. The presence of an accompanying minor complex of cross-linked dimers containing both feruloyltyramines and feruloyloctopamines was also demonstrated. This is the first characterization of cross-linked hydroxycinnamic acid amides associated with wound healing in potato (Solanum tuberosum) tubers.
Keywords: Hydroxycinnamic acid amides; Solanum tuberosum; Common scab; Streptomyces scabies; Grossamide;

Identification and heritability of fumonisin insensitivity in Zea mays by Anne E. Desjardins; Ronald D. Plattner; Richard J. Stessman; Susan P. McCormick; Mark J. Millard (2474-2480).
Most domesticated maize and wild teosintes (Zea mays species) are highly sensitive to fumonisin B1, a phytotoxic polyketide produced by fungi pathogenic to maize. In a survey of genetically diverse maize landraces, high insensitivity to fumonisin B1 was identified and shown to be an inheritable trait.Landraces of maize (Zea mays ssp. mays) and its wild teosinte relatives (Zea mays spp. parviglumis and mexicana) were surveyed for sensitivity to fumonisin B1, a phytotoxin produced by the maize pathogen Gibberella moniliformis. Only two of 42 Z. mays samples were highly insensitive to FB1 (ED50  = ca. 200 μM). The teosintes and 76% of the maize landraces were moderately or highly sensitive to FB1 (ED50  ⩽ 30 μM), which indicates that FB1 sensitivity is likely to be an ancestral trait in Z. mays. F1 generations derived from crosses between FB1-sensitive maize inbred B73 and insensitive landraces were significantly less sensitive than B73. Thus, our data indicate that FB1-insensitivity is a relatively rare but heritable trait in maize. We also report the sensitivity of maize to other Gibberella toxins – beauvericin, diacetoxyscirpenol, and moniliformin.
Keywords: Zea mays; Gramineae; Maize; Teosinte; Gibberella moniliformis; Fusarium verticillioides; Phytotoxicity; Fumonisins; Beauvericin; Diacetoxyscirpenol; Moniliformin;

Peniocerol 1 (R=H), macdougallin 2 (R=H), as well as mixtures and extracts, showed insecticidal and insect growth regulatory activities against Spodoptera frugiperda and Tenebrio molitor.A methanol extract from the roots and aerial parts of Myrtillocactus geometrizans (Cactaceae) yielded peniocerol 1, macdougallin 2, and chichipegenin 3. The natural products 1, 2 their mixtures, MeOH and CH2Cl2 extracts showed insecticidal and insect growth regulatory activity against fall armyworm [Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae)], an important insect pest of corn, and [Tenebrio molitor (Coleoptera)], a pest of stored grains in Mexico. The most active compounds were 1, 2, and a mixture (M 2 ) of 1 and 2 (6:4). All these extracts, compounds and the mixture had insect growth regulating (IGR) activity between 5.0 and 50.0 ppm and insecticidal effects between 50 and 300 ppm in diets. The extracts were insecticidal to larvae, with lethal doses between 100 and 200 ppm. These compounds appear to have selective effects on the pre-emergence metabolism of Coleoptera, because in all treatments of the larvae of T. molitor, pupation were shortened and this process show precociousness in relation to controls. In contrast to S. frugiperda larvae, onset of pupation was noticeably delayed. Emergence in both cases was drastically diminished. In both pupae and in the few adults that were able to emerge, many deformations were observed. The results of these assays indicated that the compounds were more active than other known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. Peniocerol, macdougallin and chichipegenin, as well as mixtures of these substances, may be useful as natural insecticidal agents.
Keywords: Phytoecdysteroids; Ecdysone mimics; Myrtillocactus geometrizans; Cactaceae; Sterols; Insect growth regulation; IPM; Spodoptera frugiperda; Fall armyworm; Tenebrio molitor;

γ-Glutamyl dipeptides in Petiveria alliacea by Roman Kubec; Rabi A. Musah (2494-2497).
Isolation and identification of three γ-glutamyl dipeptides from Petiveria alliacea L. roots is reported. These include (S C2 R C7)-γ-glutamyl-S-benzylcysteine together with both diastereomers of the corresponding S-oxide.Three γ-glutamyl dipeptides have been isolated from Petiveria alliacea L. roots. These dipeptides include (S C2 R C7)-γ-glutamyl-S-benzylcysteine together with two diastereomeric sulfoxides, namely (S C2 R C7 R S)- and (S C2 R C7 R S)-γ-glutamyl-S-benzylcysteine S-oxides (γ-glutamyl-petiveriins A and B, respectively). Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy, and confirmed by comparison with authentic compounds obtained by synthesis.
Keywords: Petiveria alliacea; Phytolaccaceae; Glutamyl dipeptide; Petiveriin; S-benzylcysteine sulfoxide; S-benzylcysteine;

by David A.H. Taylor (2498).

by Pietro Spanu (2498-2499).

Erratum to “Evidence for the monophyletic evolution of benzylisoquinoline alkaloid biosynthesis in angiosperms” [Phytochemistry 66 (2005) 1374–1393] by David K. Liscombe; Benjamin P. MacLeod; Natalia Loukanina; Owi I. Nandi; Peter J. Facchini (2500-2520).