aucuparin and diphenyl

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M. domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V. inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.

Topics: Amino Acid Sequence; Ascomycota; Benzoates; Biphenyl Compounds; Cell Culture Techniques; Cinnamates; Gene Expression Regulation, Plant; Genes, Plant; Ligases; Malus; Models, Molecular; Molecular Docking Simulation; Nicotiana; Phytoalexins; Plant Diseases; Plant Leaves; Protein Conformation; Sequence Alignment; Sesquiterpenes

Aucuparin is the most widely distributed biphenyl phytoalexin in the rosaceous subtribe Pyrinae, which includes the economically important fruit trees apple and pear. The biphenyl scaffold is formed by biphenyl synthase, which catalyzes biosynthesis of 3,5-dihydroxybiphenyl. Conversion of this precursor to aucuparin (3,5-dimethoxy-4-hydroxybiphenyl) was studied in cell cultures of Sorbus aucuparia after treatment with an elicitor preparation from the scab-causing fungus Venturia inaequalis. The sequence of the biosynthetic steps detected was O-methylation - 4-hydroxylation - O-methylation. The two alkylation reactions were catalyzed by distinct methyltransferases, which differed in pH and temperature optima as well as stability. Biphenyl 4-hydroxylase was a microsomal cytochrome P450 monooxygenase, whose activity was appreciably decreased by the addition of established P450 inhibitors. When fed to V. inaequalis-treated S. aucuparia cell cultures, radioactively labeled 3,5-dihydroxybiphenyl was not only incorporated into aucuparin but also into the dibenzofuran eriobofuran, the accumulation of which paralleled that of aucuparin. However, biphenyl 2'-hydroxylase activity proposed to be involved in dibenzofuran formation was detected in neither microsomes nor cell-free extracts in the presence of NADPH and 2-oxoglutarate, respectively. Nevertheless, a basis for studying biphenyl biosynthesis at the gene level is provided.

Topics: Ascomycota; Benzofurans; Biphenyl Compounds; Cells, Cultured; Cytochrome P-450 Enzyme System; Ketoglutaric Acids; Malus; Multienzyme Complexes; Phytoalexins; Pyrus; Sesquiterpenes; Sorbus

Three new biphenyls, berbekorin A (1), 2'-hydroxy-3,4,5-trimethoxybiphenyl (2), and 4,5-dihydroxy-3-methoxybiphenyl (3), together with 11 known biphenyls (4-14), were isolated from the n-hexane-soluble fraction of the MeOH extract of the trunk of Berberis koreana. The structures of 1-3 were determined by spectroscopic methods, including 1D and 2D NMR analysis. Compound 1 exhibited cytotoxic activity against the SK-MEL-2 skin melanoma cell line, in particular. Moreover, compounds 2, 5, 6, and 14 inhibited NO production in LPS-activated BV-2 cells, a microglial cell line.

Topics: Animals; Antineoplastic Agents, Phytogenic; Berberis; Biphenyl Compounds; Drug Screening Assays, Antitumor; Hexanes; Korea; Lipopolysaccharides; Mice; Microglia; Molecular Structure; Nitric Oxide; Nuclear Magnetic Resonance, Biomolecular; Plant Stems; Plants, Medicinal