Exodermis and endodermis are the sites of xanthone biosynthesis in Hypericum perforatum roots.

Xanthones are specialized metabolites with antimicrobial properties, which accumulate in roots of Hypericum perforatum. This medicinal plant provides widely taken remedies for depressive episodes and skin disorders. Owing to the array of pharmacological activities, xanthone derivatives attract attention for drug design. Little is known about the sites of biosynthesis and accumulation of xanthones in roots. Xanthone biosynthesis is localized at the transcript, protein, and product levels using in situ mRNA hybridization, indirect immunofluorescence detection, and high lateral and mass resolution mass spectrometry imaging (AP-SMALDI-FT-Orbitrap MSI), respectively. The carbon skeleton of xanthones is formed by benzophenone synthase (BPS), for which a cDNA was cloned from root cultures of H. perforatum var. angustifolium. Both the BPS protein and the BPS transcripts are localized to the exodermis and the endodermis of roots. The xanthone compounds as the BPS products are detected in the same tissues. The exodermis and the endodermis, which are the outermost and innermost cell layers of the root cortex, respectively, are not only highly specialized barriers for controlling the passage of water and solutes but also preformed lines of defence against soilborne pathogens and predators.

Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

Differential expression of biphenyl synthase gene family members in fire-blight-infected apple 'Holsteiner Cox'.

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple 'Golden Delicious', nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple 'Holsteiner Cox,' heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple 'Cox Orange,' expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.