Abstract
Hypericum perforatum (St. John’s wort; Clusiaceae) is an important medicinal plant, which is widely used as an antidepressant. The plant is characterized by the presence of different types of secretory tissue including translucent glands, black nodules and secretory canals. Hypericum species are ideal experimental systems for studying the biosynthesis of a diversity of aromatic polyketides. Two type III polyketide synthases (PKSs) involved are benzophenone synthase (BPS) and chalcone synthase (CHS), for which cDNAs had been cloned and characterized. This work describes immunochemical studies and immunofluorescence localization of these PKSs in H. perforatum. Both enzymes were heterologously expressed in E. coli as 6xHis-tagged proteins and GST-fusion proteins. Polyclonal antibodies were raised against the 6xHis-tagged PKSs in rabbits and the IgG fractions were isolated. The specificity of the antibodies was examined using immunoblotting and immunotitration techniques. Protein extracts from various H. perforatum organs were subjected to SDS-PAGE and immunoblotting. BPS was mainly immunodetected in middle-aged fruits. CHS was detected in young leaves and flower buds. The tissue-specific localization of BPS and CHS was studied with H. perforatum organs using the immunofluorescence technique and confocal laser scanning microscopy. BPS was expressed to a low extent in mesophyll cells of young leaves and strongly expressed in the glandular cells of large translucent glands present inside the leaves. In roots, BPS was located in the cortex cells. In floral parts, as far as studied, BPS was found in the secretory tissue of sepals of young buds and in middle-aged fruits. In addition, seeds present in the middle-aged fruits contained BPS. CHS was strongly expressed in the mesophyll cells of young leaves and was not present in glands. Nor was the enzyme observed in roots. In floral parts, as far as studied, CHS is located in the mesophyll of sepals of young buds.
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