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Polyketide synthases in St. John's wort (Hypericum perforatum L.) leaves: immunochemical studies and immunofluorescence localization
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 were cloned and characterized. The present work describes immunochemical studies and immunofluorescence localization of these PKSs in H. perforatum leaves. 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 different stages of H. perforatum leaves were subjected to SDS-PAGE and immunoblotting. While BPS expression was low, a high CHS level was found in young leaves. The tissue-specific localization of BPS and CHS was studied in different developmental stages of H. perforatum leaves using the immunofluorescence technique and confocal laser scanning microscopy. BPS was observed 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. CHS was strongly expressed in the mesophyll cells of young leaves and was absent from glands.