Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves.

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine's localization.