Abstract:
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.