, Volume 237, Issue 3, pp 849–858 | Cite as

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

  • Shane V. van BredaEmail author
  • Chris F. van der Merwe
  • Hannes Robbertse
  • Zeno Apostolides
Original Article


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.


Caffeine Camellia Freeze substitution High pressure freezing Immunohistochemical Plant chemical defense 



High-pressure freezing


Freeze substitution


Freeze drying


Confocal scanning microscopy




High-performance liquid chromatography



We are grateful to Alan Hall and Andre Botha from the laboratory for microscopy and microanalysis at the University of Pretoria for all their microscopy knowledge and assistance.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Shane V. van Breda
    • 1
    Email author
  • Chris F. van der Merwe
    • 2
  • Hannes Robbertse
    • 3
  • Zeno Apostolides
    • 1
  1. 1.Department of BiochemistryUniversity of PretoriaPretoriaSouth Africa
  2. 2.Laboratory for Microscopy and MicroanalysisUniversity of PretoriaPretoriaSouth Africa
  3. 3.Department of Plant Production and Soil ScienceUniversity of PretoriaPretoriaSouth Africa

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