Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves
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ORIGINAL ARTICLE
Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves Shane V. van Breda • Chris F. van der Merwe Hannes Robbertse • Zeno Apostolides
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Received: 9 July 2012 / Accepted: 30 October 2012 / Published online: 10 November 2012 Ó Springer-Verlag Berlin Heidelberg 2012
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 S. V. van Breda (&) Z. Apostolides Department of Biochemistry, University of Pretoria, Lynnwood Road, Hatfield, Pretoria 0002, Gauteng, South Africa e-mail: [email protected] C. F. van der Merwe Laboratory for Microscopy and Microanalysis, University of Pretoria, Lynnwood Road, Hatfield, Pretoria 0002, Gauteng, South Africa H. Robbertse Department of Plant Production and Soil Science, University of Pretoria, Lynnwood Road, Hatfield, Pretoria 0002, Gauteng, South Africa
with chlorogenic acid is also discussed as this may also help explain caffeine’s localization. Keywords Caffeine Camellia Freeze substitution High pressure freezing Immunohistochemical Plant chemical defense Abbreviations HPF High-pressure freezing FS Freeze substitution FD Freeze drying CSLM Confocal scanning microscopy EtOH Ethanol HPLC High-performance liquid chromatography
Introduction Tea is the agricultural product of Camellia sinensis, utilizing the top youngest leaves of the plant. The chief teas of commerce are green tea, black tea, and oolong tea and in beverage form, it is the second most widely consumed product worldwide after water (Willson and Clifford 1992). The young leaves of C. sinensis contain catechins known for their beneficial health properties such as radical scavenging, antibacterial action, and prevention of cancer (Hara 2001). A less popular compound is a xanthine alkaloid known as caffeine whose content is between 3 and 4 % (based on dry weight) and is responsible for distu
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