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

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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.

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Abbreviations

HPF:

High-pressure freezing

FS:

Freeze substitution

FD:

Freeze drying

CSLM:

Confocal scanning microscopy

EtOH:

Ethanol

HPLC:

High-performance liquid chromatography

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Acknowledgments

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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Authors and Affiliations

  1. Department of Biochemistry, University of Pretoria, Lynnwood Road, Hatfield, Pretoria, 0002, Gauteng, South Africa

    Shane V. van Breda & Zeno Apostolides

  2. Laboratory for Microscopy and Microanalysis, University of Pretoria, Lynnwood Road, Hatfield, Pretoria, 0002, Gauteng, South Africa

    Chris F. van der Merwe

  3. Department of Plant Production and Soil Science, University of Pretoria, Lynnwood Road, Hatfield, Pretoria, 0002, Gauteng, South Africa

    Hannes Robbertse

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  1. Shane V. van Breda
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  2. Chris F. van der Merwe
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  3. Hannes Robbertse
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  4. Zeno Apostolides
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Correspondence to Shane V. van Breda.

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van Breda, S.V., van der Merwe, C.F., Robbertse, H. et al. Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves. Planta 237, 849–858 (2013). https://doi.org/10.1007/s00425-012-1804-x

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