By Ellie Fung
As wild as it may sound, caffeine was not designed to be guzzled upon by sleep-deprived university students. Its stimulant activity is exploited by millions worldwide, whether it be a morning coffee or a late-night Redbull, but much less is known about its function in the plants that produce them. These genera include Coffea (coffee), Camellia (tea) and Theobroma (chocolate), but guarana (Paullinia) and some citrus (Citrus) species are also natural sources of the ubiquitous stimulant (Huang et al., 2016).
Caffeine, or 1,3,7-trimethylxanthine, is a purine alkaloid, a group of chemical compounds forming part of the wide arsenal of plant defenses against abiotic and biotic stresses (Kim, Yun-Soo, Choi & Sano, 2010). These are well-established to be directly toxic and unpalatable to pests, but it wasn’t until recently that the specific activity of caffeine and other methylxanthines was identified. In an early study, tobacco hornworm larvae exhibited dose-dependent inhibition of feeding activity when finely ground Camellia sinensis leaves and Coffea arabica beans were added to a liquid medium up to a concentration of 3% and 10% respectively. Beyond these concentrations, the larvae were killed within 24 hours. The study also found that the levels of purified caffeine naturally found in fresh tea leaves and coffee beans were lethal to most larvae, providing early evidence that caffeine may function as an insecticide. Other insect species were shown to be affected too, such as mealworm larvae, butterfly larvae and mosquitoes (Nathanson, 1984a).
Kim et al. (2006) supported these findings by creating transgenic tobacco plants that were able to synthesise caffeine naturally. Contrary to their wild counterparts, these plants were unpalatable to tobacco cutworms and lepidopteran caterpillars. The repellent effects seem to extend to pathogens as well. Later, Kim & Sano (2008) found that while wild-type and transgenic varieties constitutively expressed pathogenesis-related (PR)-1a and proteinase inhibitor II (PR-2) genes, the transgenic plants expressed much higher levels of these defense-related genes when infected with tobacco mosaic virus and Pseudomonas syringae. They suggested that caffeine elevates resistance against pathogens possibly by acting as a signalling molecule and increasing salicylic acid production immediately after infection, which stimulates a greater hypersensitive response. Caffeine that is applied exogenously to wild-type tobacco also resulted in higher PR-1a and PR-2 expression and could even disturb the reproductive potential of four moth species (Nathanson, 1984).
Further research has also demonstrated the toxicity of caffeine against insects and pathogens, but its exact physiological role in plants remains unclear. Surprisingly, caffeine may even be mildly toxic to the plant producer. By acting as a phosphodiesterase inhibitor, caffeine raises intracellular concentrations of cyclic AMP. In plants, accumulation of cAMP may impair signal transduction in pathways modulating stomatal closure, cell cycle regulation and cell channel guarding. It has been suggested that self-defence signalling pathways, such as salicylic acid production, have arisen to compensate for these detrimental effects, which performs double duty as this also provides protection against biotic stresses. In light of this, researchers have even suggested that caffeine could pave the way to developing a novel “plant vaccine”, as much like animal vaccines, the introduction of a slightly dangerous external agent would confer resistance against more deadly invaders (Kim, Choi & Sano, 2010).
With caffeine recognised as a defensive compound, its presence in the nectar of some Citrus and Coffea species came across as counterintuitive, leading researchers to believe that caffeine may also confer a reproductive advantage. Indeed, one group showed that individual honeybees rewarded with caffeine-containing nectar were three times more likely to remember the scent of the rewarding flower 24 hours later than those that were not fed with caffeine. Bees were twice as likely to retain scent memory after 72 hours. As their strongly aromatic flowers signifies, Citrus and Coffea reproduce in a pollinator-dependent manner. The group hypothesised that caffeine enhances long-term associative memory to increase the likelihood of pollinators returning to flowers with the same scent signals. Even so, the bitterness of caffeine is not lost upon bees: concentrations above 1mM was enough to be repellent, but, as testament to the remarkable ability of plants in manipulating their pollinators, caffeine concentrations in naturally-occurring nectar never exceeded 0.3mM, even if the quantity in other tissues is much higher (Wright et al., 2013). Though these findings are exciting, the experiment does not accurately reflect the real-world as it neglects the social interactions between bees, which exerts a strong influence on foraging behaviour (Thomson, Draguleasa & Tan, 2015). To date, studies regarding the role of caffeine in improving pollinator memory are very limited, thus more research needs to be conducted to confirm this hypothesis.
Given these advantages of caffeine, it is predictably produced in multiple lineages. In fact, the biosynthetic pathway seemed to have evolved independently at least 5 times in angiosperm history. Previously, it was believed that all these species utilised the same three-step biochemical pathway, but recent research has demonstrated that at least three distinct pathways exist, involving orthologous enzymes derived from ancestral methyltransferases. By reconstructing the ancestral xanthine methyltransferase (XMT) of the Citrus genus, they found that just a few amino acid substitutions allowed for substrate preference to switch to theophylline, the exact compound that is preferentially methylated by modern Citrus XMT to make caffeine. This suggests that ancient enzymes previously engaged in other biochemical pathways had undergone exaptation to synthesise caffeine in a relatively facile fashion, which could explain the many instances of convergence (Huang et al., 2016).
For something we humans so readily put into our bodies, the origin and specific role of caffeine in the context of its botanical sources is not well understood. One might wonder how many more cups of coffee it might take for scientists to come up with firm conclusions.
Huang, R., O’Donnell, A.,J., Barboline, J. J. & Barkman, T. J. (2016) Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes. Proceedings of the National Academy of Sciences of the United States of America. 113 (38), 10613-10618. Available from: doi: 10.1073/pnas.1602575113.
Kim, Y. S. & Sano, H. (2008) Pathogen resistance of transgenic tobacco plants producing caffeine. Phytochemistry. 69 (4), 882-888. Available from: doi: S0031-9422(07)00614-0 [pii].
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Kim, Y., Choi, Y. & Sano, H. (2010) Plant vaccination: Stimulation of defense system by caffeine production in planta. Null. 5 (5), 489-493. Available from: doi: 10.4161/psb.11087.
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Nathanson, J. A. (1984b) Caffeine and related methylxanthines: possible naturally occurring pesticides. Science (New York, N.Y.). 226 (4671), 184-187. Available from: doi: 10.1126/science.6207592.
Thomson, J. D., Draguleasa, M. A. & Tan, M. G. (2015) Flowers with caffeinated nectar receive more pollination. Arthropod-Plant Interactions. 9 (1), 1-7. Available from: doi: 10.1007/s11829-014-9350-z.
Wright, G. A., Baker, D. D., Palmer, M. J., Stabler, D., Mustard, J. A., Power, E. F., Borland, A. M. & Stevenson, P. C. (2013) Caffeine in floral nectar enhances a pollinator’s memory of reward. Science (New York, N.Y.). 339 (6124), 1202-1204. Available from: doi: 10.1126/science.1228806.