Date of Award

8-2017

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Todd J. Barkman

Second Advisor

Dr. Yan Lu

Third Advisor

Dr. Pamela Hoppe

Fourth Advisor

Dr. James Kiddle

Keywords

Caffeine, molecular evolution, ancestral sequence resurrection, caffeine biosynthetic enzymes, flowering plants

Abstract

Convergent evolution generally refers to the independent evolution of similar biological function more than once in unrelated species. Caffeine is thought to have evolved by convergence, and is naturally produced through secondary metabolism in plants to defend against pathogen attack and insect feeding or to attract pollinators. The same caffeine biosynthetic pathway has been elucidated in Camellia (tea) and Coffea (coffee), in which xanthosine is sequentially methylated to caffeine via 7-methylxanthine and theobromine. However, although the same catalysis pathway is used, different (paralogous) enzymes in the SAMT/BAMT/theobromine synthase (SABATH) multigene family are used in the two species. In my dissertation, first, novel three-step caffeine biosynthetic pathways were characterized in Citrus (kumquat) flowers and Paullinia (guarana) seeds, and although both species belong to Sapindales, their caffeine synthases that catalyze the new pathways are paralogous, rather than orthologous genes. Interestingly, the caffeine synthases of Paullinia are the same as (orthologous) Camellia while those in Citrus are orthologous to Coffea.

Secondly, I used a paleomolecular biology technique to resurrect ancestral caffeine synthases at different evolutionary time points to see whether the ability to synthesize caffeine was an ancestral trait that was inherited only in a small number of modern-day species while lost in many other species, or whether the caffeine biosynthetic ability evolved more recently in a limited number of plant species that we see today. Most ancestral caffeine synthases were found to possess activities with a broad range of substrates, and may therefore have been maintained long-term for possibly alternative biochemical functions before they specialized for caffeine biosynthesis, a phenomenon termed exaptation. In my experiments, ancestral caffeine synthases in Citrus and Paullinia were also experimentally mutated towards their modern-day descendants to show how the three-step caffeine biosynthetic pathways were assembled in these two species, and to test three hypotheses regarding the evolution of metabolic pathways.

Lastly, I characterized the caffeine biosynthetic enzymes and pathway in Tilia, where caffeine is produced in Tilia flowers, possibly for pollinator attraction. The Tilia transcriptome was assembled using the RNA-Seq technique, and one caffeine synthase gene, which is orthologous to the caffeine synthase genes in Theobroma (chocolate), Paullinia and Camellia, was identified and functionally characterized. Based upon xanthine alkaloids found in Tilia flowers, possible caffeine biosynthetic pathways functioning in Tilia were proposed.

Access Setting

Dissertation-Open Access

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