Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes Ruiqi Huanga, Andrew J. O’Donnella,1, Jessica J. Barbolinea, and Todd J. Barkmana,2 aDepartment of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008 Edited by Ian T. Baldwin, Max Planck Institute for Chemical Ecology, Jena, Germany, and approved July 18, 2016 (received for review March 25, 2016) Convergent evolution is a process that has occurred throughout the the evolutionary gain of traits such as caffeine that are formed via tree of life, but the historical genetic and biochemical context a multistep pathway. First, although convergently co-opted genes, promoting the repeated independent origins of a trait is rarely such as XMT or CS, may evolve to encode enzymes for the same understood. The well-known stimulant caffeine, and its xanthine biosynthetic pathway, it is unknown what ancestral functions they alkaloid precursors, has evolved multiple times in flowering plant historically provided that allowed for their maintenance over mil- history for various roles in plant defense and pollination. We have lions of years of divergence. Second, it is unknown how multiple shown that convergent caffeine production, surprisingly, has protein components are evolutionarily assembled into an ordered, evolved by two previously unknown biochemical pathways in functional pathway like that for caffeine biosynthesis. Under the chocolate, citrus, and guaraná plants using either caffeine synthase- cumulative hypothesis (26), it is predicted that enzymes catalyzing or xanthine methyltransferase-like enzymes. However, the pathway earlier reactions of a pathway must evolve first; otherwise, enzymes and enzyme lineage used by any given plant species is not predict- that perform later reactions would have no substrates with which to able from phylogenetic relatedness alone. Ancestral sequence res- react. Subsequently, duplication of the gene encoding the first urrection reveals that this convergence was facilitated by co-option enzyme would give rise to enzymes catalyzing later steps. This of genes maintained over 100 million y for alternative biochemical hypothesis assumes that, initially, the intermediates in a pathway roles. The ancient enzymes of the Citrus lineage were exapted for are advantageous, because it is unlikely that multiple enzymatic reactions currently used for various steps of caffeine biosynthesis steps in a pathway could evolve simultaneously. Alternatively, the and required very few mutations to acquire modern-day enzymatic retrograde hypothesis (27) states that enzymes catalyzing reactions characteristics, allowing for the evolution of a complete pathway. that occur at the end of a pathway evolved first. Gene duplication Future studies aimed at manipulating caffeine content of plants will of the sequence encoding the first-evolved enzyme would eventu- require the use of different approaches given the metabolic and ally result in new enzymes that perform the preceding pathway genetic diversity revealed by this study. steps. This hypothesis assumes that the intermediates of a given pathway would be produced nonenzymatically and be available for convergent evolution | caffeine biosynthesis | enzyme evolution | catalysis; as such, it may have less general explanatory application. paleomolecular biology Finally, the patchwork hypothesis (28, 29) explains the origins of novel pathways by the recruitment of enzymes from alternative EVOLUTION onvergent evolution has resulted in the independent origins of preexisting pathways. This hypothesis assumes that the older, Cmany traits dispersed throughout the tree of life. Whereas some recruited enzymes were ancestrally promiscuous with respect to the convergent traits are known to be generated via similar develop- substrates catalyzed such that they were exapted for the activities mental or biochemical pathways, others arise from different paths that they later become specialized for in the novel pathway. Unlike (1–5). Likewise, similar (orthologous) or different (paralogous or the cumulative and retrograde hypotheses, there is no prediction even unrelated) genes may encode for the regulatory or structural for the relative ages of enzymes performing each step of the novel proteins composing the components of pathways that build conver- pathway under the patchwork hypothesis. The patchwork hypoth- gent traits (6–10). One of the most prominent examples of conver- esis is compatible with the innovation, amplification, and duplication gence in plants is that of caffeine biosynthesis, which appears to have evolved at least five times during flowering plant history (11). The Significance phylogenetic distribution of caffeine, or xanthine alkaloids more generally, is highly sporadic and usually restricted to only a few Convergent evolution is responsible for generating similar traits in species within a given genus (12, 13). Caffeine accumulates in various unrelated organisms, such as wings that allow flight in birds and tissues, where it may deter herbivory (14, 15) or enhance pollinator bats. In plants, one of the most prominent examples of convergence memory (16). Numerous studies over the past 30 y have indicated is that of caffeine production, which has independently evolved in that although several possible routes exist, the same canonical numerous species. In this study, we reveal that even though the pathway to caffeine biosynthesis has evolved independently in Coffea caffeine molecule is identical in the cacao, citrus, guaraná, coffee, (coffee) and Camellia (tea) involving three methylation reactions to and tea lineages, it is produced by different, previously unknown, sequentially convert xanthosine to 7-methylxanthine to theobromine biosynthetic pathways. Furthermore, by resurrecting extinct en- to caffeine (Fig. 1) (17, 18). In Coffea, three xanthine methyl- zymes that ancient plants once possessed, we show that the novel transferase (XMT)-type enzymes from the SABATH (salicylic acid, pathways would have evolved rapidly because the ancestral en- benzoic acid, theobromine methyltransferase) family (19) are used to zymes were co-opted from previous biochemical roles to those of catalyze the methylation steps of the pathway, whereas Camellia uses caffeine biosynthesis for which they were already primed. a paralogous, convergently evolved caffeine synthase (CS)-type en- zyme (20–22) (Fig. 1). Because most SABATH enzymes catalyze the Author contributions: R.H., A.J.O., J.J.B., and T.J.B. designed research; R.H., A.J.O., and J.J.B. methylation of oxygen atoms of a wide diversity of carboxylic acids performed research; R.H., A.J.O., J.J.B., and T.J.B. analyzed data; and T.J.B. wrote the paper. such as anthranilic, benzoic, gibberellic, jasmonic, loganic, salicylic, The authors declare no conflict of interest. and indole-3-acetic acid for floral scent, defense, and hormone This article is a PNAS Direct Submission. modulation (23–25), methylation of xanthine alkaloid nitrogen atoms 1Present address: Max Planck Institute for Chemical Ecology, 07745 Jena, Germany. by XMT and CS is likely a recently evolved activity. 2To whom correspondence should be addressed. Email: [email protected]. Although convergence has been documented at multiple hier- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. archical levels, fundamental questions remain unanswered about 1073/pnas.1602575113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1602575113 PNAS | September 20, 2016 | vol. 113 | no. 38 | 10613–10618 Downloaded by guest on September 30, 2021 O O that the relative methylation preferences of these enzymes, al- CH3 H C 3 H3C though similar, have convergently evolved in Theobroma and N N N N Paullinia, likely after gene duplication independently occurred in O N NH O N N each lineage (Fig. 2 B and C and Fig. S1). No enzymes have been O H H 1-methylxanthine Paraxanthine previously reported to specialize in the methylation of xanthine or HN N [1X] [PX] 1 7 3-methylxanthine, and the biochemical route to caffeine implied by 3 O O O O N NH CH3 these enzyme activities (Fig. 2 B and C) has not been implicated as H N H3C H3C N Xanthine HN N N N the primary pathway in these or any other plants. However, there is [X] evidence for this pathway in Theobroma fruits and leaves from O N NH O N NH O N N metabolomic analyses and radiolabeled tracer studies that showed O CH3 CH3 CH3 3-methylxanthine Theophylline Caffeine 3-methylxanthine as an intermediate formed during theobromine HN N [3X] [TP] [CF] 1 7 accumulation when xanthine or various purine bases and nucleo- 3 O N O O N CH3 CH3 sides are provided as substrates (34, 35). Analyses of fruit and leaf H Ribose HN N HN N Xanthosine extracts and isotope tracer studies also report the accumulation of [XR] N O N O N N Coffea: CaDXMT 7-methylxanthine to a lesser extent (34, 35). Our liquid chroma- H Camellia: TCS1 Coffea: CaXMT CH3 tography-mass spectrometry (LC-MS) results indicate that this 7-methylxanthine Theobromine [7X] [TB] metabolite can be formed by methylation of xanthine by TcCS2 as a Coffea: CaMXMT Camellia: TCS1 secondaryactivity(Fig.2B and Fig. S3). Thus, in Theobroma,itis possible that theobromine is produced via methylation of this in- Fig. 1. Caffeine biosynthetic network has 12 potential paths. The only path termediate by BTS (36) and/or TcCS1 (Fig. 2B), in addition to characterized from plants is shown by solid black arrows and involves sequential 3-methylxanthine by TcCS2 (Fig. 2B). It is not yet