Functional genomics and molecular evolution of bioactive phenolic conjugates in plants

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Elucidating evolutionary mechanism is central to biological sciences. Gene duplications are a rich source of genome and in consequence phenotypic diversity. Many molecular evolutionary models have been developed to explain how gene duplicates may evolve new functions. But for most these models functional evidence is scarce, because most complex adaptive traits cannot be easily linked to a single gene.Plant secondary metabolism provides a notable exception. Plants produce an immense diversity of these small bioactive compounds functioning as protectants and attractants in chemical ecology. Adaptive variations of secondary metabolites can frequently be linked directly to genes encoding their biosynthetic enzymes, making them ideal test cases to validate evolutionary models.The focus here lies on a class of secondary metabolites called hydroxycinnamoyl conjugates (HCCs), which are wide spread across the plant kingdom but are particularly diverse in some species such as poplar trees. HCCs can be herbivore deterrents, antimicrobials, or UV-absorbing sunscreens. Humans exploit them as antioxidants or pharmaceuticals. One particular HCC has been recruited in seed plants as an intermediate in lignin biosynthesis. Lignin is a rigid polymer encrusted into secondary cell walls and enables upright growth. Two enzymes are responsible for HCC biosynthesis: a hydroxycinnamoyl-transferase (HCT) and a coumaroyl-conjugate 3-hydroxylase belonging to the CYP98A family. Seed plants contain multiple members, while mosses and lycopods contain only a single copy. However, the latter are not involved in lignin biosynthesis. Instead, our preliminary results suggest that the ancestral function of HCT/CYP98A was to produce a UV-absorbing and anti-microbial HCC. We hypothesize that these enzymes then continuously expanded their copy number and substrate range under positive selection to create the current HCC diversity. During this process, one of these duplicates was recruited for lignin biosynthesis and has maintained its specificity under purifying selection.To test these hypotheses, we will link evolutionary analyses of the HCT and CYP98A gene families with biochemical and chemical ecological approaches.We will use sequence data from across the plant lineage to generate phylogenetic gene trees. These pinpoint key gene duplication events and signatures of selection to either maintain a given function (expected in the lignin-related gene clade) or to diversify their functions (in protection-related clades). On a more recent evolutionary scale, poplars and related tree species provide a unique system to test ongoing adaptive radiations, because they evolved a species-specific diversity of HCCs and because we do have available sequence data from thousands of individuals covering thirty species.To experimentally test functional diversity (or uniformity) within clades, we will employ biochemical approaches using recombinant production of target proteins in bacteria or yeasts. This will determine substrate ranges (wide or narrow) of complete HCT/CYP98A families from representative species across the plant lineage. We will also use reverse genetic approaches in select species, such as the moss Physcomitrella, or the angiosperms Arabidopsis and poplar. This will facilitate metabolomic phenotyping using analytical chemistry targeting both soluble HCCs and lignin. These mutants will also allow testing the roles of these genes in chemical ecology through controlled infestations with herbivores and pathogens, and through UV exposure experiments. Together, this will link adaptive chemical traits realized through enzymatic diversity directly to gene duplications and selection signatures, thereby deciphering the modes of evolution that shaped HCC diversity.