Combinatorial Evolution of a Terpene Synthase Gene Cluster Explains Terpene Variations in

Terpenes are specialized metabolites ubiquitously produced by plants via the action of terpene synthases (TPSs). There are enormous variations in the types and amounts of terpenes produced by individual species. To understand the mechanisms responsible for such vast diversity, here we investigated the origin and evolution of a cluster of tandemly arrayed genes in In the species analyzed, genes occur as a three- cluster, a two- cluster, and a single gene in five, one, and one species, respectively. Phylogenetic analysis revealed the origins of the two- and three- clusters and the role of species-specific losses of genes. Within the three- clusters, one orthologous group exhibited conserved catalytic activities. The other two groups, both of which contained pseudogenes and/or nonfunctional genes, exhibited distinct profiles of terpene products. Sequence and structural analyses combined with functional validation identified several amino acids in the active site that are critical for catalytic activity divergence of the three orthologous groups. In the five species containing the three- cluster, their functional genes showed both conserved and species-specific expression patterns in insect-damaged and untreated plants. Emission patterns of volatile terpenes from each species were largely consistent with the expression of their respective genes and the catalytic activities of the encoded enzymes. This study indicates the importance of combinatorial evolution of genes in determining terpene variations among individual species, which includes gene duplication, retention/loss/degradation of duplicated genes, varying selection pressure, retention/divergence in catalytic activities, and divergence in expression regulation.