# Molecular and Functional Evolution of the Spermat Sesquiterpene Synthases
## 3 Discussion  
Along with the species evolution, plants have evolved to produce a different collection of terpenenes to accommodate their biotic and abiotic environment [34]. The plant sesquiterpenes gradually formed and might be a potential result of escalating defense and counter-defense between plants and specialized herbivores [35; 36]. Exploring the evolutionary origin and history of STSs can not only help to understand the evolutionary pattern and reaction mechanism, but also preliminarily predict the function of STSs. At present, there are many phylogenetic analyses of plant TPSs, most of which focus on taxonomic studies to indicate the evolutionary behavior of TPSs among and within species [16; 37], without functional selection of sequences. In this study, all 394 spermatophyte STSs were divided into five distinct groups according to structure-based phylogenetic analysis to explore the evolutionary patterns of plant STSs and sesquiterpenes. Many genes are involved in sesquiterpene biosynthesis in the genome of each plant species, which also provides a large platform for the evolution of new sesquiterpenes via gene duplication and subfunctionalization [21; 26]. Intron loss, mutations, and coevolution with natural enemies are considered to be the most important evolutionary dynamics of STSs. Evolution in STSs is often the result of intron loss or mutations that lead to subfunctionalization or function loss [38]. For example, a large fragment loss of \(\delta\)-selinene synthase 2 (_Agsel2_) from _A. grandis_ was found around intron X that led to _Agsel2_ being transcribed as a pseudogene [39]. Single amino acid W279A switch converts \(\delta\)-cadinene synthase (CAD1-A) into germacradien-4-ol synthase [40]. New sesquiterpenes keep arising in specific plant lineages, potentially as an outcome of coevolution with natural enemies [35]. Different plant lineages have evolved the ability to make additional \"specialized\" metabolites that are implicated in defense or the attraction of beneficial organisms, which indicates a dominant process dynamic evolution in STSs to the chemical diversity in plants. STSs have various evolutionary forms. It can also be expected that STSs with altered subcellular localization and new substrate specificities would have evolved. Although TPSs often have broad substrate specificity and accept GPP, FPP, or GGPP in vitro, their function may be narrower in planta due to their subcellular localization [41]. Monoterpene synthases and diterpene synthases typically contain N-terminus signal peptides and are transported into plastids, STSs, however, are usually found in the cytosol [9]. There is increasing evidence for an exchange of TPS subcellular localization, especially under stress conditions [42; 43]. Examples include the _AmNES/LIS-1/2_ and _CsLIS/NES-1/2_ analyzed above in the TPS-g subfamily. In this scenario, driven by adaptive evolution, ancestral monoterynes hasing the N-terminus signal peptide changed their substrate pool and gradually evolved into STSs. Models for gymnosperm TPS evolution proposed that STSs evolved from diterpene synthases through loss of introns, which resulted in, among other changes, the complete loss of the \(\gamma\) domain [39]. Based on this model, _Abies grandis_ a-bisabolene synthase _Ag1_ (C6-C1 closure), a three-domain plant STSs, is potentially an intermediate in the evolutionary history from diterpene to sesquiterpene synthase [44]. Distinct catalytic features of the STSs arose early in spermatophyte evolution and the reactions have become more complex over time. In the evolution of STSs, it is easier to form acyclic sesquiterpenes than cyclic sesquiterpenes according to phylogenetic analysis. Acyclic sesquiterpenes were formed directly from farnesyl cation or merididyl cation by proton loss or addition of water [9].