# Molecular and Functional Evolution of the Spermat Sesquiterpene Synthases
## 1 Introduction  
Terpenes constitute a large class of chemically and structurally diverse natural products in the plant kingdom and serve multiple physiological and ecological functions. At present, more than 25,000 terpenoid structures and 80,000 compounds have been found [1]. Sesquiterpenes are the most complex group with structural diversity, and more than 300 kinds of basic skeletons have been found, which are widely distributed in plants and microorganisms [2]. Sesquiterpenes play important roles in interactions with pollinators and seed dispersers [3], direct defenses against herbivores [4] and pathogens [5], mediate plant-plant and plant-microbe interactions [6], and help acclimation to biotic and abiotic environmental stress [7]. The main structure of sesquiterpene is biosynthesized by STSs, which convert far-nesyl diphosphate (FPP) into the sesquiterpene skeleton [8]. Common to all STSs is the formation of highly reactive carbocation intermediates which can undergo a great variety of rearrangements resulting in a huge number of different sesquiterpene structures [9]. It is initiated by the divalent metal ion-dependent ionization of the substrate FPP, and the following cyclization reactions depending on which carbon\(-\)carbon double bond reacts with the initially formed allylic carbocation (Figure 1) [10]. One type involves cyclization of farnesyl cation to yield (_E,E_)-germacradienyl cation (C10-C1 closure) or (_E_)-humulyl cation (C11-C1 closure) rings. The other type is initiated by isomerization of the C2-C3 double bond of farnesyl cation to the tertiary nerolidyl cation. Then, the cisoid conformer of nerolidyl cation can undergo cyclization to either the central or distal double bond forming bisabolyl cation (C6-C1 closure), cycloheptanyl cation (C7-C1 closure), (Z,E)-germacradienyl cation (C10-C1 closure), or (Z)-humulyl cation (C11-C1 closure). The resulting cationic intermediate undergoes deprotonation or addition of water before termination sesquiterpene [1]. Figure 1: The reaction mechanism of sesquiterpene production starting with FPP. Farnesyl cation is formatted by losing the diphosphate moiety (OPP). Then, the farnesyl cation can be converted to the nerolidyl cation. Possible cyclizations for both cations are indicated in the figure. The ancestral bifunctional diterpene synthases having \(\gamma\beta\alpha\) tri-domain architecture, presumably catalyzing production of gibberellin intermediate ent-kaurene, seem to the ancestor of all plant terpene synthases (TPS) [11]. The ancient gene duplication and sub-functionalization led to separate class II diterpene cyclases (\(\gamma\beta\alpha\) tri-domain) and subsequently class I TPSs (\(\beta\alpha\) didomain) including the STSs [12]. At present, most studies on STS evolution focused on the phylogenetic location of STSs [13; 14]. The TPS family is classified into seven subfamilies designated as TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h [11]. Among these, the STSs are predominantly distributed in the angiosperm-specific TPS-a clade and the gymnosperm-specific TPS-d clade [12], and there are few STSs in the TPS-b, TPS-g, and TPS-e/f subfamilies [11]. However, functional evolution of plant STSs has not yet been clarified. It is difficult to predict enzyme function from STS sequences, because STSs represent a very diverse set of enzymes with a wide range of sequence similarities. [PERSON] et al. gathered 262 plant STS sequences with experimentally characterized products, hoping to choose likely functional residues for mutagenesis studies; unfortunately, they did not reveal any general rules for STS function prediction [15]. In this study, we collected a dataset of spermatophyte STSs with characterized products from public databases and the literature. In total, 394 spermatophyte STSs were analyzed to explore the functional formation and evolution of plant sesquiterpenes.