**1. Introduction**

The Rosaceae family consists of more than 2500 species in more than 90 genera and the family is divided into four subfamilies based on fruits: *Spiraeoideae* (*Spirea* subfamily), *Rosoideae* (rose subfamily), *Prunoideae* (plum subfamily), and *Maloideae* (apple subfamily). A number of species in the Rosaceae family are of economic importance as food crops, such as peaches, apples, almonds, cherries, pears, raspberries, and strawberries. Some species in the Rosaceae family are grown as ornamentals, such as the spiraea and rose. For Rosaceae, the inner quality of fruits or flowers is mainly determined by aroma and flavor. The aroma components are mainly composed of volatile products and terpenoids are important components of the volatile products such as linalool, (E)-β–damarone, and β–ionone [1].

**Citation:** Zhang, A.; Xiong, Y.; Fang, J.; Jiang, X.; Wang, T.; Liu, K.; Peng, H.; Zhang, X. Diversity and Functional Evolution of Terpene Synthases in Rosaceae. *Plants* **2022**, *11*, 736. https://doi.org/10.3390/ plants11060736

Academic Editors: Wolfgang Friedt and Igor Jerkovi´c

Received: 31 December 2021 Accepted: 4 March 2022 Published: 10 March 2022

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Terpenoid compounds have been characterized in almond, apple, and peach [2–5]. Volatile compounds in almond (plum subfamily) mainly consist of fatty acid-derived volatiles, several monoterpenes, sesquiterpenes, and phenylpropanoids, and significant differences in volatile composition were observed between different tissues and various varieties [3]. Apple (apple subfamily) fruit produce more than 300 volatile organic compounds (VOCs), including alcohols, aldehyde esters, and ketones; the specific VOC composition in apple depends on several factors, including cultivar, climacteric ethylene production levels, maturity, and environmental conditions [4,5]. More than 100 volatile chemicals have been identified in peach (plum subfamily) fruit, in which linalool is a key odorant that affects fruit aroma and consumer preference; over-expression of *terpene synthase gene 3* led to linalool accumulation [2]. Thus, small changes in volatile content have the potential to affect fruit flavor quality, there is an emphasized interest to regulate fruit flavor related volatiles, and epigenetic regulation of terpenoids is also a control strategy during fruit ripening [6,7]. To facilitate the breeding of Rosaceae species with desirable sensory qualities and improve their qualities under stress conditions, a better understanding of the genetic determinants of aroma and flavor in general and terpenes in particular is required.

Terpenoids represent the largest group of natural products and make up diverse secondary metabolites. Terpenoid composition not only represents a critical attribute in determining the quality of horticultural food products, such as taste and aroma, but also functions widely in plant development and defense, such as attracting pollinators, defending against herbivores, and acting as anti-bacterial agents [8–10]. Plant terpenoids (isoprene-C5, monoterpenes-C10, sesquiterpenes-C15, diterpenes-C20, and polyterpenoids-C5xn) are some compounds derived from isomeric 5-carbon building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) [11,12]. About 50,000 terpenoid metabolites including monoterpenes, sesquiterpenes, and diterpenes have been identified in higher plants, liverworts, and fungi [11,12]. In general, for a better adaptation to a local ecological niche, each species typically synthesizes only a small fraction of terpenoid metabolites [9,13]. In plants, geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) are the precursors for monoterpenes, sesquiterpenes, and diterpenes, respectively; terpene synthase genes (TPSs) are responsible for converting them into a multitude of cyclic and acyclic terpenoids [12]. The characteristic catalytic function of TPSs is to generate multiple terpenoid products using one substrate, thus collectively contributing to numerous different structures of plant terpenoids in addition to modifying enzymes [12]. Generally, the TPS family is characterized by two large domains including the N-terminal domain (PF01397) and the C-terminal metal cofactor binding domain (PF03936) [14]. The N-terminal domain possesses a conserved RRX8W (R, arginine, W, tryptophan, and X, alternative amino acid) motif and the C-terminal domain contains two highly conserved aspartate-rich motifs, the DDxxD motif and the NSE/DTE motif. The DDxxD motif is involved in the coordination of divalent ion(s), water molecules, and the stabilization of the active site, and is found among all functional TPSs [3]. The NSE/DTE motif flanks the entrance of the active site and function in binding a trinuclear magnesium cluster [14,15]. TPSs are split into seven subgroups based on their amino acid sequence relatedness, namely TPS a-g. The majority of TPSs in most plants fall into one or two clades and the TPS-d clade was only encoded in gymnosperms [15,16]. Different TPS clades differ considerably in catalysate and sequence. As the largest clade, the TPS-a clade mainly encodes sesquiterpenes. TPS-b and TPS-g clades are clustered closely to TPS-a; the TPS-b clade contains the conserved R(R)X8W motif and usually encodes monoterpenes, while the TPS-g clade lacks the conserved R(R)X8W motif, and functions in producing mono- and sesquiterpenes [17]. The TPS-c clade is characterized by the "DXDD" motif but not the "DDXXD" that was detected in other clades, and mainly functions in producing diterpene products [17]. The TPS-d clade is only encoded in gymnosperms, and function in producing mono- and sesquiterpene products. The TPS-e/f clades are clustered closely, and mainly encode diterpene products [17]. TPS-a/b clades lack the N-terminal γ domain characteristic of diterpene synthases found in clades c, e, and f. In general, the biosynthesis

of isoprene, monoterpenes, and diterpenes occurs in the plastid and the biosynthesis of sesquiterpenes occurs in the cytosol [17].

So far, the TPS gene family members have been characterized in many plant species. The sizes of TPS families in the majority of sequenced plants genomes range from 1 to 100. The TPS families probably evolved through duplication of genes followed by functional divergence [13]. The bryophyte *Physcomitrella patens* has a single TPS gene [13]. In *Arabidopsis thaliana*, *Vitis vinifera*, *Ocimum sanctum*, *Daucus carota*, tomato, and *Camellia sinensis*, 32, 69, 81, 19, 44, and 80 TPSs have been identified, respectively [13,18–20]. However, not all of the TPSs were functional. In recent years, the genomes of Rosaceae species such as peach, plum, apple, pear, and strawberry have been sequenced, which has significantly promoted the related studies of this family. Although abundant terpenes were characterized in different tissues of Rosaceae species [3,4,21], a comprehensive study on TPSs has not been reported in Rosaceae.

In this study, we mainly focused on three subfamilies in Rosaceae that are of economic importance as food crops, and did not select the *Spirea* subfamily, as the *Spirea* subfamily is only composed of ornamental flowers; thus, we excluded it from this study. For each subfamily, we chose two representative species with available sequenced genomes of high quality (chromosome scale). Based on the annotated genomes, we carried out the identification, characterization, and metabolite pathway mapping predictions of all TPSs encoded by eight Rosaceae species, including three Prunoideae species, three Maloideae species, and two Rosoideae species. We classified these TPSs into putative TPS-like proteins containing either PF01397 or PF03936 and complete ones containing both of them, respectively. The analysis of phylogeny, gene structure, and expression patterns were conducted with a special focus on their family number distribution and tissue expression patterns among subfamilies and different TPS clades. The findings revealed the diversity and functional evolution of TPSs in Rosaceae. The results provide a foundation for the exploration of TPSs to improve the understanding of the evolution and biosynthesis of terpenoids in Rosaceae.
