# Diversity and Functional Evolution of Terpene Synthases in Rosaceae
## 3 Discussion  
In the plant kingdom, terpenes are traditionally classified as secondary metabolites. Thousands of terpenes have been found, and have proven to play significant roles not only in resistance against stress conditions but also in flavor formation [19; 20; 24; 25]. However, each species is capable of synthesizing only a small fraction, whose synthesis has evolved in plants as a result of selection for increased fitness via better adaptation to the local ecological niche of each species [12]. Terpene synthases are responsible for the synthesis of the various terpene molecules [12]; plant TPS gene families are a medium-sized group, and display varied numbers of TPS families among different species [16]. The Rosaceae family has significant economic value, including the fruit crops and ornamental flowers; however, comprehensive molecular evolutionary and function analysis of TPSs remain elusive. In this study, we screened for the TPS family from eight Rosaceae species. We identified TPSs by detecting both domains and either single domain separately, thus minimizing the chance of missing putative TPSs. We found this family in Rosaceae is a midsized family, as identified in a previous study [13], ranging from 10 TPSs in _P. mira_ to 76 in _R. chinensis_. Domain loss for either N-terminal or C-terminal occurred frequently in Rosaceae species. All the Rosaceae TPSs in this study were divided into seven known clades, TPS a-g. The family numbers of different TPS clades varied among three Rosaceae subfamilies; for example, more than two TPS-c gene copies existed in Maloideae and Rosoideae subfamilies, while only one copy was detected in Prunoideae species. The average number of TPSs in Prunoideae species is lower than that of Maloideae and Rosoideae; no recent WGD except a triplicated arrangement could limit the expansion of TPSs in Prunoideae. Additionally, fewer TPSs in the early Prunoideae species _P. mira_, but more TPSs in modern peach _P. persica_,  
Figure 7: The expression level of TPSs from clades b, c, g, e, and f in the three Rosaceae species (_P. persica, M. domestica, F. vesca_). Tissues (immature fruit, leaf) are indicated by different colors. further revealed the evident evolutionary plasticity of the TPS family. Lineage- and species-specific expansions of the TPSs were widely observed within different TPS clades in Rosaceae. The varied family number and differentiation of TPSs in Rosaceae may play roles in the specialization of essential traits and species differentiation. It has been proposed that lineage-specific genes have a greater chance to contribute to phenotypic variations because their roles are not essential. Further synteny analysis showed that segmental and tandem duplications were both the driving force for the expansion of the TPS gene family in Rosaceae; for _M. domestica_ and _P. persica_, segmental and tandem duplication contributed to family expansion; and for _F. vesca_, tandem duplication played the most important role in the family expansion. Ka/Ks calculations further revealed that TPSs genes mainly evolved under purifying selection, except for several pairs; the divergent time indicated TPS-e clade was diverged relatively anciently. Since closely related enzymes differ in their product profiles, subcellular localization, or substrates, the prediction strategy based on sequence similarity usually cannot accurately describe the specialized function of TPSs, and only roughly obtained their involved pathways. In our study, we still predicted their function based on blast searching against Uniprot and KEGG pathway databases, the results revealed the functional diversity between different TPS clades and species. For the three Prunoideae species, the functional classification of TPSs among different clades was relatively conservative except for the family number variation. Whereas for the other two subfamilies, the putative function of TPSs demonstrated wide diversity, such as TPS-a clade in _P. betulifolia_, up to seven different types of synthases were predicted.