*3.1. The Characteristics of XTH Gene Family in O. fragrans*

In this study, a total of 38 *OfXTH* genes were identified, and the corresponding proteins were analyzed for their physical–chemical properties in *O. fragrans* genome (Table 1). All of the OfXTH proteins were predicted to be located in the cell wall, which is consistent with the function where XTH proteins are involved in cell-wall reconstruction [5,16]. A signal peptide was shown in most OfXTH proteins. When the signaling peptide of *DkXTH6* was absent, the subcellular localization of *DkXTH6* protein was transformed from the cell wall to the whole cell [17]. It implied that a signal peptide played an important role in the transmembrane transport of XTH protein and guided the protein to the plant cell wall. All of the *OfXTH* genes were categorized into four subfamilies by polygenetic analysis (Figure 1). The number of 26, 5, and 6 XTHs were contained in Group I/II, IIIA, and IIIB, respectively, whereas only one XTH was contained in Ancestry Group. Compared with *A. thaliana*, the numbers of XTH proteins in Group I/II, Group IIIA, and Group IIIB were increased, while Ancestral Group was decreased in *O. fragrans* (Figure 1). This might be due to the gene duplication or loss during the evolution of the *XTH* gene family [18]. Most *OfXTHs* are involved in multiple segmental duplications, which might contribute to the expansion and evolution of the OfXTH gene family [19]. It might be the reason why the number of the *XTH* family members in *O. fragrans* exceeds other species, such as *A. thaliana* (33) [20], *O. sativa* (29) [21], and *Hordeum vulgare* (24) [22]. The Ka/Ks values of all duplicated *OfXTH* gene pairs were <1.00 (Table 2), suggesting that the evolution of duplicated *OfXTH* gene pairs occurred through purifying selection and evolved slowly in *O. fragrans*.

Cis-acting elements are specific DNA sequences connected in tandem with structural genes and are binding sites for transcription factors [23]. Through analysis of *OfXTH* promoters, we found that ABA and MeJA responsiveness elements presented in most *OfXTH* promoters (Figure 5), which suggested that *OfXTH* genes might be regulated by abscisic acid and jasmonic acid, similar to the *XTH* genes in sweet cherry [24]. Recent research showed that the expression of *PavXTH15* was elevated by the phytohormone ABA and MeJA in sweet cherry [24]. Cis-acting elements involved in SA responsiveness were found in *OfXTH* promoters, indicating that the *OfXTH* gene might be the target gene of the SA signaling pathway. Similar findings reported previously showed that the accumulation of SA suppressed cell division and elongation through reducing *AtXTH8* and *AtXTH31* expression [25]. Besides responding to plant hormones, the promoters of *OfXTHs* also contain cis-acting regulatory elements involved in drought inducibility, low-temperature responsiveness, defense, and stress responsiveness. The result suggested that *OfXTH* may respond to environmental factors and be related to biotic and abiotic stress resistance. Similar results have been reported before. Overexpression of *DkXTH1* enhances tolerance to abiotic stress salt and drought stresses in transgenic *Arabidopsis* plants with respect to root and leaf growth, and survival [26]. The *XTH19* mutant showed reduced freezing tolerance after both cold and sub-zero acclimation [27].

#### *3.2. OfXTHs Are Closely Associated with the Flower-Opening Period of O. fragrans*

The flower-opening process is accompanied by the rapid expansion of petal cells. The size of the adaxial petal epidermal cells and the abaxial petal epidermal cells increased by 37.5% and 6.5% from S1 to S2, respectively [15]. Previous works have reported that XTHs participated in flower-opening processes in many species. *DcXTH2* and *DcXTH3* transcripts were markedly accumulated in *Dianthus caryophyllus* petals of opening flowers and showed high XET activity in petal claw, which is the main part of carnation petal elongation [28]. *LhXTH1* transcript levels in the petals markedly increased during *Lilium* flower opening and were higher in adaxial epidermal cells contributing to petal expansion [29]. In addition, it is reported that *XTH* plays an important role in the rapid petal growth period of *E. grandiflorum* [10]. In our study, we focused on the *OfXTH* expression changes in the S1 and S2 periods. A total of 28 *OfXTH* genes were found and significantly upregulated in S1 and S2 periods relative to the S0 period (Figure 6B), implying that these genes might be involved in petal cell expansion and contribute to the regulation of flower opening in *O. fragrans*. In addition, it has been reported that the expression of *RbXTH1* and *RbXTH2* in *Rosa bourboniana* leads to petal abscission [30]. Overexpression of persimmon *DkXTH8* causes cells to be easily destroyed and accelerates leaf senescence in transgenic plants [31]. Six *OfXTH* genes have high expression level in the flowering periods of S4 and S5 (Figure 6C); we hypothesized that these *OfXTH* genes may contribute to the cellular senescence.
