**3. Discussion**

The SWEET gene family has been identified in many major crops such as rice [8], sorghum [10], and soybean [9], as well as important horticultural plants such as cabbage [13], potato [12], tomato [11], and apple [15]. However, little information has been reported on roses and other ornamental plants, especially their molecular regulatory mechanisms in cold response. The genome sequencing of *R. chinensis* 'Old Blush' laid the foundation for the whole genome identification and analysis of the rose gene family [48]. In this study, 25 *RcSWEET* genes were identified and classified into four subfamilies (Clades I–IV) according to their phylogenetic evolutionary relationship, including four members in Clade I, ten members in Clade II, nine members in Clade III, and two members in Clade IV (Figure 1A). The phylogenetic tree constructed with the SWEET family proteins of Arabidopsis, rice, and rose showed that the SWEET proteins of rose were more closely related to Arabidopsis, which also belongs to dicotyledons (Figure 2). Analysis of the *RcSWEET* gene structure indicated that most genes contained five or six exons, which was consistent with the analysis results in other species such as cabbage [13] and banana [17]. Moreover, similar intron and extron arrangements were found in the same subfamily members (Figure 1B), which implied a different function in each clade. In plants, SWEET proteins usually consist of seven transmembrane helices (TMHs), and two MtN3/saliva domains with three TMHs connected by the fourth transmembrane helix to form a "3-1-3" structure [3]. Compared with plants and other eukaryotes, SWEET protein in prokaryotes usually has only three TMHs, which is named SemiSWEET protein, suggesting that eukaryotic SWEET protein is likely to evolve due to gene replication or fusion [6]. Ten consensus motifs were detected in the RcSWEET proteins, and all the RcSWEET proteins contained two MtN3\_slv domains. This result suggested that the structure of RcSWEET proteins was highly conserved (Figure 1C).

Gene duplication events, including whole-genome duplication (WGD), tandem duplication, and segmental duplication events, usually result in functional segregation during gene expansion and evolution [46]. A total of 11 *RcSWEET* genes were identified as being tandemly or segmentally duplicated, which included a pair of tandemly duplicated genes (*RcSWEET10a* and *RcSWEET12*) located on Chr7, another cluster of five tandemly duplicated genes (*RcSWEET9*, *RcSWEET10b*, *RcSWEET10c*, *RcSWEET11a*, and *RcSWEET11b*) located on Chr6, and two pairs of genes (*RcSWEET4a-RcSWEET5a* and *RcSWEET12-RcSWEET15a*) were segmentally duplicated (Figure 3). These results indicated that tandem duplication and segmental duplication synergistically contribute to the expansion of the *RcSWEET* gene family, but the former is more influential than the latter. Although the duplicated *RcSWEET* genes can be derived from a common ancestor, their expression patterns and functions may have diverged. For example, the expression levels of the tandemly duplicated gene pair of *RcSWEET10a*-*RcSWEET12* identified in this work were very different in the various tissues of *R. chinensis* 'Old Blush'. In Arabidopsis, the unique *AtSWEET10* functions for early flowering [21], but there are three *RcSWEET10a/b/c* genes in rose. Moreover, *RcSWEET10a* exhibited higher expression levels in flowers, pistils, and ovaries, suggesting that *RcSWEET10a* may play an important role during the reproductive development. *AtSWEET12* was reported to be responsible for phloem loading and seed filling in Arabidopsis [11,25,28]. However, *RcSWEET12* was highly expressed in roots, suggesting that it might be involved in root development in roses.

The regulation of gene expression in higher plants usually occurs at the transcriptional level, which is coordinated by various *cis*-acting elements and trans-acting factors [49]. However, there are few studies on *SWEET* transcriptional regulation in plants. For example, the NAC transcription factor ORE1 and three abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors, ABF2 (AREB1), ABF3, and ABF4 (AREB2), were found to be involved in directly regulating senescence-associated genes *AtSWEET15* by binding to their promoters in Arabidopsis [27,50]. A waterlogging-responsive ERF (MaRAP2-4) from mint (*Mentha spp.*) was reported to specifically target the DRE or GCC box in the *AtSWEEET10* promoter to regulate soluble sugar availability and waterlogging tolerance [51]. The rice transcription factor OsDOF1 is involved in the regulation of pathogen interactions by targeting the promoters of *SWEET11/14.* In this study, a wide variety of *cis*-acting elements have been identified in the promoters of *RcSWEET* genes, including *cis*-acting elements associated with stress and hormone response, suggesting that family members are involved in complex signaling pathway regulation (Figure 4). The number of ABRE elements was the largest in the whole family. Previous studies showed that the DREB transcription factor could bind to ABRE in the gene promoter region to respond to cold stress, suggesting that *RcSWEETs* containing these *cis* elements might be involved in the regulation of the cold response in roses [52]. Moreover, 13 *RcSWEETs* contained LTR elements, which might directly participate in the regulation of low temperature response.

Low temperature is one of the typical abiotic stress factors for plants, which has an important effect on the growth and development of plants and their geographical distribution [40]. Cold acclimation at non-freezing temperatures can enhance cold resistance and induce many physiological and biochemical changes in plants, such as the accumulation of osmotic regulatory substances, the removal of reactive oxygen species, and the expression of cold responsive genes (CORs) [53–56]. It is true that the cold-induced increase of the soluble sugar content should be directly associated with the redistribution and balance of sugar in plants through the regulation of sugar transporters [57]. Previous studies showed that *SWEET* genes could be induced by low temperatures and participate in cold stress responses in many plants. For example, the overexpression of *DlSWEET1* in the tropic fruit plant longan (*Dimocarpus longan*) improved cold tolerance in transgenic *Arabidopsis* plants [58]. *BoSWEET2b*, *BoSWEET4a*, and *BoSWEET15b* were significantly upregulated in the cabbage leaves after cold stress [13]. A hexose transporter, CsSWEET2, from cucumber (*Cucumis sativus*), can improve cold tolerance in *Arabidopsis* [59]. The double mutant of *sweet11 sweet12* exhibited significantly increased freezing tolerance in the leaves of Arabidopsis, indicating that AtSWEET11 and AtSWEET12 function negatively under cold stress [60]. The Arabidopsis transgenic plants with the overexpressed *AtSWEET16*

gene exhibited higher cold resistance than wild type [29]. Moreover, overexpression of *MdSWEET16* in apple 'Orin' calli was able to increase their cold tolerance compared with *MdSWEET16* RNA interference calli [30]. Enhancing the expression of CsSWEET16/1a/17 in tea plants could improve cold tolerance in the transgenic Arabidopsis plants [14,31]. In addition, more than 90% of *MaSWEETs* in banana leaves were induced in response to cold stress in two different varieties [17].

In the present study, all eight candidate genes statistically presented significant differences in the leaves or shoots of two rose species during the different hours of cold treatment. Many of the SWEET genes that have been proven to be activated under cold stress in other species were also found in the two rose species studied. For instance, *SWEET1* has been recently reported to play an important role in cold resistance in longan [57], which is also enhanced in two rose species after cold stress. There were increased expression levels of the homologous genes *CsSWEET2* in cucumber [59], *BoSWEET2b* in cabbage [13], and *RbSWEET2a* in rose after cold treatment. Moreover, *BoSWEET4a* and *BoSWEET15b* in cabbage [13], as well as *RcSWEET4b* and *RcSWEET15a* in rose, could be activated under the cold growth temperature. We also found that the expression level of *SWEET10c* was significantly enhanced in the tissues of rose species. As far as we know, this is the first report that the *SWEET10* gene could be induced under low temperature in plants. In Arabidopsis, *AtSWEET10* acted downstream of FT to promote plant flowering [21]. *PbSWEET10* might also contribute to pollen development in the Chinese white pear [61]. Moreover, *StSWEET10b* was downregulated in the leaves of potatoes after drought treatment [62]. Recently, *GmSWEET10a/b* were reported to participate in the domestication of seed development in soybean [63].

Analysis using qRT-PCR further demonstrated that the expression levels of *SWEET* genes varied between the two species during cold treatment. The similar upregulated expression patterns of *SWEET1* and *SWEET4b* were observed in leaves and shoots of both species, implying that these two genes should be relatively conserved for the cold response in the genus *Rosa* L. Since *RbSWEET1* was localized in the plasma membrane, it may function on the cellular level for the accumulation of soluble sugars after cold stress. *RbSWEET2a* and *RbSWEET10c* were significantly upregulated in the leaves of *R. beggeriana*, while *RcSWEET2a* and *RcSWEET10c* sharply declined at 1 h and then recovered in the leaves of *R. chinensis* 'Old Blush' after cold treatment. The results suggested that *RcSWEET2a* and *RcSWEET10c* could be the crucial candidate genes for the cold-induced transportation of soluble sugars in the leaves of the deciduous and extremely cold-tolerant species *R. beggeriana*. As for *R. chinensis* 'Old Blush', *RcSWEET2a* and *RcSWEET10c* might be more sensitive to cold stress than their homologues in *R. beggeriana*, where low temperatures could initially impair their activities but then recover after the cold adaptation in this evergreen and moderately cold-tolerant species. Moreover, RcSWEET2a was localized in the tonoplast membrane, but RcSWEET10c was localized in the plasma membrane, implying that *RbSWEET2a* and *RbSWEET10c* may have different functions for the acquisition of cold tolerance in *R. beggeriana*.
