*2.4. Cis-Elements Analysis in the Promoters of the RcSWEET Genes*

To further study the potential regulatory mechanisms of the *RcSWEET* genes in response to abiotic stress, the 2000 bp upstream sequences from the translation start sites of the *RcSWEET* genes were submitted into PlantCARE to detect the cis-elements. Seventeen cis-acting elements associated with stress and hormones were identified, including ABRE, ARE, AuxRR core, Box 4, CGTCA-motif, circadian, ERE, GARE-motif, LTR, MBS, P-box, TATC-box, TCA-element, TC-rich, TGACG-motif, TGA-element, and WUN-motif (Figure 4). The details of the cis-acting elements contained in each *RcSWEET* gene have been listed in Table S5. All the *RcSWEET* genes possessed at least one stress-relevant cis-element; ABRE, ARE, and Box 4 were the most abundant genes in the *SWEET* family, indicating that abscisic acid, light, and anaerobic conditions had a great influence on the expression of *RcSWEET* genes, and these cis-acting elements were also related to stress response. In particular, 13 elements (52%) contained one or more LTR, suggesting a potential cold-stress response under low temperature conditions.

**Figure 4.** Predicted cis-elements in the promoters of the *RcSWEET* genes. Promoter sequences (−2000 bp) of 25 *RcSWEET* genes were analyzed by PlantCARE. Seventeen stress-relevant cis-acting elements are indicated in different colored boxes.

## *2.5. Expression Patterns of RcSWEET Genes in Different Tissues*

The spatial expression patterns of *RcSWEET* genes in different tissues have been analyzed according to the published transcriptome sequencing (RNA-seq) data in *R. chinensis* 'Old Blush' under the growth condition of a 12 h (25 ◦C) day/12 h (18 ◦C) night cycle [47]. There were 13 *RcSWEET* genes (52%) expressed in roots, 10 genes (40%) in stems, 9 genes (36%) in prickles and leaves, 18 genes (72%) in stamens, and 15 genes (60%) in pistils and ovaries (Figure 5). Six *RcSWEET* genes, including *RcSWEET1*, *RcSWEET2a*, *RcSWEET2b*, *RcSWEET4b*, *RcSWEET5h*, and *RcSWEET17a*, were expressed in all tissues. Especially, *Rc-SWEET1* was highly expressed (FPKM > 20) in prickles, leaves, stamens, pistils, and ovaries. *RcSWEET4b* was highly expressed (FPKM > 20) in roots, stems, prickles, stamens, pistils, and ovaries. Some of the *RcSWEET* genes exhibited similar expression patterns in the same tissues. For example, *RcSWEET5d* and *RcSWEET10a* showed extremely high expression levels (FPKM > 95) in stamen. Moreover, the highly expressed levels (FPKM > 20) were presented in *RcSWEET2a* and *RcSWEET3* in leaves, as well as *RcSWEET12* and *RcSWEET15a* in roots.

#### *2.6. Expression Patterns of Cold-Response SWEET Genes in Two Rosa Species*

Identically as in *R. chinensis*, 25 *RbSWEET* genes have been identified in *R. beggeriana* based on the transcriptome analysis of leaves and shoots in response to cold stress (Figure 6). At least 12 genes were statistically detected under the growth temperature of 23/18 ◦C, including *RbSWEET1*, *RbSWEET2a*, *RbSWEET2b*, *RbSWEET3*, *RbSWEET4b*, *RbSWEET10c*, *RbSWEET11a*, *RbSWEET15a*, *RbSWEET17a*, and *RbSWEET17b* in both tissues, as well as *RbSWEET5f* and *RbSWEET5h* in shoots. The above results presented very similar expression

patterns in the leaves and shoots of *R. chinensis* 'Old Blush'. After cold stress at 4 ◦C, the differentially expressed genes (DEGs) were screened out of the leaves and shoots of *R. beggeriana*. In leaves, *RbSWEET2a* and *RbSWEET10c* were upregulated, while *RbSWEET4b* was downregulated during cold stress. *RbSWEET1* and *RbSWEET15a* were downregulated at 1 h and then upregulated until 24 h of cold stress. On the contrary, *RbSWEET3* and *RbSWEET2b* presented the upregulated expression level at 1 h and then the downregulated expression level at 6 h and 24 h during cold stress. In shoots, *RbSWEET10c*, *RbSWEET4b*, *RbSWEET1*, and *RbSWEET2b* exhibited very similar expression trends as those genes in leaves. In contrast to leaves, there were much higher expression levels of *RbSWEET10c* and *RbSWEET4b* in shoots. *RbSWEET2a* and *RbSWEET15a* exhibited the downregulated expression levels, but *RbSWEET3* fluctuated in shoots during cold stress. In addition, *RbSWEET17a* did not show a different effect on growth temperature but maintained the relatively higher expression levels in leaves and shoots under both growth temperatures.

**Figure 5.** Expression patterns of *RcSWEET* genes in different tissues. Expression levels are shown as the log2 FPKM values obtained from the RNA-Seq data.

The expression patterns of DEGs or highly expressed *SWEET* genes in leaves and shoots of *R. chinensis* 'Old Blush' and *R. beggeriana* during cold treatment are shown in Figure 7. In the leaves of *R. chinensis* 'Old Blush', significantly increased relative expression levels were found in *RcSWEET1* at 6 h and *RcSWEET15a* at 24 h during cold stress. *Rc-SWEET2a* and *RcSWEET10c* showed a significant decline at 1 h but recovered at 6 h and 24 h. *RcSWEET2b* did not significantly change during cold stress. The relative expression levels of *RcSWEET3* and *RcSWEET17a* fluctuated during cold stress, significantly decreasing at 1 h, increasing at 6 h, and declining at 24 h. *RcSWEET4b* exhibited the exact opposite trend as *RcSWEET3*, but maintained significantly higher relative expression levels during cold treatment than those at 0 h. In shoots of *R. chinensis* 'Old Blush', *RcSWEET1* was significantly upregulated at 1 h and 24 h, but *RcSWEET2a* was unchanged during cold treatment. The increased expression levels were observed in *RcSWEET2b* at 6 h and in *RcSWEET10c* at 1 h. *RcSWEET3* was dramatically upregulated at 1 h, then significantly downregulated at 6 h, but recovered at 24 h. The relative expression levels of *RcSWEET4b*, *RcSWEET15a*, and *RcSWEET17a* significantly increased at 1 h and 6 h but sharply declined at 24 h.

As for the leaves and shoots of *R. beggeriana*, the expression patterns of most *RbSWEETs* determined using qRT-PCR detection were similar to the transcriptome results. However, the qRT-PCR analysis showed the expression levels of *RbSWEET4b* increased at 1 h and 24 h in leaves and enhanced at 1 h and 6 h in shoots, instead of the decreased results in the RNA-seq data. In addition, *RbSWEET17a* did not alter in the transcriptome data but significantly declined in leaves by qRT-PCR analysis during cold stress.

The further comparison analysis disclosed that *SWEET* genes showed various expression patterns in two species during cold treatment (Figure 7). For instance, *SWEET1* and *SWEET4b* exhibited upregulated trends in leaves and shoots of both species. The expression patterns of *SWEET2a* and *SWEET10c* were quite similar in the shoots of both species. In leaves, *RcSWEET2a* and *RcSWEET10c* sharply declined at 1 h, then recovered to the same expression levels as those genes at 0 h, while *RbSWEET2a* and *RbSWEET10c* stably enhanced and reached the significant differences after 6 h and 24 h of cold treatment, respectively. Moreover, *SWEET2b*, *SWEET3*, and *SWEET15a* exhibited upregulated trends in the shoots of *R. chinensis* 'Old Blush' at the beginning of cold stress, but downregulated in the shoots of *R. beggeriana* during the whole cold treatment.
