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Article

Genome-Wide Analysis of the SWEET Transporters and Their Potential Role in Response to Cold Stress in Rosa rugosa

by
Ronghui Li
1,†,
Peng Gao
1,†,
Tao Yang
1,2,
Jie Dong
1,
Yunting Chen
1,
Yangyang Xie
1,
Yvtong Yang
1,
Chengzhi Liu
1,
Jinzhu Zhang
1,2,* and
Daidi Che
1,2,*
1
College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin 150030, China
2
Key Laboratory of Cold Region Landscape Plants and Applications, Harbin 150030, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2023, 9(11), 1212; https://doi.org/10.3390/horticulturae9111212
Submission received: 3 October 2023 / Revised: 3 November 2023 / Accepted: 6 November 2023 / Published: 8 November 2023
(This article belongs to the Special Issue Germplasm Resources and Genetic Breeding of Ornamental Plants)
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Abstract

:
Sugar Will Eventually be Exported Transporter (SWEET) proteins are a recently discovered group of efflux transporters that play essential roles in sugar efflux, phloem loading, reproductive tissue development and stress responses. To date, there have been no reports on the Rosa rugosa (R. rugosa) SWEET genes. In this study, we conducted a comprehensive genomic analysis of the SWEET genes, including chromosome localization, phylogenetic comparison, cis-regulatory element analysis, expression pattern analysis in different tissues, expression pattern analysis under cold stress and subcellular localization analysis. A total of 33RrSWEET members were identified and classified into four distinct clades (Clade I, Clade II, Clade III and Clade IV). They were distributed across seven chromosomes and contained cis-regulatory elements associated with hormone and stress responses. The expression of RrSWEETs showed tissue specificity, with higher expression in roots, flowers or pistils compared to other tissues. Furthermore, during the entire cold stress process, the relative expression levels of RrSWEET4, 16 and 20 were significantly upregulated, especially in the roots and stems of R. rugosa. Subcellular localization analysis revealed that RrSWEET4, 16 and 20 were located on the cell membrane. In summary, the results of this study provide a theoretical basis for future research on the functions of RrSWEET genes in R. rugosa and their role in cold tolerance responses.

1. Introduction

R. rugosa is an important horticultural and ornamental plant with high commercial value in the perfume and pharmaceutical industries [1]. However, most R. rugosa are restricted by unfavorable environmental conditions. Low temperature is one of the most important factors limiting R. rugosa growth, development and survival. Every year, R. rugosa grown in cold regions require winter pruning and covering to survive the winter, which results in significant manpower and financial resources being wasted.
Low temperature is a typical abiotic stress factor that has a significant impact on plant growth, development and geographical distribution [2]. Cold acclimation at non-freezing temperatures enhances plant cold tolerance and induces various physiological and biochemical changes, such as the accumulation of osmoprotectants, removal of reactive oxygen species and expression of cold-responsive genes (CORs) [3,4,5]. The increase in solute sugar content induced by cold is directly related to the distribution and balance of sugars in plants. Sugars (mainly Suc) are transported from source to sink, a process that is important for plant development. As relatively large polar solutes, soluble sugars require corresponding transporters to assist in transmembrane transport. The efficient transportation of sugar across membranes requires the manipulation of multiple transporters [6]. Sucrose transporters (SUTs), monosaccharide transporters (MSTs) and SWEETs are commonly used for sugar translocation [7,8].
SWEET proteins are a newly discovered group of sugar transporters that facilitate the diffusion and transmembrane transport of sugars along concentration gradients. Belonging to the MtN3_saliva family, SWEET proteins are characterized by seven alpha-helical transmembrane domains (TMs), consisting of two conserved 3-TM repeat sequences and a less conserved single TM, forming a 3-1-3 symmetrical structure. The three TMs within each MtN3_saliva domain are arranged as TM1-TM3-TM2, forming three helical bundles (THBs) [9,10]. SWEET gene family members play a role in intercellular sugar transport and photosynthetic carbon transport throughout the plant and are classified into four groups based on their phylogenetic relationships. Group III is mainly involved in the uptake of sucrose, whereas groups I, II and IV mainly transport monosaccharides. However, members from the same lineage can localize to different cellular compartments, including vacuoles, Golgi membranes, plasma membranes and chloroplasts [11].
Studies have shown that SWEET sugar transporters regulate the transport, distribution and storage of sugar compounds within plants [12]. For instance, AtSWEET1 functions as a glucose uniporter in various plant tissues [12,13]. AtSWEET11 and AtSWEET12 mediate sucrose export from phloem parenchymal cells into the apoplast prior to sucrose uptake by sieve element companion cell complexes [12]. SWEET genes also participate in important physiological processes during plant growth and development. In Arabidopsis, the coordinated action of AtSWEET11/12/15 is required for seed filling, exhibiting specific spatiotemporal expression patterns in developing seeds [14]. Mutants of ZmSWEET4c and its rice homolog OsSWEET4 exhibit defects in seed filling [15]. Mutants of AtSWEET8 show a severe decrease in male fertility, indicating the role of AtSWEET8 in pollen nutrition [11,16]. GmSWEET15 functions in soybean embryo development by mediating sucrose transport from endosperm to embryo during early seed development [17]. SWEET genes also play important roles in response to biotic and abiotic stresses. Loss of function of AtSWEET17 may affect lateral root development and lead to impaired drought resistance [18]. Overexpression of AtSWEET16 and its homologs in tea and apple can enhance the cold tolerance of transgenic calli or plants [19,20,21]. Overexpression of CsSWEET1a and CsSWEET17 in tea leaves can also improve the cold tolerance of transgenic Arabidopsis plants [22]. AtSWEET2 inhibits Fusarium infection in Arabidopsis roots by reducing sugar supply [23,24]. In rice, OsSWEET11, 13 and 14 participate in resistance against bacterial blight by regulating upstream transcription factors [25,26,27]. Overexpression of VvSWEET4 in grapes increases the content of hexose in roots and enhances resistance to powdery mildew [28].
So far, the SWEET genes have been well studied in model plants [11], but a comprehensive exploration and characterization of the SWEET gene family in R. rugosa has not been conducted. In this study, we performed a genome-wide analysis of the SWEET genes in R. rugosa, including chromosome localization, phylogenetic comparison, cis-acting elements and expression pattern analysis in different tissues. Furthermore, we investigated the expression patterns of the SWEET gene family in R. rugosa during cold treatment in different tissues. Finally, we conducted a subcellular localization analysis of the selected R. rugosa genes responsive to cold stress. Our research findings will contribute to a better understanding of the SWEET gene family in R. rugosa and provide valuable information for further functional analysis of the RrSWEET genes in cold tolerance response.

2. Materials and Methods

2.1. Genome-Wide Identification and Analysis of RrSWEET

The protein sequences and genomic sequences of R. rugosa were obtained from the Rosaceae Genome Database (GDR) [29]. The SWEET domain (PF03083) was retrieved from the Pfam database [30] and used as an HMMER profile for HMMER searches [31], with an e-value threshold set at 1 × 10−10. After confirming the presence of the SWEET domain in RrSWEET and removing duplicates using Pfam [30] and ExPASy [32], each RrSWEET gene was named based on its position on the reference chromosomes. The distribution of transmembrane (TM) helices in RrSWEET was predicted using TMHMM [33]. Subcellular localization was predicted using Cell-PLoc [34]. The SWEET protein and genomic sequences of Arabidopsis thaliana (A. thaliana) and Triticum aestivum (T. aestivum) were obtained from the Ensemble database [35]. The genomic sequences of Fragaria vesca (F. vesca), Rosa wichuraiana (R. wichuraiana) and Rosa. Chinensis (R. chinensis) were obtained from the GDR database [29]. Multiple peptide sequences were aligned using ClustalW (Figure S1) [36]. A phylogenetic tree of the genes among species was constructed using the neighbor-joining method in MEGA X [36] and subjected to 1000 bootstrap replicates for statistical support.
Motifs in the SWEET protein sequences of RrSWEET were detected outside the SWEET domain, with e-values smaller than 1 × 10−20 and lengths ranging from 10 to 50 amino acids. The gene sequences were numbered based on predictions using the MEME software [37]. The exon/intron structures of RrSWEETs were analyzed and displayed using TBtools [38]. The promoter regions of RrSWEETs were set to a length of 2000 bp and extracted from the GDR database using PlantCARE [39] and TBtools [38]. The collinearity analysis of RrSWEETs was performed using the default settings of the MCScanX program [40] and visualized using Circos [41].
In order to analyze the repetitive events occurring between the inferred RrSWEET genes, a similarity matrix was constructed using TBtools [38], with a nucleotide-level similarity greater than 70% as the criterion for comparing the genetic relationship between two genomes. Subsequently, segmental or tandem repeat events were defined based on the positions of duplicated RrSWEET genes located either on different chromosomes or within a 20kb region on the same chromosome [38].

2.2. Expression Analysis of the RrSWEETs

In this study, R. rugosa Thunb. obtained from the Key Laboratory of Cold Region Landscape and Application, Northeast Agricultural University, Harbin, China, was grown under standard greenhouse conditions with 16 h of light, 8 h of darkness and 70% relative humidity. During the flowering period, samples were collected from the stems, roots, leaves, flowers, stamens and pistils of R. rugosa Thunb. Approximately 0.2 g of each sample was collected. For cold treatment, one-year-old R. rugosa Thunb. cuttings from four-year-old R. rugosa Thunb. seedlings were placed in a variable temperature (23 °C) climate chamber (16 h of light, 8 h of darkness, 70% relative humidity) one week prior to the cold treatment. The temperature of −20 °C treatment group was gradually lowered by 2 °C to 4 °C per hour until reaching −20 °C, which was maintained for 12 h, followed by a gradual increase in temperature at a rate of 2 °C per hour until reaching 23 °C. Similarly, the temperature of 4 °C treatment group was gradually lowered by 2 °C to 4 °C per hour until reaching 4 °C, which was also maintained for 12 h, followed by a gradual increase in temperature at a rate of 2 °C per hour until reaching 23 °C. Controls were left untreated in an artificial climate chamber at 23 °C. Subsequently, roots, stems, leaves, petals, sepals, pistils and stamens were rapidly frozen in liquid nitrogen and stored at −80 °C. RNA was extracted from the samples using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). Reverse transcription was performed using the Hifair® III 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen, Shanghai, China). Primers for RrSWEETs used in quantitative reverse transcription PCR (qRT-PCR) were designed using Primer Premier 5.0 software [42] (Table S1). qRT-PCR was carried out using the BIO-RAD CFX96 Touch System (CFX Touch; Bio-Rad, Hercules, CA, USA) and Taq Pro Universal SYBR qPCR Master Mix (SYBR Green I) (Vazyme, Nanjing, China), with three biological replicates and three technical replicates for each sample. RrACTIN was used as a reference gene. Statistical analysis of the gene expression data was performed using IBM SPSS Statistics ver.19.0 (IBM Corp., Armonk, NY, USA) with Duncan’s test. Tissue-specific analysis of the relative expression levels of RrSWEETs was calculated using the 2−ΔCT method [43]. The relative expression levels of RrSWEETs under cold stress were calculated using the 2−ΔΔCT method [44].

2.3. Cloning and Subcellular Localization Analysis of RrSWEETs

RrSWEET4, RrSWEET16 and RrSWEET20 open reading frames (ORFs) were amplified using primers (Table S1) and inserted into pCAMBIA1300-GFP. Agrobacterium-mediated constructs were transiently transfected into 5-week-old Tobacco leaf [45] and samples were observed using a fluorescence microscope (Olympus, FV3000, Nagano-ken, Japan) after 2 days of cultivation.

3. Results

3.1. Identification of RrSWEET Family Members

A total of 33 sequences containing the conserved SWEET domain were identified from the R. rugosa genome, each of which contained at least one MtN3_slv domain (Table S2). These 33 sequences were named RrSWEET1-RrSWEET33 based on their chromosomal positions. The physicochemical properties of the 33 RrSWEET genes (Table 1) showed that the number of amino acids encoded by RrSWEET genes ranged from 91 to 311. The theoretical isoelectric points ranged from 4.63 to 9.76, with 78.8% of the members having an isoelectric point greater than 7, indicating that most of them are alkaline proteins. RrSWEET29 had the largest molecular weight of 34.85 kDa, while RrSWEET33 had the smallest molecular weight of 11.02 kDa. Most members of the RrSWEET gene family were located in the cell membrane, while a small portion were located in the chloroplast (RrSWEET19 and RrSWEET33).

3.2. Phylogenetic Analysis, Conserved Motifs, Gene Structure Compositions and Cis-Regulatory Element Analysis of RrSWEETs

To better understand the evolutionary process of RrSWEET genes, the newly identified 33 RrSWEET genes were combined with all reported SWEET sequences from A. thaliana to construct a phylogenetic tree. The phylogenetic tree analysis revealed that the 33 newly identified RrSWEET genes were clearly divided into four clades (Clade I, Clade II, Clade III and Clade IV) (Figure 1).
The 33 identified RrSWEET proteins were clearly divided into four clades (Figure 2A). Among them, 72.73% of RrSWEETs contained 10 motifs, 15.15% contained 9 motifs, 9.09% contained 2 motifs and 3.03% contained 4 motifs. Except for a few special proteins, members of the same clade in the RrSWEET gene family showed similarities in motif positions and motif numbers, indicating that they may have similar functions(Figure S2).
To further investigate the structural characteristics of the RrSWEET genes, we analyzed their exon–intron structures (Figure 2C). The number of introns in the RrSWEET genes varied from zero to five, with most genes having five introns. All RrSWEETs in Clade IV contained five introns. In Clade III, except for RrSWEET3, all RrSWEETs had five introns. In Clade I, RrSWEET33 and RrSWEET19 were exceptions, as they had a different number of introns. In Clade II, RrSWEET27, RrSWEET31, RrSWEET26 and RrSWEET32 had five introns; RrSWEET28 had four introns; RrSWEET21 and RrSWEET24 had two introns; and RrSWEET18, RrSWEET2, RrSWEET16 and RrSWEET27 had no introns. These results indicate that, except for some unique gene family members, genes within the same evolutionary branch exhibit similar conserved domains and intron distribution, suggesting relative conservation of SWEETs in R. rugosa.
To investigate the potential regulatory mechanisms of the RrSWEET genes in plant growth, development and biotic/abiotic stress responses, we analyzed the cis-regulatory elements in their promoter regions (Figure 3). We observed that the promoter regions of RrSWEET genes contain hormone-responsive elements (ABRE, TGA-element, as-1, etc.), stress-responsive elements (CAT-box, ARE, GCN4_motif, etc.), light-responsive elements (MER, GATA-motif, Box 4, etc.) and plant growth and development-related elements (F-box, G-Box, MBS, etc.). These elements enable the RrSWEET genes to respond to plant growth, light and environmental stresses. All RrSWEET genes possess at least one stress-related cis-element, with MYB, ARE and MYC being the most abundant in the SWEET gene family. Members of the RrSWEET gene family may be sensitive to low temperature, drought, ABA and auxin. Combining these findings with the results of phylogenetic analysis, we observed that gene members within the same evolutionary branch have similar cis-elements.

3.3. Chromosomal Distribution and Duplication Analysis of RrSWEET Genes

The analysis of the chromosomal distribution and tandem duplication of RrSWEET genes revealed uneven distribution across the chromosomes of R. rugosa. The highest number of SWEET genes was found on chromosomes 2, 3 and 7, with a total of nine genes. Chromosome 1 contained four SWEET genes, chromosome 5 contained five SWEET genes, chromosome 6 contained four SWEET genes and chromosome 4 contained only one SWEEET gene. Two pairs of tandem repeat sequences were identified, namely RrSWEET8/RrSWEET9 and RrSWEET16/RrSWEET17 (Figure 4A). These results indicate that a few members of the RrSWEET gene family are generated through tandem gene duplication. Additionally, the analysis of collinearity using the TBtools Circos program revealed four pairs of duplicated segments, namely RrSWEET27/RrSWEET3, RrSWEET29/RrSWEET5, RrSWEET6/RrSWEET11 and RrSWEET7/RrSWEET11, suggesting that some RrSWEET genes may be generated through gene segment duplication (Figure 4B).

3.4. Collinearity Analysis

To further investigate the interspecies homology of SWEET genes, intergenic collinearity analysis was performed between R. rugosa and various other plants, including A. thaliana, T. aestivum, F. vesca, R. wichuraiana and R. chinensis (Figure 5). A total of 17 pairs of homologous genes were detected between R. rugosa and R. wichuraiana. Similarly, 15 pairs of homologous genes were detected between R. rugosa and R. chinensis, 12 pairs between R. rugosa and F. vesca, and 8 pairs between R. rugosa and A. thaliana. Only two pairs of homologous genes were found between R. rugosa and T. aestivum. The homology between R. rugosa and R. wichuraiana was significantly higher compared to the other four plants. Through the analysis of interspecies homology, the functional characteristics of SWEET members with collinearity relationships can be inferred.

3.5. Specific-Tissue Expression Analysis of RrSWEETs

The 16 randomly distributed RrSWEET genes across all cladesshowed expression in leaves, stems, roots, petals, sepals, stamens and pistils, with significant differences in expression levels among different tissues (Figure 6). RrSWEETs exhibited higher expression in roots, petals and pistils compared to other tissues, suggesting that roots, flowers and pistils may be target tissues for future studies on the functions of RrSWEETs. Based on the expression level results, genes from Clade I (RrSWEET20, RrSWEET22, RrSWEET23, RrSWEET25 and RrSWEET33) showed specific expression in pistils and stamens, while genes from Clade II (RrSWEET2, RrSWEET16 and RrSWEET18) were predominantly expressed in petals. Additionally, genes from Clade III (RrSWEET4) exhibited high expression levels in stems and leaves. Furthermore, genes from Clade IV (RrSWEET7, RrSWEET8, RrSWEET9, RrSWEET11, RrSWEET12 and RrSWEET13) displayed diverse expression patterns, primarily in roots and stems. Most RrSWEET genes showed expression in all tissues.

3.6. Expression Patterns of RrSWEETs under Cold Treatment

To evaluate the expression patterns of RrSWEET genes in response to cold stress in R. rugosa, we subjected R. rugosa to low-temperature treatments of 4 °C and −20 °C to simulate chilling and freezing stress, respectively. We analyzed the RrSWEET gene expression patterns in different tissues of R. rugosa in response to low temperatures using qRT-PCR (Figure 7).
After 12 h of 4 °C treatment, the relative expression levels of some RrSWEET genes were upregulated in certain tissues, with upregulation ranging from 2 to 22 times compared to the control. Twelve genes (RrSWEET4, 7, 9, 11, 12, 13, 18, 20, 22, 23, 25 and 28) showed upregulation in the roots after 4℃ treatment, with RrSWEET4, 11 and 20 exhibiting the most significant upregulation. Two genes (RrSWEET2 and RrSWEET16) showed downregulation in the roots. The expression patterns in the stems were similar to those in the roots, with upregulated expression of eleven genes (RrSWEET4, 7, 9, 11, 12, 13, 18, 20, 22, 23 and 25). RrSWEET20 in the stems continued to exhibit a significant upregulation. In the leaves, the expression of 12 genes (RrSWEET2, 7, 11, 12, 13, 16, 18, 20, 22, 23, 25 and 33) was upregulated under 4 °C treatment, with RrSWEET18 and RrSWEET23 showing the most significant upregulation. In the petals, most genes (RrSWEET 2, 4, 7, 8, 9, 11, 12 and 20) exhibited downregulation under 4 °C treatment. Four genes (RrSWEET 16, 18, 22 and 23) were upregulated in the petals, with RrSWEET22 and RrSWEET16 showing the most significant upregulation. In the sepals, except for RrSWEET12, 13, 16, 22, 23 and 25, all other genes exhibited downregulation. Similar to what was observed in the petals, almost all RrSWEET genes showed downregulation in the pistils or stamens. In the pistils, RrSWEET4, 8, 13 and 20 exhibited upregulation, while RrSWEET9 and RrSWEET22 showed no significant changes. All other genes demonstrated downregulation under the 4 °C treatment. In the stamens, except for RrSWEET12, 13, 16, 18, 22, 25 and 33, all other genes exhibited downregulation.
After 12 h of −20 °C treatment, ten genes (RrSWEET2, 4, 9, 13, 18, 20, 22, 23, 25 and 33) showed upregulated expression in the roots compared to the control, with RrSWEET4, 20 and 25 exhibiting the most significant upregulation. In the stems, most genes (RrSWEET2, 7, 9, 11, 13, 18, 20, 23 and 25) showed significant upregulation under −20 °C treatment, with RrSWEET11 and RrSWEET20 showing the most significant upregulation. In the leaves, the expression of eight genes (RrSWEET2, 16, 18, 20, 22, 23, 25 and 33) was upregulated under −20 °C treatment, while the expression of seven genes (RrSWEET4, 7, 8, 9, 11, 12 and 13) was downregulated under −20 °C treatment, with no significant differences observed for the remaining genes. In the petals, most genes exhibited downregulation under 4 °C treatment, but some genes showed upregulated expression under −20 °C treatment (RrSWEET4, 8, 9, 11, 13, 16, 18, 20, 22, 23, 25 and 33). Under −20 °C treatment, except for RrSWEET8 and RrSWEET20, which showed upregulated expression in the pistils, and RrSWEET22 and RrSWEET33, which showed upregulated expression in the stamens, the remaining RrSWEET genes showed downregulated expression.
It is worth noting that, compared to the 4 °C treatment, the expression patterns of some genes significantly changed after 12 h of −20 °C treatment. In the roots, RrSWEET7 showed downregulation after −20 °C treatment, which was significantly different from the upregulation observed under 4 °C treatment. In the stems, leaves and petals, the expression patterns were similar to those under 4 °C treatment after 12 h. In the pistils and stamens, most genes remained negatively regulated after −20 °C treatment, but the regulation was not as active compared to the 4 °C treatment. Considering the gene specificity and potential applications in gene research, we propose that RrSWEET4, 16 and 20 exhibit responsiveness to plant cold stress. Upon exposure to 4 °C treatment, RrSWEET4 expression was significantly upregulated in roots, stems and pistils, with a remarkable fold increase of over 20-fold in pistils. Conversely, under −20 °C treatment, its expression was upregulated in petals and roots, with a particularly pronounced upregulation in roots. This intriguing observation suggests that RrSWEET4 primarily functions in reproductive organs under 4 °C cold stress, while it assumes a role in nutrient organs under −20 °C freezing stress. Such organ-specific gene activities in response to low-temperature and freezing stress hold significant value for further investigation. Additionally, RrSWEET16, which exhibits specific expression in petals under −20 °C stress, is believed to play a pivotal role in the freezing stress response of reproductive organs. Moreover, RrSWEET20 displayed upregulation in roots, stems, leaves and pistils under both −20 °C and 4 °C treatments. These three genes, belonging to distinct evolutionary branches with diverse expression patterns, thus warrant consideration as candidate genes for elucidating the cold stress response of SWEET genes in R. rugosa.

3.7. Subcellular Localization Analysis of RrSWEETs

In this study, it was predicted that almost all RrSWEET proteins localize to the cell membrane (Table 1). To confirm the subcellular localization of RrSWEET proteins, we cloned the genes RrSWEET4, RrSWEET16 and RrSWEET20, which have been shown to regulate cold stress potential in R. rugosa. The results showed that 35S::RrSWEET4-GFP, 35S::RrSWEET16-GFP and 35S::RrSWEET20-GFP were detected on the cell membrane (Figure 8), consistent with the predicted results (Table 1). In addition, as a control, the GFP alone (35S::GFP) was localized to the nuclear membrane and cell membrane.

4. Discussion

The SWEET family is a novel type of sugar transporter that is involved in the regulation of plant growth, development and stress responses in many plant species. Using a whole-genome identification approach, 33 SWEET genes were identified in R. rugosa. This number is similar to that of other species such as tomato (29 genes) [46], cabbage (30 genes) [47], potato (35 genes) [48] and rubber tree (36 genes) [49]. Based on phylogenetic analysis (Figure 1), these genes were classified into four clades (Clades I-IV), consistent with the classification of SWEETs in A. thaliana. However, there were differences in the size of the clades, for example, Clade II contained as many as 11 RrSWEET members, which is twice the number of Clade II members in AtSWEETs (Figure 1).
Analysis of the gene structure of RrSWEETs revealed that most genes contained five introns, which is consistent with the results of other species such as cabbage [47] and banana [50]. Each member in each branch of RrSWEETs had a similar number of introns (Figure 2). For example, each member in Clades I (except RrSWEET33 and RrSWEET19), IV (except RrSWEET3) and III contained five introns, while members in Clade II contained fewer introns, with RrSWEET27, RrSWEET31, RrSWEET26 and RrSWEET32 having five introns; RrSWEET28 having four introns; RrSWEET21 and RrSWEET24 having two introns; and RrSWEET18, RrSWEET2, RrSWEET16 and RrSWEET27 having no introns. According to previous reports, the loss rate of introns is faster than the gain rate after gene duplication. Therefore, we believe that Clades I, III and IV may contain the ancestral RrSWEET genes, while Clade II may have arisen through gene duplication, differentiation and subsequent intron loss. This structural feature has also been observed in other plants, such as S. lycopersicum [51]. Additionally, the analysis of conserved motifs showed that most RrSWEETs contain seven or more conserved motifs, while gene members within each subfamily share other more specific motifs. This observation is consistent with observations in Arabidopsis [11] and tomato [46]. Promoter analysis indicated that these RrSWEET genes play a key role in environmental stress and plant hormones.
Gene duplication events, including whole-genome duplication (WGD), tandem duplication and segmental duplication, often lead to functional divergence during gene amplification and evolution [52]. In this study, we identified two pairs of tandem duplicated genes in RrSWEETs, including a pair on Chr2 (RrSWEET8 and RrSWEET9) and two pairs on Chr3 (RrSWEET16 and RrSWEET17). We also found four pairs of segmentally duplicated genes (RrSWEET27/RrSWEET3, RrSWEET29/RrSWEET5, RrSWEET6/RrSWEET11 and SWEET7/SWEET11) (Figure 4). These results suggest that both tandem and segmental duplications have contributed to the expansion of the RrSWEET gene family, but the impact of segmental duplication appears to be greater. The duplicated RrSWEET genes may have originated from a common ancestor, and their expression patterns and functions are likely to be similar. Our analysis of tissue-specific expression data for 16 randomly selected genes showed that the expression levels of tandem duplicated genes RrSWEET8/RrSWEET9 and segmentally duplicated genes SWEET7/SWEET11 were highly similar in different tissues of R. rugosa. In response to cold stress, the expression patterns of segmentally duplicated genes SWEET7/SWEET11 were also highly similar, with significant responses to 4 °C stress in roots and −20 °C stress in stems. The tandem duplicated genes RrSWEET8/RrSWEET9 showed slight differences under cold stress, with RrSWEET8 showing a significant response in petals and RrSWEET9 showing a significant response in pistils, indicating functional divergence between these two genes during evolution.
This study also revealed tissue-specific expression patterns of RrSWEETs [53]. We found that RrSWEET genes were predominantly expressed in the roots, petals and pistils of R. rugosa (Figure 6). Previous studies have shown that SWEET genes in evolutionary Branch IV are highly expressed in the root epidermis and encode proteins that function as fructose-specific uniporters in root vacuolar membranes [54]. Our tissue-specific expression results showed significant expression of RrSWEET8, RrSWEET9 and RrSWEET13, which belong to Clade IV, in roots, suggesting that they may have similar functions. It has been reported that AtSWEET5 plays an important role in seed germination and pollen development [55]. The high expression of RrSWEET18, 20, 22, 23, 28 and 33 in stamens suggests that they may have similar functions to AtSWEET5. It has been reported that AtSWEET4 plays a crucial role in mediating sugar supply to the axis tissue, and knocking out AtSWEET4 expression leads to decreased glucose and fructose levels and the collapse of the vein network [56]. In our study, RrSWEET20, 22 and 23, which belong to the same branch as AtSWEET4, showed high expression in leaves, suggesting that they may have similar functions to AtSWEET4.
Under low-temperature stress, plants generally reduce their osmotic potential to ensure normal growth in adverse conditions. Low temperature can induce the expression of SWEET genes in plants, leading to a rapid increase in soluble sugar content in tissues, thereby reducing plant osmotic potential and ensuring normal growth under stress [57,58]. In our study, almost all RrSWEET genes showed varying degrees of regulation under cold stress in different tissues (Figure 7). After 12 h of treatment at −20 degrees Celsius, RrSWEET4, 20 and 25 exhibited significant upregulation in the roots. RrSWEET20 showed significant upregulation in the stems. In the leaves, RrSWEET12 and RrSWEET23 were dramatically upregulated. RrSWEET9 and RrSWEET16 showed the most pronounced upregulation in the petals. In the pistils, RrSWEET9, 20 and 13 were upregulated. In the stamens, RrSWEET12, 13, 16, 18 and 22 were upregulated. Additionally, our results also showed that the roots, stems and leaves had stronger responses to cold stress, while the flowers, pistils and stamens had relatively weaker responses. This is consistent with previous research. Many studies have shown that SWEETs play a role in maintaining sugar homeostasis in plant microorgans and promote tolerance to low temperatures. In bananas, the MaSWEET-4b, -4c, -4d and -14b genes were significantly upregulated under cold stress [50]. Similar results were observed in P. mume [59], wheat [60] and common bean [43]. The expression of DlSWEET1 in the tropical fruit plant longan improved the cold tolerance of transgenic arabidopsis plants [50]. CsSWEET2, a hexose transporter in cucumber, enhanced cold tolerance in Arabidopsis [61]. AtSWEET11 and AtSWEET12 negatively regulate cold stress in leaves [62]. Furthermore, previous studies have shown that AtSWEET16 [20] and AtSWEET17 [57] can enhance plant resistance to low-temperature stress by regulating plant osmotic potential. The RrSWEET4 gene, which is closely related to AtSWEET16 and AtSWEET17 and belongs to the same evolutionary branch, showed significant upregulation in the roots under cold stress, indicating that it may play a similar role to that of AtSWEET16 and AtSWEET17 in R. rugosa.

5. Conclusions

This study identified and analyzed a total of 33 RrSWEET genes in R. rugosa. Among them, there were two pairs of tandem duplicated genes (RrSWEET8/RrSWEET9, RrSWEET16/RrSWEET17) and four pairs of segmentally duplicated genes (RrSWEET27/RrSWEET3, RrSWEET29/RrSWEET5, RrSWEET6/RrSWEET11, SWEET7/SWEET11). Through analysis and construction of an evolutionary tree, the RrSWEET gene family was divided into four clades. The gene structures of the 33 genes were analyzed, and it was suggested that Clades I, III and IV may contain the ancestral RrSWEET genes, while Clade II may have arisen through gene duplication, differentiation and subsequent intron loss. Conserved motif analysis revealed that most RrSWEETs contained seven or more conserved motifs, while gene members within each subfamily shared additional specific motifs. Further predictions were made using cis-acting element analysis, suggesting potential roles for RrSWEET gene family members in hormone response and stress reactions. Tissue-specific expression analysis showed that 16 randomly selected RrSWEET gene members exhibited tissue-specific expression in seven different tissues, with similar expression patterns within the same clade. Cold treatment at 4 °C and −20 °C was conducted to simulate low-temperature and freezing stress, leading to the identification of three genes (RrSWEET4, RrSWEET16 and RrSWET20) that may be associated with cold stress. Subcellular localization analysis of the selected three genes confirmed their presence on the cell membrane, consistent with the predicted results. The above analysis provides direction and insights for future studies on RrSWEET gene family members and offers prospects for the molecular breeding of cold tolerance in R. rugosa.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9111212/s1, Figure S1: The multiple peptide sequence alignment of RrSWEETs using ClustalW; Figure S2: RrSWEET protein motif logos; Table S1: Gene accession numbers and primer sequences of SWEET gene family; Table S2: Gene accession numbers and protein sequences of SWEET gene family.

Author Contributions

Conceptualization, J.Z. and R.L.; methodology, P.G. and R.L.; formal analysis, R.L., T.Y., Y.C., Y.Y., Y.X. and C.L.; results validation, R.L. and P.G.; writing—original draft preparation, R.L.; writing—review and editing, R.L., J.D., P.G., J.Z. and D.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, (Grant No. 31971700) and the Joint Guiding Project of the Natural Science Foundation of Heilongjiang Province, (Grant No. LH2020C014).

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy concerns.

Acknowledgments

We appreciate all the people who have collaborated in this project.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic relationship of SWEET family genes in R. rugosa and A. thaliana. Proteins from R. rugosa and A. thaliana are denoted by Rr and At prefixes, respectively. Four major phylogenetic groups designated from Clade I to Clade IV are indicated. The four clades are indicated with different colors.
Figure 1. Phylogenetic relationship of SWEET family genes in R. rugosa and A. thaliana. Proteins from R. rugosa and A. thaliana are denoted by Rr and At prefixes, respectively. Four major phylogenetic groups designated from Clade I to Clade IV are indicated. The four clades are indicated with different colors.
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Figure 2. Phylogenetic relationship, conserved domains and gene structure of RrSWEETs in R. rugosa. (A) Neighbor-joining phylogenetic tree of RrSWEET proteins, with Clades I, II, III and IV indicated. (B) Motif composition of RrSWEET proteins, with different colors representing ten different motifs. (C) Exon–intron structures analyzed using Tbtools. Exons are represented by green boxes, upstream and downstream regions are represented by blue boxes and introns are represented by black lines.
Figure 2. Phylogenetic relationship, conserved domains and gene structure of RrSWEETs in R. rugosa. (A) Neighbor-joining phylogenetic tree of RrSWEET proteins, with Clades I, II, III and IV indicated. (B) Motif composition of RrSWEET proteins, with different colors representing ten different motifs. (C) Exon–intron structures analyzed using Tbtools. Exons are represented by green boxes, upstream and downstream regions are represented by blue boxes and introns are represented by black lines.
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Figure 3. Analysis of cis-elements in the promoter regions of RrSWEET gene family members. Different cis-regulatory elements in RrSWEET promoter regions are indicated with different colors.
Figure 3. Analysis of cis-elements in the promoter regions of RrSWEET gene family members. Different cis-regulatory elements in RrSWEET promoter regions are indicated with different colors.
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Figure 4. Analysis of the chromosomal distribution and tandem duplication of RrSWEET genes in R. rugosa. (A) Chromosomal distribution and tandem duplication analysis of RrSWEET genes. Genes within the boxes represent tandem duplicated genes. Scale bar represents megabases (Mb). (B) Chromosomal relationships of RrSWEET genes. Gray lines represent all homologous blocks in the R. rugosa genome. Red lines represent collinear blocks of SWEET genes in the R. rugosa genome. Collinear RrSWEET gene pairs have the same font color.
Figure 4. Analysis of the chromosomal distribution and tandem duplication of RrSWEET genes in R. rugosa. (A) Chromosomal distribution and tandem duplication analysis of RrSWEET genes. Genes within the boxes represent tandem duplicated genes. Scale bar represents megabases (Mb). (B) Chromosomal relationships of RrSWEET genes. Gray lines represent all homologous blocks in the R. rugosa genome. Red lines represent collinear blocks of SWEET genes in the R. rugosa genome. Collinear RrSWEET gene pairs have the same font color.
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Figure 5. Comparative analysis of the SWEET gene family between R. rugosa and A. thaliana, T. aestivum, F. vesca, R. wichuraiana and R. chinensis. Gray lines represent collinear gene pairs between R. rugosa and other species, while blue lines represent collinear SWEET gene pairs.
Figure 5. Comparative analysis of the SWEET gene family between R. rugosa and A. thaliana, T. aestivum, F. vesca, R. wichuraiana and R. chinensis. Gray lines represent collinear gene pairs between R. rugosa and other species, while blue lines represent collinear SWEET gene pairs.
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Figure 6. The relative expression levels of 16 randomly selected RrSWEET genes in different organs. Abscissa represents different organs; ordinate represents the value of a relative expression. Error bars represent ± SD. The letters above the columns represent the significant differences in transcript levels at p < 0.05 (Duncan’s test).
Figure 6. The relative expression levels of 16 randomly selected RrSWEET genes in different organs. Abscissa represents different organs; ordinate represents the value of a relative expression. Error bars represent ± SD. The letters above the columns represent the significant differences in transcript levels at p < 0.05 (Duncan’s test).
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Figure 7. Relative expression levels of RrSWEETs in different organs after cold treatments (4 °C and −20 °C). Error bars represent ± SD. Asterisks represent significant differences in transcript level compared with control (Student’s t-test: **, p < 0.01, ***, p < 0.001).
Figure 7. Relative expression levels of RrSWEETs in different organs after cold treatments (4 °C and −20 °C). Error bars represent ± SD. Asterisks represent significant differences in transcript level compared with control (Student’s t-test: **, p < 0.01, ***, p < 0.001).
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Figure 8. Subcellular localization of RrSWEET4, RrSWEET16 and RrSWEET20 in tobacco leaves. 35S::GFP was used as the empty control.
Figure 8. Subcellular localization of RrSWEET4, RrSWEET16 and RrSWEET20 in tobacco leaves. 35S::GFP was used as the empty control.
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Table 1. Predicted gene list and related information including gene name, gene locus, molecular details, TM, subcellular location, AA (number of amino acids), MW (molecular weight in kDa), PI (isoelectric point).
Table 1. Predicted gene list and related information including gene name, gene locus, molecular details, TM, subcellular location, AA (number of amino acids), MW (molecular weight in kDa), PI (isoelectric point).
Gene NameGene LocusStandAA(aa)PIMW (kDa)TM
RrSWEET1evm.model.Chr1.60−1279.2814.303
RrSWEET2evm.model.Chr1.1695+2438.9427.147
RrSWEET3evm.model.Chr1.2350−1814.6320.214
RrSWEET4evm.model.Chr1.2452−2415.8526.987
RrSWEET5evm.model.Chr1.3067+2817.731.537
RrSWEET6evm.model.Chr2.1813+2946.7332.817
RrSWEET7evm.model.Chr2.1816+3099.6834.487
RrSWEET8evm.model.Chr2.1817+2499.3927.837
RrSWEET9evm.model.Chr2.1819+2738.7230.237
RrSWEET10evm.model.Chr2.1921+2935.4832.707
RrSWEET11evm.model.Chr2.1926+3049.533.927
RrSWEET12evm.model.Chr2.1927+2969.4632.777
RrSWEET13evm.model.Chr2.1933+2738.7230.237
RrSWEET14evm.model.Chr2.3499−2479.2827.287
RrSWEET15evm.model.Chr3.2084−2438.9126.897
RrSWEET16evm.model.Chr3.3219+2439.0227.296
RrSWEET17evm.model.Chr3.3233−2438.9127.297
RrSWEET18evm.model.Chr3.3287+2399.0826.896
RrSWEET19evm.model.Chr3.3681−1365.4215.681
RrSWEET20evm.model.Chr3.4823+2518.9228.327
RrSWEET21evm.model.Chr4.1820+1927.5321.544
RrSWEET22evm.model.Chr5.4695+2358.6126.307
RrSWEET23evm.model.Chr5.5986+2359.3326.427
RrSWEET24evm.model.Chr6.3424+2258.5325.446
RrSWEET25evm.model.Chr6.3661+1706.5519.081
RrSWEET26evm.model.Chr6.4775+2359.0226.316
RrSWEET27evm.model.Chr6.6650+2439.3826.996
RrSWEET28evm.model.Chr7.760−2539.2828.137
RrSWEET29evm.model.Chr7.1004+3116.6534.857
RrSWEET30evm.model.Chr7.1007+2888.1532.707
RrSWEET31evm.model.Chr7.1376−2388.8926.977
RrSWEET32evm.model.Chr7.1378−2379.3226.577
RrSWEET33evm.model.Chr7.1609+919.7611.021
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MDPI and ACS Style

Li, R.; Gao, P.; Yang, T.; Dong, J.; Chen, Y.; Xie, Y.; Yang, Y.; Liu, C.; Zhang, J.; Che, D. Genome-Wide Analysis of the SWEET Transporters and Their Potential Role in Response to Cold Stress in Rosa rugosa. Horticulturae 2023, 9, 1212. https://doi.org/10.3390/horticulturae9111212

AMA Style

Li R, Gao P, Yang T, Dong J, Chen Y, Xie Y, Yang Y, Liu C, Zhang J, Che D. Genome-Wide Analysis of the SWEET Transporters and Their Potential Role in Response to Cold Stress in Rosa rugosa. Horticulturae. 2023; 9(11):1212. https://doi.org/10.3390/horticulturae9111212

Chicago/Turabian Style

Li, Ronghui, Peng Gao, Tao Yang, Jie Dong, Yunting Chen, Yangyang Xie, Yvtong Yang, Chengzhi Liu, Jinzhu Zhang, and Daidi Che. 2023. "Genome-Wide Analysis of the SWEET Transporters and Their Potential Role in Response to Cold Stress in Rosa rugosa" Horticulturae 9, no. 11: 1212. https://doi.org/10.3390/horticulturae9111212

APA Style

Li, R., Gao, P., Yang, T., Dong, J., Chen, Y., Xie, Y., Yang, Y., Liu, C., Zhang, J., & Che, D. (2023). Genome-Wide Analysis of the SWEET Transporters and Their Potential Role in Response to Cold Stress in Rosa rugosa. Horticulturae, 9(11), 1212. https://doi.org/10.3390/horticulturae9111212

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