Characterization of the SWEET Gene Family in Longan (Dimocarpus longan) and the Role of DlSWEET1 in Cold Tolerance

Sugars will eventually be exported transporters (SWEET), a group of relatively novel sugar transporters, that play important roles in phloem loading, seed and fruit development, pollen development, and stress response in plants. Longan (Dimocarpus longan), a subtropic fruit tree with high economic value, is sensitive to cold. However, whether the SWEET gene family plays a role in conferring cold tolerance upon longan remains unknown. Here, a total of 20 longan SWEET (DlSWEET) genes were identified, and their phylogenetic relationships, gene structures, cis-acting elements, and tissue-specific expression patterns were systematically analyzed. This family is divided into four clades. Gene structures and motifs analyses indicated that the majority of DlSWEETs in each clade shared similar exon–intron organization and conserved motifs. Tissue-specific gene expression suggested diverse possible functions for DlSWEET genes. Cis-elements analysis and quantitative real-time PCR (qRT-PCR) analysis revealed that DlSWEET1 responded to cold stress. Notably, the overexpression of DlSWEET1 improved cold tolerance in transgenic Arabidopsis, suggesting that DlSWEET1 might play a positive role in D. longan’s responses to cold stress. Together, these results contribute to a better understanding of SWEET genes, which could serve as a foundation for the further functional identification of these genes.


Introduction
Sugars are not only the predominant carbon and energy sources for both eukaryotes and prokaryotes, but also the essential signaling molecules for diverse physiological processes [1]. In addition, sugars are the main determiners of fruit quality and flavor [2,3]. In plants, sugars are the main products of photosynthesis, which occurs mainly in the stromal cells of the chloroplast, and are translocated to the sink organs via long-distance transport [4,5]. However, the movement of sugar between tissues via the phloem requires the assistance of sugar transporters [6,7]. To date, various sugar transporters have been identified in plants and they can be grouped into three families: monosaccharide transporter-like (MST), sucrose transporters (SUTs/SUCs), and sugars will eventually be exported transporters (SWEETs) [8].
The SWEET families are a recently discovered protein family of carbohydrate transporters in plants and mammals [9], which feature the MtN3/Saliva motif (PF03083) and seven transmembrane helices (TMHs) [10]. The eukaryotic SWEETs have a 3-1-3 TMH structure, which is organized as tandem repeats of two 3-TMH domains separated by a single transmembrane domain [9,[11][12][13]. On the contrary, prokaryotic SWEETs (designated as SemiSWEETs) contain only a single triple helix bundle (THB), indicating that SemiSWEETs might have evolved from the duplication of the THB [10,14,15].
Longan (Dimocarpus longan L.), which belongs to the family of Sapindaceae, is an economically important evergreen fruit crop [35]. China is the largest producer of longan and account for 70% and more than 50% of the world's acreage and production, respectively [36]. Sugar composition and content in the aril of longan fruit is an important factor determining the quality of longan fruit. Consequently, high sweetness has become a major objective for longan breeding. Cold stress is a major environmental factor that adversely affects plant growth and development, as well as the product quality and yield. Longan, which originated in South China or Southeast Asia, is sensitive to cold, although longan is widely cultivated in the tropical and subtropical regions of the world [37]. In recent years, unpredictable frost-inducing weather has occurred in southern China, resulting in severe economic losses in the longan industry; thus, a better understanding of the molecular response mechanisms of longan cold stress is urgent.
In the present study, 20 SWEET genes in longan (termed DlSWEET) were identified, and their phylogenetic relationship, gene structures, protein motifs, cis-acting elements, and tissue-specific expression patterns were analyzed. We also determined the expression patterns of some selected DlSWEET genes during fruit development stages and in response to different abiotic stress treatments. Additionally, the DlSWEET1 gene was introduced into A. thaliana by Agrobacterium-mediated method to investigate its function. The results will serve to facilitate our understanding of the function of SWEET genes.

Identification and Phylogenetic Analysis of SWEET Proteins in Longan
A total of 20 SWEET genes were identified and renamed from DlSWEET1 to Dl-SWEET16b according to their phylogenetic relationship with 17 AtSWEET (Table 1). Gene characteristics, including gene size, coding sequence size, protein length, isoelectric point (pI), molecular weight (MW), and grand average of hydropathicity (GRAVY) value were analyzed. The predicted size of 20 DlSWEET proteins ranged from 140 (DlSWEET16b, 15.48 KDa) to 440 (DlSWEET2b, 49.29 KDa) amino acids, and the predicted isoelectric points ranged from 5.77 (DlSWEET15) to 9.54 (DlSWEET4). The predicted GRAVY value of DlSWEET proteins ranged from 0.236 to 0.914, indicating hydrophobic properties. The evolutionary relationships of DlSWEET family members were assessed by constructing an NJ phylogenetic tree ( Figure 1). The results showed that DlSWEET proteins were clearly grouped into four different clades. Clade III is the largest groups, which contained eight DlSWEETs (DlSWEET9a/9b/9c/9d/10a/10b/10c/15), while clade IV had only two members (DlSWEET16a/16b). Clades I and II contained six and four members, respectively.

Analysis of Transmembrane Domains and Conserved Motifs
To further investigate the features of DlSWEET proteins, TM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) (Copenhagen, Dan predict the transmembrane domains (Table 1 and Figure S1). The resu ten DlSWEET proteins contain seven TMHs, and five DlSWEET pro In addition, four DlSWEET proteins had five TMHs. Interest contained three TMHs, which is observed in eukaryotes.

Analysis of Transmembrane Domains and Conserved Motifs
To further investigate the features of DlSWEET proteins, TMHMM Server v2.0 (http: //www.cbs.dtu.dk/services/TMHMM-2.0/) (Copenhagen, Danmark) was used to predict the transmembrane domains (Table 1 and Figure S1). The results showed that only ten DlSWEET proteins contain seven TMHs, and five DlSWEET proteins had six TMHs. In addition, four DlSWEET proteins had five TMHs. Interestingly, DlSWEET16b contained three TMHs, which is observed in eukaryotes.
The conserved motifs of the DlSWEET proteins were examined using MEME program in DlSWEET proteins (Figure 2 and Figure S2 and Table S1). The result showed that 10 conserved motifs were identified among the 20 DlSWEETs. Motifs 1 and 2 were observed in 20 DlSWEETs, while motif 7 and motif 10 only appeared in two DlSWEETs each.

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The conserved motifs of the DlSWEET proteins were examined using MEME program in DlSWEET proteins (Figures 2 and S2 and Table S1). The result showed that 10 conserved motifs were identified among the 20 DlSWEETs. Motifs 1 and 2 were observed in 20 DlSWEETs, while motif 7 and motif 10 only appeared in two DlSWEETs each.

Exon-Intro Organization of DlSWEET Genes
To gain insights into the structure of DlSWEET genes, the introns and exons organization, which play important roles in the evolution of multiple gene families, were analyzed ( Figure 3). The results showed that all DlSWEET genes contained introns, ranging from two to seven. Furthermore, nine DlSWEET genes (DlSWEET1/2a/3a/3b/3c/7a/10a/10c/16a) contained six exons, while seven DlSWEET genes (DlSWEET7b/7c/9a/9c/9d/10b/15) harbored five exons, two genes (DlSWEET4/9b) displayed four exons, and DlSWEET2b and DlSWEET16b possessed eight and three exons, respectively. Although some similar DlSWEET genes in the same clade shared a similar gene structure, for example, DlSWEET3a/3b/3c contains 6 exons and 5 introns, differences also existed in their length due to the introns.

Exon-Intro Organization of DlSWEET Genes
To gain insights into the structure of DlSWEET genes, the introns and exons organization, which play important roles in the evolution of multiple gene families, were analyzed ( Figure 3). The results showed that all DlSWEET genes contained introns, ranging from two to seven. Furthermore, nine DlSWEET genes (DlSWEET1/2a/3a/3b/3c/7a/10a/10c/16a) contained six exons, while seven DlSWEET genes (DlSWEET7b/7c/9a/9c/9d/10b/15) harbored five exons, two genes (DlSWEET4/9b) displayed four exons, and DlSWEET2b and Dl-SWEET16b possessed eight and three exons, respectively. Although some similar DlSWEET genes in the same clade shared a similar gene structure, for example, DlSWEET3a/3b/3c contains 6 exons and 5 introns, differences also existed in their length due to the introns.

Exon-Intro Organization of DlSWEET Genes
To gain insights into the structure of DlSWEET genes, the introns and exons organization, which play important roles in the evolution of multiple gene families, were analyzed ( Figure 3). The results showed that all DlSWEET genes contained introns, ranging from two to seven. Furthermore, nine DlSWEET genes (DlSWEET1/2a/3a/3b/3c/7a/10a/10c/16a) contained six exons, while seven DlSWEET genes (DlSWEET7b/7c/9a/9c/9d/10b/15) harbored five exons, two genes (DlSWEET4/9b) displayed four exons, and DlSWEET2b and DlSWEET16b possessed eight and three exons, respectively. Although some similar DlSWEET genes in the same clade shared a similar gene structure, for example, DlSWEET3a/3b/3c contains 6 exons and 5 introns, differences also existed in their length due to the introns.

Cis-Acting Elements in the Promoters of DlSWEETs
The regulatory role of DlSWEETs was studied by gathering the 2000 bp upstream regions of 19 DlSWEETs (DlSWEET15 is located from 142 to 2474 bp in scaffold601, so we could not obtain the promoter sequence from the longan genome), and the transcriptional response elements of DlSWEETs were predicted using the PLACE database ( Figure 4 and Table S2). A series of cis-elements involved in plant growth regulation processes were identified, such as GCN4 motifs found in the promoters of four genes (DlSWEET2b/9b/9c/16a). Additionally, 20 light-responsive cis-elements were identified; the BOX 4 element, in particular, existed in all promoters of DlSWEETs genes, suggesting that the light may play important roles in regulating the expression of DlSWEETs genes. Notably, many plant hormones and stress-responsive cis-elements, such as ABRE, AuxRR-core, LTR, and MBS were present in the promoter region of DlSWEETs, suggesting that some DlSWEET genes may responsed to plant hormone and stress treatments.

Cis-Acting Elements in the Promoters of DlSWEETs
The regulatory role of DlSWEETs was studied by gathering the 2000 bp upstream regions of 19 DlSWEETs (DlSWEET15 is located from 142 to 2474 bp in scaffold601, so we could not obtain the promoter sequence from the longan genome), and the transcriptional response elements of DlSWEETs were predicted using the PLACE database ( Figure 4 and Table S2). A series of cis-elements involved in plant growth regulation processes were identified, such as GCN4 motifs found in the promoters of four genes (DlSWEET2b/9b/9c/16a). Additionally, 20 light-responsive cis-elements were identified; the BOX 4 element, in particular, existed in all promoters of DlSWEETs genes, suggesting that the light may play important roles in regulating the expression of DlSWEETs genes. Notably, many plant hormones and stress-responsive cis-elements, such as ABRE, AuxRR-core, LTR, and MBS were present in the promoter region of DlSWEETs, suggesting that some DlSWEET genes may responsed to plant hormone and stress treatments.

Tissue-Specific Expression Patterns of DlSWEET Genes
To understand the tissue-specific-expression patterns of DlSWEET genes, transcripts abundance of DlSWEET genes in different tissues, including root, stem, leaf, flower, fruit, and seed, were analyzed based on publicly available RNA-seq datasets (GSE84467) [38] ( Figure 5). As is shown in Figure 5a, the SWEET genes showed different expression patterns in different longan tissues (DlSWEET10b had no record in the datasets). Among the 19 DlSWEETs, 16 were expressed at relatively high levels (FPKM value > 1) in at least one tissue, including 11, 4, 7, 14, 6, and 11 DlSWEETs in root, stem, leaf, flower, fruit, and seed, respectively ( Figure 5b). Interestingly, the FPKM values of some DlSWEETs were higher than 50, demonstrating that they may be important for longan development. For instance, three genes (DlSWEET2a/10c/16a) and six genes (DlSWEET1/2a/7b/9a/10c/15) showed a higher expression level in leaf and flower, respectively, indicating their potential roles in regulating longan leaf and flower development. Notably, DlSWEET1 showed higher expression levels in flower, fruit and seeds, indicating that it may play an important role in longan growth and development.

Tissue-Specific Expression Patterns of DlSWEET Genes
To understand the tissue-specific-expression patterns of DlSWEET genes, transcripts abundance of DlSWEET genes in different tissues, including root, stem, leaf, flower, fruit, and seed, were analyzed based on publicly available RNA-seq datasets (GSE84467) [38] ( Figure 5). As is shown in Figure 5a, the SWEET genes showed different expression patterns in different longan tissues (DlSWEET10b had no record in the datasets). Among the 19 DlSWEETs, 16 were expressed at relatively high levels (FPKM value > 1) in at least one tissue, including 11, 4, 7, 14, 6, and 11 DlSWEETs in root, stem, leaf, flower, fruit, and seed, respectively (Figure 5b). Interestingly, the FPKM values of some DlSWEETs were higher than 50, demonstrating that they may be important for longan development. For instance, three genes (DlSWEET2a/10c/16a) and six genes (DlSWEET1/2a/7b/9a/10c/15) showed a higher expression level in leaf and flower, respectively, indicating their potential roles in regulating longan leaf and flower development. Notably, DlSWEET1 showed higher expression levels in flower, fruit and seeds, indicating that it may play an important role in longan growth and development.

Soluble Sugar and Expression Patterns of DlSWEET Genes during Fruit Development
As is shown in Figure 6a, the sucrose content increased rapidly from 60 DAF to DAF, with concentrations ranging from 4.72 to 46.81 g·kg −1 fresh weight (FW). T concentrations of fructose exhibited a slightly increasing trend as the fruit develop Additionally, the glucose content increased slightly from 60 DAF to 90 DAF and th slightly decreased at the later stage of ripening. Overall, sucrose is the main compon of soluble sugars in mature longan fruit. Considering the potential roles of DlSWEETs the soluble sugar accumulation, we investigated the transcript abundance of six ge during longan fruit development using the qRT-PCR method (Figure 6b). Two ge (DlSWEET1/10c) contained extremely high expression levels in fruit at 60 DAF, showed a decrease in transcript abundance throughout fruit development. On contrary, the other four genes (DlSWEET2a/2b/3a/16a) showed relatively low express levels at 60 DAF and then increased during fruit development, which are clos connected to the change in sugar content during longan fruit development.

Soluble Sugar and Expression Patterns of DlSWEET Genes during Fruit Development
As is shown in Figure 6a, the sucrose content increased rapidly from 60 DAF to 120 DAF, with concentrations ranging from 4.72 to 46.81 g·kg −1 fresh weight (FW). The concentrations of fructose exhibited a slightly increasing trend as the fruit developed. Additionally, the glucose content increased slightly from 60 DAF to 90 DAF and then slightly decreased at the later stage of ripening. Overall, sucrose is the main component of soluble sugars in mature longan fruit. Considering the potential roles of DlSWEETs in the soluble sugar accumulation, we investigated the transcript abundance of six genes during longan fruit development using the qRT-PCR method (Figure 6b). Two genes (DlSWEET1/10c) contained extremely high expression levels in fruit at 60 DAF, but showed a decrease in transcript abundance throughout fruit development. On the contrary, the other four genes (DlSWEET2a/2b/3a/16a) showed relatively low expression levels at 60 DAF and then increased during fruit development, which are closely connected to the change in sugar content during longan fruit development.

Expression Patterns of DlSWEET Genes under Abiotic Stress Condition
To detect whether the DlSWEET genes were induced by different abiotic stresses, qRT-PCR was performed to determine the expression levels of the seven DlSWEET genes responding to cold, heat, salt, and drought treatments in seedlings (Figure 7). Overall, the levels of expression of the seven DlSWEET genes differed among the four stress treatments. In detail, all the genes's expression levels were significantly changed by cold stresses, and only DlSWEET1 was up-regulated by cold stress, while the other genes were downregulated by cold, indicating that DlSWEET1 may play an important role in longan cold response. Four genes (DlSWEET2b/3a/10c/16b) were down-regulated by heat and salt stresses, respectively; two genes (DlSWEET2b/3a) were down-regulated by drought stress, whereas two genes (DlSWEET1/10c) were up-regulated by drought stress.

Expression Patterns of DlSWEET Genes under Abiotic Stress Condition
To detect whether the DlSWEET genes were induced by different abiotic stresse qRT-PCR was performed to determine the expression levels of the seven DlSWEET gene responding to cold, heat, salt, and drought treatments in seedlings ( Figure 7). Overall, th levels of expression of the seven DlSWEET genes differed among the four stres treatments. In detail, all the genes's expression levels were significantly changed by col stresses, and only DlSWEET1 was up-regulated by cold stress, while the other genes wer down-regulated by cold, indicating that DlSWEET1 may play an important role in longa cold response. Four genes (DlSWEET2b/3a/10c/16b) were down-regulated by heat and sa stresses, respectively; two genes (DlSWEET2b/3a) were down-regulated by drought stres whereas two genes (DlSWEET1/10c) were up-regulated by drought stress.

Overexpression of DlSWEET1 Enhances Cold Tolerance in Transgenic Arabidopsis Plants
To further explore the roles of DlSWEET1 in cold tolerance in plants, the 35S: DlSWEET1: pSAK277 construct was transferred into A. thaliana, and four independent transgenic lines (T1 generation) were obtained based on kanamycin resistance selection and genomic PCR verification. T3 homozygous transgenic lines were used for cold tolerance assessments. Among the T3 generation transformed lines, four lines (OE-5, OE-6, OE-7, and OE-8) presented high expression of DlSWEET1 by qRT-PCR with a wild-type

Overexpression of DlSWEET1 Enhances Cold Tolerance in Transgenic Arabidopsis Plants
To further explore the roles of DlSWEET1 in cold tolerance in plants, the 35S: Dl-SWEET1: pSAK277 construct was transferred into A. thaliana, and four independent transgenic lines (T1 generation) were obtained based on kanamycin resistance selection and genomic PCR verification. T3 homozygous transgenic lines were used for cold tolerance assessments. Among the T3 generation transformed lines, four lines (OE-5, OE-6, OE-7, and OE-8) presented high expression of DlSWEET1 by qRT-PCR with a wild-type (WT) Arabidopsis line as a negative control (Figure 8a). Meanwhile, we further observed the frost damage to leaves of transgenic plants (OE-7 and OE-8) and WT plants. As is shown in Figure 8b, there was no apparent difference in phenotype between the transgenic and WT plants. When they were treated at −4 • C for 12 h, after which they were allowed to recover for 6 days at 25 • C, all WT plants died, while most transgenic plants stayed alive and resumed growing (Figure 8b). The survival rate of transgenic Arabidopsis lines was significantly higher than the WT line at 60.00% for OE-7 and 86.67% for OE-8, compared to 0% for WT (Figure 8c). These results indicated that the overexpression of DlSWEET1 could improve the freezing resistance of plants.

Discussion
SWEET proteins play key roles in plant growth and development by regulating sugar translocation and allocation [39,40]. To date, genome-wide analyses have identified a variable number of SWEET genes in over 20 plant species, and the number of reported SWEET gene members varies from 7 to 108, which may be caused by gene duplication, including tandem or segmental duplication [41][42][43]. In this study, 20 SWEET genes were identified in longan, similar to in Arabidopsis [9], rice [13], lychee [16], and grape [44]. These genes were classified into four clades according to their phylogenetic evolutionary relationship (Figure 2), which was consistent with previous studies [45][46][47][48]. Notably, a gene structure analysis indicated that nine DlSWEET genes contained six exons. Similar results were also observed in soybean [41], tomato [49], pear [50], and cucumber [51], suggesting that SWEET gene members are highly conserved during evolution.
Plant SWEET proteins play diverse physiological functions and can transport different sugar molecules [5,10,13]. Based on tissue-specific expression patterns of

Discussion
SWEET proteins play key roles in plant growth and development by regulating sugar translocation and allocation [39,40]. To date, genome-wide analyses have identified a variable number of SWEET genes in over 20 plant species, and the number of reported SWEET gene members varies from 7 to 108, which may be caused by gene duplication, including tandem or segmental duplication [41][42][43]. In this study, 20 SWEET genes were identified in longan, similar to in Arabidopsis [9], rice [13], lychee [16], and grape [44]. These genes were classified into four clades according to their phylogenetic evolutionary relationship (Figure 2), which was consistent with previous studies [45][46][47][48]. Notably, a gene structure analysis indicated that nine DlSWEET genes contained six exons. Similar results were also observed in soybean [41], tomato [49], pear [50], and cucumber [51], suggesting that SWEET gene members are highly conserved during evolution.
Plant SWEET proteins play diverse physiological functions and can transport different sugar molecules [5,10,13]. Based on tissue-specific expression patterns of DlSWEET genes, DlSWEET9a was highly expressed in flowers ( Figure 5), and its homologous gene was found to be involved in nectar secretion in Arabidopsis, Brassica, and Nicotiana [18]. The results indicated that DlSWEET9a appeared to play a key role in longan nectar secretion. The stem participates in many physiological and biochemical processes in plants. Sugar is an essential organic substance in the growth and development of the stem. DlSWEET16a was expressed highly in the stem (Figure 5), and the same result was observed in its homologous gene (AT-SWEET16-1) in Annona squamosa L. [52], suggesting that DlSWEET16a may be involved in stem growth and development. DlSWEET15 had higher expression levels in flowers and seeds than other tissues, suggesting its function in specialized organs. In Arabidopsis, AtSWEET15 was expressed in both the seed coat and endosperm, and acts as a sucrose transporter in seed coat efflux and seed filling [53]. These results suggest that DlSWEET15 may participate in longan seed development.
Sugar content is an important factor for determining fruits' organoleptic quality [2]. As is shown in Figure 6, the content of soluble sugars increased during fruit development and sucrose was the dominating soluble sugar in mature longan fruit. Although 20 SWEET gene members were identified in longan, we only detected the transcripts of six DlSWEETs in longan fruit. Among these six DlSWEETs, four genes (DlSWEET1/2a/2b/3a) belong to clade I, in which SWEET proteins mainly transport monosaccharides [9]. In addition, the expression level of DlSWEET1 declined during fruit development, but the transcripts level of the other three genes increased, suggesting that DlSWEET1 may play a role in transporting monosaccharides in the early stages of fruit development, while DlSWEET2a/2b/3a comes to play the roles at the fruit expansion and mature stages. In clade III, SWEETs in Arabidopsis and rice transport disaccharides, mainly sucrose [9,12]. Although DlSWEET10c belongs to clade III, its transcript abundance is inconsistent with the sucrose content during fruit development. The cause of the discrepancy may be a rapid conversion of sucrose to glucose or fructose at the early fruit development stage.
Longan is frequently challenged by abiotic stressors, for instance, extreme temperatures, drought, and high salinity. Sugars accumulate in plant cells to reduce the osmotic potential, which is conducive to the normal growth of plants under abiotic stress conditions [54,55]. Previous reports have shown that the SWEET genes were involved in abiotic stress [25,26,49]. The results showed that seven genes showed significantly up-or downregulated expression in at least one tested treatment (Figure 7). Under drought conditions, two genes' (DlSWEET1/10c) levels were significantly increased. Similar findings were observed in cabbage [31]. Although the seven genes were significantly induced by cold stress, only DlSWEET1 showed a significantly up-regulated trend. The same result was also observed in tea, in which the expression level of CsSWEET1 increased markedly during cold acclimation [56]. Cis-elements analysis showed that a TC-rich repeats element, which is involved in defense and stress responsiveness, existed in the promoter of DlSWEET1, indicating that DlSWEET1 may play an important role in the longan cold response. Moreover, the overexpression of DlSWEET1 conferred its tolerance to cold stress in transgenic Arabidopsis. However, due to the difference between Arabidopsis and longan, the function of DlSWEET1 in longan cold-response warrants further investigation.

Plant Materials, Stress Treatments, and Measurement of Soluble Sugars Content
To investigate the function of DlSWEETs involved in longan fruit sugar accumulation, we used fruits of Longan (Dimocarpus longan L.) cultivar "Qingkebaoyuan". The trees were grown in the Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, China. The fruit samples were randomly picked from three trees, under standard cultivation conditions, at 60, 90, and 120 days after flowering (DAF). Soluble sugars in longan pulp were extracted and measured according to the previously reported protocol [57].
"Honghezi" seedlings were grown for six month in Fujian Agriculture and Forestry University, Fuzhou, China, which were used for abiotic stress treatments. For heat and cold stresses, seedlings were grown at 40 • C or 4 • C for 4 h, respectively. For salt and drought stresses, seedlings were treated with NaCl (200 mM) or PEG6000 (20%) for 4 h at 28 • C, respectively. Each treatment had three replicates of three plants. In addition, three seedlings grown at 28 • C were used as a control. After treatment, the leaf samples of the same leaf position were collected and immediately frozen in liquid nitrogen and stored at −80 • C for further analysis.
The MEME (Multiple EM for Motif Elicitation) program (http://meme.nbcr.net/ meme/cgi-bin/meme.cgi) (accessed on 31 March 2019) was used to analyze the motifs of DlSWEET proteins, with the parameters set to the following: maximum number of motifs 600, motif width 15-60, and number 15 [59].

Cis-Elements Search and Exon-Intron Organization of DlSWEET Genes
The exon-intron organization of the DlSWEETs were analyzed and visualized in GSDS 2.0 (gene structure display server) [60]. Furthermore, the 2000 bp promoter regions of DlSWEET were analyzed in PLACE (https://www.dna.affrc.go.jp/PLACE/?action= newplace) (accessed on 1 April 2019), and the distribution of four categories of cis-acting elements was visualized in the TBtools toolkit [61].

Expression Pattern Analysis of DlSWEET Genes
To gain insights into the DlSWEET genes expression patterns, we utilized the transcriptome data, which included different longan tissues (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE84467) (accessed on 31 March 2019) [38]. The gene expression values were normalized by fragments per kilobase million (FPKM) and log2-transformed. The heat maps were plotted using the software TBtools [61].

RNA Isolation and qRT-PCR Analysis
Total RNA was extracted using an RNAprep Pure Plant Kit (TIANGEN, Beijing, China) with three biological replicates. Then, approximately 1 µg of total RNA per sample was used as the template for reverse-transcription via TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANS, Beijing, China). The RT-qPCR was performed using SYBR Green I Master Mix (Takara, Dalian, China) and a LightCycler 96 Real-Time PCR Systems (Roche, Basel, Switzerland). The relative expression levels were calculated with the formula 2 −∆∆CT method with three biological and three technical replicates [62]. All primer sequences used in this study are listed in Table S3.

Functional Analysis of DlSWEET1-Overexpressing Transgenic Arabidopsis Thaliana
The full-length cDNA sequence of DlSWEET1 was cloned into the pSAK277 vector. The 35S: DlSWEET1 plasmid was transferred into Agrobacterium strain GV3101 by the freeze-thaw method, and transferred into the Arabidopsis plants using the floral dip method. The Arabidopsis ecotype Columbia-0 (col-0) was used as the wild type (WT) in this study. Transformed plants were selected on the basis of their resistance to kanamycin, and 3-weeksold T3 homozygous plants and WT seedlings were used for further experiments. Two transgenic lines and WT seedlings were transferred to 4 • C growth chambers with the same ambient conditions for 48 h and then were exposed to −4 • C for 12 h, followed by 12 h of darkness at 4 • C, after which they were returned to normal conditions for recovery for 6 days. The plant survival rates and phenotypes were recorded.

Statistical Analyses
All reported values are presented as mean ± standard error (SE, n = 3). The means are compared using the Student's t-test at the 0.05 and 0.01 levels of significance.

Conclusions
In summary, 20 longan SWEET genes (DlSWEETs) were identified in the D. longan genome. A phylogenetic analysis showed that DlSWEETs were grouped into four different subfamilies. Tissue-specific expression patterns analysis showed that DlSWEET genes may play various roles in the development of longan tissues. qRT-PCR analyses implied that three and four DlSWEET genes might be involved in abiotic stress response and fruit sugar accumulation, respectively. Furthermore, the overexpression of the DlSWEET1 gene improved cold stress tolerance in transgenic Arabidopsis. This work will contribute to the follow-up study of the functional characteristics of DlSWEET genes and the cultivation of high-quality D. longan varieties.