Identification of HuSWEET Family in Pitaya (Hylocereus undatus) and Key Roles of HuSWEET12a and HuSWEET13d in Sugar Accumulation

The sugar composition and content of fruit have a significant impact on their flavor and taste. In pitaya, or dragon fruit, sweetness is a crucial determinant of fruit taste and consumer preference. The sugars will eventually be exported transporters (SWEETs), a novel group of sugar transporters that have various physiological functions, including phloem loading, seed filling, nectar secretion, and fruit development. However, the role of SWEETs in sugar accumulation in pitaya fruit is not yet clear. Here, we identified 19 potential members (HuSWEET genes) of the SWEET family in pitaya and analyzed their conserved motifs, physiochemical characteristics, chromosomal distribution, gene structure, and phylogenetic relationship. Seven highly conserved α-helical transmembrane domains (7-TMs) were found, and the HuSWEET proteins can be divided into three clades based on the phylogenetic analysis. Interestingly, we found two HuSWEET genes, HuSWEET12a and HuSWEET13d, that showed strong preferential expressions in fruits and an upward trend during fruit maturation, suggesting they have key roles in sugar accumulation in pitaya. This can be further roughly demonstrated by the fact that transgenic tomato plants overexpressing HuSWEET12a/13d accumulated high levels of sugar in the mature fruit. Together, our result provides new insights into the regulation of sugar accumulation by SWEET family genes in pitaya fruit, which also set a crucial basis for the further functional study of the HuSWEETs.


Introduction
Sugars are the predominant product of photosynthesis and play crucial roles in signal transduction, osmotic regulation, molecule transport, transient energy storage, and stress resistance during plant growth and development [1,2].The sugars are synthesized in leaves and transported to non-photosynthesis organs (seeds, roots, and fruits) to fulfill sufficient plant growth [2][3][4].So far, three families of eukaryotic sugar transporters have been identified, including sucrose transporters (SUTs), monosaccharide transporters (MSTs), and sugars will eventually be exported transporters (SWEETs) [1,5].
SWEETs (also known as the PQ-loop-repeat) are a new type of sugar transporters, which belong to the MtN3/saliva family (Pfam code PF03083) [6,7].Three α-helical transmembrane domains (3-TMs) were found in the MtN3/saliva domain.Eukaryotic SWEET proteins typically comprise seven TMs that are composed of tandem repeats of two 3-TMs separated by a single TM [8].SWEETs function as uniporters and participate in the uptake and efflux of different sugar substrates across cell membranes in pace with a concentration gradient [3,9,10].Up to now, SWEET genes in many plant species have been reported, such as Arabidopsis thaliana [6], Oryza sativa [11], Glycine max [12], Vitis vinifera [13], Citrus sinensis [14], Malus pumila [15], Lycopersicon esculentum [16], Litchi chinensis [17], and Eriobotrya japonica [18].In general terms, SWEET proteins are divided into four clades (clade I, clade II, clade III, and clade IV) with distinct characteristics.SWEETs in clade I and clade II preferentially transport hexoses; members of clade III mainly prefer sucrose; and members of clade IV are efficient fructose transporters [5,6].
The SWEET gene family is usually involved in different physiological processes in plants, for example, phloem loading [19], nectar secretion [20][21][22], seed filling [23,24], plant-pathogen interaction [6], biotic and abiotic stresses [25][26][27], pollen nutrition [28], and fruit development [29].The accumulation of sugar is the core factor to determining fruit quality and yield.And, sugar transporters play integral roles in sugar partitioning and accumulation [30].Many studies have shown that SWEET proteins were involved in sugar accumulation in fruit.In watermelon fruit, ClSWEET3 could help hexose uptake into fruit cells from the intercellular space, and overexpression of CISWEET3 could improve sugar contents [31].DlSWEET2a/2b/3a/16a showed close relationships to alterations in sugar contents during longan fruit development and might be involved in fruit sugar accumulation [32].SlSWEET1a participated in sugar regulation in tomato fleshy fruit [33].In addition, SlSWEET7a and SlSWEET14 were highly expressed in the fruit and silencing SlSWEET7a and SlSWEET14 increased hexose contents in tomato fruit [29].In cucumber, the hexose transporter CsSWEET7a mediated phloem unloading in companion cells for fruit development [34].Overexpressing VvSWEET10 in grapevine calyx and tomatoes could significantly increase hexose and total sugar contents [35].Additionally, sucrose is the main carbon form transported from source to sink, which is important for sugar accumulation in the fruit and some SWEETs can specifically transport sucrose [36].For example, PuSWEET15 and CitSWEET11d were significantly positively correlated with sucrose concentration and promoted accumulation of sucrose in pear and citrus fruit, respectively [37,38].These studies showed that SWEETs play key roles in sugar distribution and accumulation in fruit.However, no information is available about the molecular characteristics and gene functions of SWEETs in pitaya.
Pitaya belongs to Hylocereus and Selenicereus in the Cactaceae family of Caryophyllales.Based on the color of the peel and flesh, it can be divided into three main types: red peel with red pulp (H.monacanthus or H. polyrhizus), red peel with white pulp (H.undatus), and yellow peel with white pulp (H.undatus, H. megalanthus, or S. megalanthus) [39].It has a delicate taste and is rich in sugars, organic acids, vitamins, betalain, and other nutrients, among which soluble sugar content is a critical factor affecting the flavor and taste quality of pitaya [39].Pitaya is classified as a hexose accumulation fruit.The main soluble sugars in the fruit of pitaya are glucose, fructose, and sucrose.And, in mature pitaya fruit, glucose is the predominant soluble sugar.The concentration of sugar gradually increased during fruit development and reached a maximum level at the mature stage [40][41][42].In addition, sugar accumulation varied in different sections of pitaya pulp, and the sugar content in the center section was significantly higher than that of the outside section [42].The accumulation of soluble sugars in fruits is determined by synthesis, degradation, transport, and storage [43].HpINV2 and HpSuSy1 were well correlated with the content of glucose and fructose in pitaya, and HpWRKY3 activated the expressions of HpINV2 and HpSuSy1 [44].These results showed that HpINV2, HpSuSy1, and HpWRKY3 play important roles in sugar metabolism during fruit development.However, there is little information available about sugar transport in pitaya.
In this study, the SWEET family genes (HuSWEET) were identified across the whole pitaya genome.Expression profiles of HuSWEETs were analyzed in different tissue/organs and fruit development stages of the 'Guanhuahong' pitaya and functions of two members, HuSWEET12a and HuSWEET13d, were preliminarily validated.The present study aims to elucidate the functional roles of HuSWEET genes in sugar accumulation in pitaya and identify candidate genes that could contribute to fruit quality improvement in pitaya breeding programs.

Identification and Phylogenetic Analyses of the HuSWEET Family
A total of 19 candidate SWEET genes with two MtN3/saliva domains were identified in the H. undatus genome [45].These SWEET genes were named HuSWEET1 to HuSWEET13e according to their homologous genes in A. thaliana.Multiple sequence alignments of these 19 HuSWEET proteins showed that the 7-TMs were highly conserved (Figure S1), which is consistent with results in other reported plants [46], as well as the characteristics of other plant SWEET proteins.The physicochemical characteristics of HuSWEETs were also analyzed, including the length of open reading frames (ORFs), the numbers of amino acids (aa), molecular weights (MW), isoelectric points (pI), hydrophilicity, and instability index (Table 1).The ORFs of the HuSWEET genes ranged from 708 to 1029 bp in length, encoding amino acids from 235 to 342 aa.The MW ranged from 26.30 (HuSWEET2) to 38.40 kDa (HuSWEET13f ) and their pI-values ranged from 5.71 to 9.62, with an average of 8.76.The hydrophilicity of the 19 HuSWEET proteins is more than 0, indicating that these are hydrophobic proteins.Nine HuSWEET proteins (HuSWEET2/7a/7b/11/12a/12b/12d/13c/13d) have an instability index of more than 40, suggesting that these HuSWEETs are unstable proteins.To explore the phylogenetic relationship of HuSWEETs, we downloaded SWEET proteins from pitaya, A. thaliana, rice, and grape to construct a phylogenetic tree using the maximum likelihood (ML) method.As shown in Figure 1, SWEET proteins from A. thaliana, rice, and grape were classified into four clades (clade I to clade IV), while the HuSWEET proteins were clustered into three clades.There is no HuSWEET member in clade IV.

Conserved Motif and Gene Structure Analyses of HuSWEETs
We further constructed a phylogenetic tree only using the 19 SWEET family prote in pitaya, and classified them into three groups (Figure 2A) which is consistent with t result in Figure 1.We identified eight motifs based on the Multiple Em for Motif Elicitati (MEME) analysis (Figure 2B).There are five motifs shared by all HuSWEET protei indicating they are the key motifs that affect the function of the HuSWEET proteins.W also analyzed the exon-intron structures of the HuSWEET genes using TBtools [47], a found that HuSWEET genes in the same clade always exhibited similar exon-intr structure.

Conserved Motif and Gene Structure Analyses of HuSWEETs
We further constructed a phylogenetic tree only using the 19 SWEET family proteins in pitaya, and classified them into three groups (Figure 2A) which is consistent with the result in Figure 1.We identified eight motifs based on the Multiple Em for Motif Elicitation (MEME) analysis (Figure 2B).There are five motifs shared by all HuSWEET proteins, indicating they are the key motifs that affect the function of the HuSWEET proteins.We also analyzed the exon-intron structures of the HuSWEET genes using TBtools [47], and found that HuSWEET genes in the same clade always exhibited similar exon-intron structure.

Chromosomal Localizations and Synteny Analyses of HuSWEETs
According to the location information of the pitaya genome database, the distribution of each HuSWEET gene on the chromosome was determined.The results showed that HuSWEET genes were unequally distributed on seven of the eleven chromosomes (Chrs) of pitaya (Figure 3).HuSWEET genes were mainly concentrated at both ends of the chromosomes; the most HuSWEET genes were distributed on Chr4 with seven (36.8%), followed by Chr1 with three genes (15.8%).Notably, Chr2, Chr7, Chr8, and Chr9 all contained two genes, but Chr3 had only one HuSWEET gene.

Chromosomal Localizations and Synteny Analyses of HuSWEETs
According to the location information of the pitaya genome database, the distribution of each HuSWEET gene on the chromosome was determined.The results showed that HuSWEET genes were unequally distributed on seven of the eleven chromosomes (Chrs) of pitaya (Figure 3).HuSWEET genes were mainly concentrated at both ends of the chromosomes; the most HuSWEET genes were distributed on Chr4 with seven (36.8%), followed by Chr1 with three genes (15.8%).Notably, Chr2, Chr7, Chr8, and Chr9 all contained two genes, but Chr3 had only one HuSWEET gene.Gene/segmental duplication is a major mechanism driving gene family expansio with potential to create new functions.Results from synteny analyses of HuSWEE showed that there was one pair of segmental duplicate events (HuSWEET11 an HuSWEET12d) occurring on Chr1 and Chr4 (Figure 4).Gene/segmental duplication is a major mechanism driving gene family expansion with potential to create new functions.Results from synteny analyses of HuSWEETs showed that there was one pair of segmental duplicate events (HuSWEET11 and HuSWEET12d) occurring on Chr1 and Chr4 (Figure 4).

Expression Profiles of HuSWEETs in Different Pitaya Tissues and Organs
To study the roles of HuSWEET genes in pitaya, we analyzed the expression patterns of HuSWEETs in different tissues and organs of 'Guanhuahong' pitaya (Figure 5).The results showed that most HuSWEET gene members expressed in all detected tissues and organs of pitaya, but the expression level varied dramatically.Most HuSWEET genes were strongly expressed in flowers and fruits.HuSWEET4a, HuSWEET4b, HuSWEET10, HuSWEET12b, HuSWEET12c, and HuSWEET13c had the highest expression in stamens, and HuSWEET9 was highly expressed in styles.Comparatively, HuSWEET7a, HuSWEET12a, HuSWEET13d, and HuSWEET13e were highly expressed in pulps.In addition, HuSWEET12a, HuSWEET13a, HuSWEET13b, HuSWEET13d, and HuSWEET13f were strongly expressed in stems.These results indicated that HuSWEETs have diverse functions and HuSWEET7a, HuSWEET12a, HuSWEET13d, and HuSWEET13e are involved in sugar accumulation of pitaya

Expression Profiles of HuSWEETs in Different Pitaya Tissues and Organs
To study the roles of HuSWEET genes in pitaya, we analyzed the expression patterns of HuSWEETs in different tissues and organs of 'Guanhuahong' pitaya (Figure 5).The results showed that most HuSWEET gene members expressed in all detected tissues and organs of pitaya, but the expression level varied dramatically.Most HuSWEET genes were strongly expressed in flowers and fruits.HuSWEET4a, HuSWEET4b, HuSWEET10, HuSWEET12b, HuSWEET12c, and HuSWEET13c had the highest expression in stamens, and HuSWEET9 was highly expressed in styles.Comparatively, HuSWEET7a, HuSWEET12a, HuSWEET13d, and HuSWEET13e were highly expressed in pulps.In addition, HuSWEET12a, HuSWEET13a, HuSWEET13b, HuSWEET13d, and HuSWEET13f were strongly expressed in stems.These results indicated that HuSWEETs have diverse functions and HuSWEET7a, HuSWEET12a, HuSWEET13d, and HuSWEET13e are involved in sugar accumulation of pitaya

Changes in Soluble Sugars during Pitaya Fruit Development
To investigate the sugar accumulation pattern in pitaya fruit, we measured the soluble sugar content in pulps of 'Guanhuahong' pitaya during fruit development (Figure 6A,B).Total soluble sugar content increased throughout fruit development process, along with gradual increases in glucose, fructose, and sucrose levels.Glucose and fructose contents in pulp showed rapid accumulation from 23 days after flowering (DAF) to 25 DAF, reaching maximum levels at full maturation (35 DAF).At this stage, glucose and fructose contents were 57.97 mg/g and 36.71mg/g, respectively, significantly higher than sucrose levels (19.74 mg/g).These findings suggest that glucose is the main soluble sugar in mature pitaya fruit, consistent with previous studies [40,42].

Changes in Soluble Sugars during Pitaya Fruit Development
To investigate the sugar accumulation pattern in pitaya fruit, we measured the soluble sugar content in pulps of 'Guanhuahong' pitaya during fruit development (Figure 6A,B).Total soluble sugar content increased throughout fruit development process, along with gradual increases in glucose, fructose, and sucrose levels.Glucose and fructose contents in pulp showed rapid accumulation from 23 days after flowering (DAF) to 25 DAF, reaching maximum levels at full maturation (35 DAF).At this stage, glucose and fructose contents were 57.97 mg/g and 36.71mg/g, respectively, significantly higher than sucrose levels (19.74 mg/g).These findings suggest that glucose is the main soluble sugar in mature pitaya fruit, consistent with previous studies [40,42].

Expression Analyses of HuSWEETs in Pitaya Fruits
To explore the relationship between HuSWEET genes and fruit sugar accumulation, we further analyzed the expression patterns of HuSWEETs in the pulps at different fruit developmental stages of 'Guanhuahong' pitaya.As shown in Figure 7A, HuSWEET1, HuSWEET9, HuSWEET10, and HuSWEET13a showed irregular expression patterns during fruit development of 'Guanhuahong' pitaya.Ten HuSWEET genes (HuSWEET2, HuSWEET4b, HuSWEET7a, HuSWEET7b, HuSWEET12b, HuSWEET12c, HuSWEET13b, HuSWEET13c, HuSWEET13e, and HuSWEET13f) exhibited gradually down-regulated expression patterns in pulps during fruit development of 'Guanhuahong' pitaya; HuSWEET7a, HuSWEET7b, and HuSWEET12b showed higher expression level and change among the ten HuSWEETs.On the contrary, the expression levels of HuSWEET4a, HuSWEET11, HuSWEET12a, HuSWEET12d, and HuSWEET13d showed up-regulated trends.Notably, HuSWEET4a had the highest expression level at 32 DAF while HuSWEET11/12d and HuSWEET12a/13d reached the highest expression at 27 and 35 DAF, respectively.Furthermore, higher expression levels of HuSWEET12a, HuSWEET12d, and

Expression Analyses of HuSWEETs in Pitaya Fruits
To explore the relationship between HuSWEET genes and fruit sugar accumulation, we further analyzed the expression patterns of HuSWEETs in the pulps at different fruit developmental stages of 'Guanhuahong' pitaya.As shown in Figure 7A, HuSWEET1, HuSWEET9, HuSWEET10, and HuSWEET13a showed irregular expression patterns during fruit development of 'Guanhuahong' pitaya.Ten HuSWEET genes (HuSWEET2, HuSWEET4b, HuSWEET7a, HuSWEET7b, HuSWEET12b, HuSWEET12c, HuSWEET13b, HuSWEET13c, HuSWEET13e, and HuSWEET13f ) exhibited gradually down-regulated expression patterns in pulps during fruit development of 'Guanhuahong' pitaya; HuSWEET7a, HuSWEET7b, and HuSWEET12b showed higher expression level and change among the ten HuSWEETs.On the contrary, the expression levels of HuSWEET4a, HuSWEET11, HuSWEET12a, HuSWEET12d, and HuSWEET13d showed up-regulated trends.Notably, HuSWEET4a had the highest expression level at 32 DAF while HuSWEET11/12d and HuSWEET12a/13d reached the highest expression at 27 and 35 DAF, respectively.Further-more, higher expression levels of HuSWEET12a, HuSWEET12d, and HuSWEET13d were detected in the pulps of mature fruit than those of the other HuSWEETs (Figure 7B).

Plasma Membrane-Localized HuSWEET12a and HuSWEET13d Proteins
To further investigate the potential functions of HuSWEET12a and HuSWEET13d in Additionally, we also investigated the correlation between the expression of HuSWEETs and the content of main sugars during fruit development of 'Guanhuahong' pitaya.As shown in Table 2, the expression pattern of most HuSWEETs exhibited negative correlations with the contents of major sugars in pitaya fruit.Specifically, HuSWEET12c and HuSWEET13f showed significantly negative correlations with total soluble sugar, sucrose, glucose, and fructose.On the other hand, the expressions of five HuSWEETs (HuSWEET4a, HuSWEET11, HuSWEET12a, HuSWEET12d, and HuSWEET13d) showed positive correlations with the contents of major sugars in pitaya fruit.Of them, the expressions of HuSWEET12a and HuSWEET13d showed significant and positive correlation with the accumulation of total soluble sugar, sucrose, glucose, and fructose during fruit maturation.Results from gene expressions and sugar contents suggested that HuSWEET12a and HuSWEET13d are likely contributed to sugar accumulation during the fruit development of pitaya and were selected as the candidate genes for further analyses.

Plasma Membrane-Localized HuSWEET12a and HuSWEET13d Proteins
To further investigate the potential functions of HuSWEET12a and HuSWEET13d in the accumulation of soluble sugars in the pitaya fruit, we analyzed their subcellular localization (Figure 8).We first constructed 35S-HuSWEET12a-GFP and 35S-HuSWEET13d-GFP vectors and then co-expressed them with mCherry-labeled plasma membrane marker protein (pCAM35S::At5g19750-mCherry) in N. benthamiana leaves.The fluorescence of HuSWEET12a-GFP and HuSWEET13d-GFP was found to overlap with the red fluorescence of PM-mCherry, suggesting that HuSWEET12a and HuSWEET13d are localized in the plasma membrane.
membrane marker protein (pCAM35S::At5g19750-mCherry) in N. benthamiana leave fluorescence of HuSWEET12a-GFP and HuSWEET13d-GFP was found to overlap w red fluorescence of PM-mCherry, suggesting that HuSWEET12a and HuSWEET1 localized in the plasma membrane.

Overexpression of HuSWEET12a and HuSWEET13d Could Increase Sugar Contents Tomato Fruits
To further study the function of HuSWEET12a and HuSWEET13d, they transferred into the 'Micro-Tom' tomato (Solanum lycopersicum) cotyledons usi Agrobacterium-mediated method, and the transformed plants were selected bas kanamycin resistance.Two independent T1 generation lines were obtained for f analyses with overexpression of either HuSWEET12a (12a-OE-4/11) or HuSWEET13d OE-24/29).There were no significant differences in morphology between the tran lines and the wild-type (WT) plants (Figure 9A,B).Transcriptome analysis confi successful expression of HuSWEET12a and HuSWEET13d in tomatoes (Figure 9C,D) DAF, the total sugar, glucose, and fructose contents in HuSWEET12a-OE-4/1 HuSWEET13d-OE-24/29 lines were significantly higher than those in WT lines.In add sucrose content was also significantly increased in the HuSWEET12a-OE-4 HuSWEET13d-OE-29 lines compared with WT (Figure 9E,F).

Overexpression of HuSWEET12a and HuSWEET13d Could Increase Sugar Contents of Tomato Fruits
To further study the function of HuSWEET12a and HuSWEET13d, they were transferred into the 'Micro-Tom' tomato (Solanum lycopersicum) cotyledons using an Agrobacterium-mediated method, and the transformed plants were selected based on kanamycin resistance.Two independent T 1 generation lines were obtained for further analyses with overexpression of either HuSWEET12a (12a-OE-4/11) or HuSWEET13d (13d-OE-24/29).There were no significant differences in morphology between the transgenic lines and the wild-type (WT) plants (Figure 9A,B).Transcriptome analysis confirmed successful expression of HuSWEET12a and HuSWEET13d in tomatoes (Figure 9C,D).At 50 DAF, the total sugar, glucose, and fructose contents in HuSWEET12a-OE-4/11 and HuSWEET13d-OE-24/29 lines were significantly higher than those in WT lines.In addition, sucrose content was also significantly increased in the HuSWEET12a-OE-4 and HuSWEET13d-OE-29 lines compared with WT (Figure 9E,F).

Discussion
Exported transporters or SWEETs can mediate the uptake and efflux of sugars, which play critical roles in plants [6].Genome-wide identification of SWEET genes has been widely studied in many plants, and the SWEET gene members vary from 7 to 108 [6,13,48,49].In different plants, the number of SWEET genes is various, which may be caused by tandem or segmental duplication events [12,48,50].In our study, a total of 19 SWEET genes were identified in pitaya, and one segmentally duplicated event (HuSWEET11 and HuSWEET12d) was identified in pitaya.The number of SWEET genes in pitaya is similar to that of A. thaliana [6] and litchi [17].The length of 19 HuSWEET proteins ranged from 235 aa to 352 aa, similar to cucumber [40], banana [51], and litchi [17].HuSWEET proteins are similar in structure to SWEET proteins in most other plants which contain seven TMs including two typical MtN3 domains.Gene structure analyses showed that most HuSWEET genes had six exons; the similar results were also found in cucumber [52], litchi [17], and apple [53].In general, the acquisition or loss of introns and the distribution pattern of exonintron may lead to the complexity of gene structure, thereby obtaining new gene functions, which are considered key factors affecting the evolutionary mechanism of gene families [54].HuSWEET genes of the same clade shared similar conserved motif composition and exonintron structure; these results suggested that the SWEET gene family in pitaya is relatively conservative in the evolutionary process.
SWEET genes have different expression patterns in different plant organs and tissues, which are involved in a variety of physiological processes [55].In our study, most of the HuSWEET genes were expressed in the all-detected tissues and organs with different expression patterns.Jeena et al. [55] found that the SWEET gene family plays an important role in the reproductive development of plants.In our study, all HuSWEET genes were expressed in flowers, similar to the results in A. thaliana [6,22,56], M. domestica [53], V. vinifera [13], and Jasminum sambac [57].Additionally, HuSWEET9 was highly expressed in the style and ovary, and its orthologous gene, AtSWEET9, was involved in nectar secretion in A. thaliana [22].The results suggested that HuSWEET9 may be responsible for nectar secretion in pitaya.In A. thaliana, AtSWEET2 was abundantly expressed in roots and involved in the transfer of sucrose from the root tip epidermal cells to the root margins.The highest level of HuSWEET2 was detected in roots compared with other tissues and organs of pitaya, which was similar to the expression pattern of AtSWEET2.Studies have shown that SWEETs can regulate sugar partitioning and accumulation in leaves and fruits of plants [58].In tomato, SlSWEET1a was highly expressed in leaves, and SlSWEET7a and SlSWEET14 were highly expressed in fruit, which played key roles in the sugar accumulation of leaves and fruit, respectively [29,33].In pitaya, HuSWEET12a, HuSWEET13a, HuSWEET13b, HuSWEET13d, HuSWEET13e, and HuSWEET13f were highly expressed in the stem or mature fruit, suggesting that they may play important roles in sugar accumulation in the stem or mature fruit of pitaya.
Sugar content is a major factor of fruit quality [32], and the functional identification of SWEET in sugar accumulation of fruit has been extensively studied.In apple, MdSWEET9b and MdSWEET15a were significantly correlated to fruit sugar contents and may be involved in sugar accumulation [59].Notably, PuSWEET15 showed positive correlation with sucrose content, and overexpression of PuSWEET15 in pear fruit increased significantly sucrose levels [38].In this study, we found that the contents of soluble sugars increased during pitaya fruit development; expression levels of HuSWEET7a, HuSWEET7b, HuSWEET12b, HuSWEET13c, and HuSWEET13e showed strong down-regulated expression patterns during fruit development.These results suggested that HuSWEET7a, HuSWEET7b, HuSWEET12b, HuSWEET13c, and HuSWEET13e may play roles in sugar accumulation in the early stages of pitaya fruit development, and similar results were also reported for pineapple [60].In addition, higher expression levels of HuSWEET12a, HuSWEET12d, and HuSWEET13d were detected in mature pitaya fruits.Furthermore, HuSWEET12a and HuSWEET13d were significantly and positively correlated with the accumulation of soluble sugar in pitaya fruits.Compared with WT, overexpression of HuSWEET12a and HuSWEET13d in tomato resulted in higher contents of sucrose, glucose, fructose, and total sugar.These results suggested that HuSWEET12a and HuSWEET13d are possibly involved in the sugar accumulation of pitaya.

Plant Materials
'Guanhuahong' pitaya (H.monacanthus) and 'Micro-Tom' tomato (Solanum lycopersicum) were selected to be materials in this study.'Guanhuahong' is an excellent pitaya variety with red peel and red pulp which were selected by the Dongguan Institute of Forestry Science and South China Agricultural University (SCAU, Guangzhou, China) [61].'Guanhuahong' were grown in the orchard of SCAU, the stem, root, flower (petal, stamen, style, and ovary), fruit (peel and pulp), and fruits from the 17th, 19th, 23rd, 25th, 27th, 32nd, and 35th (DAF) of pitaya were collected for gene expression.All samples were frozen in liquid nitrogen immediately and kept at −80 • C for further study.
'Micro-Tom' tomatoes were grown in a plastic container (9 cm × 9 cm) with nutritious soil (Jiffy), and cultivated in the greenhouse (16 h/8 h (light/dark); a temperature of 25 ± 1 • C and relative humidity of 65%) were used for genetic transformation.

Measurement of Soluble Sugars
The soluble sugar (including fructose, glucose, and sucrose) contents were measured according to the method of Zhang et al. [42] with minor modifications.A total of 0.5 g of samples were used to extract soluble sugars with 4 mL and 2 mL of 90% alcohol (v/v), respectively.The supernatants were dried using Concentrator plus (Eppendorf AG, Hamburg, Germany).C18 column and 0.45 µm filters were used to filter supernatants.Samples were determined in an Agilent 1200LC HPLC system (Agilent Technologies, Waldbronn, Germany).The content of total soluble sugar was measured according to Xie et al. [41].

Identification of HuSWEET Family Genes in Pitaya
The HMMER profile [62] of the MtN3/saliva motif (PF03083) was downloaded from PFAM (https://pfam.xfam.org/)(accessed on 20 October 2022) [63] and was used as a query to identify SWEET genes from the pitaya genome database (http://www.pitayagenomic.com)(accessed on 20 October 2022) [45].Putative SWEET family genes were then confirmed through PFAM and SMART with an e-value less than 1 × 10 −5 (https://smart.embl-heidelberg.de/)(accessed on 20 October 2022) to eliminate genes without the reported conserved domains and motifs of the MtN3/saliva/SWEET family members.The final SWEET gene family members were identified using the two MtN3/saliva conserved domains.ExPASy (http://web.expasy.org/potparam/)(accessed on 20 October 2022) was used to predict the characteristic of the pitaya SWEET proteins (the numbers of amino acids, MW, pI, hydrophilicity, and stability).

Gene Structure and Conserved Motifs Analyses of HuSWEETs
The exon/intron organizations of HuSWEETs were drawn by TBtools software (v 1.098769) [47].The MEME (https://meme-suite.org/) (accessed on 20 October 2022) was used to analyze the conserved motifs.The optimized parameters of MEME were set up as follows: the maximum number was eight identified motifs and the optimum width was from 1 to 50 residues of each motif.DNAMan software (v 6.0.3.99) was used to obtain the multiple sequence alignments of the amino acid sequences.

Locations in the Chromosome, Phylogenetic, and Duplication Pattern of HuSWEETs
We downloaded the complete amino acid sequences of SWEETs for the phylogenetic analysis from the Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/) (accessed on 20 October 2022) and NCBI databases (https://www.ncbi.nlm.nih.gov)(accessed on 20 October 2022), respectively.The phylogenetic trees of pitaya, grape, rice, and A. thaliana SWEET proteins were generated using the ML method in MEGA X (v 10. 1.6) with 1000 bootstrap replicates [46].The sequences were listed in Table S2.The EVOLVIEW online tool (https://evolgenius.info//evolview-v2/#login) (accessed on 20 October 2022) was used to exhibit all the evolutionary trees [64].The chromosomal locations of HuSWEET genes were identified from the pitaya genome database.The MapChart software (v 1.0.0.0) was used to draw the locations of HuSWEETs.TBtools software was used for the synteny analyses of each HuSWEET.

RNA Isolation and Gene Expression Analyses
The EASYspin Plus Complex Plant RNA Kit (RN53) (Aidlab Biotechnology, Beijing, China) was used for the RNA isolation.The PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan) was used to synthesize the single-stranded cDNA.Quantitative real-time PCR (qRT-PCR) was performed using an CFX384-Real-Time System (C1000 Touch Thermal Cycler, Bio-Rad, Irvine, CA, USA) with the RealUniversal Color PreMix (SYBR Green) (TIANGEN, Beijing, China).The specific primers for qRT-PCR were designed at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)(accessed on 20 October 2022).Actin was selected to be the internal control for gene expression analyses [65].The primers were listed in Table S1.All experiments were conducted in three biological replicates.The comparative 2 −∆∆C T method was used to calculate the relative expression levels of each transcript [66].

Subcellular Localization
The coding regions of the HuSWEET12a/13d genes (without stop codons) with Xba I and Kpn I restriction sites were cloned into the pC18-35S:GFP vector.The primers are listed in Table S1.The recombinants were induced into Agrobacterium tumefaciens strain GV3101 (pSoup-p19) and used for transient expression in N. benthamiana.The infected N. benthamiana leaves were examined 48 h after infiltration.Fluorescence microscopes (ZEISS LCM-800, Oberkochen, Germany) were used to capture the GFP signal.All assays were repeated three times.

Genetic Transformation
The coding sequences of HuSWEET12a/13d were cloned into the pPZP6K90 vector and transformed into A. tumefaciens strain GV3101.Cotyledons from seven-to ten-day-old seedlings of 'Micro-Tom' tomato were selected as explants.Both ends of each cotyledon were removed to allow it to absorb the bacterial suspension.The explants were dipped in the bacterial suspension (OD 600 = 0.5-0.6)for 20 min and blotted dry on a sterilized paper towel.Then, the explants were placed in co-culture medium (MS medium with 30 g/L sucrose, 6 g/L agar, 2.0 mg/L 6-BA, and 0.2 mg/L IAA, pH 5.4) and incubated in darkness for two days at 24 • C.After co-culture, the explants were transferred to screening medium (30 g/L sucrose, 6 g/L agar, 2.0 mg/L 6-BA, 0.2 mg/L IAA, 100 mg/L kanamycin, and 300 mg/L timentin, pH 5.8).When calli with adventitious buds developed from the explants, the cotyledons were cut off and transferred to a new screening medium with 50 mg/L kanamycin.Buds higher than one cm were cut and transferred to rooting medium (MS medium with 30 g/L sucrose, 6 g/L agar, 0.5 mg/L IBA, 25 mg/L kanamycin and 150 mg/L timentin, pH 5.8).The transformed 'Micro-Tom' tomato plantlets were analyzed using PCR and qRT-PCR.The primers were listed in Table S1.Fruits from the T 1 generation of overexpressing HuSWEET12a and HuSWEET13d lines were used for sugar concentration analyses.

Conclusions
In summary, nineteen HuSWEET genes were identified in pitaya and distributed unevenly across seven of the eleven chromosomes.HuSWEET12a and HuSWEET13d were found to be significantly and positively correlated with the accumulation of total soluble sugar, sucrose, glucose, and fructose during pitaya fruit maturation.HuSWEET12a and HuSWEET13d were located on the plasma membrane and overexpressing them in

Figure 1 .
Figure 1.The maximum likelihood phylogenetic tree of SWEET proteins from pitaya and A. thalia rice, and grape.Four clades labeled with different colors.HuSWEETs are indicated by a red asteri

Figure 1 .
Figure 1.The maximum likelihood phylogenetic tree of SWEET proteins from pitaya and A. thaliana, rice, and grape.Four clades labeled with different colors.HuSWEETs are indicated by a red asterisk.

Figure 2 .
Figure 2. Phylogenetic relationship, conserved motif, and gene structure analyses of HuSWEET genes.(A) The neighbor-joining phylogenetic tree of HuSWEETs was generated using MEGA Ⅹ with 1000 bootstrap replicates.(B) The motif compositions of HuSWEET proteins.Eight motifs were displayed in rectangles of different colors.(C) Exon-intron structure of HuSWEET genes.Yellow rectangles indicate exons, black lines indicate introns, and green rectangles represent the UTR region.(D) Amino acid sequences of the eight motifs of HuSWEET proteins.

Figure 2 .
Figure 2. Phylogenetic relationship, conserved motif, and gene structure analyses of HuSWEET genes.(A) The neighbor-joining phylogenetic tree of HuSWEETs was generated using MEGA X with 1000 bootstrap replicates.(B) The motif compositions of HuSWEET proteins.Eight motifs were displayed in rectangles of different colors.(C) Exon-intron structure of HuSWEET genes.Yellow rectangles indicate exons, black lines indicate introns, and green rectangles represent the UTR region.(D) Amino acid sequences of the eight motifs of HuSWEET proteins.

Figure 3 .
Figure 3. Distribution of HuSWEET members on the eleven chromosomes of pitaya, Bars = 20 Mb

Figure 3 .
Figure 3. Distribution of HuSWEET members on the eleven chromosomes of pitaya, Bars = 20 Mb.

Figure 4 .
Figure 4. Synteny analyses of HuSWEETs.Gray lines represent all synteny blocks in the pitaya genome, and the red lines indicate the duplicated HuSWEET gene pair.

Figure 4 .
Figure 4. Synteny analyses of HuSWEETs.Gray lines represent all synteny blocks in the pitaya genome, and the red lines indicate the duplicated HuSWEET gene pair.

Figure 5 .
Figure 5. Expression analyses of 19 HuSWEET genes in different pitaya tissues and organs.The data are shown as the means ± SDs of three independently biological replicates.The different letters above bars indicate significant differences at the p < 0.05 level according to Duncan's multiple comparison tests.

Figure 5 .
Figure 5. Expression analyses of 19 HuSWEET genes in different pitaya tissues and organs.The data are shown as the means ± SDs of three independently biological replicates.The different letters above bars indicate significant differences at the p < 0.05 level according to Duncan's multiple comparison tests.

Figure 6 .
Figure 6.Changes in sugar contents in pulp during fruit development of pitaya.(A) Pitaya fruit (whole fruit and longitudinal sections) at seven developmental stages.(B) Contents of total sugar, glucose, fructose, and sucrose in pulps during fruit development.The data were shown as the means ± SDs of three independently biological replicates.The different letters above bars indicate significant differences at the p < 0.05 level according to Duncan's multiple comparison tests.

Figure 6 .
Figure 6.Changes in sugar contents in pulp during fruit development of pitaya.(A) Pitaya fruit (whole fruit and longitudinal sections) at seven developmental stages.(B) Contents of total sugar, glucose, fructose, and sucrose in pulps during fruit development.The data were shown as the means ± SDs of three independently biological replicates.The different letters above bars indicate significant differences at the p < 0.05 level according to Duncan's multiple comparison tests.

20 Figure 7 .
Figure 7. Expression patterns of HuSWEETs in pitaya pulps.(A) Expression analyses of 19 HuSWEET genes in pitaya pulps during fruit development.(B) RT-qPCR analyses of 19 HuSWEETs in mature fruits of pitaya.The data were shown as the means ± SDs of three independently biological replicates.The different letters above bars indicate significant differences at the p < 0.05 level according to Duncan's multiple comparison tests.

Figure 7 .
Figure 7. Expression patterns of HuSWEETs in pitaya pulps.(A) Expression analyses of 19 HuSWEET genes in pitaya pulps during fruit development.(B) RT-qPCR analyses of 19 HuSWEETs in mature fruits of pitaya.The data were shown as the means ± SDs of three independently biological replicates.The different letters above bars indicate significant differences at the p < 0.05 level according to Duncan's multiple comparison tests.

Figure 9 .
Figure 9. Analysis of sugars content and morphological phenotypes in HuSWEET12a/13d transgenic lines (OE) and the wild-type (WT) tomato plants.(A,B) Morphological phenotypes of HuSWEET12a-OE and HuSWEET13d-OE (Bar = 1 cm).(C,D) Transcript levels of HuSWEET12a/13d in OE and WT plants.(E) The content of total sugar in mature fruit of OE and WT plants.(F) The contents of sucrose, glucose, and fructose in mature fruit of OE and WT tomato plants.The data represent mean values ± SDs.The asterisks indicate p-values (* p < 0.05; ** p < 0.01, and *** p < 0.001) according to Student's t test.

Figure 9 .
Figure 9. Analysis of sugars content and morphological phenotypes in HuSWEET12a/13d transgenic lines (OE) and the wild-type (WT) tomato plants.(A,B) Morphological phenotypes of HuSWEET12a-OE and HuSWEET13d-OE (Bar = 1 cm).(C,D) Transcript levels of HuSWEET12a/13d in OE and WT plants.(E) The content of total sugar in mature fruit of OE and WT plants.(F) The contents of sucrose, glucose, and fructose in mature fruit of OE and WT tomato plants.The data represent mean values ± SDs.The asterisks indicate p-values (* p < 0.05; ** p < 0.01, and *** p < 0.001) according to Student's t test.

Table 1 .
Physiochemical characteristics of the HuSWEETs in pitaya.

Table 2 .
Correlation analyses of expression levels of HuSWEETs and sugar content during fruit development.