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Article

Identification and Expression Analysis of Putative Sugar Transporter Gene Family during Bulb Formation in Lilies

by
Ziyang Huang
1,
Cong Gao
2,
Yunchen Xu
2,
Jie Liu
1,
Jie Kang
1,
Ziming Ren
1,
Qi Cui
1,
Dongze Li
1,
Si Ma
3,
Yiping Xia
2,* and
Yun Wu
1,*
1
Laboratory of Flower Bulbs, Department of Landscape Architecture, Zhejiang Sci-Tech University, Hangzhou 310018, China
2
Genomics and Genetic Engineering Laboratory of Ornamental Plants, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
3
College of Horticulture, China Agricultural University, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(6), 3483; https://doi.org/10.3390/ijms25063483
Submission received: 5 February 2024 / Revised: 7 March 2024 / Accepted: 14 March 2024 / Published: 20 March 2024
(This article belongs to the Special Issue The Role of Sugars in Plant Responses to Stress and Their Regulatory Function during Development–3rd Edition)
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Versions Notes

Abstract

:
Sugar transporters play important roles in plant growth and development, flowering and fruiting, as well as responses to adverse abiotic and biotic environmental conditions. Lilies (Lilium spp.) are some of the most representative ornamental bulbous flowers. Sugar metabolism is critical for bulb formation in lilies; therefore, clarifying the amount and expression pattern of sugar transporters is essential for further analyzing their roles in bulb formation. In this study, based on the transcriptome data of the Lilium Oriental hybrid ‘Sorbonne’ and Lilium × formolongi, a total of 69 and 41 sugar transporters were identified in ‘Sorbonne’ and Lilium × formolongi, respectively, by performing bioinformatics analysis. Through phylogenetic analysis, monosaccharide transporters (MSTs) can be divided into seven subfamilies, sucrose transporters (SUTs) can be divided into three subgroups, and sugars will eventually be exported transporters (SWEETs) can be divided into four clades. According to an analysis of conserved motifs, 20, 14, and 12 conserved motifs were predicted in MSTs, SUTs, and SWEETs, respectively. A conserved domain analysis showed that MSTs and SUTs contained a single domain, whereas most of the SWEETs harbored two MtN3/saliva domains, also known as a PQ-loop repeat. The LohINT1, which was predicted to have a smaller number of transmembrane structural domains, was cloned and analyzed for subcellular localization. It was found that the LohINT1 protein is mainly localized in the cell membrane. In addition, the expression analysis indicated that 22 LohMSTs, 1 LohSUTs, and 5 LohSWEETs were upregulated in ‘Sorbonne’ 1 day after scale detachment treatment, suggesting that they may regulate the initiation of the bulblet. A total of 10 LflMSTs, 1 LflSUTs, and 6 LflSWEETs were upregulated 4~6 months after sowing, which corresponds to the juvenile-to-adult transition phase of Lilium × formolongi, suggesting that they may also play a role in the accompanying bulb swelling process. Combined with quantitative real-time PCR (qRT-PCR) analysis, LohSTP8 and LohSTP12 were significantly overexpressed during the extremely early stage of bulblet initiation, and LflERD6.3 was significantly overexpressed during the growth of the underground bulblet, suggesting that they may be key sugar transporters in the formation of lily bulbs, which needs further functional verification.

1. Introduction

Sugars are the main product of plant photosynthesis, which not only provide energy and carbon skeletons for processes such as plant growth and development and various stress responses but also play a signaling role [1,2,3]. In higher plants, sugars are mainly synthesized in source organs (such as mature leaves) and transported over long distances via the phloem to sink organs (such as plant roots, reproductive structures, storage, and development organs) that depend on nutrient supply. It is thus clear that the transportation and distribution of sugars are important for maintaining the metabolic balance between source and sink [4]. As the main form of long-distance transport of assimilates from source to sink, the loading, unloading, and distribution of sucrose are mainly dependent on the involvement of sugar transporters on the cell membrane [5,6].
The sugar transporters that have been identified in plants so far include three main classes: the monosaccharide transporters (MSTs), sucrose transporters (SUTs), and sugars will eventually be exported transporters (SWEETs) [7,8]. MSTs are Sugar_tr domain-containing members of the major facilitator superfamily (MFS) class of transporters. Structurally, MFS transporters usually contain 12 transmembrane domains (TMDs) [8,9,10]. A total of 53 MSTs were identified in the model plant Arabidopsis (Arabidopsis thaliana) and were further separated into seven subfamilies: sugar transport protein/hexose transporter (STP/HT); polyol/monosaccharide transporter (PLT/PMT); vacuolar glucose transporter (VGT); plastidic glucose transporter/suppressor of g protein beta1 (PGlcT/SGB1); tonoplastic monosaccharide transporter/tonoplast sugar transporter (TMT/TST); inositol transporter (INT); and early responsive to dehydration six-like (ERD6L). In addition, the ERD6L family includes two closely related homologous MST genes: SFP1 (sugar-porter family protein) and SFP2 [11,12]. These members play a variety of roles, including participating in the transport, uptake, utilization, and accumulation of monosaccharides as well as affecting sugar accumulation in plants via means such as pollen tube growth and fruiting [8,13,14]. MSTs have been identified in many species; for instance, 46 MSTs were identified in ‘Furongli’ (Prunus salicina) [15], 35 MSTs were identified in lotuses (Nelumbo nucifera) [16], 69 MSTs were identified in pears (Pyrus bretschneideri) [17], and 52 MSTs were identified in longans (Dimocarpus longan) [18]. SUTs, like MSTs, are Sugar_tr domain members of the MFS superfamily [10]. SUTs and MSTs share little homology at the amino acid level, although there are structural similarities. The structural difference between the two is usually the length of the central loop between transmembrane domains 6 and 7 [19]. According to their amino acid sequence homology and sucrose affinity, SUTs can be divided into five subgroups, among which SUT2 and SUT4 are common to monocotyledons and dicotyledons, SUT3 and SUT5 are unique to monocotyledons, and SUT1 is specific to dicotyledons [20]. SUTs play important roles in the plastid transport of sucrose and the unloading of sink organs and they have certain effects on the growth and development of plants such as flowers and fruits [21]. SUTs from a large number of plants have been identified since the discovery of SUTs in the first species; for instance, there are 9 in Arabidopsis [22], 5 in rice (Oryza sativa) [23], 5 in pears [17], 5 in petunias (Petunia hybrida) [24], 10 in pomegranates (Punica granatum) [25], and 22 in orchids (Orchidaceae) [26]. SWEETs, unlike MSTs and SUTs, are a newly discovered class of sugar transporters that can be bidirectional and do not rely on proton dynamic potential, characterized by containing two MtN3/saliva domains with seven transmembrane regions [7]. There are four clades of SWEETs exclusive to plants, numbered I through IV. In Arabidopsis, members of clade I (AtSWEET1~3) and clade II (AtSWEET4~8) transport hexose, members of clade III (AtSWEET9~15) mainly transport sucrose, whereas members of clade IV (AtSWEET16~17) are located in vesicle membranes and are mainly responsible for the transport of fructose. AtSWEET16 also transports glucose and sucrose [27,28,29]. SWEETs are involved in various physiological processes during plant growth and development, such as phloem unloading, hormone transport, pollen development, fruit development, and resistance to different stresses. Currently, SWEETs have been found in a variety of plants, including grapes (Vitis vinifera) [30], tomatoes (Solanum lycopersicum) [10], pears [31], and lychees (Litchi chinensis) [32].
Lilies (Lilium spp.) are a bulbous perennial plant belonging to Liliaceae, with high ornamental and economic value [33]. However, the low reproduction coefficient of bulbs and the long production cycle have become the bottleneck of bulb propagation, which seriously constrains the renewal, popularization, and application of new varieties of lilies [34]. Therefore, unraveling the mechanism of lily bulb formation and improving the coefficient and rate of bulb formation are crucial for improving the yield and quality of bulbous flowers. The Lilium Oriental hybrid ‘Sorbonne’ (abbreviated Loh) is one of the most important cut flower varieties in China, which has great market potential, but still faces the problem of bulb localization [35]. Lilium × formolongi (abbreviated Lfl), an interspecies hybrid of Lilium longiflorum and Lilium formosanum, completes the transition from its juvenile stage to adult stage in 4~6 months and is a typical short juvenile stage germplasm [36]. Juvenile-to-adult phase transition is related to bulb growth [37], which might be further utilized to improve bulb production. Our previous studies have shown that sucrose unloading mediated by cell wall invertases (CWINs) is crucial in the early stage of lily bulb formation [38]. Since the lily’s genome is large (~36 Gb) [39], no genome-wide information has been published, and the gene families of MSTs, SUTs, and SWEETs are not yet known. Therefore, in this study, based on the transcriptome data of lilies previously determined, we used bioinformatics to characterize the sugar transporter gene family of lilies; comprehensively analyzed their physicochemical properties, conserved motifs, and expression patterns; and screened out the potential key sugar transporter genes during bulb formation using qRT-PCR. These results will provide new information for us to verify the gene functions and the role of sugar transporter genes in lily bulb formation.

2. Results

2.1. Identification and Phylogenetic Analysis of the Sugar Transporters in Lilies

Based on the preliminary HMM search and Blastp comparison as well as the validation and de-redundancy of the sequences, a total of 49 LohMSTs, 5 LohSUTs, and 15 LohSWEETs were finally identified in the ‘Sorbonne’ transcriptome, and 27 LflMSTs, 2 LflSUTs, and 12 LflSWEETs in the Lilium × formolongi transcriptome. The identified lily sugar transporters were renamed according to the previous studies on Arabidopsis, and a phylogenetic tree was constructed together with the homologous sequences of sugar transporters from Arabidopsis and rice. The MST family can be divided into seven independent subfamilies. Among the 49 MSTs identified in ‘Sorbonne’, STP contains 16 members, ERD6L contains 9 members, and INT, pGlcT, PLT, TMT, and VGT contain 7, 6, 6, 3, and 2 members, respectively. Among the 27 MSTs identified in Lilium × formolongi, STP remained the subfamily with the most MSTs, which contains eight members, and the subsequent subfamilies in descending order of the number of members were ERD6L, INT, pGlcT, PLT, TMT, and VGT, with five, four, three, three, two, and two members, respectively. It is evident that STP and ERD6L constitute the two largest branches of MSTs of the lilies used (Figure 1). The amino acid lengths of a total of 76 MSTs in ‘Sorbonne’ and Lilium × formolongi ranged from 203 aa (LohERD6.3) to 753 aa (LflTMT1), the number of transmembrane regions ranged from 1 (LohTMT1) to 13 (LohSTP7), and the isoelectric points ranged from 4.80 (LohTMT1) to 10.19 (LflSTP2); the cell membrane is the preferred subcellular localization for all MSTs, in addition to which LohTMT1 may also localize to the nucleus (Table 1).
In agreement with previous reports, the SUTs of lilies, as monocotyledons, were distributed in the SUT2 and SUT4 subgroups common to monocotyledons and dicotyledons and the SUT3 subgroup specific to monocotyledons, whereas the SUT5 subgroup was not found to be distributed in our transcriptome, which may be related to the fact that they are not expressed during the biological process tested. Among the five SUTs identified in ‘Sorbonne’, LohSUT3 was located in the SUT2 subgroup, LohSUT1 in the SUT4 subgroup, and LohSUT2, LohSUT4, and LohSUT5 in the SUT3 subgroup. The two SUTs, LflSUT1 and LflSUT2, identified in Lilium × formolongi, on the other hand, were located in the SUT2 and SUT4 subgroups, respectively (Figure 2). The amino acid lengths of a total of seven SUTs from ‘Sorbonne’ and Lilium × formolongi ranged from 213 aa (LohSUT2) to 590 aa (LohSUT3), the number of transmembrane regions ranged from 5 to 12, and the isoelectric points ranged from 6.74 (LflSUT1) to 9.40 (LohSUT5), with subcellular localization all located at the cell membrane (Table 2).
Based on the phylogenetic analysis, SWEETs of ‘Sorbonne’ and Lilium × formolongi could be divided into four different clades. Clade I contained two LohSWEETs and three LflSWEETs. Clade II contained three LohSWEETs and three LflSWEETs each. Clade III clustered the highest number of SWEETs, containing eight LohSWEETs and five LflSWEETs, while clade IV contained only two LohSWEETs and one LflSWEET (Figure 3). Of the 27 SWEETs predicted for ‘Sorbonne’ and Lilium × formolongi, the amino acid lengths ranged from 77 aa (LflSWEET10) to 331 aa (LohSWEET6), the number of transmembrane regions ranged from one to seven, and the isoelectric points ranged from 5.29 (LohSWEET6) to 10.01 (LflSWEET8), with most of the subcellular localizations at the cell membrane and a few at the chloroplasts (LohSWEET11, LohSWEET15, LflSWEET4, LflSWEET5, LflSWEET10) and the peroxisomes (LflSWEET4) (Table 3).

2.2. Analysis of Conserved Motifs and Domains of Lily Sugar Transporters

After that, we analyzed the conserved motifs of lily sugar transporters through the MEME server. A total of 20 conserved motifs were predicted in the MSTs. Motif3 was present in almost all MSTs, indicating that it is important in lily MSTs. Motif9, motif15, and motif17 were only present in the STP subfamily. Motif14 was only present in the INT subfamily, suggesting that they may be necessary for STP and INT subfamilies, respectively. Conserved domain analysis showed that all structurally similar members clustered in the same subfamily (Figure 4). A total of 14 conserved motifs were identified for SUTs. Motif2 and motif10 were present in all SUTs, indicating that they are conserved domains of SUTs. Although MSTs and SUTs have the same transmembrane domains according to previous reports, the conserved motifs between them are quite different, suggesting that MSTs and SUTs are functionally distinct from each other (Figure 5). A total of 12 conserved motifs were identified in SWEETs. Motif1 was present in almost all SWEETs. Motif7, motif8, and motif10 were present only in clade III, and motif9 was unique to clade IV. In addition, the protein sequences of members of the lily SWEETs family are relatively conserved, with most SWEETs containing two MtN3/slv domains (CDD accession No. pfam03083) or the PQ-loop superfamily (CDD accession No. pfam03083) in similar positions, whereas the smaller portion of SWEETs (LohSWEET14, LohSWEET15, LflSWEET10, LflSWEET8, and LflSWEET5) had only one MtN3/slv domain or PQ-loop superfamily, which is possibly due to the fact that all of these sequences were derived from lily unigenes rather than full-length genes (Figure 6)

2.3. Cloning and Subcellular Localization Analysis of LohINT1 Gene

To verify the robustness of the transcriptome data, we selected the LohINT1 gene (6TMDs) with a small number of TMDs as an example of MSTs to carry out cloning and subcellular localization analysis. Firstly, a LohINT1-specific band was obtained under 1% agarose gel electrophoresis analysis (Figure S1A), and the sequencing result was highly consistent with the original sequence from the transcriptome. To examine the subcellular localization of the LohINT1 protein, the open reading frame (ORF) of the LohINT1 gene was fused to the N-terminal of the GFP reporter, driven by the CaMV35S promoter. Both the recombined (LohINT1-GFP) and unrecombined (free GFP) vectors were transferred into maize yellowing seedling protoplasts. The results showed that LohINT1-GFP subcellularly localized without a signal (Figure 7A). The experiments were carried out several times to exclude the technical issues and still no signal could be observed. We, therefore, proposed that the ORF might be incomplete as the predicted protein is only 285 aa in length while its orthologous gene in Arabidopsis thaliana is 582 aa in length. We then carefully checked the original sequence in the NCBI database by the basic local alignment search tool (BLAST) and found a base deletion (T) probably resulting the early termination of the protein.
Afterwards, by designing the primer according to the new coding sequence, we amplified LohINT1 using the previously obtained ‘Sorbonne’ complementary DNA (cDNA) as a template (Figure S1B). The sequencing results revealed that the total length of the LohINT1 gene is 1743 bp, encoding 580 amino acids. The recombinant LohINT1-YFP was constructed with mCherry-labeled cell membranes as a marker. The results showed that LohINT1-YFP was mainly localized in the cell membrane, with a small distribution in other endomembrane systems (Figure 7B). Based on the above-mentioned observations, we speculated that the low number of TMDs contained in some sugar transporters may also be due to incomplete transcriptome data.

2.4. Expression Patterns of Sugar Transporter Genes at Different Stages of Lily Bulb Initiation and Development

Based on the expression of transcriptome data, we mapped the expression patterns of different sugar transporter genes during bulb initiation and development and clustered them logarithmically. The expression of sugar transporter genes at the stage of bulblet initiation was observed by aeroponic ‘Sorbonne’ scales. The outer scale from the mother bulblet was applied detachment treatment and the previous studies in the laboratory divided the process of lily bulb formation into four key stages: the stage of scale detachment (0 days after treatment (DAT)), the stage of wound response and early regeneration competence (1 DAT), the stage of adventitious bud initiation (8 DAT), and the stage of adventitious bud swelling and bulblet formation (14 DAT). Among them, 1 DAT is the early stage of ontogeny and is critical for bulblet initiation, so this stage was chosen to explore the situation of sugar transporter-related genes. The results showed that 22 LohMSTs, 1 LohSUTs, and 5 LohSWEETs were upregulated about 1.5-fold at 1 DAT (Figure 8). Similarly, based on previous studies, the bulb swelling and development process could be divided into three main stages: the juvenile stage (2~4 months (M)), the transition stage (4~6 M), and the adult stage (6~24 M). We focused on the expression of sugar transporter genes during bulb swelling accompanied by the simultaneous transition stage of lilies by sampling the shoot apical meristem (SAM) of Lilium × formolongi at 4 M, 6 M, and 24 M after sowing. Sugar transporter genes upregulated at 4~6 M and downregulated at 6~24 M were highlighted, which contained 10 LflMSTs, 1 LflSUTs, and 6 LflSWEETs; they were upregulated nearly 2-fold around 4~6 M (Figure 9).

2.5. Validation of Lily Sugar Transporter Genes Expression by qRT-PCR

The expression levels of some genes associated with the initial and developmental process of lily bulbs were chosen and analyzed by qRT-PCR, and the quantitative analysis results were compared with the expression trends of FPKM (fragments per kb per million) values. In ‘Sorbonne’ we verified all 28 genes (Figure 10), and the expression of two sugar transporter genes, LohSTP8 and LohSTP12, were upregulated and expressed at least 3-fold at 1 DAT, which was significantly higher than at the other stages, and this was consistent with the results by the FPKM expression pattern. The qRT-PCR analysis of other genes showed that the expression levels of LohERD6s, LohTMTs, LohpGlcT4, LohSTP10, and LohSWEET6 decreased gradually from 0 DAT. The expression levels of LohVGT2, LohSTP16, and LohSWEET10 were significantly lower at 1 DAT. The expression levels of LohSWEET3, LohSWEET4, and LohSWEET5 were significantly increased at 8 DAT. The expression levels of LohSTP15 and LohSUT4 were significantly increased at 14 DAT. There were no significant differences in the expression levels of LohINT3, LohpGlcT1, LohPLT6, LohSTP7, and LohSTP9 between 0 DAT and 1 DAT, while LohPLT1, LohSTP1, LohSTP2, and LohSTP4 were not significantly different at any of these four stages. Some genes were selected for validation in Lilium × formolongi (Figure 11). The qRT-PCR results showed that one sugar transporter gene, LflERD6.3, was significantly overexpressed at 4~6 M and upregulated about 5.5-fold, which was also in line with the results of the FPKM expression pattern. In addition, LflINT2, LflERD6.5, and LflSTP1 showed no significant change at 4~6 M and then decreased significantly at 6~24 M. On the contrary, LflSTP8 and LflSWEET11 were significantly elevated at 4~6 M and had no significant change at 6~24 M. LflINT3, LflSTP12, and LflSWEET3 had no significant differences.

3. Discussion

The initiation and development of bulbs are crucial to the growth cycle of the lily, which will further affect the yield and quality of the lily [34]. As an important storage organ, the bulb of a lily mainly accumulates substances through starch synthesis, by which the decomposition and transport of sucrose provide important precursor substances for the synthesis of starch [40]. Previous studies have shown that sucrose is the main component of phloem transport of lilies [41]; so, sucrose metabolism, especially sucrose unloading, plays an important role in carbon allocation during the initiation and development of lily bulbs. The main ways through which sucrose enters the sink cells are the symplastic pathway and the apoplastic pathway. Among them, the sucrose in the apoplastic pathway is transported by SWEETs located in the cell membrane, and then unloaded directly to the cytoplasm via SUTs, or hydrolyzed to glucose and fructose by CWINs and then transported to the cytoplasm via MSTs. It can be seen that sugar transporters are key substances in the apoplastic unloading pathway [40,42,43].
Previous studies of sugar transporter genes in the allocation of assimilates of Lilium Oriental hybrid ‘Sorbonne’ have been carried out to observe the assimilates’ allocation by determining the carbohydrate contents in different tissues of five critical stages during lily development, including the bulb setting stage, the plant height of 30 cm with leaf-spread stage, the budding stage, the flowering stage, and the final flowering stage. Finally, three sugar transporter genes that play key roles in the accumulation and transportation of assimilates in lilies were further identified among 16 sugar transporter genes related to sugar transport and metabolism [44]. Additionally, the importance of carbohydrates during flowering has also been explored through the study of ‘Sorbonne’, which mainly focused on the effect of SUT genes [45]. Unlike the previous biological processes from bulb sowing to flowering and vernalization to flower bud vernalization, the biological process we focused on is the initial and developmental stages of lily bulblets. Our systematic study will provide a basis for the functional validation and further investigation of the mechanism of action by mining the candidate genes related to the bulb formation process.
In this study, 69 sugar transporters were identified in the ‘Sorbonne’ transcriptome, including 49 MSTs, 5 SUTs, and 15 SWEETs. Likewise, 41 sugar transporters, including 27 MSTs, 2 SUTs, and 12 SWEETs, were identified in the Lilium × formolongi transcriptome. According to the identification of different sugar transporter families, MSTs tended to contain the largest number of gene family members, while SUTs belonged to a relatively small gene family, which may be related to the fact that MSTs contained more subfamilies and could transport more types of sugars. Similar results have been observed in other plants. For example, 69 MSTs and 6 SUTs were found in pears [17], 64 MSTs and 9 SUTs were found in apples (Malus domestica) [46], and 46 MSTs and 6 SUTs were found in longans [18]. The number of SWEETs was usually intermediate between MSTs and SUTs. Phylogenetic analysis showed that both LohMSTs and LflMSTs could be divided into seven independent subfamilies, and STP and ERD6L constituted the two largest branches of the MSTs (Figure 1), which is consistent with previous reports on other plants, such as strawberries (Fragaria × ananassa) [47], longans [18], lotuses [16], jujubes (Ziziphus jujuba) [48], etc. Most of the LohSUTs and LflSUTs are highly homologous with rice and were distributed on SUT2, SUT3, and SUT4, where LflSUTs were not found in SUT3. LohSWEETs and LflSWEETs were divided into four clades (clade I to clade IV), which is consistent with Arabidopsis [7], rice [49], grapes [50], lychees [32], daylilies (Hemerocallis citrina) [51], etc. In addition, SWEETs aggregated in clade III are the most numerous, similar to the results for lychees [32], bananas (Musa acuminata) [52], and alfalfa (Medicago truncatula) [53]. It is worth noting that a batch of sugar transporter genes (18) has already been reported in ‘Sorbonne’ during the process of vernalization and flowering [44,45], and our results would be a fine supplement to this.
Conserved motif analysis showed that different sugar transporter families all contained some essential conserved motifs, which is consistent with the results for pears [17], longans [18], and peppers (Capsicum annuum) [54], suggesting that they are of special significance for different sugar transporters. These results also imply that they have different functions in different clades of different sugar transporters in lilies. At the same time, some studies have found that those with similar motifs in general not only belong to a subfamily but also may be correlated in their biological functions [55]. As for the situation that some motifs were not included in some sequences despite the significant level of conservation, we speculate that there may be two reasons; the first one may be that some motifs were missing due to incomplete transcriptome data, and such sequences were retained because they still have conserved domains that can validate them as sugar transporters despite the incomplete motifs included in the sequences. We also confirmed this speculation by subcellular localization analysis. The second possibility is that many sugar transporters have undergone TMD deletion events at the N-terminal and C-terminal during evolution. In terms of conserved domains, MFS transporters in plants possess a common structure of 12 transmembrane domains (TMD1–TMD12), which are separately contained within the N-terminal (TMD1–TMD6) and the C-terminal (TMD7–TMD12), and each of these domains contains five to seven transmembrane-spanning α helices, with six being the most common number [56,57,58]. In the current study, 10 LohMSTs, 8 LflMSTs, and 3 LohSUT, 1 LflSUT had fewer than 10 TMDs. Consistent with this, similar protein structures are also observed in tomatoes [10] and grapes [30]. A related speculation is that the loss of the N-terminal or C-terminal regions may have occurred in some MSTs and SUTs during evolution. Taking LohINT1, an MST gene containing fewer TMDs, as an example for clonal sequencing and subcellular localization, its sequencing results were highly consistent with the transcripts, but there was no signal for subcellular localization, and further amplification of the full length of LohINT1 sequence showed that its subcellular localization was mainly located in the cell membrane, which was in agreement with the previous prediction in Table 1. This verified that the transcriptome data were not completely reliable. It suggests that the low number of TMDs in some MSTs and SUTs may be due to incomplete transcriptome data (Table S4). However, a low number of TMDs still possibly exists as reported in [53,59]. It is impossible to check all the incomplete sequences experimentally in the current study. We recommend further BLAST to check the base deletion/insertion or the rapid amplification of cDNA ends method could be considered to obtain the full-length sequence when verifying the upstream biological function for those genes marked as incomplete in Table S4; the original sequence has been shown in Table S5. The same situation may also exist in SWEETs, making the number of TMDs in some SWEETs fewer than seven. Notably, 13 TMDs were predicted in LohSTP7, which may arise from the duplication of adjacent genes [9]. Comparing different sugar transporters, although MSTs and SUTs have relatively similar domains, the conserved motifs of the two are quite different, indicating that MSTs and SUTs play different functions in the process of sugar transport. Furthermore, among MSTs, different subfamilies have similar single domains, indicating that members of the same subfamily have the same function. In contrast, the domains contained in SWEETs are relatively conservative, with most of them containing two MtN3/slv domains (also known as a PQ-loop repeat), which also implies that SWEETs have functional diversity during plant growth and development.
Sugar transporter genes constitute a versatile gene family, and they play critical roles in many biological processes during plant growth and responses to environmental stimuli [7,12]. LoSWEET14 is a sugar transporter that has been identified in recent years as potentially involved in the abscisic acid (ABA) signaling pathway to regulate sugar accumulation under abiotic stresses in lilies [60]. In our study, the counterpart to LoSWEET14 is LohSWEET4, which has >90% sequence similarity and was significantly expressed during bulblet initiation 8 days after aeroponics, which suggests LohSWEET4 might be crucial for adventitious bud initiation and needs further verification. By contrast, we are most interested in genes that play important roles in the formation of lily bulbs. In ‘Sorbonne’, 28 sugar transporter genes (including 22 LohMSTs, 5 LohSUTs, and 5 LohSWEETs) were upregulated at the stage of wound response and early regeneration competence, indicating that starch accumulation and sucrose metabolism are active in this bulblet initiation stage, and these genes may also be relevant to bulb formation. STPs are responsible for the transport of monosaccharides and proton cotransport into the cell and also play an important role in sugar transport in Arabidopsis and rice [14,61]. Apple MdSTP13a regulates apple pollen tube growth by taking up both hexose and sucrose [62]. Lupinus polyphyllus LpSTP1 can transport a variety of hexose substrates [63]. In this study, 10 of the 22 LohMSTs upregulated at the stage of wound response and early regeneration competence belonged to STPs. After a subsequent qRT-PCR verification, LohSTP8 and LohSTP12 were significantly overexpressed during the initiation of small bulblets. Phylogenetic relationships revealed that LohSTP8 and LohSTP12 are most closely related to AtSTP1, which is mainly expressed in germinating seeds, young seedlings, and guard cells and mainly mediates the transport of monosaccharides such as hexose and glucose in addition to fructose [64]. Therefore, it can be speculated that ‘Sorbonne’ LohSTP8 and LohSTP12 may be involved in the transport of monosaccharides after unloading the hydrolysis of sucrose in the apoplastic pathway. Seventeen sugar transporter genes (including LflMSTs10, LflSUTs1, and LflSWEETs6) were upregulated at the transition stage in Lilium × formolongi, indicating that there possibly may be a transition in this stage where sucrose was unloaded from the symplast pathway to the apoplast pathway, and these genes may also be involved in the growth of the underground bulblet. ERD6L is one of the least studied subfamilies with very few members characterized. It has been shown to be involved in keeping a balance of glucose between the inside and outside of the vacuole [65]. In this study, 3 of the 10 LflMSTs upregulated at the transition stage belonging to ERD6L. LflERD6.3 was significantly overexpressed during the bulb swelling that accompanies the transition of lilies from juvenile to adult after verification by qRT-PCR, suggesting that this gene may be involved in bulb carbohydrate accumulation. As a result, LohSTP8, LohSTP12, and LflERD6.3 were selected as key sugar transporter genes during bulb formation in lilies for subsequent functional verification.

4. Materials and Methods

4.1. Plant Material and Culture Conditions

Lilium Oriental hybrids of ‘Sorbonne’ and Lilium × formolongi were selected as materials to investigate the process of lily bulb initiation and development by using aeroponics and potted planting, respectively. The bulbs of ‘Sorbonne’ were cleaned and sterilized by soaking in carbendazim (1:1000) for 30 min before aerial cultivation, and the outer 1~2 layers of full scales were peeled off from the basal plate and laid on 3~4 layers of moist sterilized gauze near the axial surface, and then they were placed on a plastic tray within a plant growth chamber in Zhejiang University (118°21′–120°30″, 29°11′–30°33″) with temperatures at 24 °C, humidity at 90–95%, and light/darkness = 12/12 h. Plump and uniform seeds of Lilium × formolongi were sown aseptically and then propagated in seed media (4.43 g/L MS, 1.0 mg/L 6-BA, 0.2 mg/L NAA, 30 g/L Sucrose, 3 g/L Phytagel, PH 5.8) at (24 ± 2) °C. After 15 days of dark culture, germinated seeds with strong growth consistency were selected and transferred to 50-well cavity trays with temperatures of 24 °C. After about two months of growth, they were transferred to planting pots with temperatures of 24 °C, 16/8 h of light/darkness, and a light intensity of 80 μmoL m−2s−1 PPFD. The seedling medium of the basin plate was peat: perlite: vermiculite = 2:1:1 (v/v/v). The samples were selected from the critical periods of bulbs initiation and development, sampling points included 0 DAT, 1 DAT, 8 DAT, and 14 DAT under the condition of ‘Sorbonne’ aerial cultivation and 4 M, 6 M, 24 M after the sowing of Lilium × formolongi. All samples were rapidly frozen with liquid nitrogen and stored at −80 °C for subsequent analysis, and three biological replicates were carried out for each treatment to ensure that the data were accurate and reliable.

4.2. Identification of Sugar Transporters

The transcriptome data of ‘Sorbonne’ and Lilium × formolongi were obtained by the previous sequencing of our research group, and the library for ‘Sorbonne’ is a merged one including PacBio full-length sequencing (Table S6). To identify lily sugar transporters, the HMMER profiles of Sugar_tr domain (PF00083), MFS-1 (PF07690), MFS-2 (PF13347), and MtN3_slv (PF03083) were first downloaded from Pfam (http://pfam.xfam.org/, accessed on 9 April 2023) [66]. The hmmsearch command in TBtools version 2.0 was used to detect the ‘Sorbonne’ and Lilium × formolongi transcriptome databases [67]. The search results were manually checked to remove the redundancy initially, where E-value < 1 × 10−3. The Arabidopsis sugar transporters sequences were downloaded from the TAIR website (https://www.arabidopsis.org/, accessed on 9 April 2023) and used as query sequences to perform Blastp sequence comparison to find the best matching sequences and remove redundancy initially.
The above candidate sequences were validated by online analysis using the NCBI conserved domain database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 9 April 2023) [68] and selected Uniprot for reverse Blastp in the NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 9 April 2023) to comprehensively identify whether they have the family’s characteristic domains. Sequences that do not contain conserved domains were removed; similar sequences were clustered to remove redundancies using the CD-HIT web server (https://www.bioinformatics.org/cd-hit/, accessed on 10 April 2023) with the parameters set as identity 0.9, threads 10, and word_length 5 [69]; and finally, the sequences of lily sugar transporters family members were obtained.

4.3. Phylogenetic Analysis

A multiple sequence comparison of the identified lily sugar transporters with those of Arabidopsis and rice was performed using the MUSCLE Wrapper tool in the TBtools version 2.0 (Table S1). Phylogenetic trees were constructed using the maximum likelihood (MJ) method in FastTree version 10.0.19045.4170 [70], and the constructed evolutionary trees were collapsed and formatted with using the online tool iTOL version 6 (https://itol.embl.de/, accessed on 12 April 2023) [71].

4.4. Physicochemical Property Analysis and Prediction of Subcellular Localization

Properties such as isoelectric point (pI), protein molecular weight (MW), and other attributes of the obtained protein sequences were predicted using the online analysis tool EXPASY ProtParam (https://web.expasy.org/protparam/, accessed on 15 April 2023) [72]. Signal peptide prediction was accomplished using SignalP-4.1 (https://services.healthtech.dtu.dk/services/SignalP-4.1/, accessed on 15 April 2023) [73]. The number of transmembrane regions was predicted using TMHMM2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/, accessed on 15 April 2023) [74]. The proteins encoded by candidate genes were analyzed for subcellular localization prediction using the online Plant mPLoc website (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 15 April 2023) [75]. Protein secondary structure was predicted using SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html, accessed on 15 April 2023) [76].

4.5. Conserved Domain Analysis and Motif Distribution Analysis

Motif prediction of proteins was performed using MEME Suite version 5.5.3 (https://meme-suite.org/meme/, accessed on 17 April 2023) [77], and the conserved regions of the key conserved domains were preserved. All parameter settings were set as default, except the maximum number of predicted motifs which was set to 20, and the motif distribution was plotted in combination with TBtools version 2.0.

4.6. Cloning and Subcellular Localization Analysis of LohINT1 Gene

Taking MSTs as an example, based on the number of TMDs of the 76 MSTs identified, LohINT1 (6TMDs) was selected from the proteins with the number of TMDs fewer than 10 for clone sequencing and subcellular localization analysis. The transcript DNA (cDNA) of the LohINT1 gene was extracted from the ‘Sorbonne’ transcriptome database. cDNA cloning primers (Table S3) were designed by the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, accessed on 7 May 2023) according to the transcript sequence data of ‘Sorbonne’, and synthesized by Sangon Biotech (Sangon, Shanghai, China). The LohINT1 gene was amplified from the cDNA using PrimeSTAR®Max DNA polymerase (R045, TaKaRa, Dalian, China). Amplified PCR products were electrophoresed in 1% agarose gel and then purified with MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (9762, TaKaRa, Dalian, China). Sequencing of the PCR products was performed by Sangon Biotech (Sangon, Shanghai, China).
The transcript and the full-length CDS region of LohINT1 were amplified with primers LohINT1-GFP-F/R and LohINT1-YFP-F/R, respectively (Table S3). The amplified fragments were inserted into the Kpnl-linearized p221-GFP vacuole and EcoRI/Spel-linearized pUC-35S-YFP vacuole, respectively, by the seamless cloning method. Plasmids were transfected with maize yellowing seedling protoplasts prepared using the PEG-mediated method and observed with a confocal microscope (TCS SP8, Leica, Wetzlar, Germany) after overnight incubation. The cell membranes were labeled with an mCherry marker. Excitation/emission wavelengths for GFP, YFP, and mCherry were 488/(510–550) nm, 514/(525–575) nm, and 587/(607–650) nm, respectively.

4.7. Expression Analysis of Lily Sugar Transporter Gene

Expression in the transcriptome database was expressed as log2 transformed values of FPKM, and the expression value of each stage was the average of three biological replicates (Table S2). The data were normalized by Minitab version 20.3 and then the expression heat map was plotted using TBtools version 2.0 [78], which was used to identify the expression patterns of different sugar transporters during the initiation and development stage of bulblets.

4.8. Quantitative Real-Time PCR (qRT-PCR) Analysis

The total RNA of ‘Sorbonne’ and Lilium × formolongi were extracted from scale samples using an EASYspin Plus Complex RNA Kit (RN53 and RN40, Aidlab Bio, Beijing, China). RNA concentration was measured by NanoDrop2000 (Thermo Scientific, Waltham, MA, USA), and RNA quality was verified by 1% agarose gel electrophoresis using PowerPacTM Basic (Bio-Rad, Hercules, CA, USA). Total RNA (1 μg and 1.6 μg) of each sample of ‘Sorbonne’ and Lilium × formolongi were reverse transcribed by the PrimeScriptTM RT reagent Kit with gDNA Eraser (RR047A, TaKaRa, Dalian, China) and PrimeScriptTM II 1st Strand cDNA Synthesis Kit with DNase I (6210A, TaKaRa, Dalian, China), respectively. Gene-specific primers were designed (Table S3) according to the previously described method (Section 4.6). Then, qRT-PCR was performed in a Bio-Rad ConnectTM optical module (Bio-Rad, Hercules, CA, USA) using the TB GreenTM Premix Ex TaqTM kit (RR420A, TaKaRa, Dalian, China). All reactions were performed in three replicates in a 10 μL system at 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The relative expression was calculated by the 2−ΔΔCt method using GAPDH in the transcriptome of ‘Sorbonne’ and Unigene0053935_UBC22 in the transcriptome of Lilium × formolongi as the internal reference genes [79], and the correlation between transcriptome expression patterns and fluorescence quantitative results was compared separately.

4.9. Statistical Analysis

Values for three biological replicates were calculated as mean ± SEM. Differences between the groups were analyzed by one-way ANOVA with Duncan tests by SPSS Statistics version 17.0, and p-values less than 0.05 were considered statistically significant. Results were visualized by GraphPad Prism version 8.3.0.

5. Conclusions

In this study, we identified members of the sugar transporter family in the Lilium Oriental hybrid ‘Sorbonne’ and Lilium × formolongi. A total of 69 LohSTs and 41 LflSTs were found in the transcriptomes of ‘Sorbonne’ and Lilium × formolongi, respectively. Phylogenetic analyses showed that the MSTs could be categorized into seven subfamilies, SUTs into three subgroups, and SWEETs into four clades. According to the conserved motif analysis, different families of sugar transporters contain some essential or special conserved motifs, indicating that there are some functional differences among members of different families of sugar transporters. Conserved domain analysis showed that most SWEETs had two MtN3/saliva domains (also known as a PQ-loop repeat), which was significantly different from the single domain contained in MSTs and SUTs. Further expression analysis showed that 28 LohSTs were upregulated and expressed during the process of bulblet initiation, and 17 LflSTs were upregulated and expressed during the bulb swelling process that accompanies the transition of lilies from juvenile to adult. Finally, verified by qRT-PCR, we screened LohSTP8, LohSTP12, and LflERD6.3 as key sugar transporter genes during lily bulb formation. Our study laid the foundation for elucidating the biological functions of sugar transporter genes in lily bulb formation.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms25063483/s1. Reference [80] is cited in the supplementary materials.

Author Contributions

Z.H. and Y.W. conceived the project and designed the experiments; Z.H., C.G., Y.X. (Yunchen Xu), J.L. and J.K. collected the samples and completed the experiments together; Z.R., Q.C., D.L. and S.M. helped to complete the experiments; Z.H. and C.G. conducted the data analysis; Z.H. drafted the manuscript; Y.W. and Y.X. (Yiping Xia) revised the manuscript. 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. 32372743), the Young Scientists Fund (Grant No. 32002071), the Zhejiang Sci-Tech University Research Program Start-up Funding (Grant No. 21052103-Y), the Zhejiang Province First-Class Discipline (Civil Engineering) Construction Project (Grant No. 11141131282001), and the 2022 Hangzhou Agricultural and Social Development Scientific Research Guidance Project (Grant No. 20220919Y1).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Lastdrager, J.; Hanson, J.; Smeekens, S. Sugar signals and the control of plant growth and development. J. Exp. Bot. 2014, 65, 799–807. [Google Scholar] [CrossRef] [PubMed]
  2. O’Hara, L.E.; Paul, M.J.; Wingler, A. How do sugars regulate plant growth and development? New insight into the role of trehalose-6-phosphate. Mol. Plant 2013, 6, 261–274. [Google Scholar] [CrossRef] [PubMed]
  3. Rolland, F.; Baena-Gonzalez, E.; Sheen, J. Sugar sensing and signaling in plants: Conserved and novel mechanisms. Annu. Rev. Plant Biol. 2006, 57, 675–709. [Google Scholar] [CrossRef] [PubMed]
  4. Ludewig, F.; Fluegge, U.I. Role of metabolite transporters in source-sink carbon allocation. Front. Plant Sci. 2013, 4, 231. [Google Scholar] [CrossRef]
  5. Lemoine, R.; La Camera, S.; Atanassova, R.; Dedaldechamp, F.; Allario, T.; Pourtau, N.; Bonnemain, J.L.; Laloi, M.; Coutos-Thevenot, P.; Maurousset, L.; et al. Source-to-sink transport of sugar and regulation by environmental factors. Front. Plant Sci. 2013, 4, 272. [Google Scholar] [CrossRef]
  6. Reinders, A.; Sivitz, A.B.; Ward, J.M. Evolution of plant sucrose uptake transporters. Front. Plant Sci. 2012, 3, 20960. [Google Scholar] [CrossRef] [PubMed]
  7. Chen, L.Q.; Hou, B.H.; Lalonde, S.; Takanaga, H.; Hartung, M.L.; Qu, X.Q.; Guo, W.J.; Kim, J.G.; Underwood, W.; Chaudhuri, B.; et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 2010, 468, 527–532. [Google Scholar] [CrossRef]
  8. Doidy, J.; Grace, E.; Kuhn, C.; Simon-Plas, F.; Casieri, L.; Wipf, D. Sugar transporters in plants and in their interactions with fungi. Trends Plant Sci. 2012, 17, 413–422. [Google Scholar] [CrossRef]
  9. Reddy, V.S.; Shlykov, M.A.; Castillo, R.; Sun, E.I.; Saier, M.H. The major facilitator superfamily (MFS) revisited. FEBS J. 2012, 279, 2022–2035. [Google Scholar] [CrossRef]
  10. Reuscher, S.; Akiyama, M.; Yasuda, T.; Makino, H.; Aoki, K.; Shibata, D.; Shiratake, K. The sugar transporter inventory of tomato: Genome-wide identification and expression analysis. Plant Cell Physiol. 2014, 55, 1123–1141. [Google Scholar] [CrossRef]
  11. Buettner, M. The monosaccharide transporter(-like) gene family in Arabidopsis. FEBS Lett. 2007, 581, 2318–2324. [Google Scholar] [CrossRef] [PubMed]
  12. Wen, S.; Neuhaus, H.E.; Cheng, J.; Bie, Z. Contributions of sugar transporters to crop yield and fruit quality. J. Exp. Bot. 2022, 73, 2275–2289. [Google Scholar] [CrossRef]
  13. Wormit, A.; Trentmann, O.; Feifer, I.; Lohr, C.; Tjaden, J.; Meyer, S.; Schmidt, U.; Martinoia, E.; Neuhaus, H.E. Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell 2006, 18, 3476–3490. [Google Scholar] [CrossRef] [PubMed]
  14. Buttner, M. The Arabidopsis sugar transporter (AtSTP) family: An update. Plant Biol. 2010, 12, 35–41. [Google Scholar] [CrossRef]
  15. Jiang, C.C.; Fang, Z.Z.; Zhou, D.R.; Lin, Y.J.; Ye, F.X. Identification and expression analysis of sugar transporter family genes in ‘Furongli’ (Prunus salicina) based on genome. Acta Hortic. Sin. 2022, 49, 252–264. [Google Scholar]
  16. Wu, P.; Zhang, Y.Y.; Zhao, S.P.; Li, L.J. Comprehensive analysis of evolutionary characterization and expression for monosaccharide transporter family genes in Nelumbo nucifera. Front. Ecol. Evol. 2021, 9, 537398. [Google Scholar] [CrossRef]
  17. Li, J.M.; Zheng, D.M.; Li, L.T.; Qiao, X.; Wei, S.W.; Bai, B.; Zhang, S.L.; Wu, J. Genome-wide function, evolutionary characterization and expression analysis of sugar transporter family genes in pear (Pyrus bretschneideri Rehd). Plant Cell Physiol. 2015, 56, 1721–1737. [Google Scholar] [CrossRef]
  18. Fang, T.; Peng, Y.; Rao, Y.; Li, S.H.; Zeng, L.H. Genome-wide identification and expression analysis of sugar transporter (ST) gene family in longan (Dimocarpus longan L.). Plants 2020, 9, 342. [Google Scholar] [CrossRef]
  19. Williams, L.E.; Lemoine, R.; Sauer, N. Sugar transporters in higher plants-a diversity of roles and complex regulation. Trends Plant Sci. 2000, 5, 283–290. [Google Scholar] [CrossRef]
  20. Kuehn, C.; Grof, C.P.L. Sucrose transporters of higher plants. Curr. Opin. Plant Biol. 2010, 13, 287–298. [Google Scholar] [CrossRef]
  21. Liesche, J. Sucrose transporters and plasmodesmal regulation in passive phloem loading. J. Integr. Plant Biol. 2017, 59, 311–321. [Google Scholar] [CrossRef]
  22. Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 2000, 408, 796–815. [Google Scholar] [CrossRef]
  23. Aoki, N.; Hirose, T.; Scofield, G.N.; Whitfeld, P.R.; Furbank, R.T. The sucrose transporter gene family in rice. Plant Cell Physiol. 2003, 44, 223–232. [Google Scholar] [CrossRef]
  24. Iftikhar, J.; Lyu, M.; Liu, Z.; Mehmood, N.; Munir, N.; Ahmed, M.A.A.; Batool, W.; Aslam, M.M.; Yuan, Y.; Wu, B. Sugar and hormone dynamics and the expression profiles of SUT/SUC and SWEET sugar transporters during flower development in Petunia axillaris. Plants 2020, 9, 1770. [Google Scholar] [CrossRef]
  25. Luo, X.; Cao, S.Y.; Li, H.X.; Zhao, D.G.; Zhang, F.H.; Chen, L.N.; Krishna, P.; Jing, D.; Tang, L.Y. Identification of SUT genes and their expression models during the development of seed in pomegranate. J. Fruit Sci. 2020, 37, 485–494. [Google Scholar]
  26. Wang, Y.Z.; Chen, Y.; Wei, Q.Z.; Wan, H.J.; Sun, C.B. Phylogenetic relationships of sucrose transporters (SUTs) in plants and genome-wide characterization of SUT genes in Orchidaceae reveal roles in floral organ development. PeerJ 2021, 9, e11961. [Google Scholar] [CrossRef] [PubMed]
  27. Chardon, F.; Bedu, M.; Calenge, F.; Klemens, P.A.W.; Spinner, L.; Clement, G.; Chietera, G.; Leran, S.; Ferrand, M.; Lacombe, B.; et al. Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis. Curr. Biol. 2013, 23, 697–702. [Google Scholar] [CrossRef] [PubMed]
  28. Eom, J.S.; Chen, L.Q.; Sosso, D.; Julius, B.T.; Lin, I.W.; Qu, X.Q.; Braun, D.M.; Frommer, W.B. SWEETs, transporters for intracellular and intercellular sugar translocation. Curr. Opin. Plant Biol. 2015, 25, 53–62. [Google Scholar] [CrossRef] [PubMed]
  29. Klemens, P.A.W.; Patzke, K.; Deitmer, J.; Spinner, L.; Le Hir, R.; Bellini, C.; Bedu, M.; Chardon, F.; Krapp, A.; Neuhaus, H.E. Overexpression of the vacuolar sugar carrier AtSWEET16 modifies germination, growth, and stress tolerance in Arabidopsis. Plant Physiol. 2013, 163, 1338–1352. [Google Scholar] [CrossRef] [PubMed]
  30. Afoufa-Bastien, D.; Medici, A.; Jeauffre, J.; Coutos-Thevenot, P.; Lemoine, R.; Atanassova, R.; Laloi, M. The Vitis vinifera sugar transporter gene family: Phylogenetic overview and macroarray expression profiling. BMC Plant Biol. 2010, 10, 245. [Google Scholar] [CrossRef] [PubMed]
  31. Li, J.M.; Qin, M.F.; Qiao, X.; Cheng, Y.S.; Li, X.L.; Zhang, H.P.; Wu, J. A new insight into the evolution and functional divergence of SWEET transporters in chinese white pear (Pyrus bretschneideri). Plant Cell Physiol. 2017, 58, 839–850. [Google Scholar] [CrossRef]
  32. Xie, H.; Wang, D.; Qin, Y.; Ma, A.; Fu, J.; Qin, Y.; Hu, G.; Zhao, J. Genome-wide identification and expression analysis of SWEET gene family in Litchi chinensis reveal the involvement of LcSWEET2a/3b in early seed development. BMC Plant Biol. 2019, 19, 499. [Google Scholar] [CrossRef]
  33. Feng, J.; Wu, Z.; Wang, X.; Zhang, Y.; Teng, N. Analysis of pollen allergens in lily by transcriptome and proteome data. Int. J. Mol. Sci. 2019, 20, 5892. [Google Scholar] [CrossRef]
  34. Wu, Y.; Li, Y.; Ma, Y.D.; Zhang, L.; Ren, Z.M.; Xia, Y.P. Hormone and antioxidant responses of Lilium Oriental hybrids ‘Sorbonne’ bulblets to humic acid treatments in vitro. J. Hortic. Sci. Biotechnol. 2017, 92, 155–167. [Google Scholar] [CrossRef]
  35. Kumar, S.; Awasthi, V.; Kanwar, J.K. Influence of growth regulators and nitrogenous compounds on in vitro bulblet formation and growth in oriental lily. Hortic. Sci. 2007, 34, 77–83. [Google Scholar] [CrossRef]
  36. Li, Y.F.; Zhang, M.F.; Zhang, M.; Jia, G.X. Analysis of global gene expression profiles during the flowering initiation process of Lilium × formolongi. Plant Mol. Biol. 2017, 94, 361–379. [Google Scholar] [CrossRef] [PubMed]
  37. Langens-Gerrits, M.M.; De Klerk, G.J. Micropropagation of flower bulbs. Lily and narcissus. Methods Mol. Biol. 1999, 111, 141–147. [Google Scholar] [PubMed]
  38. Ren, Z.M.; Zhang, D.; Jiao, C.; Li, D.Q.; Wu, Y.; Wang, X.Y.; Gao, C.; Lin, Y.F.; Ruan, Y.L.; Xia, Y.P. Comparative transcriptome and metabolome analyses identified the mode of sucrose degradation as a metabolic marker for early vegetative propagation in bulbs of Lycoris. Plant J. 2022, 112, 115–134. [Google Scholar] [CrossRef] [PubMed]
  39. Du, F.; Wu, Y.; Zhang, L.; Li, X.W.; Zhao, X.Y.; Wang, W.H.; Gao, Z.S.; Xia, Y.P. De novo assembled transcriptome analysis and SSR marker development of a mixture of six tissues from Lilium Oriental Hybrid ‘Sorbonne’. Plant Mol. Biol. Report. 2015, 33, 281–293. [Google Scholar] [CrossRef]
  40. Wu, Y.; Ren, Z.; Gao, C.; Sun, M.; Li, S.; Min, R.; Wu, J.; Li, D.; Wang, X.; Wei, Y.; et al. Change in sucrose cleavage pattern and rapid starch accumulation govern lily shoot-to-bulblet transition in vitro. Front. Plant Sci. 2021, 11, 564713. [Google Scholar] [CrossRef]
  41. Sun, H.M.; Li, T.L.; Li, Y.F. Changes of carbohydrate and amylase in lily bulb during bulb development. Bull. Bot. Res. 2005, 25, 59–63. [Google Scholar]
  42. Ruan, Y.L. Sucrose metabolism: Gateway to diverse carbon use and sugar signaling. Annu. Rev. Plant Biol. 2014, 65, 33–67. [Google Scholar] [CrossRef] [PubMed]
  43. Geng, Y.Q.; Dong, X.C.; Zhang, C.M. Recent progress of sugar transporter in horticultural crops. Acta Hortic. Sin. 2021, 48, 676–688. [Google Scholar]
  44. Zeng, Z.; Lyu, T.; Jia, X.; Chen, Y.; Lyu, Y. Expression patterns of sugar transporter genes in the allocation of assimilates and abiotic stress in lily. Int. J. Mol. Sci. 2022, 23, 4319. [Google Scholar] [CrossRef] [PubMed]
  45. Gu, J.; Zeng, Z.; Wang, Y.; Lyu, Y. Transcriptome analysis of carbohydrate metabolism genes and molecular regulation of sucrose transport gene LoSUT on the flowering process of developing Oriental hybrid lily ‘Sorbonne’ bulb. Int. J. Mol. Sci. 2020, 21, 3092. [Google Scholar] [CrossRef] [PubMed]
  46. Watari, J.; Kobae, Y.; Yamaki, S.; Yamada, K.; Toyofuku, K.; Tabuchi, T.; Shiratake, K. Identification of sorbitol transporters expressed in the phloem of apple source leaves. Plant Cell Physiol. 2004, 45, 1032–1041. [Google Scholar] [CrossRef] [PubMed]
  47. Liu, H.T.; Ji, Y.; Liu, Y.; Tian, S.H.; Gao, Q.H.; Zou, X.H.; Yang, J.; Dong, C.; Tan, J.H.; Ni, D.A.; et al. The sugar transporter system of strawberry: Genome-wide identification and expression correlation with fruit soluble sugar-related traits in a Fragaria × ananassa germplasm collection. Hortic. Res. 2020, 7, 132. [Google Scholar] [CrossRef]
  48. Zhang, C.; Bian, Y.; Hou, S.; Li, X. Sugar transport played a more important role than sugar biosynthesis in fruit sugar accumulation during Chinese jujube domestication. Planta 2018, 248, 1187–1199. [Google Scholar] [CrossRef]
  49. Yuan, M.; Wang, S. Rice MtN3/Saliva/SWEET family genes and their homologs in cellular organisms. Mol. Plant 2013, 6, 665–674. [Google Scholar] [CrossRef]
  50. Chong, J.; Piron, M.C.; Meyer, S.; Merdinoglu, D.; Bertsch, C.; Mestre, P. The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea. J. Exp. Bot. 2014, 65, 6589–6601. [Google Scholar] [CrossRef] [PubMed]
  51. Huang, D.M.; Chen, Y.; Liu, X.; Ni, D.A.; Bai, L.; Qin, Q.P. Genome-wide identification and expression analysis of the SWEET gene family in daylily (Hemerocallis fulva) and functional analysis of HfSWEET17 in response to cold stress. BMC Plant Biol. 2022, 22, 211. [Google Scholar] [CrossRef]
  52. Miao, H.; Sun, P.; Liu, Q.; Miao, Y.; Liu, J.; Zhang, K.; Hu, W.; Zhang, J.; Wang, J.; Wang, Z.; et al. Genome-wide analyses of SWEET family proteins reveal involvement in fruit development and abiotic/biotic stress responses in banana. Sci. Rep. 2017, 7, 3536. [Google Scholar] [CrossRef] [PubMed]
  53. Hu, B.; Wu, H.; Huang, W.; Song, J.; Zhou, Y.; Lin, Y. SWEET gene family in Medicago truncatula: Genome-wide identification, expression and substrate specificity analysis. Plants 2019, 8, 338. [Google Scholar] [CrossRef] [PubMed]
  54. Wei, H.; Liu, J.; Zheng, J.; Zhou, R.; Cheng, Y.; Ruan, M.; Ye, Q.; Wang, R.; Yao, Z.; Zhou, G.; et al. Sugar transporter proteins in Capsicum: Identification, characterization, evolution and expression patterns. Biotechnol. Biotechnol. Equip. 2020, 34, 341–353. [Google Scholar] [CrossRef]
  55. Wu, Y.; Wen, J.; Xia, Y.; Zhang, L.; Du, H. Evolution and functional diversification of R2R3-MYB transcription factors in plants. Hortic. Res. 2022, 9, uhac058. [Google Scholar] [CrossRef] [PubMed]
  56. Yan, N. Structural advances for the major facilitator superfamily (MFS) transporters. Trends Biochem. Sci. 2013, 38, 151–159. [Google Scholar] [CrossRef]
  57. Hirai, T.; Heymann, J.A.W.; Shi, D.; Sarker, R.; Maloney, P.C.; Subramaniam, S. Three-dimensional structure of a bacterial oxalate transporter. Nat. Struct. Biol. 2002, 9, 597–600. [Google Scholar] [CrossRef]
  58. Nino-Gonzalez, M.; Novo-Uzal, E.; Richardson, D.N.; Barros, P.M.; Duque, P. More transporters, more substrates: The Arabidopsis major facilitator superfamily revisited. Mol. Plant 2019, 12, 1182–1202. [Google Scholar] [CrossRef]
  59. Fakher, B.; Jakada, B.H.; Greaves, J.G.; Wang, L.; Niu, X.; Cheng, Y.; Zheng, P.; Aslam, M.; Qin, Y.; Wang, X. Identification and expression analysis of pineapple sugar transporters reveal their role in the development and environmental response. Front. Plant Sci. 2022, 13, 964897. [Google Scholar] [CrossRef]
  60. Zeng, Z.; Lyu, T.; Lyu, Y. LoSWEET14, a sugar transporter in lily, is regulated by transcription factor LoABF2 to participate in the ABA signaling pathway and enhance tolerance to multiple abiotic stresses in tobacco. Int. J. Mol. Sci. 2022, 23, 15093. [Google Scholar] [CrossRef]
  61. Slewinski, T.L. Diverse functional roles of monosaccharide transporters and their homologs in vascular plants: A physiological perspective. Mol. Plant 2011, 4, 641–662. [Google Scholar] [CrossRef] [PubMed]
  62. Li, C.; Meng, D.; Pineros, M.A.; Mao, Y.; Dandekar, A.M.; Cheng, L. A sugar transporter takes up both hexose and sucrose for sorbitol-modulated in vitro pollen tube growth in apple. Plant Cell 2020, 32, 449–469. [Google Scholar] [CrossRef] [PubMed]
  63. Szenthe, A.; Schafer, H.; Hauf, J.; Schwend, T.; Wink, M. Characterisation and expression of monosaccharide transporters in lupins, Lupinus polyphyllus and L. albus. J. Plant Res. 2007, 120, 697–705. [Google Scholar] [CrossRef]
  64. Sherson, S.M.; Hemmann, G.; Wallace, G.; Forbes, S.; Germain, V.; Stadler, R.; Bechtold, N.; Sauer, N.; Smith, S.M. Monosaccharide/proton symporter AtSTP1 plays a major role in uptake and response of Arabidopsis seeds and seedlings to sugars. Plant J. Cell Mol. Biol. 2000, 24, 849–857. [Google Scholar] [CrossRef]
  65. Yu, X.; Ali, M.M.; Li, B.; Fang, T.; Chen, F. Transcriptome data-based identification of candidate genes involved in metabolism and accumulation of soluble sugars during fruit development in ‘Huangguan’ plum. J. Food Biochem. 2021, 45, e13878. [Google Scholar] [CrossRef]
  66. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.E.; Paladin, L.; Raj, S.; Richardson, L.J.; et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021, 49, D412–D419. [Google Scholar] [CrossRef] [PubMed]
  67. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  68. Marchler-Bauer, A.; Lu, S.; Anderson, J.B.; Chitsaz, F.; Derbyshire, M.K.; DeWeese-Scott, C.; Fong, J.H.; Geer, L.Y.; Geer, R.C.; Gonzales, N.R.; et al. CDD: A conserved domain database for the functional annotation of proteins. Nucleic Acids Res. 2011, 39, D225–D229. [Google Scholar] [CrossRef]
  69. Li, W.; Godzik, A. Cd-hit: A fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 2006, 22, 1658–1659. [Google Scholar] [CrossRef]
  70. Price, M.N.; Dehal, P.S.; Arkin, A.P. FastTree 2-approximately maximum-likelihood trees for large alignments. PLoS ONE 2010, 5, e9490. [Google Scholar] [CrossRef]
  71. Letunic, I.; Bork, P. Interactive tree of life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021, 49, W293–W296. [Google Scholar] [CrossRef] [PubMed]
  72. Artimo, P.; Jonnalagedda, M.; Arnold, K.; Baratin, D.; Csardi, G.; de Castro, E.; Duvaud, S.; Flegel, V.; Fortier, A.; Gasteiger, E.; et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012, 40, W597–W603. [Google Scholar] [CrossRef]
  73. Petersen, T.N.; Brunak, S.; von Heijne, G.; Nielsen, H. SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat. Methods 2011, 8, 785–786. [Google Scholar] [CrossRef]
  74. Krogh, A.; Larsson, B.; von Heijne, G.; Sonnhammer, E.L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 2001, 305, 567–580. [Google Scholar] [CrossRef]
  75. Chou, K.C.; Shen, H.B. Plant-mPLoc: A top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS ONE 2010, 5, e11335. [Google Scholar] [CrossRef]
  76. Geourjon, C.; Deleage, G. SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput. Appl. Biosci. CABIOS 1995, 11, 681–684. [Google Scholar] [CrossRef]
  77. Bailey, T.L.; Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proceedings. Int. Conf. Intell. Syst. Mol. Biol. 1994, 2, 28–36. [Google Scholar]
  78. Chen, C.; Xia, R.; Chen, H.; He, Y. TBtools, a toolkit for biologists integrating various HTS-data handling tools with a user-friendly interface. BioRxiv 2018, 289660. [Google Scholar]
  79. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  80. Gao, C.; Zhang, L.; Xu, Y.; Xia, Y.; Wu, Y.; Ren, Z. Full-length transcriptome analysis revealed that 2,4-dichlorophenoxyacetic acid promoted in vitro bulblet initiation in lily by affecting carbohydrate metabolism and auxin signaling. Front. Plant Sci. 2023, 14, 1236315. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic analysis of MSTs in lilies, Arabidopsis, and rice. The sequences of 191 MSTs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method. STP, sugar transport protein; VGT, vacuolar glucose transporter; TMT, tonoplastic monosaccharide transporter; INT, inositol transporter; PLT, polyol transporter; pGlcT, plastidic glucose transporter; ERD6L, plastidic glucose transporter.
Figure 1. Phylogenetic analysis of MSTs in lilies, Arabidopsis, and rice. The sequences of 191 MSTs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method. STP, sugar transport protein; VGT, vacuolar glucose transporter; TMT, tonoplastic monosaccharide transporter; INT, inositol transporter; PLT, polyol transporter; pGlcT, plastidic glucose transporter; ERD6L, plastidic glucose transporter.
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Figure 2. Phylogenetic analysis of SUTs in lilies, Arabidopsis, and rice. The sequences of 21 SUTs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method.
Figure 2. Phylogenetic analysis of SUTs in lilies, Arabidopsis, and rice. The sequences of 21 SUTs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method.
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Figure 3. Phylogenetic analysis of SWEETs in lilies, Arabidopsis, and rice. The sequences of 65 SWEETs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method.
Figure 3. Phylogenetic analysis of SWEETs in lilies, Arabidopsis, and rice. The sequences of 65 SWEETs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method.
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Figure 4. Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi MSTs. (A) Phylogenetic trees of LohMSTs and LflMSTs were constructed using the maximum likelihood method. Seven subfamilies were labeled. (B) Motif compositions of LohMSTs and LflMSTs. A total of 20 motifs are shown as rectangles with different colors. (C) Domain compositions of LohMSTs and LflMSTs. (D) Amino acid sequences of the 20 conserved motifs of LohMSTs and LflMSTs are shown.
Figure 4. Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi MSTs. (A) Phylogenetic trees of LohMSTs and LflMSTs were constructed using the maximum likelihood method. Seven subfamilies were labeled. (B) Motif compositions of LohMSTs and LflMSTs. A total of 20 motifs are shown as rectangles with different colors. (C) Domain compositions of LohMSTs and LflMSTs. (D) Amino acid sequences of the 20 conserved motifs of LohMSTs and LflMSTs are shown.
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Figure 5. Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi SUTs. (A) Phylogenetic trees of LohSUTs and LflSUTs were constructed using the maximum likelihood method. Three subgroups were labeled. (B) Motif compositions of LohSUTs and LflSUTs. A total of 14 motifs are shown as rectangles with different colors. (C) Domain compositions of LohSUTs and LflSUTs. (D) Amino acid sequences of the 14 conserved motifs of LohSUTs and LflSUTs are shown.
Figure 5. Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi SUTs. (A) Phylogenetic trees of LohSUTs and LflSUTs were constructed using the maximum likelihood method. Three subgroups were labeled. (B) Motif compositions of LohSUTs and LflSUTs. A total of 14 motifs are shown as rectangles with different colors. (C) Domain compositions of LohSUTs and LflSUTs. (D) Amino acid sequences of the 14 conserved motifs of LohSUTs and LflSUTs are shown.
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Figure 6. Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi SWEETs. (A) Phylogenetic trees of LohSWEETs and LflSWEETs were constructed using the maximum likelihood method. Four clades were labeled. (B) Motif compositions of LohSWEETs and LflSWEETs. A total of 12 motifs are shown as rectangles with different colors. (C) Domain compositions of LohSWEETs and LflSWEETs. (D) Amino acid sequences of the 12 conserved motifs of LohSWEETs and LflSWEETs are shown.
Figure 6. Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi SWEETs. (A) Phylogenetic trees of LohSWEETs and LflSWEETs were constructed using the maximum likelihood method. Four clades were labeled. (B) Motif compositions of LohSWEETs and LflSWEETs. A total of 12 motifs are shown as rectangles with different colors. (C) Domain compositions of LohSWEETs and LflSWEETs. (D) Amino acid sequences of the 12 conserved motifs of LohSWEETs and LflSWEETs are shown.
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Figure 7. Subcellular localization analysis of LohINT1. (A) Subcellular localization of LohINT1-GFP and free GFP. (B) Subcellular localization of LohINT1-YFP and free YFP.
Figure 7. Subcellular localization analysis of LohINT1. (A) Subcellular localization of LohINT1-GFP and free GFP. (B) Subcellular localization of LohINT1-YFP and free YFP.
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Figure 8. Expression patterns of ‘Sorbonne’ sugar transporter genes at four periods under aeroponic conditions. (A) Expression patterns of LohMSTs during four periods of bulblet initiation. (B) Expression patterns of LohSUTs during four periods of bulblet initiation. (C) Expression patterns of LohSWEETs during four periods of bulblet initiation. Color scale represents reads per kilobase per million normalized log2 transformed counts, where dark red indicates high level, dark blue indicates low level, and white indicates medium. DAT, days after treatment.
Figure 8. Expression patterns of ‘Sorbonne’ sugar transporter genes at four periods under aeroponic conditions. (A) Expression patterns of LohMSTs during four periods of bulblet initiation. (B) Expression patterns of LohSUTs during four periods of bulblet initiation. (C) Expression patterns of LohSWEETs during four periods of bulblet initiation. Color scale represents reads per kilobase per million normalized log2 transformed counts, where dark red indicates high level, dark blue indicates low level, and white indicates medium. DAT, days after treatment.
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Figure 9. Expression patterns of Lilium × formolongi sugar transporter genes at three periods after sowing. (A) Expression patterns of LflMSTs at three periods during the growth of the underground bulblet. (B) Expression patterns of LflSUTs at three periods during the growth of the underground bulblet. (C) Expression patterns of LflSWEETs at three periods during the growth of the underground bulblet. Color scale represents reads per kilobase per million normalized log2 transformed counts, where dark red indicates high level, dark blue indicates low level, and white indicates medium. M, months.
Figure 9. Expression patterns of Lilium × formolongi sugar transporter genes at three periods after sowing. (A) Expression patterns of LflMSTs at three periods during the growth of the underground bulblet. (B) Expression patterns of LflSUTs at three periods during the growth of the underground bulblet. (C) Expression patterns of LflSWEETs at three periods during the growth of the underground bulblet. Color scale represents reads per kilobase per million normalized log2 transformed counts, where dark red indicates high level, dark blue indicates low level, and white indicates medium. M, months.
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Figure 10. Expression profiles of 28 LohSTs genes during the extremely early stage of bulblet initiation. All data are presented as mean ± standard error of mean (SEM). Lowercase letters above the bars indicate significant differences between periods (p-value < 0.05, LSD, Duncan).
Figure 10. Expression profiles of 28 LohSTs genes during the extremely early stage of bulblet initiation. All data are presented as mean ± standard error of mean (SEM). Lowercase letters above the bars indicate significant differences between periods (p-value < 0.05, LSD, Duncan).
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Figure 11. Expression profiles of 9 LflSTs genes during the growth of the underground bulblet. All data are presented as mean ± SEM. Lowercase letters above the bars indicate significant differences between periods (p-value < 0.05, LSD, Duncan).
Figure 11. Expression profiles of 9 LflSTs genes during the growth of the underground bulblet. All data are presented as mean ± SEM. Lowercase letters above the bars indicate significant differences between periods (p-value < 0.05, LSD, Duncan).
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Table 1. Physicochemical properties and structural analysis of lily MSTs.
Table 1. Physicochemical properties and structural analysis of lily MSTs.
NameGene IDPhysicochemical PropertySecond-Level StructureSignal PeptideTMD g
AA aMW bPI cII dAI eGRAVY fSubcellular Locationα-HelixExtension Chainβ-CornerAperiodical Coil
STP (sugar transport protein/hexose transporter)
LohSTP1Isoform_3595450856,512.579.7539.44108.620.380Cell membrane47.0515.756.6930.51No10
LohSTP2Isoform_3764351857,031.779.1334.51108.050.498Cell membrane47.3017.574.8330.31No12
LohSTP3Unigene002494150755,105.699.0035.58107.320.585Cell membrane48.3217.555.3328.80No12
LohSTP4Unigene003503452056,842.798.9839.92107.620.559Cell membrane54.6213.465.7726.15No11
LohSTP5Unigene005389852457,736.859.1940.79106.770.463Cell membrane48.4716.224.3930.92No12
LohSTP6Unigene009067451156,022.088.8039.65111.820.537Cell membrane48.7317.616.8526.81No12
LohSTP7Unigene01124954860,062.558.0036.52107.100.599Cell membrane50.7315.335.2928.65No13
LohSTP8Unigene01125852357,544.029.3132.02105.720.610Cell membrane51.4315.305.9327.34No11
LohSTP9Unigene01178452357,406.708.9736.82106.290.610Cell membrane51.2416.834.7827.15No12
LohSTP10Unigene01187849453,558.238.9238.19117.590.717Cell membrane51.6217.214.8626.32No12
LohSTP11Unigene01243251055,947.656.4143.50111.840.554Cell membrane50.9815.695.6927.65No10
LohSTP12Unigene01324550355,493.619.0934.43109.680.624Cell membrane51.6915.514.7728.03Yes12
LohSTP13Unigene01534450854,585.889.4032.22105.390.588Cell membrane48.6216.934.7229.72No11
LohSTP14Unigene02890826630,490.108.7036.42103.980.507Cell membrane53.3814.294.5127.82No6
LohSTP15Unigene25955
_L-Tis6-Transc		50755,230.149.3736.62109.050.561Cell membrane47.9316.575.3330.18No12
LohSTP16Unigene26933
_L-Tis6-Transc		31335,346.229.5150.90111.730.592Cell membrane54.319.585.7530.35Yes6
LflSTP1Unigene000964252456,408.338.8232.2499.900.462Cell membrane44.0817.375.1533.40No9
LflSTP2Unigene000964436639,004.8810.1938.82112.980.620Cell membrane51.0918.854.6425.41No8
LflSTP3Unigene000969150655,597.378.3343.20110.810.536Cell membrane52.3714.235.3428.06No10
LflSTP4Unigene002602149353,656.289.4536.54113.940.655Cell membrane52.5415.825.0726.57No12
LflSTP5Unigene007640950054,447.339.5036.74110.000.596Cell membrane50.0016.205.8028.00No12
LflSTP6Unigene008593352257,288.839.5334.37103.120.594Cell membrane48.0817.435.5628.93No12
LflSTP7Unigene008593453658,873.197.6138.36108.400.601Cell membrane47.3917.916.5328.17No12
LflSTP8Unigene008593532636,501.988.1651.5697.450.225Cell membrane47.2413.504.2934.97No3
ERD6-like (early responsive to dehydration six-like)
LohERD6.1CL1228.Contig1
_L-Tis6-Transc		49653,277.369.1439.64114.440.586Cell membrane53.2353.237.4621.98No12
LohERD6.2Isoform_4295847150,332.018.3534.14109.300.624Cell membrane46.0722.086.7925.05No12
LohERD6.3Unigene002730520321,680.505.2539.82127.641.036Cell membrane52.7122.175.4219.70No6
LohERD6.4Unigene006271848651,640.588.4141.32111.600.651Cell membrane44.4420.996.3828.19No11
LohERD6.5Unigene008575747851,283.115.7933.97112.660.632Cell membrane48.5419.875.6525.94No12
LohERD6.6Unigene00996649653,054.268.8744.90117.360.640Cell membrane49.6019.567.2623.59No12
LohERD6.7Unigene013250849052,929.097.4628.93114.160.652Cell membrane43.2724.496.5325.71No12
LohERD6.8Unigene01389248151,582.698.3231.62111.500.680Cell membrane46.5721.006.6525.78No11
LohERD6.9Unigene01545747051,168.346.3635.34108.230.631Cell membrane54.4719.366.8119.36No12
LflERD6.1Unigene003080446649,711.147.6432.45110.040.620Cell membrane47.6421.676.4424.25No11
LflERD6.2Unigene004319350254,520.948.9442.7997.110.295Cell membrane41.2422.915.9829.88No8
LflERD6.3Unigene008099949653,074.278.8744.94115.990.629Cell membrane49.6019.567.2623.59No12
LflERD6.4Unigene008100040042,864.099.1741.62114.570.534Cell membrane52.0016.507.2524.25No9
LflERD6.5Unigene008485258467,140.988.5350.2794.380.351Cell membrane22.0940.418.7328.77No8
INT (inositol transporter)
LohINT1Isoform_1674928530,965.095.4745.11107.820.400Cell membrane47.7220.357.0224.91No6
LohINT2Isoform_2545757762,331.898.8341.70101.960.404Cell membrane43.6718.374.6833.28No12
LohINT3Unigene00683452155,817.135.0035.35109.520.590Cell membrane49.9018.625.3726.10No12
LohINT4Unigene00847657462,622.098.7439.78103.500.383Cell membrane43.7318.644.5333.10No12
LohINT5Unigene01029057462,063.298.2337.17105.910.408Cell membrane45.3018.125.5731.01No12
LohINT6Unigene010536440443,968.267.4047.0696.040.259Cell membrane44.8014.854.7035.64No7
LohINT7Unigene013051057962,779.188.7940.22104.440.382Cell membrane42.6618.485.1833.68No12
LflINT1Unigene004832156961,606.128.9241.85102.040.417Cell membrane40.9519.165.4534.45No12
LflINT2Unigene006047029332,107.806.7835.03112.490.705Cell membrane55.2914.684.7825.26No7
LflINT3Unigene006661857462,073.288.2336.20105.730.403Cell membrane44.0818.994.8832.06No12
LflINT4Unigene006662051956,053.328.5438.45105.840.426Cell membrane42.0018.695.3933.91No10
pGlcT (plastidic glucose transporter)
LohpGlcT1Isoform_3413948351,883.108.7432.69113.020.652Cell membrane53.4215.534.7626.29No10
LohpGlcT2Isoform_4123630032,479.908.5540.63105.330.496Cell membrane50.6718.338.0023.00No6
LohpGlcT3Unigene00748653457,757.745.3445.07104.440.399Cell membrane48.1314.795.2431.84No9
LohpGlcT4Unigene00925153456,342.169.4635.05112.320.586Cell membrane51.8714.985.0628.09No10
LohpGlcT5Unigene012856626528,883.138.7339.97112.230.618Cell membrane55.8517.366.0420.75No6
LohpGlcT6Unigene26459
_L-Tis6-Transc		50654,258.608.3041.59105.420.535Cell membrane53.7513.445.7327.08No10
LflpGlcT1Unigene004245048251,956.098.7533.98115.080.660Cell membrane56.4313.904.9824.69No10
LflpGlcT2Unigene006729156059,162.019.4835.60107.120.476Cell membrane51.0715.716.7926.43No10
LflpGlcT3Unigene007473345749,271.254.9938.13112.650.598Cell membrane56.0214.445.2524.29No10
PLT (polyol/monosaccharide transporter)
LohPLT1Isoform_3809737540,222.699.9755.2394.880.179Cell membrane37.6013.076.6742.67No5
LohPLT2Unigene003910953557,479.249.3936.04107.590.370Cell membrane49.7215.145.2329.91No10
LohPLT3Unigene00860949953,758.007.6540.55111.780.575Cell membrane50.9016.635.6126.85No11
LohPLT4Unigene01261153056,691.289.3534.85108.920.492Cell membrane50.0014.915.0930.00No11
LohPLT5Unigene01363551254,854.369.7535.74109.610.510Cell membrane51.7614.655.6627.93No12
LohPLT6Unigene013653751054,679.075.7746.96117.750.603Cell membrane53.5314.515.2926.67No12
LflPLT1Unigene004666152556,159.619.2735.02108.840.506Cell membrane48.5715.625.3330.48No10
LflPLT2Unigene006031252156,049.548.7040.88110.420.551Cell membrane49.3315.365.9529.37No9
LflPLT3Unigene007914750654,175.455.6048.60118.870.626Cell membrane52.9616.405.7324.90No12
TMT (tonoplast sugar transporter)
LohTMT1Isoform_1872138642,178.394.8059.2673.50−0.403Cell membrane, Nucleus25.6514.512.5957.25No1
LohTMT2Unigene00228274980,237.275.2043.78104.220.363Cell membrane35.1117.226.0141.66No11
LohTMT3Unigene00311775481,213.055.0546.65105.600.311Cell membrane35.6816.455.7042.18No11
LflTMT1Unigene003011775381,178.985.0946.78105.740.303Cell membrane36.1217.005.9840.90No10
LflTMT2Unigene003011874679,852.725.1146.43104.520.364Cell membrane34.8517.025.9042.23No10
VGT (vacuolar glucose transporter)
LohVGT1Unigene00963555158,545.629.1441.93119.550.579Cell membrane48.2816.523.8131.40No11
LohVGT2Unigene26230
_L-Tis6-Transc		49653,012.545.4140.02123.290.782Cell membrane52.4217.345.0425.20No12
LflVGT1Unigene005574749452,774.245.5438.31123.990.784Cell membrane47.9819.436.0726.52No12
LflVGT2Unigene005730648952,010.055.6435.18124.680.685Cell membrane49.2819.025.1126.58No11
a Length of the amino acid sequence. b Molecular weight of the amino acid sequence, kDa is kilo Daltons. c Isoelectric point. d Instability index. e Aliphatic index. f Grand average of hydropathicity. g Number of transmembrane helices, as predicted by the TMHMM Server 2.0.
Table 2. Physicochemical properties and structural analysis of lily SUTs.
Table 2. Physicochemical properties and structural analysis of lily SUTs.
NameGene IDPhysicochemical PropertySecond-Level StructureSignal PeptideTMD g
AA aMW bPI cII dAI eGRAVY fSubcellular Locationα-HelixExtension Chainβ-CornerAperiodical Coil
SUT2
LohSUT3Unigene00671559063,479.287.1636.1994.530.367Cell membrane35.5916.274.7543.39No11
LflSUT1Unigene002776837240,336.416.7437.9396.990.269Cell membrane35.4815.053.7645.70No5
SUT3
LohSUT2Unigene001668321322,336.178.5220.49113.050.771Cell membrane43.1920.197.5129.11No5
LohSUT4Unigene01868527029,486.609.1728.87102.890.440Cell membrane46.3017.046.3030.37No5
LohSUT5Unigene02175731234,055.889.4028.05102.210.519Cell membrane55.7716.035.7722.44No6
SUT4
LohSUT1Isoform_3989349753,094.439.3033.35113.860.616Cell membrane44.4716.504.0235.01No12
LflSUT2Unigene005788049252,451.729.2130.92117.600.654Cell membrane46.1414.433.6635.77No12
a Length of the amino acid sequence. b Molecular weight of the amino acid sequence, kDa is kilo Daltons. c Isoelectric point. d Instability index. e Aliphatic index. f Grand average of hydropathicity. g Number of transmembrane helices, as predicted by the TMHMM Server 2.0.
Table 3. Physicochemical properties and structural analysis of lily SWEETs.
Table 3. Physicochemical properties and structural analysis of lily SWEETs.
NameGene IDPhysicochemical PropertySecond-Level StructureSignal PeptideTMD g
AA aMW bPI cII dAI eGRAVY fSubcellular Locationα-HelixExtension Chainβ-CornerAperiodical Coil
Clade I
LohSWEET2CL5864.Contig1
_L-Tis6-Transc		25127,820.219.6631.32111.040.639Cell membrane44.2216.733.1935.86No7
LohSWEET13Unigene02702023025,686.819.1542.13119.480.887Cell membrane45.6518.73.0432.61No7
LflSWEET3Unigene003559522925,654.779.3941.381200.889Cell membrane45.8519.213.9331No7
LflSWEET8Unigene006168615116,952.4510.0135.97118.680.874Cell membrane41.7224.57.9525.83No5
LflSWEET9Unigene006168725127,968.289.6339.32108.330.554Cell membrane45.4220.323.9830.28No7
Clade II
LohSWEET9Unigene01983225828,713.419.1337.33122.330.792Cell membrane37.622.873.8835.66No7
LohSWEET11Unigene02497523425,912.358.8941.17123.970.93Cell membrane, Chloroplast45.322.222.5629.91No7
LohSWEET12Unigene02515025728,632.489.2635.63129.260.808Cell membrane42.821.013.532.68No7
LflSWEET5Unigene00560668710,105.855.6746.92141.030.841Cell membrane, Chloroplast51.7220.694.622.99No1
LflSWEET11Unigene008110921924,471.58.9833.49128.130.801Cell membrane33.3321.922.7442.01No6
LflSWEET12Unigene008623021524,143.019.2733.61125.020.838Cell membrane41.8621.43.7233.02No6
Clade III
LohSWEET1CL469.Contig2
_L-Tis6-Transc		27230,486.598.9842.67125.040.891Cell membrane37.521.321.4739.71No7
LohSWEET3Unigene002693928031,065.579.0224.51105.820.558Cell membrane39.6416.072.8641.43No7
LohSWEET4Unigene002694025428,783.357.5928.72113.90.65Cell membrane41.3415.752.7640.16No6
LohSWEET5Unigene002694126930,164.787.5929116.650.744Cell membrane43.8718.222.9734.94No7
LohSWEET6Unigene006676633136,516.115.2940.76114.530.58Cell membrane45.0215.412.1137.46No7
LohSWEET10Unigene02476827731,108.88.6328.83110.870.631Cell membrane44.7719.492.1733.57No7
LohSWEET14Unigene04010119421,758.88.6142.85120.520.436Cell membrane38.6613.921.5545.88No4
LohSWEET15Unigene04831712314,174.516.5649.0889.43−0.107Chloroplast30.0814.631.6353.66No2
LflSWEET1Unigene002698025628,865.729.144.97123.710.905Cell membrane42.9722.273.9130.86No7
LflSWEET2Unigene002698125628,737.479.3739.19120.310.78Cell membrane41.822.663.1232.42No7
LflSWEET6Unigene005747526329,746.28.3433.34112.280.644Cell membrane40.319.392.6637.64No7
LflSWEET7Unigene005976526529,955.76.8226.53118.750.765Cell membrane36.620.383.439.62No7
LflSWEET10Unigene0059765778829.719.0618.11120.131.021Chloroplast36.3636.362.624.68No2
Clade IV
LohSWEET7Unigene012268429232,135.199.5627.55122.710.621Cell membrane46.5815.075.1433.22No7
LohSWEET8Unigene012268424327,016.78.4531.94107.450.514Cell membrane38.6824.282.4734.57No6
LflSWEET4Unigene004633828931,809.99.7826.8122.630.639Cell membrane, Chloroplast, Peroxisome42.9115.923.8137.37No7
a Length of the amino acid sequence. b Molecular weight of the amino acid sequence; kDa is kilo Daltons. c Isoelectric point. d Instability index. e Aliphatic index. f Grand average of hydropathicity. g Number of transmembrane helices, as predicted by the TMHMM Server 2.0.
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MDPI and ACS Style

Huang, Z.; Gao, C.; Xu, Y.; Liu, J.; Kang, J.; Ren, Z.; Cui, Q.; Li, D.; Ma, S.; Xia, Y.; et al. Identification and Expression Analysis of Putative Sugar Transporter Gene Family during Bulb Formation in Lilies. Int. J. Mol. Sci. 2024, 25, 3483. https://doi.org/10.3390/ijms25063483

AMA Style

Huang Z, Gao C, Xu Y, Liu J, Kang J, Ren Z, Cui Q, Li D, Ma S, Xia Y, et al. Identification and Expression Analysis of Putative Sugar Transporter Gene Family during Bulb Formation in Lilies. International Journal of Molecular Sciences. 2024; 25(6):3483. https://doi.org/10.3390/ijms25063483

Chicago/Turabian Style

Huang, Ziyang, Cong Gao, Yunchen Xu, Jie Liu, Jie Kang, Ziming Ren, Qi Cui, Dongze Li, Si Ma, Yiping Xia, and et al. 2024. "Identification and Expression Analysis of Putative Sugar Transporter Gene Family during Bulb Formation in Lilies" International Journal of Molecular Sciences 25, no. 6: 3483. https://doi.org/10.3390/ijms25063483

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