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7 February 2026

19 Pages

Genome-Wide Identification of the SWEET Gene Family and Functional Analysis of LpSWEET13 in Perennial Ryegrass Under Drought Stress

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College of Landscape and Architecture, Zhejiang A&F University, Hangzhou 311300, China
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Authors to whom correspondence should be addressed.
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These authors contributed equally to this work.
Plants2026, 15(4), 526;https://doi.org/10.3390/plants15040526
This article belongs to the Special Issue Abiotic Stress Responses in Plants—Second Edition
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Abstract

SWEETs (Sugars Will Eventually be Exported Transporters) represent a distinct category of proteins responsible for mediating intracellular sugar transport. These transporters are involved in sugar allocation processes, contribute to plant growth and development, and mediate adaptive responses to abiotic and biotic stresses. Despite extensive research on SWEET genes in many plant species, their functions in perennial ryegrass have not been clearly characterized. In this study, genome-wide bioinformatic analyses were conducted to identify SWEET family members in perennial ryegrass (Lolium perenne; LpSWEETs) and to explore their potential involvement in drought stress responses. Twenty-three LpSWEET genes were identified based on whole-genome sequence data, and phylogenetic inference indicated that these genes clustered into four clades. Comprehensive analyses of gene organization, conserved motifs, cis-regulatory elements, and protein features demonstrated strong evolutionary conservation across LpSWEET members, while quantitative real-time PCR analysis demonstrated that LpSWEET13 exhibited drought-responsive expression, and subcellular localization analysis showed that LpSWEET13 was localized in the plasma membrane. Under drought stress, transgenic Arabidopsis thaliana lines overexpressing LpSWEET13 presented a substantially higher survival rate than their corresponding wild-type controls. In addition, ectopic expression of LpSWEET13 increased expression levels of AtP5CS1, AtRD22, AtRD29A, and AtRD29B expression. These findings offer insights into the cladistic characteristics of the LpSWEET family and establish a useful framework for subsequent functional studies of LpSWEET genes.

1. Introduction

Sugars, as primary photosynthetic assimilates, are fundamental to plant growth and development, and play pivotal roles in regulating plants’ adaptation to biotic and abiotic stresses [1]. Eukaryotic sugar transport systems are generally classified into three evolutionarily conserved families: the monosaccharide transporter (MST) and sucrose transporter (SUT), as well as Sugars Will Eventually be Exported Transporters (SWEETs) [2]. Sucrose serves as the principal form of carbon transported over long distances in plants, with its phloem loading and transport predominantly facilitated by SUTs. Upon reaching phloem unloading sites, this disaccharide is delivered to sink tissues, where it is hydrolyzed into monosaccharides (glucose and fructose) for subsequent transport via MSTs. A new sugar transporter family, the SWEET family, has recently been identified and characterized, and it appears to mediate the transport of both sucrose and monosaccharides. In contrast to most SUTs and MSTs, which rely on energy-dependent transport mechanisms driven by H+-ATPase activity, SWEET proteins facilitate bidirectional sugar movement across membranes along concentration gradients without direct energy consumption and with minimal pH dependence [3].
SWEETs belong to the MtN3/saliva family (Pfam PF03083) [4], and A total of three α-helical transmembrane domains (3-TMs) have been identified within the MtN3/saliva domain. Characteristically, eukaryotic SWEET proteins consist of seven transmembrane domains (TMs), which are structurally organized as two tandem-repeated 3-TM motifs on either side of a single intervening TM segment [5]. Phylogenetic analyses have been performed on the SWEET genes family in Arabidopsis thaliana, Oryza sativa (rice), Sorghum bicolor (sorghum), Zea mays (maize), and other plant species, and they reveal that the plant SWEET sugar transporter family can be categorized into four distinct evolutionary clades. Taking Arabidopsis as an example, Clade I includes AtSWEET1–3, Clade II includes AtSWEET4–8, Clade III includes AtSWEET9–15, and Clade IV includes AtSWEET16–17. Notably, Clades I, II, and IV are primarily responsible for the transport of monosaccharides such as glucose, galactose, and fructose, whereas Clade III exhibits a preference for disaccharide transport, with sucrose as a typical substrate [2]. SWEET proteins have been extensively reported across diverse plant species, including Arabidopsis [3], rice [6], maize [7,8], Glycine max (soybean) [9], Fragaria × ananassa Duch. (strawberry) [10], and Vitis vinifera L. (grapevine) [11].
The SWEET gene family has been shown to contribute to multiple physiological processes in plants, including phloem loading [12], seed filling [13,14], fruit development [15], and biotic [4] and abiotic stresses [16,17]. Accumulating evidence indicates that SWEET genes across diverse plant species exhibit transcriptional responses to abiotic stresses, highlighting their potential roles in plant stress-response pathways. Arabidopsis AtSWEET15 (SAG29) is localized to the plasma membrane and regulates plant salt stress responses, which is supported by the observation that AtSWEET15-overexpressing transgenic lines exhibit increased salt sensitivity, while AtSWEET15-deficient mutants show enhanced salt tolerance [18]. AtSWEET16 is localized to the tonoplast, and overexpression of this gene enhances the cold tolerance of Arabidopsis under cold stress [19]. Similarly, its homologous gene CsSWEET16 is also localized to the tonoplast and improves the cold tolerance of Camellia sinensis (tea plant) by increasing the contents of sucrose and glucose [20]. AtSnRK2 can phosphorylate the serine residue at position 237 of the AtSWEET11 protein and the serine residue at position 248 of the AtSWEET12 protein. This modification is induced by drought stress and dependent on the core abscisic acid (ABA signaling pathway), which enhances the oligomerization and sucrose transport activity of SWEET proteins, thereby promoting the long-distance translocation of sucrose from shoots to roots, increasing the plant root–shoot ratio, and ultimately enhancing drought tolerance [21].
Perennial ryegrass (Lolium perenne L.) is extensively grown in temperate climates. As an excellent cool-season turfgrass, ecological grass, and forage grass, it plays a crucial role in landscape greening and ecological restoration. In the present study, a comprehensive genome-scale investigation of 23 members of the SWEET gene family was performed in perennial ryegrass, and their evolutionary relationships were elucidated through comparison with rice Arabidopsis, and maize. The phylogeny, gene structure, chromosomal distribution, and regulatory cis-elements associated with the SWEET gene family, as well as the transcriptional patterns of LpSWEET genes in response to drought stress, were systematically evaluated. This study provides insights into the involvement of LpSWEET genes during abiotic stress conditions and identifies promising SWEET candidates for developing stress-tolerant perennial ryegrass cultivars.

2. Results

2.1. Identification and Bioinformatics Analysis of LpSWEET Genes

The genome-wide analysis determined 23 SWEET candidate genes in Lolium perenne. L. (perennial ryegrass). Among all LpSWEETs, 19 were mapped to chromosomes, whereas 4 (KYUSt_contig_605.398, KYUSt_contig_49.9, KYUSt_contig_528.645, KYUSt_contig_1537.307) were not assigned to any definitive linkage group. These SWEET genes were designated LpSWEET1–LpSWEET16 according to their homology with O. sativa (rice) genes (Table S1). The predicted proteins encoded by these genes vary in length from 85 (LpSWEET16c) to 348 amino acids (aa) (LpSWEET1a), with relative molecular weights (MWs) between 20.12 kDa (LpSWEET16c) and 37.10 kDa (LpSWEET1a), and isoelectric point (pI) values ranging from 4.70 (LpSWEET16b) to 9.74 (LpSWEET16a). Nearly 73.91% of LpSWEET proteins showed pI values greater than seven, suggesting that the majority of this gene family’s members exhibit alkaline properties. Subcellular localization analysis improved our understanding of molecular functions, revealing that LpSWEET18 was localized to the chloroplast; LpSWEET2b and LpSWEET16b were localized to the vacuole; and the remaining LpSWEET proteins were localized to the plasma membrane (Table S1).

2.2. Phylogenetic Analysis of LpSWEETs

A Maximum Likelihood (ML) phylogenetic tree was obtained, based on the multiple sequence alignment of LpSWEET sequences with AtSWEETs, OsSWEETs, and ZmSWEETs, to infer the evolutionary relationships among SWEET genes (Figure 1). Our analysis indicated that SWEET genes are grouped into four distinct subfamilies (Clades I-IV). Specifically, Clade I contained six LpSWEETs, six OsSWEETs, three AtSWEETs, and five ZmSWEETs; Clade II comprised seven LpSWEETs, nine OsSWEETs, five AtSWEETs, and five ZmSWEETs; Clade III included six LpSWEETs, five OsSWEETs, seven AtSWEETs, and eleven ZmSWEETs; and Clade IV harbored four LpSWEETs, one OsSWEET, two AtSWEETs, and three ZmSWEETs. Notably, Clades II and III harbored more SWEET genes than Clades I and IV.
Figure 1. Phylogenetic analysis of SWEET family genes derived from A. thaliana, O. sativa, Z. mays, and L. Perenne. Four clades are marked with different colors. AtSWEET, OsSWEET, ZmSWEETs, and LpSWEET proteins are denoted by purple dots, brown dots, yellow dots, and green dots, respectively.

2.3. Gene Structure, Conserved Motifs and Transmembrane Helices of LpSWEETs

Conserved motifs provide a more in-depth understanding of the structural properties of LpSWEETs. Analysis using Multiple Em for Motif Elicitation (MEME) uncovered 12 conserved motifs in the LpSWEET family, with motifs 1–5 present in all members except LpSWEET16c (lacking motif 1) and LpSWEET1a (lacking motif 2). LpSWEET16c harbored the fewest motifs (Figure 2A). Conserved-domain analysis revealed that all LpSWEETs possess two MtN3 domains, indicating that the SWEET protein family of perennial ryegrass was relatively conservative in evolution [22]. Gene structure analysis indicated that LpSWEET genes varied in intron number (Figure 2B), indicating structural diversity among the SWEET genes of perennial ryegrass. Exon numbers varied from one to seven, with approximately 30.78% of LpSWEET genes containing five exons, and about 30.43% harboring four exons (Figure 2C). Transmembrane helix (TMH) analysis revealed that LpSWEET16c contained only four TMHs, while LpSWEET1a and LpSWEET16d each possessed five TMHs. Five proteins, including LpSWEET1b, LpSWEET2a, LpSWEET14, LpSWEET16a, and LpSWEET16b, harbored six TMHs, and the remaining LpSWEET proteins were characterized by the presence of seven TMHs (Figure S1). These findings highlight substantial variation among the 23 members of the SWEET gene family.
Figure 2. Analysis of conserved motifs, conserved domains, and gene structures of LpSWEET members. (A) Motif composition of LpSWEET proteins. Twelve motifs are displayed as rectangles of different colors. (B) Conserved domains of LpSWEET proteins. Green boxes denote the MtN3 domain. (C) Exon-intron structures of LpSWEET genes. Green rectangles stand for exons, and black lines stand for introns.

2.4. Analysis of Secondary and Tertiary Structures of LpSWEETs Proteins

The LpSWEET proteins contained α-helices (34.43 to 57.88%), extended strands (10.26 to 23.28%), and random coils (29.45 to 42.86%). Notably, no β-turns were detected in any LpSWEET proteins (Table S2). By folding their secondary structures, all 23 LpSWEET proteins formed similar tertiary structures (Figure 3), while structural differences existed between LpSWEET2a and LpSWEET2b.
Figure 3. Predicted tertiary structure of LpSWEET proteins.

2.5. Chromosomal Localizations and Synteny Analysis of the LpSWEET Gene Family Members

Chromosomal localization analysis indicated that LpSWEET loci showed an uneven distribution across the genome, with 23 LpSWEET genes mapped to six out of the seven chromosomes (Figure 4). A total of 13 genes were detected on Chromosome 3, accounting for the highest proportion of genes among all chromosomes. Chromosomes 1, 2, 6, and 7 each harbored one LpSWEET gene, and Chromosome 5 contained two LpSWEET genes, while no LpSWEET genes were identified on Chromosome 4 (Figure 4).
Figure 4. Distribution of LpSWEET genes on the six chromosomes.
When exploring the evolutionary trajectory of the SWEET gene family in the perennial ryegrass genome, gene duplication events are regarded as the molecular basis driving the evolution of this family. Therefore we conducted a detailed gene duplication investigation on 23 LpSWEET members in perennial ryegrass (Figure 5A). Our results identified two pairs of segmental duplication genes pairs (Table S3), suggesting that segmental duplications could contribute to the expansion, functional diversification, and evolutionary development of the LpSWEET gene family. In addition, this study assessed the syntenic relationships among SWEET genes of perennial ryegrass and those of Arabidopsis, rice, and maize (Figure 5B,C). The results showed that there was one pair of homologous genes between perennial ryegrass and Arabidopsis. Perennial ryegrass shares 11 pairs of homologous genes with rice and 12 pairs with maize, and these genes had a relatively high frequency of occurrence on Chromosome 3 of perennial ryegrass.
Figure 5. Synteny analyses of LpSWEETs. (A) Synteny analysis of LpSWEET genes in the perennial ryegrass genome. Gray lines: all synteny blocks in the perennial ryegrass genome. Red lines: duplicated LpSWEET gene pairs. The innermost circos shows chromosome positions; the middle circos displays a line chart of GC content ratio across syntenic regions; the outermost circos presents a gene density heatmap, with color gradients corresponding to relative gene abundance. (B) Syntenic relationships across the genomes of perennial ryegrass, Arabidopsis, and rice. Gray lines represent synteny between all members, and blue lines represent synteny between SWEET family members. (C) Syntenic relationships across the genomes of perennial ryegrass and maize. Gray lines represent synteny between all members, and red lines represent synteny between SWEET family members.

2.6. Cis-Element Analysis for the LpSWEETs Gene Family

To elucidate the regulatory mechanisms and functional roles of LpSWEET genes, cis-acting elements within their 2000-base-pairs (bp) promoter regions were systematically analyzed. The cis-acting elements present in LpSWEETs promoter regions were related to light responses and processes of growth and development, hormone responses, and abiotic stress responses (Table S4). Focusing on stress-responsive cis-acting elements, we observed that all 23 genes harbored MYB elements within their promoter regions, 23 genes contained ABRE (abscisic-acid-responsive element) elements (with the exception of LpSWEET4a), and 23 genes contained MYC elements (excluding LpSWEET1c and LpSWEET16b) in their promoter regions. All three categories of the aforementioned cis-acting elements were linked to ABA-induced responses and occur in the promoters of various stress-resistance genes. LTRs (low-temperature responses) were identified in 15 LpSWEET genes; AREs (antioxidant response elements), which are generally involved in hypoxic conditions, were identified in 14 LpSWEET genes; MBSs (MYB binding sites involved in drought inducibility) were detected in 12 LpSWEET genes, whereas EREs (ethylene-responsive elements) were detected in 9; and W-box elements (binding sites for WRKY transcription factors), which are typically regulated by salicylic acid (SA) and occur in disease-resistance genes, were detected in 8 LpSWEET genes (Figure 6).
Figure 6. Analysis of cis-acting elements in the promoter regions of LpSWEET genes. PlantCARE was used to identify cis-acting elements using the 2000 bp upstream sequences of LpSWEET genes. TBtools (version 2.400) was employed to illustrate the location, type, and number of response elements in the LpSWEET promoters.

2.7. Expression Pattern of LpSWEET Genes Under Drought Stress

To examine their drought responsiveness we analyzed 12 LpSWEET genes with MBS or ABRE cis-acting elements in their promoter regions. The results showed that the transcript levels of LpSWEET4d, LpSWEET5a, and LpSWEET13 increased after 6 h (hours) of drought treatment and reached a peak at 12 h. The expression levels of LpSWEET2a and LpSWEET16a genes showed no significant difference under drought treatment. The transcripts of LpSWEET2c and LpSWEET16c were induced after 12 h of drought treatment, while that of LpSWEET2b significantly decreased after 2 h of drought treatment (Figure 7). These results indicate that LpSWEET genes exhibit drought responsiveness.
Figure 7. Expression analysis of the 12 LpSWEET genes under drought stress. The relative expression levels of the LpSWEET genes were determined using the 2−ΔΔCT method. The same letter above each column denotes the absence of significant differences at p < 0.05 on the basis of Duncan’s test.

2.8. Overexpression of LpSWEET13 Increases Drought Stress Tolerance in A. thaliana

The expression of LpSWEET13 was markedly increased under drought stress, indicating its potential involvement in drought tolerance regulation. Multiple sequence alignments of LpSWEET13 with OsSWEET13 (Os12g29220), ZmSWEET13a (Zm00001d023677), ZmSWEET13b (Zm00001d023673), and ZmSWEET13c (Zm00001d041067) revealed high sequence identities of 76.79%, 73.31%, 72.05%, and 69.76%, respectively (Figure S2). First, predictive analysis of the subcellular localization of LpSWEET13 indicated that this gene was localized to the plasma membrane (Table S1). To verify this hypothesis, we evaluated the localization of LpSWEET13 by co-expressing LpSWEET13-GFP and AtAKT1-mCherry in tobacco leaves. The results displayed that the fluorescence signal of LpSWEET13 was colocalized with that of AtAKT1, whereas the fluorescence signals of the control GFP was not colocalized with that of AtAKT1. These findings confirm that LpSWEET13 is localized to the plasma membrane (Figure 8).
Figure 8. Analysis of subcellular localization of LpSWEET13. Co-localization analysis of GFP (A) and LpSWEET13-GFP (B) with AtAKT1-mCherry in tobacco leaf cells. The images show GFP fluorescence, mCherry fluorescence, bright field, and the merged image of the three. Bar = 20 µm.
To gain deeper insight into the function of LpSWEET13 under drought stress, two LpSWEET13-overexpressing Arabidopsis lines (OE17, OE24) were generated via the floral dip method, and their expression levels were examined via semi-quantitative RT-PCR (Figure 9A) and qRT-PCR (Figure S3). Both the overexpression lines and wild-type (WT) Arabidopsis plants were cultivated in soil for 4 weeks, after which seedlings representing different genotypes were exposed to an 18 d (days) drought treatment. Phenotypic changes were monitored until wilting symptoms appeared, and survival rates were recorded after 5 d of rehydration. The transgenic lines displayed a higher survival rate than the WT, accompanied by lower electrolyte leakage (EL) levels (Figure 9). These results indicate that overexpression of LpSWEET13 enhances plant drought resistance.
Figure 9. Evaluation of drought tolerance in LpSWEET13 overexpressing A. thaliana at the rosette stage. Representative images of 3-week-old seedling of WT (Col-0) and LpSWEET13 OE lines following 18 d drought treatment. (A) Semi quantitation RT-PCR analysis of LpSWEET13 transcription levels in plants. (B) Plants photographed after 5 d of rehydration. (C) Survival rate calculation. (D) EL analysis of leaves from WT (Col-0) and LpSWEET13 OE lines. Values are mean ± SD (n = 3 replicates, 5 seedling/replicate). The same letter above each column denotes no significant difference at p < 0.05 on the basis of Duncan’s test.
To investigate the effect of LpSWEET13 ectopic expression on transgenic A. thaliana, four drought-responsive genes, namely AtP5CS1, AtRD22, AtRD29A, and AtRD29B, were selected for analysis. Under normal conditions, we observed relatively similar gene expression patterns between WT and transgenic Arabidopsis plants. In contrast, under drought stress conditions, the relative expression levels of AtP5CS1, AtRD22, AtRD29A, and AtRD29B were significantly higher in the transgenic lines than in the WT plants (Figure 10).
Figure 10. Expression levels of drought-responsive genes in WT and LpSWEET13-overexpressing Arabidopsis lines. The relative expression levels of AtP5CS1, AtRD22, AtRD29A, and AtRD29B were determined using the 2−ΔΔCT method. The same letter above each column denotes the absence of significant differences at p < 0.05 on the basis of Duncan’s test.

3. Discussion

SWEET genes are broadly distributed across plants, animals, and prokaryotes and participate in diverse physiological processes, including the modulation of plant growth and development, mediation of plant–pathogen interactions, and adaptation to stress. This gene family has low-affinity and high-capacity transport characteristics, and comprises an average of 20 paralogs in angiosperms [23]. Research on the SWEET family dates back to the discovery of its first member, MtN3, which was revealed to be a nodule-specific EST in the leguminous plant Medicago truncatula [24]. In 2010, scientists further discovered a novel class of sugar transporters using optical glucose sensors and named them SWEETs [4]. Their core function is to participate in sugar efflux, and based on this characteristic, studies have reported that some genes of the SWEET family act as pivotal determinants of phloem loading [3] and nectar secretion [25]. Recently, many studies have focused on the identification of SWEET members and their potential functions in various plant species [23,26,27,28]. However, the regulatory functions of the SWEET gene family are divergent across a range of species, necessitating further species-specific data to unravel the biological functions of these genes.
In the present study, 23 LpSWEET genes were characterized in perennial ryegrass. Predictions showed that most SWEET proteins contain seven transmembrane helices (TMs) constituting two MtN3 domains [4,6,29]. The present study demonstrated that most LpSWEET proteins contained seven TMs, with TM1–3 and TM5–7 forming two MtN3 domains. The region exhibited strong evolutionary conservation. Phylogenetic analysis indicated that LpSWEETs could be classified into four clades, consistent with those identified in A. thaliana, O. sativa, and Z. mays. Clades II and III represented the predominant groups across plant lineages, a trend that was consistently observed in the present study. Studies on sugar transport characteristics have shown that SWEET proteins in Clades I, II, and IV facilitate the transport of hexoses, such as glucose, fructose, and galactose, whereas members of Clade III are involved in SUT [2,3,25].
Gene and segmental duplications represent key evolutionary processes that drive gene copy number expansion and contribute to the emergence of novel gene functions [30]. In this study, we examined duplication events within the LpSWEET gene family of perennial ryegrass, identifying two pairs of gene or segmental duplication pairs among the 23 SWEET genes. These results suggest that gene duplication serves as a key mechanism in expanding the SWEET gene family. Additionally, there was one pair of homologous genes between perennial ryegrass and Arabidopsis, and shares 11 pairs of homologous genes with rice and 12 pairs with maize, with these genes occurring more frequently on Chromosome 3. Notably, this chromosome was a hotspot region containing 11 LpSWEET genes.
Regulatory elements located within promoter regions play key roles in controlling gene expression [31]. This study identified nine types of stress-related response elements within the promoter regions of LpSWEET genes, including three ABA-responsive elements (MYB elements, MYC elements, and ABREs) [32]. We also identified other stress-responsive elements: MBSs, LTRs, W-box, EREs, and AREs mediate the drought, low-temperature, SA, ethylene, and anaerobic responses, respectively [33,34]. These results indicate that LpSWEET genes might be involved in plant adaptation to abiotic stress through hormonal regulation. The promoter regions of all 12 selected SWEET genes contained MBSs or ABREs elements, and four of these LpSWEET genes can be upregulated in response to drought stress.
Research has demonstrated that SWEET proteins participate in plants’ responses to drought. In Arabidopsis, AtSWEET17 functions as a carbohydrate transporter, facilitating vacuolar sugar transport in roots, and is essential for drought tolerance [35]. In Brassica juncea, the transcript levels of BjSWEET17.2, BjSWEET17.4, BjSWEET12.2, and BjSWEET12.3 are significantly upregulated under drought conditions [26]. In O. sativa, the ABA-responsive transcription factor OsbZIP72 binds to the promoters to induce high expressions of OsSWEET13 and OsSWEET15, which may potentially regulate sucrose transport and distribution under drought and salt stress [36]. By contrast, the transcription levels of ZmSWEET3a and ZmSWEET3b genes are significantly repressed under drought stress [8]. Sequence alignment analysis revealed that the homology between LpSWEET13 and OsSWEET13 was 76.79%, which was higher than that between LpSWEET13 and ZmSWEET3a (73.31%) or ZmSWEET3b (72.05%). Further promoter analysis of ZmSWEET3a, ZmSWEET3b, ZmSWEET3c, and OsSWEET13 genes demonstrated that only ZmSWEET3a had a fewer number of cis-acting elements, while the characteristics of cis-acting elements in the promoters of the other genes showed no significant differences (Figure S4). To verify the function of LpSWEET13 in drought tolerance, we generated LpSWEET13-overexpressing Arabidopsis lines and investigated their phenotypic performance under drought conditions. The results showed that LpSWEET13-overexpressing Arabidopsis plants exhibited a markedly increased survival percentage compared with the WT controls, while their EL levels were substantially reduced relative to the WT plants. The results imply that LpSWEET13 can enhance plant drought tolerance. In addition, the transcripts of AtP5CS1, AtRD22, AtRD29A, and AtRD29B were induced after drought treatment, with AtRD22, AtRD29A, and AtRD29B representing stress- and ABA-responsive marker genes that are involved in drought adaptation [37]. We found that the expression of AtP5CS1, AtRD22, AtRD29A, and AtRD29B was significantly increased in LpSWEET13-overexpressing Arabidopsis lines compared to WT plants after drought treatment.

4. Materials and Methods

4.1. Identification of the SWEET Gene Family in Perennial Ryegrass

The high-quality genome-wide dataset of Lolium perenne L. (perennial ryegrass) was retrieved from the dedicated Ensembl Plants platform (https://plants.ensembl.org/Lolium_perenne/Info/Index, accessed on 1 November 2025). The Hidden Markov Model (HMM) [38] profile associated with the SWEET domain (PF03083) was obtained from the Pfam database (http://pfam.xfam.org/, accessed on 1 November 2025), and the HMMER software (version 3.3.2) was adopted to scour the MtN3_saliva domain in the perennial ryegrass proteome. Using rice SWEET protein sequences as queries, BLASTP searches were conducted against the perennial ryegrass protein dataset under a stringent threshold of 1 × 10−10 for the E-value and a sequence identity requirement of no less than 50%. After systematically comparing and rigorously parsing the hits generated by HMMER and BLAST, a self-BLAST analysis was subsequently conducted on the resulting sequences to eliminate redundant entries. Alternative splice variants were not taken into account throughout the entire analytical process. Following manual curation and verification, 23 candidate SWEET proteins from perennial ryegrass were successfully identified. To further validate the reliability of the predicted LpSWEET genes, two specialized bioinformatics resources were employed: the NCBI-CDD web server (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 10 November 2025) and the SMART database (https://smart.embl.de/, accessed on 10 November 2025). To characterize the physicochemical properties property characterization of the retrieved proteins, the ExPASy server (http://www.expasy.org, accessed on 11 November 2025/) was utilized, with its compute pI/Mw tool enabling the accurate assessment of MW and pI. Additionally, the Cello web server (https://wolfpsort.Hgc.Jp/, accessed on 11 November 2025) was employed to infer the subcellular localization of the identified SWEET proteins.

4.2. Phylogenetic Analysis of LpSWEET Genes

Phylogenetic relationships were inferred from the complete amino acid sequences of AtSWEETs, OsSWEETs, and ZmSWEETs, together with LpSWEETs (Table S5). Multiple sequence alignment was carried out using ClustalW, and evolutionary relationships were reconstructed using the ML method implemented in MEGA11 (https://www.megasoftware.net/, accessed on 13 November 2025) [39]. All parameters employed were set to the default values, with bootstrap support assessed using 1000 replicates. Based on phylogenetic tree topology, as well as reference classifications of AtSWEETs and OsSWEETs, LpSWEETs and ZmLpSWEETs were assigned to four clades.

4.3. Transmembrane Topology, Conserved Motifs, and Gene Structure Analysis

Using TBtools (version 2.400) [40], we systematically analyzed and visualized the exon–intron structures and splicing patterns of LpSWEET genes (Table S6, Figure 2). To identify and characterize conserved motifs and domains, the web-based MEME Suite platform (http://meme-suite.org/, accessed on 15 November 2025) was employed with parameter configurations as follows: up to 12 motifs were allowed to be detected, and motif lengths were constrained within a range of 5–200 aa. For the computational prediction of transmembrane helical architectures in LpSWEET family proteins, TMHMM version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 5 November 2025), was employed, with the respective aa sequences of these LpSWEET proteins serving as input data.

4.4. Chromosomal Location and Gene Duplication of LpSWEET Genes

A circos diagram was constructed using the genome annotation file to characterize the chromosomal distribution of LpSWEET genes, and the loci of LpSWEET genes on their respective chromosomes were precisely mapped via the TBtools software package. Furthermore, synteny correlation analysis among SWEET genes of perennial ryegrass and those of Arabidopsis, rice, and maize was validated, and the resulting syntenic relationships were visualized using TBtools.

4.5. Structural Analysis of LpSWEET Proteins at Multiple Levels

Secondary structural characteristics in LpSWEET proteins were computationally inferred via the SOPMA platform (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html, accessed on 16 November 2025), with a similarity threshold of eight. For tertiary-structure modeling, the SWISS-MODEL platform (https://swissmodel.expasy.org/, accessed on 16 November 2025) was utilized to screen for documented protein structures sharing a ≥30% sequence identity with LpSWEET sequences. The structures with the highest match that met this criterion were designated as templates for constructing the target protein models. Subsequently, the three-dimensional (3D) structures of all 23 LpSWEET proteins were generated and visualized based on these templates.

4.6. Promoter Analysis for LpSWEETs

To elucidate the functionalities of SWEET genes, promoter alignments spanning 2000 bp upstream of the ATG initiation codon were subjected to analysis via the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 17 November 2025) [41] for the detection of cis-regulatory elements in perennial ryegrass, and the resulting information was visualized using TBtools.

4.7. Expression Patterns of LpSWEET13 in Perennial Ryegrass and Drought-Responsive Genes in Arabidopsis Following Drought Treatment

Uniform perennial ryegrass seedlings pre-cultured in 1/2 Hoagland nutrient solution for 3 weeks were selected and transplanted into fresh Hoagland nutrient solution for further hydroponic culture for another 2 weeks, with the nutrient solution renewed every 3 d. To investigate the expression pattern of the LpSWEET13 gene under drought stress, the seedlings were subjected to simulated drought treatment with 15% (w/v) PEG6000 (commonly used to mimic drought stress) for 6, 12, and 24 h, respectively. Leaf samples were collected at each treatment time point for total RNA extraction, and three biological replicates were carried out.
To further verify the response of the LpSWEET13 gene to drought stress, homozygous T3 lines and WT plants were grown on 1/2 MS medium for 10 d, and then transferred to 1/2 Hoagland nutrient solution alone or supplemented with 15% (w/v) PEG6000. After 3 d of treatment, quantitative real-time reverse transcription PCR (qRT-PCR) was performed to detect the expression levels of drought-responsive genes in seedlings, including AtP5CS1, AtRD22, AtRD29A, and AtRD29B.

4.8. Quantitative Real-Time PCR Analysis

Real-time quantitative polymerase chain reaction assays were conducted following the experimental protocol described in a previous study [42], and total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen Inc., Beijing, China). For first-strand cDNA synthesis, 1 μg of the isolated total RNA was reverse-transcribed with the HiScript IV 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China). Reverse-transcription–quantitative polymerase chain reaction (RT-qPCR) was performed using a Takara Thermal Cycler Dice Real-Time System (Takara, Otsu, Japan), with three biological replicates for all independent samples. The 10 μL RT-qPCR mixture was composed of 0.2 μM each of the forward and reverse primers, 200 ng of cDNA template, and 5 μL of SYBR qPCR Master Mix. The amplification protocol was programmed as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s. The gene-specific primers utilized for the qRT-PCR assay are presented in Table S7, with LpeIF4A selected as the reference genes for normalization [43,44].

4.9. Subcellular Localization

The coding sequence (CDS) of LpSWEET13, with the termination codon excluded, was PCR-extended and subsequently cloned into the pCAMBIA-1305 vector to generate a green fluorescent protein (GFP)-tagged fusion protein, which was regulated transcriptionally by the CaMV 35S promoter. By combining the constructs 35S::GFP and 35S::LpSWEET13-GFP with 35S::AtAKT1-mCherry, we conducted co-transformation on the leaves of 4-week-old Nicotiana benthamiana plants using the Agrobacterium-mediated transformation method. After 48–72 h of transformation, fluorescent signals were illustrated using a confocal laser scanning microscope (CLSM; Carl Zeiss SAS, Jena, Germany).

4.10. Generation of Transgenic Plants Drought Tolerance Assays

The CDS of LpSWEET13 was cloned into the pCAMBIA3301 vector, which was placed transcriptionally by the CaMV 35S promoter to construct the recombinant expression plasmid. Young four-week-old A. thaliana ecotype Col-0 plants were genetically engineered via the A. tumefaciens strain EHA105 carrying the aforementioned recombinant plasmid, with a modified floral dip protocol adopted for stable transformation [45]. The transgenic plants were subsequently subjected to Basta resistance screening to identify positive transformants, and homozygous T3 transgenic lines were ultimately obtained and harvested for subsequent experimental analyses. The expression levels of the transgenic lines were quantified by semi-quantitative RT-PCR using the specific primers 5′-CCCCTGGGCATTTGCTTTTG-3′ and 5′-TGGACTTTACCAGCGCGTAG-3′, with AtActin2 serving as the internal control. After generating homozygous T3 lines exhibiting high expression abundances, seeds of both the overexpression lines and WT were sown separately on 1/2 MS medium. The seeds were vernalized at 4 °C for 3 d, and subsequently transferred to a 16/8 h (light/dark) photoperiod cycle for 7 d, followed by transplantation into a soil–perlite mixture at a volume ratio of 2:1. The plants were grown for 1 month under the following conditions: 23 °C, an irradiance of 200 μmol m−2 s−1, and a 16/8 h (light/dark) photoperiod cycle. Following this, the growth performance of diverse genotypes under control conditions was imaged and documented. One day before the drought treatment, the different genotypes of Arabidopsis seedlings with different genotypes were thoroughly watered at a single time. Seeding of different genotypes were incubated under drought treatment for 18 d, and after 5 d of rehydration, the survival rate was calculated and the seedlings were photographed.

5. Conclusions

Through bioinformatics analysis, 23 LpSWEET family members were identified in perennial ryegrass. All LpSWEET proteins possessed the typical MtN3 domains, which was consistent with the conserved characteristics of SWEET proteins in most plant species. Phylogenetic analysis classified the LpSWEET genes into four subfamilies; promoter analysis demonstrated that the LpSWEET genes were enriched in cis-acting elements responsive to ABA, cold, drought, and other factors, implying that the expression pattern of LpSWEET genes might be dictated by multiple environmental signals and hormone pathways; and qRT-PCR analysis demonstrated that LpSWEETs could respond to drought stress. In addition, overexpression of LpSWEET13 improved the drought tolerance of transgenic A. thaliana. The results of this study will facilitate further elucidation of the roles of the LpSWEET gene family and provide a reference for the molecular breeding of perennial ryegrass.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants15040526/s1, Figure S1: The transmembrane domain of LpSWEET proteins. Figure S2: Multiple sequence alignment of LpSWEET13, OsSWEET13, ZmSWEET13a, ZmSWEET13b, and ZmSWEET13c was performed. Figure S3: qRT-PCR analysis of LpSWEET13 transcription levels in plants. Figure S4. Analysis of cis-acting elements in the promoter regions of ZmSWEET13a, ZmSWEET13b, ZmSWEET13c, and OsSWEET13 genes. Table S1: Complete list of LpSWEET genes identified in the perennial ryegrass genome. Table S2: Secondary structure classification of LpSWEET proteins. Table S3: List of perennial ryegrass with perennial ryegrass, A. thaliana, O. sativa, and Z. may SWEET syntenic gene pairs. Table S4: Summary of cis-elements is in the promoter regions of SWEET family genes in perennial ryegrass. Table S5: SWEET aa sequences of A. thaliana, O. sativa, and Z. mays. Table S6: The details of the motifs, conserved domains, and exons/introns of LpSWEET members. Table S7: Primer sequences in this study.

Author Contributions

Conceptualization, Y.X.; Methodology, Y.M. (Yuxi Miu) and Y.M. (Yingying Mao); software, Y.M. (Yuxi Miu), S.P. and Y.M. (Yingying Mao); validation, Y.M. (Yuxi Miu), Y.M. (Yingying Mao) and M.C.; formal analysis, Y.M. (Yuxi Miu), S.P., Y.M. (Yingying Mao) and Y.X.; investigation, S.P., M.C. and Y.X.; resources, Y.M. (Yuxi Miu), S.P., Y.M. (Yingying Mao) and M.C.; data curation, Y.M. (Yuxi Miu), M.C. and Y.X.; writing—original draft, S.Y.; writing—review & editing, S.P., L.Z. and S.Y.; visualization, L.Z. and S.Y.; supervision, L.Z. and S.Y.; project administration, L.Z. and S.Y.; funding acquisition, L.Z. and S.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Zhejiang Provincial Natural Science Foundation of China (Grant No. LQN25C170004), Personnel Startup Project of the Scientific Research and Development Foundation of Zhejiang A&F University (Grant Nos 2021FR041 and 2023LFR039), the National Natural Science Foundation of China (Grant Nos 32503274 and 32171687), and Zhejiang Provincial Innovation and Entrepreneurship Project (Grant No. S202510341012).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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