Identification and Characterization of SWEET Gene Family in Peanuts and the Role of AhSWEET50 in Sugar Accumulation

Abstract

The SWEET (sugars will eventually be exported transporter) gene family represents a novel class of sugar transporters capable of bidirectionally transporting sugars along the concentration gradient. In this study, we identified 50 SWEET genes from the peanut cultivar Shitouqi, which were phylogenetically classified into four clades. Promoter analysis revealed that the AhSWEET genes contain multiple cis-acting elements associated with stress responses, growth regulation, and hormone signaling, suggesting their potential roles in plant development and adaptation to environmental challenges. Transcriptome profiling highlighted AhSWEET50 as the most highly expressed member during early seed development stages in both low- and high-sucrose peanut cultivars and also highly expressed at the mature stage. Subcellular localization confirmed the presence of AhSWEET50 in both the plasma membrane and cytoplasm, with predominant expression observed in embryos. The heterologous overexpression of AhSWEET50 in Arabidopsis significantly increased soluble sugar accumulation when compared to wild-type plants. These results validate the functional role of AhSWEET50 in sugar transport and provide a foundation for understanding the mechanisms of sugar allocation in peanuts, which has implications for improving seed quality through metabolic engineering.

1. Introduction

As the fourth largest oil crop globally, peanut (Arachis hypogaea) possesses significant nutritional and economic value. The seed kernels are abundant in oil, protein, and functional secondary metabolites [1]. Peanuts are extensively utilized in food processing and edible oil extraction and as industrial raw materials [1]. Notably, carbohydrates are recognized as a crucial component of the dry matter in peanut kernels and play an essential role in flavor quality [2]. Despite this, research on peanuts has predominantly concentrated on quality traits such as oil content, protein levels, and fatty acid composition, with limited studies addressing the sucrose traits of peanuts.

In recent years, the breeding and research of high-sucrose sweet edible peanut varieties have garnered increasing attention. Plant sugar transporters are key proteins that regulate the transport and distribution of carbohydrates between source and sink organs, as well as within organs [3]. Understanding their functional mechanisms is of great significance. Research has identified three main types of sugar transporter proteins involved in sugar transport in plants: monosaccharide transporters (MSTs) [4], sucrose transporters (SUTs) [5], and sugars will eventually be exported transporters (SWEETs) [6]. Among these, the SWEET gene family consists of transmembrane proteins that are energy-independent, facilitating the bidirectional transport of saccharides through concentration gradients [6]. This family was first identified by Chen et al. using fluorescence resonance energy transfer (FRET) technology in the model plant Arabidopsis thaliana [6]. Plant SWEET proteins belong to the MtN3/saliva family (PF03083), with the N-terminal and C-terminal located on the outer and inner sides of the cell cytoplasm, respectively. They typically contain seven α-helical transmembrane domains (TMs), with the fourth TM being less conserved and primarily functioning as a linker. This linker divides the protein into two MtN3/saliva structural domains, each containing three TMs, forming a 3-1-3 structure [7]. In Arabidopsis, the SWEET family members can be systematically categorized into four subfamilies (I–IV), with different subfamilies exhibiting selective recognition abilities for substrates such as glucose, sucrose, and fructose [8]. Notably, members of branch III (such as AtSWEET11 and AtSWEET12) play a crucial regulatory role in seed filling [8].

SWEET proteins facilitate the uptake and efflux of sucrose in cells, operating down a concentration gradient through bidirectional transmembrane transport [9]. The SWEET gene family has been identified in many species, including wheat, pepper, sweet potato, and so on [10,11,12]. Transcriptome analysis and the comparison of gene expression profiles of peanut varieties ICG 12625 and Zhonghua 10 during seed development revealed that 30 SWEET genes were involved in sucrose metabolism [13]. In Arabidopsis, members of Clades III are primarily responsible for sucrose transport, while those in Clades I, II, and IV predominantly transport hexose sugars [14]. Furthermore, Clade IV SWEET proteins are localized to the vesicular membrane and specifically export fructose in Arabidopsis [15]. The knockout of OsSWEET4 and OsSWEET11 in rice (Oryza sativa) leads to a decrease in starch content and an increase in glucose and sucrose levels in rice seeds [16]. In barley (Hordeum vulgare), the knockdown of HvSWEET11b resulted in smaller seeds and reduced starch content [17]. The silencing of SlSWEET7a and SlSWEET14 in tomato (Solanum lycopersicum) significantly reduces the starch content of mature tomato fruit [18]. In apple (Malus domestica), the MdSWEET9b gene promotes sucrose transport, leading to increased sugar accumulation in fruit [19].

The sucrose content not only determines the flavor of peanuts but also directly affects the quality of deep-processed products, such as candy and baked goods. However, the sucrose content of most peanut varieties in China is low. Although a small number of high-sugar varieties have emerged in recent years, they still struggle to meet market demand. Therefore, the cultivation of new edible peanut varieties with a high sucrose content and excellent taste has become an urgent key challenge. In recent years, SWEET family sugar transporters have been shown to play a significant role in plant growth and physiological regulation. In this study, based on the genomic data of the peanut cultivar Shitouqi, we systematically mined and identified the members of the peanut SWEET family and comprehensively analyzed their phylogeny, chromosome distribution, gene structure, conserved motifs, cis-acting elements, and expression patterns in high- and low-sugar peanut varieties. Additionally, we cloned the AhSWEET50 gene and explored its expression characteristics and subcellular localization in different tissues and across high- and low-sugar varieties. By constructing AhSWEET50 overexpression transgenic plants and measuring soluble sugar content, this study provides a solid theoretical basis for elucidating the function of the SWEET gene family in regulating peanut sugar metabolism and offers key gene resources for the cultivation of new high-sugar peanut cultivars.

2. Materials and Methods

2.1. Plant Materials and Methods

The Nanbeitian cultivar, which had a sucrose content of 7.00%, was provided by Zhongkai University of Agriculture and Engineering. In addition, the low-sugar cultivar Minhua 16 (MH16; sucrose content of 3.25%) and high-sugar cultivar Minhua 175 (MH175; sucrose content of 9.38%) were supplied by Research Center of Leguminous Oil Plant Genetics and Systems Biology, Fujian Agriculture and Forestry University. These peanuts were cultivated in a light incubator set to 28°C, adhering to a photoperiod of 16 h of light and 8 h of darkness to fulfill the photoperiodic requirements necessary for optimal growth and development.

2.2. Identification of Members of the Peanut SWEET Gene Family

The genomic information of the tetraploid peanut cultivar Shitouqi was downloaded from the Peanut Genome Resource (PGR) website (http://peanutgr.fafu.edu.cn/Download.php, accessed on 6 March 2024) for subsequent data analysis [20]. The hidden Markov model (HMM) file PF03083 was obtained from the Pfam database (http://pfam.xfam.org/, accessed on 6 March 2024) to serve as a search model for SWEET sequences in the genome of the peanut cultivar Shitouqi [21]. Additionally, the Blast function in TBtools software (v2.210) was employed to retrieve sequences in the genome of Shitouqi that were similar to the known SWEET protein sequence in Arabidopsis, using the latter as a reference [22]. Based on these two approaches, the SWEET family members were identified. The CD-Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 10 March 2024) from National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/, accessed on 10 March 2024) was utilized to examine the domain of the SWEET protein.

2.3. Phylogenetic Tree Construction and Prediction of Physicochemical Properties

The phylogenetic tree of SWEET proteins in the peanut cultivar Shitouqi was constructed using SWEET proteins from Arabidopsis thaliana and rice as references. The MUSCLE Wrapper function in TBtools software (v2.210) was employed to perform multiple sequence comparisons of SWEET proteins, and the trimAL Wrapper was utilized to refine the resulting multiple sequence comparison files [22]. The phylogenetic tree was constructed using IQ-tree based on the maximum likelihood method [23]. The physicochemical properties of the identified SWEET gene family members, including the number of amino acids, molecular weight, isoelectric point, instability coefficient, and average hydrophobicity, were predicted using the ProtParam online tool available at ExPASy (https://www.expasy.org/resources/protparam, accessed on 20 March 2024) [24].

2.4. Chromosome Localization Analysis

The GFF3 file of the peanut cultivar Shitouqi was downloaded from the PGR online database (http://peanutgr.fafu.edu.cn/Download.php, accessed on 21 March 2024) to obtain the length and positional information of the SWEET gene [20]. Subsequently, the location of the peanut SWEET gene on the chromosome was visualized using TBtools software (v2.210) [22].

2.5. Conserved Motif and Gene Structure Analysis

The conserved motifs of the SWEET gene family were analyzed using the MEME Suite (https://meme-suite.org/meme/tools/meme, accessed on 22 March 2024), with parameters configured to identify 10 conserved motifs while retaining the default settings for all other parameters. The exon–intron structural information of the SWEET gene was sourced from the GFF3 file of the peanut cultivar Shitouqi [20]. The visualization of the conserved motifs and the gene structure of SWEET was accomplished using TBtools software (v2.210) [22].

2.6. Promoter Cis-Acting Element Analysis

The 2000 bp promoter sequence upstream of the SWEET gene was extracted based on the genome and GFF3 file of the peanut cultivar Shitouqi [20]. The function of cis-acting elements in the SWEET gene upstream promoter was predicted using the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 22 March 2024) [25].

2.7. GO Enrichment Analysis

The Shitouqi protein sequence file of the peanut cultivar was downloaded from the PGR website (http://peanutgr.fafu.edu.cn/Download.php, accessed on 23 March 2024) [20]. Subsequently, this protein file was uploaded to the eggNOG-mapper website (http://eggnog-mapper.embl.de, accessed on 23 March 2024) to obtain the functional annotation files for all genes in the peanut cultivar Shitouqi. The functional annotation results generated by TBtools software (v2.210) were employed to produce the background files necessary for GO enrichment analysis [22].

2.8. Expression Pattern Analysis

The expression patterns of the AhSWEET genes were analyzed using transcriptome data collected at six different time points (10, 20, 30, 40, 50, and 60 d) for both the low-sugar cultivar MH16 (sucrose content of 3.25%) and the high-sugar cultivar MH175 (sucrose content of 9.38%). The transcriptional abundance of the SWEET gene was assessed using fragments per kilobase of exon model per million mapped fragments (FPKM), and a heat map was generated using TBtools software (v2.210) [22].

2.9. RNA Extraction and cDNA Synthesis

When the peanuts reached the 5-to-7-leaf stage, new leaf tissues were frozen in liquid nitrogen and subsequently stored in a −80 °C refrigerator for future use. RNA was extracted from the peanut leaves using a modified CTAB method and reverse-transcribed into cDNA. The reverse transcription kit (Ev0M-MLV Reverse Transcription Kit) was obtained from Accurate Biology (Changsha, China), and the cDNA, exhibiting good integrity, was preserved in a −20 °C refrigerator for future use.

2.10. Gene Cloning and Bioinformatic Analysis

The full-length sequence of the AhSWEET50 gene was obtained from the PGR (http://peanutgr.fafu.edu.cn/Download.php, accessed on 30 March 2024) as reported by Zhuang et al. [20]. Specific primers for AhSWEET50 were designed to amplify its cDNA fragment. The conserved domain of the protein was predicted using CD-Search in the NCBI database. The ExPASy online analysis tool was employed to assess the physicochemical properties of the protein encoded by the target gene. The secondary and tertiary structures of the target gene’s protein were predicted using the SOPMA and SWISS-MODEL online analysis tools, respectively. The primers utilized in this study are detailed in Supplementary Table S1.

2.11. Subcellular Localization Analysis

The subcellular localization vector of AhSWEET50 was constructed using the Gateway method. The resulting pEarleyGate103-AhSWEET50-GFP plant expression vector was transformed into competent Agrobacterium tumefaciens (GV3101) cells. The vector was transiently overexpressed in tobacco leaves following the protocol established by Wang et al. [26]. After two days, observations and photographs were taken using a fluorescence confocal microscope. The primers utilized in this study are detailed in Supplementary Table S1.

2.12. Determination of Soluble Sugar Content in Transgenic A. thaliana

Transgenic Arabidopsis plants overexpressing the peanut AhSWEET50 gene were obtained by the Agrobacterium-mediated flower-dipping method [27]. The soluble sugar content in transgenic Arabidopsis tissues was determined by high-performance liquid chromatography (HPLC). The primers used are shown in the Supplementary Table S1.

3. Results

3.1. Identification, Phylogenetic Evolution, and Physicochemical Properties of AhSWEET Gene Family in Peanut

A total of 50 AhSWEET genes were identified in the genome of the peanut cultivar Shitouqi (Supplementary Table S2). The phylogenetic tree of AhSWEET proteins was constructed based on the SWEET proteins from Arabidopsis and rice (Figure 1). The results indicated that the AhSWEET proteins clustered into four branches (I–IV), with branch III containing the most members, totaling 18 proteins; branches I and II contained 11 and 16 proteins, respectively, while branch IV had the fewest members, with only 5 (Figure 1). The physicochemical properties of the identified AhSWEET proteins were predicted, including the number of amino acids, molecular weight, isoelectric point, instability coefficient, and average hydrophobicity (Supplementary Table S2). The number of amino acids in AhSWEET proteins ranged from 94 to 962; the lowest molecular weight recorded was 10.43 kDa, while the highest was 108.37 kDa. The isoelectric point varied from 5.81 to 99.73. The instability coefficient ranged from 24.33 to 57.28, with 12 AhSWEET proteins exhibiting coefficients higher than 40, indicating that they are unstable proteins, whereas the remaining AhSWEET proteins are stable. The average hydrophobicity of the 50 identified AhSWEET proteins was greater than 0, suggesting that AhSWEET proteins are hydrophobic.

3.2. Chromosomal Localization of Peanut AhSWEET Genes

A total of 50 AhSWEET genes exhibited an uneven distribution across 18 chromosomes, excluding Chr02 and Chr12. The number of genes ranged from 1 (on Chr01, Chr04, Chr07, Chr09, Chr10, Chr11, and Chr12) to 7 (on Chr13), with significant gene clustering observed on Chr16 and Chr19 (Figure 2). In terms of chromosomal localization, the AhSWEET gene family displayed similarities to those in other species, characterized by gene clustering, which suggests potential analogous functions.

3.3. Conserved Motifs and Gene Structure of Peanut AhSWEET Genes

Phylogenetic analysis classified the AhSWEET proteins into four distinct evolutionary branches (I–IV) (Figure 3A). Conserved motif prediction indicated that members of each branch exhibited characteristic motif composition patterns: all members of branch I contained Motifs 1–5 (with AhSWEET43 containing two instances of Motif 1); only AhSWEET21, AhSWEET40, and AhSWEET49 in branch II lacked Motif 1; and branch III was characterized by the presence of Motif 10 in AhSWEET4 and AhSWEET25, while all members retained Motifs 3, 4, and 6 (with AhSWEET8 containing two instances of Motif 5) (Figure 3B). Gene structure analysis revealed significant variation in the number of exons among the AhSWEET genes, ranging from 1 to 8, with 6 exons being the predominant configuration (Figure 3C). Notably, structural differences were also observed within the same evolutionary branch, exemplified by the contrast between AhSWEET19 (1 exon) and AhSWEET43 (8 exons) in branch I. The findings from this structural analysis suggest that the variation in exon number among AhSWEET genes may underlie the functional differentiation of SWEET gene family members.

3.4. Promoter Cis-Acting Elements of Peanut AhSWEET Genes

By analyzing the cis-acting elements in the promoter region (2000 bp upstream of the transcription start site) of 50 peanut AhSWEET genes, we identified multiple regulatory elements associated with stress response, growth and development, and hormone response (Figure 4). Specifically, in terms of stress responsiveness, AhSWEET31 and AhSWEET22 contained the wound-responsive element WUN-motif and the dehydration/cold/salt stress-responsive element DRE, respectively. Furthermore, 36% of the genes carried the drought-responsive element MBS, 16% possessed the low-temperature-responsive element LTR, and 64% included the defense-related element TC-rich. Regarding growth and developmental regulation, 80% of AhSWEET genes exhibited a phloem tissue expression-related CAT-box element in their promoter region. Hormone response analysis indicated that 62% of the genes contained the methyl jasmonate (MeJA) response elements TGACG-motif and CGTCA-motif, while 70% had the salicylic acid (SA) response element TCA-element. These results systematically reveal that the AhSWEET gene family may play a significant role in the transcriptional regulation mechanisms of various biological processes.

3.5. GO Enrichment Analysis of Peanut AhSWEET Gene Family

To further analyze the biological function of the SWEET gene in the peanut cultivar Shitouqi, we conducted a systematic Gene Ontology (GO) enrichment analysis (Figure 5 and Supplementary Table S3). The GO analysis revealed that, at the molecular functional level, 49 AhSWEET genes were significantly enriched in sugar transmembrane transporter activity (GO:0051119), with 13 genes specifically enriched in sucrose transmembrane transporter activity (GO:0008515). At the cellular component level, 13 genes were significantly enriched in sucrose transport (GO:0015770). These enrichment results underscore the core function of the SWEET gene family in carbohydrate transport at the molecular level. Notably, certain members may be specifically involved in the transmembrane transport of sucrose, providing important insights for subsequent functional studies.

3.6. Expression Pattern of Peanut AhSWEET Genes in High- and Low-Sugar Peanut Cultivars

To investigate the expression characteristics of the AhSWEET gene family members, we analyzed the transcriptomes of low- and high-sugar peanut cultivars at various developmental stages to determine their expression patterns (Figure 6). The results indicated that the expression patterns of the AhSWEET genes clustered within the same branch exhibited significant differences. Notably, AhSWEET9 and AhSWEET10, which clustered in branch I, were expressed at different developmental stages in both low- and high-sugar sucrose peanut cultivars. Furthermore, AhSWEET20 and AhSWEET50, clustered in branch III, showed high expression levels during the early developmental stages (10 d and 20 d) in the low-sugar cultivar MH16, which were significantly reduced during the later developmental stages. However, the expression levels of AhSWEET20 and AhSWEET50 were highly expressed at the 10 d stage and mature stage (60 d) in the high-sugar cultivar MH175 (Figure 6). These findings indicate that AhSWEET20 and AhSWEET50 might play important roles in sugar transport in peanut.

3.7. Characteristics of AhSWEET50 Gene in Peanut

We successfully cloned the AhSWEET50 gene from the peanut cultivar Nanbeitian. The amino acid sequence similarity between this protein and AhSWEET50 in the peanut cultivar Shitouqi was found to be 100%. The full length of AhSWEET50 was 879 bp, encoding 293 amino acids with a molecular weight of 73,317.86 Da. The isoelectric point, aliphatic index, and instability index of AhSWEET50 were 5.81, 27.65, and 38.17, respectively. The secondary structure of the AhSWEET50 protein was analyzed using an online tool. The results indicated that the secondary structure of the AhSWEET50 protein was relatively complete, with α-helix and random coil constituting a significant proportion; α-helix comprised approximately half of the secondary structure, serving as the predominant structural element of the protein.

The expression pattern of the AhSWEET50 gene in different tissues of peanut was analyzed utilizing transcriptome data. The results indicated that the AhSWEET50 gene exhibited high expression in peanut embryos, followed by expression in flowers, while the lowest expression was observed in the pericarp (Figure 7A). Expression was undetectable in other parts, including leaves, roots, root tips, and stems (Figure 7A). The expression of the AhSWEET50 gene was higher in the low-sugar peanut cultivar (MH16) at 20 and 40 d, demonstrating a trend of elevated expression during the early stages of kernel development, followed by a decrease in later stages (Figure 7B). Conversely, in the high-sugar cultivar (MH175), a trend of low expression was observed during the early stages of kernel development, with increased expression in later stages, peaking at 50 and 60 d of kernel development (Figure 7B). Subcellular localization results indicated that the AhSWEET50 protein was predominantly distributed in the cell membrane and cytoplasm (Figure 7C), suggesting that it may perform specific functions within these cellular structures.

3.8. AhSWEET50 Gene Increased the Soluble Sugars

There are three primary types of soluble sugars found in plants: sucrose, glucose, and fructose. To investigate the role of the AhSWEET50 gene in plant sugar transport, we transferred the AhSWEET50 gene into wild-type A. thaliana (WT) using the Agrobacterium-mediated flower-dipping method and subculture to T₃ generation. Fourteen days post-pollination, we measured the soluble sugar content in the tissues of both WT and transgenic plants (Figure 8). Compared to the WT, the total sugar content in the leaves of AhSWEET50-overexpressing plants exhibited significant alterations, with the levels of sucrose, glucose, and fructose markedly higher than those in WT. These results indicate that the overexpression of AhSWEET50 has a profound impact on the accumulation pattern of soluble sugars in the leaves of Arabidopsis, suggesting that this gene may play a crucial role in the regulatory network of plant carbon distribution by modulating sugar transport and metabolic processes.

4. Discussion

The SWEET gene family, a significant class of sugar transporter proteins, plays a crucial role in key physiological processes such as carbohydrate transport, distribution, and storage in plants [28,29]. Notably, there are substantial differences in the number of SWEET gene family members across various plant species, including Arabidopsis [8], wheat (108) [30], and tomato (29) [31]. Based on the classification criteria derived from the phylogenetic trees of Arabidopsis and rice, AhSWEETs could be distinctly classified into four evolutionary branches (Figure 1). In alignment with the distributional characteristics observed in most plant species, the peanut SWEET family was predominantly represented by Clade II (32%) and Clade III (36%). This finding further corroborates the functional conservation of the SWEET gene family throughout the evolutionary process.

Studies have demonstrated that most SWEET proteins are localized within the plasma membrane system and play a crucial role in regulating sugar transmembrane transport [28]. However, different SWEET family members exhibit significant substrate-specific differences. Current research has confirmed that the majority of identified SWEET family members function as plasma membrane-localized glucose or sucrose transporters. Notably, Valifard et al. discovered that SWEET17 possessed unique vacuolar membrane localization characteristics and exhibited bidirectional fructose transport activity in heterologous systems, thereby revealing a new dimension of SWEET protein functionality [32]. Additionally, SlSWEET7a and SlSWEET14 had been confirmed as plasma membrane-localized sugar transporters with dual functions in transporting hexose and sucrose [18]. In this study, the plasma membrane localization characteristics of AhSWEET50 were verified through the confocal microscopy of the 35S::AhSWEET50::GFP fusion protein (Figure 7C), suggesting its potential role in plasma membrane-mediated sugar transport. Collectively, these findings indicate that the SWEET protein family exhibits significant functional differentiation in terms of subcellular localization and substrate selectivity.

The expression patterns of SWEET genes vary across different plant species. In tomato, S1SWEET7a and S1SWEET14 were primarily expressed in fruits, indicating their significant roles in fruit sucrose unloading [18]. AnmSWEET5 and AnmSWEET11 exhibited high expression levels during the early stages of fruit development, which subsequently declined as development progressed. Conversely, AnmSWEET21 and AnmSWEET6 displayed an initial increase in expression followed by a decrease [33]. This suggests that different SWEET gene members may sequentially regulate fruit sugar transport and accumulation. In this study, the peanut AhSWEET50 gene was found to be highly expressed in embryonic tissues (Figure 7A) and exhibited distinct expression patterns in high-sugar versus low-sugar cultivars. In low-sugar cultivars, AhSWEET50 was highly expressed during the early stages of kernel development but was down-regulated later. In contrast, high-sugar cultivars showed low expression initially, followed by up-regulation in later stages (Figure 7B). This differential expression pattern indicates that AhSWEET50 may have varying regulatory roles in the development of high- and low-sugar peanut kernels, potentially influencing sugar accumulation during kernel maturation. These findings provide important insights into the functional differentiation of SWEET genes during peanut seed development.

In higher plants, sucrose, glucose, and fructose are the primary soluble sugars, and their distribution involves source–sink transport and organelle transport, which are strictly regulated by the physiological needs of the plant [34]. The SWEET protein family serves as a crucial regulator of sugar transport, directly influencing seed size, weight, and crop yield by mediating the transport of soluble sugars to developing seeds [8]. Several studies have substantiated this mechanism: the ZmSWEET4c gene was selected during the domestication process, with its mutants exhibiting defects in endosperm development, an “empty seed coat” phenotype, and significantly reduced starch content [35]; Arabidopsis AtSWEET11/12/15 genes were specifically expressed in seeds, and mutations in these genes lead to delayed embryo development [36]. The OsSWEET11 mutation in rice resulted in endosperm defects and abnormal starch accumulation [37]; the knockout of GmSWEET15a/b in soybean led to reduced embryo sugar content and seed abortion [38]. Collectively, these findings reveal the conserved function of SWEET genes in seed development. In this study, we analyzed AhSWEET50 transgenic Arabidopsis and found that the total sugar content in the leaves of overexpressing plants was significantly increased. The contents of sucrose, glucose, and fructose were approximately 10% higher compared to the WT (Figure 8). Given the specific expression pattern of AhSWEET50 in peanut kernels, we speculate that this gene may play a role in kernel development by regulating sugar transport. Therefore, in subsequent studies, we will develop a genetic transformation system for peanut to further characterize the role of the candidate gene AhSWEET50 in sucrose transport, as well as to observe whether agronomic traits are altered in overexpressing transgenic peanut plants. These results provide critical evidence for elucidating the molecular function of AhSWEET50 in peanut seed sugar accumulation and also suggest potential target genes for improving crop yield.

5. Conclusions

In this study, a total of 50 SWEET genes were identified from the peanut cultivar Shitouqi, which could be categorized into four distinct groups. The upstream promoter of the AhSWEET gene contained multiple cis-acting elements associated with stress response, growth, and hormonal regulation, suggesting that the AhSWEET gene may play a significant role in plant responses to various stresses, as well as in the regulation of growth and development. Transcriptomic analysis revealed that AhSWEET50 was highly expressed during the early developmental stages of peanut varieties with both high and low sucrose levels. Furthermore, we successfully cloned the AhSWEET50 gene from peanut varieties, which was predominantly expressed in embryos and was localized within the cell membrane and cytoplasm. The overexpression of the AhSWEET50 gene in Arabidopsis resulted in an increased soluble sugar content in the plants. Preliminary findings indicate that the AhSWEET50 gene may be involved in the sugar metabolism of peanuts. This study provides a theoretical basis for further exploring the molecular mechanism of sugar transport in peanut and also lays a foundation for regulating sucrose metabolism and creating a new functional peanut germplasm with high-sugar characteristics and a high sensory quality.