Sucrose transport gene FaSWEET9a regulated by FaDOF2 transcription factor promotes sucrose accumulation in strawberry

Sucrose transport gene FaSWEET9a regulated by FaDOF2 transcription factor promotes sucrose accumulation in strawberry | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sucrose transport gene FaSWEET9a regulated by FaDOF2 transcription factor promotes sucrose accumulation in strawberry Yan Xu, Shuang Liu, Hongying Sun, Jian Zang, Chao Zhang, Wei Guo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6529445/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract This study identified and characterized 25 members of the SWEET gene family in the genome of cultivated strawberry ( Fragaria × ananassa cv. ‘Yanli’), focusing on their potential roles in fruit development. Notably, FaSWEET9a , a specific member of the SWEET family, was found to be uniquely expressed in ‘Yanli’ fruit. Functional analysis via heterologous expression in Saccharomyces cerevisiae confirmed that FaSWEET9a acts as a sucrose transporter. To further investigate its role, we generated FaSWEET9a overexpression lines and demonstrated that FaSWEET9a not only enhances sucrose accumulation in strawberry fruits but also influences plant growth and development. We identified FaDOF2 that could bind to the promoter of FaSWEET9a and enhance its transcription by conducting yeast one-hybrid assays, electrophoretic mobility shift assays, β-glucuronidase assays, and luciferase reporter gene assays. Moreover, transient transformation experiments revealed that FaDOF2 could elevate sucrose content in strawberry fruits by regulating FaSWEET9a . This research brings new viewpoints on the molecular mechanisms that govern sucrose regulation in strawberry fruits, spotlighting the functional significance of the FaSWEET9a -FaDOF2 regulatory module in the aspects of fruit quality and development. FaSWEET9a FaDOF2 Sucrose transport Fruit sweetness Gene regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Message FaSWEET9a, an important sucrose transport gene regulated by transcription factor FaDOF2, regulates the accumulation of sucrose in strawberry fruits and affects the growth of strawberry plants. Introduction Sucrose, glucose, and fructose are key products in the carbon metabolism of plants. In the process of fruit development, an adequate supply of sucrose is essential for promoting fruit growth and maturation. Sucrose modulates gene expression related sugar transport and metabolism, improving fruit quality. Sucrose metabolism represents the most common and efficient sugar metabolic pathway. Within the cytoplasm of fruit cells, rapid and incessant repetitions of sucrose breakdown and rebuilding take place. Sucrose synthase (SUS) facilitates both the breakdown and formation of sucrose, while sucrose phosphate synthase (SPS) specifically catalyzes sucrose synthesis (Li et al. 2020 ). Among these enzymes, SUS plays a pivotal regulatory role in this cycle. Within the vacuole, invertase (INV) catalyzes the hydrolysis of sucrose into hexoses, although some of these hexoses are recycled back into the cytoplasm for sucrose synthesis. This cycle is thought to enhance the efficiency of sugar storage and provide sucrose equivalents (hexoses) in a stored form. In the apoplast, cell wall invertases (CWINV) facilitate the hydrolysis of sucrose, with the majority of the resulting hexoses being recycled into the cytoplasm for sucrose synthesis (Lu et al. 2024 ). The metabolic cycles of sucrose synthesis and degradation work together to regulate sugar accumulation in fruits (Nguyen et al. 2001). Fruits serve as vital ‘sink’ organs within plants, and sucrose transport and metabolism are of great significance in their growth and development. The sucrose transport pathway in fruits encompasses phloem transport, unloading, intercellular transfer, and metabolic utilization. Sucrose is primarily transported from source organs such as leaves to ‘sink’ organs like fruits through the phloem sieve tubes. Photosynthesis in leaves leads to the synthesis of sucrose. This newly-formed sucrose is then actively moved into the sieve tubes with the help of the sieve tube-companion cell complex. Subsequently, it travels to the fruits as a result of the pressure flow gradient (Wang et al. 2021 ). Key transporters in this process are sucrose transporters (SUT/SUC), monosaccharide transporters (MST), hexose transporters (HT), tonoplast monosaccharide transporters (TMT), tonoplast sugar transporters (TST), and sugars will eventually be exported transporters (SWEET) (Braun, 2022 ; Chen et al. 2012 ; Moore et al. 2015 ). Sucrose is transferred from the sieve tubes to fruit cells through two primary pathways. The first is the symplastic pathway, where sucrose enters fruit cells through plasmodesmata, which are cytoplasmic channels connecting adjacent plant cells. In this pathway, sucrose molecules move directly between cells via the cytoplasmic continuum, bypassing potential interference from enzymes responsible for hydrolysis within the apoplastic space (Lucas et al. 2013 ). In the second pathway, sucrose is unloaded from the sieve tubes into the apoplast. This process is facilitated by specific unloading mechanisms, likely involving certain transporter proteins that transfer sucrose into the apoplastic space (Milne et al. 2017 ). The extracellular region called the apoplast provides an environment where acid invertase acts to split sucrose into its component sugars, glucose and fructose. Simultaneously, sucrose transporters located on the cell membranes of fruit cells utilize the proton motive force or ATP hydrolysis to actively transport sucrose into the cells against its concentration gradient. The hydrolysis-transport process differs across fruit types and is subject to varying levels of regulation (Lalonde et al. 2004 ; Ruan et al. 2012). In strawberries, a typical non-climacteric fruit, symplastic unloading predominates during the early developmental stages. As strawberry fruits mature, apoplastic unloading gradually becomes the dominant pathway. It is well known that sucrose is formed in leaves with photosynthetic activity (source organs) by combining glucose and fructose. Subsequently, it is conveyed and loaded into the phloem’s sieve tubes via the symplastic or apoplastic route. However, the transport pathway of sucrose from mesophyll cells However, the transport pathway of sucrose from mesophyll cells to the apoplast had been unclear before the discovery of SWEET transporters. The SWEET gene family was initially discovered in Arabidopsis thaliana , encoding a novel type of transmembrane protein. These proteins can transport both monosaccharides and disaccharides in an energy-independent manner along a concentration gradient (Chen et al. 2010 ; Xuan et al. 2013 ). Seven transmembrane helix domains are presented in SWEET proteins (7-TMS) which mediate passive sugar transport through the unique MtN3/saliva repeat structure (Xuan et al. 2013 ). The SWEET family is divided into four subgroups. Clade I and Clade II mainly transport hexoses; Clade III preferentially transports sucrose; and Clade IV participates in intracellular fructose transport. In plants, SWEET members from different subgroups exhibit diverse gene functions, contributing to various physiological processes. Triple mutants ( atsweet11 ; 12 ; 15 ) show increased seed coat starch, decreased seed fat, delayed embryo development, and reduced seed weight with a shriveled appearance, suggesting these proteins aid in sucrose transport from the seed coat to the embryo, supporting seed growth (Chen et al. 2015 ). AtSWEETs play roles in both above-ground and root development, and they modulate abiotic stress responses through sugar transport allocation (Chen et al. 2012 ; Yang et al. 2006 ). SWEET-mediated sugar efflux serves as a nutrient acquisition pathway for pathogens, first observed in rice. Rice with recessive OsSWEET11 / Os8N3 alleles shows resistance to bacterial leaf blight. In contrast, dominant allele lines allow bacterial TAL effectors to induce OsSWEET expression, enhancing sugar efflux and pathogen nutrition acquisition (Yang et al. 2006 ). DOF (DNA-binding with One Finger) transcription factors, which were initially identified in maize in 1993 (Yanagisawa et al. 1993), are plant-specific zinc finger proteins. Target gene expression is regulated by transcription factors that bind to the cis-elements 5’-(T/A)/AAAG-3’ (Ma et al. 2017 ; Wu et al. 2018 ; Yanagisawa, 1998 ). DOF proteins regulate carbon-nitrogen metabolism, hormone signaling, photoperiod responses, and stress adaptation (Kim et al. 2021 ; Yanagisawa, 1998 ). For example, ZmDOF2 enhances DNA-binding efficiency through interactions with high-mobility group proteins, whereas HvBPBF activates endosperm-specific genes by collaborating with MYB (Cai et al. 2016 ; Gupta et al. 2014 ). DOF transcription factors play a role in plant development, influencing root growth, endosperm development, stomatal formation, and vascular differentiation. OsDOF11 in rice enhances OsSUT1 and OsSWEET14 expression (Kim et al. 2021 ). MdDOF54 enhances drought resistance by improving photosynthesis efficiency and water transport in branches (Chen et al. 2010 ; Guo et al. 2014 ). This study identified SWEET gene family members in the cultivated strawberry genome (Mao et al. 2023 ) and discovered that FaSWEET9a displays distinct expression patterns during fruit development. Transient overexpression and silencing of FaSWEET9a in ‘Yanli’ fruits significantly altered sucrose accumulation. Consistently, stable genetic transformation experiments in the strawberry cultivar ‘Chulian’ demonstrated similar regulatory effects on sucrose metabolism (Zhang et al. 2024 ). Mechanistically, we found that the DOF transcription factor FaDOF2 bound to the FaSWEET9a promoter, leading to an upregulation of its expression. This finding holds significant implications for the study of strawberry quality and its growth and development. Materials and methods Plant materials and growth conditions The strawberry ( Fragaria × ananassa ) cultivars ‘Yanli’ and ‘Chulian’ were cultivated in a greenhouse at Shenyang Agricultural University, China. ‘Chulian’ served as the stable genetic transformation, and the non-transformed ‘Chulian’ functioned as the wild‐type (WT) plant. ‘Yanli’ plants were subjected to transient genetic transformation. Tobacco ( Nicotiana benthamiana ) plants were selected and then planted in a growth chamber where the temperature was kept at 25°C, and the lighting regime consisted of 16 hours of light followed by 8 hours of darkness. The growth medium employed was potting medium. The samples were frozen using liquid nitrogen and kept at -80°C until they were analyzed. RNA extraction and RT-qPCR analysis We employed the optimized CTAB method described in the research of Wang et al. ( 2023 ). The total RNA was extracted from the fruit samples. After that, the extracted RNA was subjected to reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian). The cDNA was diluted to one-fourth of its original concentration and served as the template for real-time quantitative polymerase chain reaction (RT-qPCR) analysis. RT-qPCR was performed using the UltraSYBR Mixture (CWBIO, Taizhou, China). As reported in the study by Mao et al. ( 2024 ), the GAPC2 gene was deliberately chosen to function as the internal control for the experiment. The 2 −∆∆Ct method was used to precisely estimate the relative gene expression levels. For each individual sample, the analysis was meticulously conducted with three technical replicates. Each quantitative real-time polymerase chain reaction (qPCR) assay was performed with three separate biological replicates. The Primer3 software ( http://frodo.wi.mit.edu/ ) was used to design all primers, which are presented in Table S1 . Identification of the FaSWEET gene family Haplotype-resolved genomic sequence and annotation information for the octoploid strawberry ‘Yanli’ were sourced from the Rosaceae database ( https://www.rosaceae.org/Analysis/147231070 ). We conducted an HMMER search using the ‘Yanli’ protein database. The Pfam database ( https://pfam.xfam.org/ ) provided the Hidden Markov Model (HMM) profile for the SWEET domain (PF03083). BLASTp analysis was employed to look for homologous sequences between the Arabidopsis SWEET proteins obtained from TAIR at https://www.arabidopsis.org/ and the ‘Yanli’ protein database. An assessment of the candidate proteins was performed through the utilization of the Conserved Domain Database (CDD)( https://www.ncbi.nlm.nih.gov/ ) to verify SWEET structural conservation, and non-conforming proteins were eliminated. Chromosomal distribution and structure analysis Chromosomal localization and gene structure analysis of the SWEET family genes were performed using the GFF3 annotation file with TBtools software (v2.149) (Chen et al. 2020 ). Gene expression level analysis We analyzed the transcriptome data gathered from different ‘Yanli’ fruit development stages: small green, big green, white, turning, and ripening (Wang et al. 2024 ). The RNA-sequencing data were deposited in the NCBI database. The accession number assigned to this data set is PRJNA975298. Each experiment utilized three biological replicates, and the heatmap was generated with TBtools (v2.149). Cis-regulatory elements of the FaSWEET9a To conduct an analysis of the cis-acting elements, the PlantCARE online resource ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) was utilized. By utilizing PlantCARE, a 2000-bp sequence upstream of the coding sequence of the FaSWEET9a genes was obtained. Then, the results were presented visually using TBtools (v2.149). Upstream transcription factor prediction of FaSWEET9a A comprehensive prediction of the transcription factor (TF)-binding sites in the promoter regions of the candidate genes was performed using the PlantTFDB database, which can be accessed at http://planttfdb.cbi.pku.edu.cn/ . The predicted TF-binding sites were presented in Table S2 for detailed reference. Subcellular localization The CELLO website ( http://cello.life.nctu.edu.tw/ ) was utilized to predict the intracellular localization of FaSWEET9a . According to Dong et al. ( 2021 ), the pRI101- FaSWEET9a ‐GFP fusion vector was constructed. Refer to Table S1 for the associated primers. The bacterial suspension of Agrobacterium tumefaciens strain GV3101 containing different vectors was transferred to N. benthamiana leaves. In order to observe the GFP fluorescence signals, confocal fluorescence microscopy (TCS SP8, Leica, Germany) was utilized. Phylogenetic analysis The phylogenetic tree was constructed via the maximum-likelihood approach. For the calculation of phylogenetic distances, expressed as the number of amino acid substitutions per site, the Poisson correction method was employed. The entire phylogenetic analysis was carried out within the MEGA11 software. Transient expression in strawberry fruits The CDS regions of FaSWEET9a or FaDOF2 were cloned separately into the pRI101-AN vector to construct the pFaSWEET9a-OE and pFaDOF2-OE vectors for overexpression in ‘Yanli’ fruits. To silence FaSWEET9a expression in ‘Yanli’ fruit, the forward and reverse fragments of the FaSWEET9a gene were amplified and inserted into the pRNAi-E vector (Chen et al. 2019 ) to construct the pFaSWEET9a-RNAi vector. Refer to Table S1 for the associated primers. These plasmids were introduced into the A . tumefaciens strain GV3101. Then we prepared the infiltration buffer and infiltrated the fruits as per the methods of Li et al. ( 2017 ). A 1 mL sterile syringe was used to inject 1 mL of infiltration buffer into large green fruit. One fruit specimen was selected and utilized for the purpose of infiltrating the target constructs into it, while another served as a control with empty vectors. Seven days subsequent to the infiltration procedure, the fruits that had undergone infiltration were harvested. A biological replicate was composed of a set of six fruits, and at least three such biological replicates were carried out in this study. Strawberry stable genetic transformation The in vitro plants of cultivated strawberry cultivar ‘Chulian’ were subcultured on MS medium with 0.2 mg/L 6-Benzylaminopurine (6-BA), 0.1 mg/L Gibberellic Acid 3 (GA 3 ), and 0.01 mg/L Indole-3-butyric acid (IBA). The FaSWEET9a -overexpression (OE) construct was introduced into the A . tumefaciens strain GV3101 by means of the freeze-thaw technique (Wang et al. 2011 ). First, a single colony from the bacterial strain was selected and inoculated into the prepared liquid yeast extract peptone (YEP) medium. This medium was enhanced with 25 mg/L kanamycin and 100 mg/L spectinomycin. The medium that had been inoculated was placed on an orbital shaker set at a rotational speed of 180 rpm and incubated at 28°C for a duration of 16 hours. After incubation, the OD₆₀₀ of the bacterial solution was carefully adjusted to 0.1 in 50 mL of YEP medium. The solution was incubated at 28°C with agitation for 5 to 6 h until the OD600 of 0.5 was achieved. Next, each petiole was sectioned into 1 cm segments. These segments were then submerged in the bacterial suspension and gently swirled for 8 min. After retrieval from the suspension, the excess liquid was carefully blotted off using a sterile filter paper. Subsequently, the petiole segments were placed in a bud regeneration medium, consisting of MS medium with 2.0 mg/L thidiazuron (TDZ), and 0.2 mg/L IBA, and kept in total darkness for 3 days for co-cultivation. After the co-cultivation phase, the petiole segments were moved to a new prepared bud regeneration medium containing 250 mg/L cefotaxime and 25 mg/L kanamycin for selection. Once the selection was finalized, the petiole explants were relocated to a well-lit area for a 4 week cultivation period. When the regenerated buds attained a length of 1 cm, they were delicately excised and transferred into a development medium containing MS medium with 0.2 mg/L 6-BA, 0.1 mg/L GA3, 0.01 mg/L IBA, 250 mg/L cefotaxime, and 25 mg/L kanamycin. To sustain the selection pressure throughout the entire process, sub-culturing was performed at three week intervals. Sugar measurement Four fruit ripening stages defined as big green, white, turning, and ripening were harvested from stable transgenic strawberry plants for measuring soluble sugar content. Soluble sugar content was measured in new leaves, mature leaves, petioles, and roots collected from stable genetically transformed plants grown in MS medium, ensuring samples were taken from consistent positions on each plant. The soluble sugar contents were determined by the anthrone colorimetric method (Li et al. 2025 ). Each group consisted of three biological replicates. Fructose, glucose and sucrose were analyzed by high-performance liquid chromatography (1290 infinity II, Agilent, USA) following the method described by Li et al. ( 2025 ). Each group had three biological replicates. Heterologous Expression of FaSWEET9a in Yeast Cells According to Li et al. ( 2020 ), the pDR196- FaSWEET9a vector was constructed. The heterologous expression of FaSWEET9a in yeast was performed using the native gene sequence without codon optimization. Refer to Table S1 for the associated primers. The constructs were transferred into the yeast mutant strain SUSY7/ura via the lithium acetate-mediated transformation technique (Li et al. 2020 ; Riesmeier et al. 1992 ; Soni et al. 1993 ). FaSWEET9a was expressed in yeast strain SUSY7/ura. Yeast cells with pDR196-FaSWEET9a or the pDR196 vector (negative control) were grown on solid Synthetic Defined Medium without uracil using 2% (w/v) glucose or sucrose as the only carbon source. Yeast one-hybrid (Y1H) assay The FaDOF2 coding sequence was cloned into the pGADT7 vector, creating the pGADT7-FaDOF2 fusion construct. The 1000 bp promoter region of FaSWEET9a containing two (A/T)AAAG elements (located at -270 bp and − 842 bp) was segmented to construct the reporter vectors pAbAi- ProFaSWEET9a -P1 (0~ -432 bp) and pAbAi- proFaSWEET9a -P2 (-742 ~ -1000 bp). Refer to Table S1 for the associated primers. According to Li et al. ( 2017 ), the pGADT7-FaDOF2 and pAbAi‐ proFaSWEET9a fusion vectors were transformed into Y1H yeast cells for cultivation. Electrophoretic mobility shift assay (EMSA) The coding region of FaDOF2 was linked to the pCold vector with the His label to construct the FaDOF-pCold vector. The probe sequence (40 bp) was isolated from FaSWEET9a promoter and consisted of the (A/T)AAAG element. Refer to Table S1 for the associated primers. FaSWEET9a -probe represents the probe sequence labelled with biotin, and competitor represents the probe sequence that was not labelled by biotin. FaSWEET9 -probe and its competitor were synthesized by Sangon Biotechnology Co. LTD (Shanghai, China). The Chemiluminescent EMSA Kit (Beyotime, Shanghai, China) was used for performing the EMSA technique. Luciferase (LUC) reporter assay According to Luo et al. ( 2024 ), the pRI101- FaDOF2 vector and the pro FaSWEET9a ::LUC vector containing a 1000-bp FaSWEET9a promoter were constructed. The bacterial suspension of A. tumefaciens strains GV3101 containing the vectors mentioned above was mixed according to the requirements and then injected into N. benthamiana leaves. Luciferase signals were detected using the live fluorescence imager (Lb985, Berthold, Germany) after a 2-day dark incubation. The transcriptional activity was evaluated by means of the dual luciferase reporter assay kit (Beyotime, Shanghai, China). Refer to Table S1 for the associated primers. β-glucuronidase (GUS) reporter assay According to Luo et al. ( 2024 ), the proFaSWEET9a ::GUS vector containing FaSWEET9a promoter (1000 bp) was constructed. Refer to Table S1 for the associated primers. The bacterial suspension of GV3101 containing the aforementioned vectors was prepared and subsequently injected into N. benthamiana leaves as per experimental requirements. The infected tobacco leaves were subjected to GUS staining in strict accordance with the protocol stipulated by the GUS stain kit (Coolaber, Beijing, China). According to Li et al. ( 2017 ), the GUS activity and GUS protein concentration of infected tobacco leaves were detected. Statistical analysis The data are presented in the form of means accompanied by standard deviations (SDs). Significance analyses were conducted via the Student’s t-tests using SPSS software (version 17.0), with significance thresholds set at P < 0.01 or P < 0.05. Accession Numbers The sequence data presented in this article can be retrieved from the Rosaceae database ( https://www.rosaceae.org/Analysis/14723107 ) using the following accession numbers: FaSWEET1a (FxaYL_241g0513140), FaSWEET1b (FxaYL_241g0513130), FaSWEET1c (FxaYL_221g0474270), FaSWEET1d (FxaYL_242g0463630), FaSWEET2a (FxaYL_341g0266030), FaSWEET2b (FxaYL_311g0354740), FaSWEET3 (FxaYL_721g0968830), FaSWEET4 (FxaYL_511g0685950), FaSWEET5a (FxaYL_511g0691570), FaSWEET5b (FxaYL_611g0109740), FaSWEET6a (FxaYL_611g0095590), FaSWEET6b (FxaYL_611g0095800), FaSWEET6c (FxaYL_612g0094050), FaSWEET7 (FxaYL_441g0239180), FaSWEET9 a (FxaYL_231g0391340), FaSWEET9b (FxaYL_211g0416320), FaSWEET9c (FxaYL_211g0416280), FaSWEET9d (FxaYL_222g0414840), FaSWEET10 (FxaYL_541g0616130, FaSWEET14a (FxaYL_211g0416270), FaSWEET14b (FxaYL_541g0616120), FaSWEET15a (FxaYL_431g0644640), FaSWEET15b (FxaYL_411g0805500), FaSWEET16 (FxaYL_741g0928650), FaSWEET17 (FxaYL_411g0802810). SPSA1 (FxaYL_142g0861950), SPSA3 (FxaYL_431g0636300), SPSA4 (FxaYL_221g0488530), CWIVN (FxaYL_612g0121980), HXK1 (FxaYL_112g0686890), HXK2 (FxaYL_221g0450840), SUS (FxaYL_121g0719220), SUS2 (FxaYL_221g0462810), SUS7 (FxaYL_121g0701630), SUC3 (FxaYL_431g0652490), SUC4 (FxaYL_511g0663990), CINV (FxaYL_531g0564800), STP1 (FxaYL_221g0482960), STP13 (FxaYL_441g0240880), STP14 (FxaYL_122g0775850), STP7 (FxaYL_141g0906560). Results Characteristic analysis of FaSWEET9a Our laboratory has previously developed a high-quality haplotype-resolved genome assembly for the cultivated octoploid strawberry cultivar ‘Yanli’, comprising two haplotype assemblies (Hap1 and Hap2) with a total of 56 chromosomes (Mao et al. 2023 ). Using the genome annotation, we identified SWEET family members through Pfam database ( https://pfam.xfam.org/ ) searches for the MtN3 (PF03083) domain. From the ‘Yanli’ genome, we identified 25 FaSWEET family members. These were systematically named FaSWEET1 ~ 17 based on their homology to A. thaliana SWEET genes. The FaSWEET genes are distributed across chromosomes 2 through 7 (Supplement Fig. S1 ). Gene structure analysis showed that FaSWEET genes contained 2–6 exons (Fig. 1 a). We analyzed the expression patterns of FaSWEET genes across five developmental stages of ‘Yanli’ fruits using RNA-seq. FaSWEET9a , exhibiting the highest transcript levels during the white and turning stages, was selected for further characterization (Fig. 1 b). The structural analysis predicted seven transmembrane domains in FaSWEET9a (Fig. 1 c). After conducting a comparative analysis of the amino acid sequences and stage-specific expression profiles among FaSWEET9a-d, it was found that the alleles of FaSWEET9a with the highest expression levels form an allelic pair located on Hap1 and Hap2, and both possess two MtN3 domains (Supplement Fig. S2 , S3). Subsequent analyses and experiments were conducted using the FaSWEET9a allele (FxaYL_231g0391340) from Hap1. Subcellular localization prediction via the CELLO database indicated potential plasma membrane (Table S3 ). Transiently expressing FaSWEET9a -GFP fusion constructs in N . benthamiana leaves confirmed that the protein was located on the plasma membrane (Fig. 1 d). We identified FaSWEET genes from the ‘Yanli’ genome and constructed a phylogenetic tree together with functionally characterized SWEET genes from model plant A. thaliana (AtSWEETs) and important crops including rice ( Oryza sativa , OsSWEETs), tomato ( Solanum lycopersicum , SlSWEETs) and apple ( Malus domestica , MdSWEETs) to comprehensively resolve the evolutionary relationships of the SWEET family in both dicot and monocot plants (Fig. 1 e). The maximum-likelihood phylogenetic tree revealed that SWEET family members clustered into four distinct clades (I–IV) with characterized AtSWEETs. Clade III contained FaSWEET9a-d, FaSWEET10, FaSWEET14a-b, FaSWEET15a-b alongside AtSWEET9-15, OsSWEET11-15, MdSWEET9a-b, MdSWEET14, SlSWEET10a-c, SlSWEET11a-d, SlSWEET12a-d and SlSWEET14, suggesting conserved functions in sugar allocation. FaSWEET9a acts as a sucrose transporter Among the four distinct clades within the SWEET gene family, SWEET9 is categorized as a member of clade III. Functionally, this particular clade is predominantly responsible for facilitating sucrose transport. To assess the sucrose transport activity of FaSWEET9a, we heterologously expressed the FaSWEET9a in the yeast ( Saccharomyces cerevisiae ) mutant SUSY7/ura. In the experiment, yeast mutant cells transfected with the empty pDR196 vector served as negative controls. The results showed that yeast mutant cells harboring FaSWEET9a grew faster and more robustly on synthetic deficient media supplemented with sucrose than yeast cells containing the empty vector. This result demonstrates that FaSWEET9a exhibits substrate specificity for sucrose transport (Fig. 2 ). Transient overexpression or silencing of FaSWEET9a alters sucrose content in strawberry fruits We explored the role of FaSWEET9a in strawberry fruits by overexpressing it in ‘Yanli’ fruits through A . tumefaciens -mediated infiltration. The control utilized was the empty pRI101-AN vector. In the fruits infiltrated with pFaSWEET9a-OE, the relative expression of FaSWEET9a was significantly higher than that in the control (Fig. 3 a). Moreover, the sucrose content exhibited a significant increase compared to that of the control. In contrast, there were no statistically significant differences detected in the levels of fructose and glucose (Fig. 3 b). We used A . tumefaciens -mediated infiltration to silence FaSWEET9a expression in ‘Yanli’ fruits. The expression of FaSWEET9a in fruits treated with pFaSWEET9a-RNAi showed a statistically significant decrease compared to the expression level in the control (Fig. 3 c). The sucrose content was significantly reduced compared to the control. At the same time, no significant difference was detected in fructose and glucose levels (Fig. 3 d). The transient transformation experiments carried out on strawberry fruits showed that FaSWEET9a could influence the changes in sucrose content within the strawberry fruits. Overexpression of FaSWEET9a alters morphological phenotypes and increases sucrose content in strawberries To investigate the impact of FaSWEET9a on the phenotype of strawberry plants, we obtained two FaSWEET9a -overexpressing lines ( FaSWEET9a -OE: OE-6 and OE-7). Morphological parameters, such as height, leaf area, the number of leaves, length and width of the third leaf, and petiole length of the third leaf were assessed during both vegetative and reproductive growth stages. During the vegetative growth stage, FaSWEET9a -OE plants exhibited significantly greater plant height than WT plants. Similarly, in terms of leaf area, length, width, and petiole length of the third leaf, FaSWEET9a -OE plants showed significant increases relative to WT plants. However, no significant differences in the number of leaves were found between FaSWEET9a -OE and WT plants (Supplement Fig. S4 a-f). In the reproductive growth stage, FaSWEET9a -OE plants were also significantly bigger than WT plants regarding plant height, leaf area, third leaf length, third leaf width, and petiole length. However, no significant differences in the number of leaves were found between FaSWEET9a -OE plants and WT plants (Supplement Fig. S4 g-l). The morphological investigations and analyses indicated that overexpression of FaSWEET9a can influence various phenotype traits in strawberry plants. We investigated the impact of FaSWEET9a on sucrose content variations in strawberries by examining the soluble sugar levels in various organs of in vitro -cultured FaSWEET9a -OE plants. The study found that soluble sugar levels in new leaves, mature leaves, petioles, and roots were markedly elevated compared to WT plants (Fig. 4 a). After transplanting the transgenic plants and WT, we successfully obtained fruits at various developmental stages, including big green, white, turning, and ripening. To validate the elevated transcript levels of FaSWEET9a at these stages, we examined FaSWEET9a expression in fruits from FaSWEET9a -overexpressing ( FaSWEET9a -OE) plants. RT-qPCR analysis revealed that the relative expression of FaSWEET9a was significantly higher in the big green, white, turning, and ripening fruits compared to that in WT (Fig. 4 b). We examined the impact of FaSWEET9a overexpression on sucrose levels in strawberries by analyzing soluble sugars (fructose, glucose, and sucrose) at different developmental stages in both FaSWEET9a -OE and WT fruits. The findings demonstrated that FaSWEET9a -OE fruits exhibited a significantly higher soluble sugar content compared to WT fruits at various stages of fruit development (Fig. 4 c). Further analysis of the glucose, fructose, and sucrose contents at various stages of fruit development revealed that all these sugars were present at significantly higher levels in the FaSWEET9a -OE fruits compared to WT counterparts throughout all examined stages (Fig. 4 d-f). Notably, the sucrose content peaked during the white and turning phases (Fig. 4 f). The present findings strongly imply that FaSWEET9a exerts a pivotal influence on promoting the accumulation of sucrose within the fruits of strawberry plants. We selected genes associated with sucrose metabolism and transport to analyze their relative expression in FaSWEET9a -OE fruits at the white stage and turning stage, aiming to better understand the variations in sucrose content. The results indicated a significant increase in the relative expression of genes linked to sucrose metabolism, including SPS4 (sucrose phosphate synthases), CWINV (cell wall invertase), HXK2 (hexokinase), SUS , SUS7 (sucrose synthase), during the white stage. Additionally, we observed a marked elevation in the relative expression of sucrose transport-related genes, including STP7 , STP10 , STP13 , STP14 (sugar transporter protein), SUC4 (sucrose carrier) (Fig. 4 g). Notably, SUS exhibited the highest level of relative expression among these genes. During the turning stage, we found a significant upregulation of sucrose metabolism-related genes such as SPSA1 , SPSA3 , CWINV , HXK1 , SUS , SUS7 , CIN (cytoplasm invertase). Moreover, the relative expression of sucrose transport-related genes, including STP10 , STP13 , and STP14 , showed a significant increase (Fig. 4 h). These results indicate that FaSWEET9a has the potential to enhance fruit sucrose content by modulating specific gene expression critical for sucrose degradation and resynthesis processes. FaSWEET9a is regulated by FaDOF2 To further investigate which signals regulate the expression of FaSWEET9a , based on the analysis of the FaSWEET9a promoter cis-acting elements (Supplement Fig. S5) and the predictions from the plantTFDB website ( http://planttfdb.gao-lab.org/ ) (Table S2 ), we selected the FaDOF2 transcription factor for further analysis, whose family members have been reported to regulate SWEET transporters (Wu et al. 2018 ). FaDOF2 CDS was cloned into the pGADT7 vector, and we identified two (T/A)AAAG motifs at positions − 270 bp (P1) and − 842 bp (P2) within the 1000-bp promoter region of FaSWEET9a and subsequently constructed two corresponding reporter vectors, pAbAi- ProFaSWEET9a -P1 and pAbAi- ProFaSWEET9a -P2. Subsequently, the recombinant plasmid pAbAi- ProFaSWEET9a -P1/2 was utilized in the Y1H assay. Yeast cells co-transformed with pGADT7 and pAbAi- ProFaSWEET9a -P1/2 failed to grow on Leu medium containing 75 ng/ml aureobasidin A (AbA). Yeast cells co-transformed with pGAD-FaDOF2 and pAbAi- ProFaSWEET9a -P2 grew normally on the medium, while the result of pAbAi- ProFaSWEET9a -P1 was consistent with that of the negative control. The result shows that FaDOF2 can bind to the promoter of FaSWEET9a (Fig. 5 a). Additionally, we analyzed the FaSWEET9a promoter and identified a ~ AAAG motif located at − 842 bp. To confirm the binding of FaDOF2 to the FaSWEET9a promoter, an EMSA was performed. The hot probe contains biotin-labeled P2 fragments, and EMSA was performed using the counterpart unlabeled fragments as competitors (cold probe). In the experiment, we progressively increased the amount of cold probes (10×, and 50× fold probe concentration). Specific binding of FaSWEET9a in fragments containing FaDOF2-binding elements (TAAAG) was shown by their upward shift. The results demonstrated that competitive cold probes significantly reduced the radioactive signal, including the binding signal between FaDOF2 and the FaSWEET9a promoter. Furthermore, binding was disrupted when mutated probes were used (Fig. 5 b). GUS assays were conducted to investigate whether FaDOF2 influences the transcriptional activity of the FaSWEET9a promoter. The GUS analysis indicated that p ProFaSWEET9a ::GUS expression levels were significantly elevated when co-expressed with p35S:: FaDOF2 than co-expression with pRI101-AN. Likewise, the GUS histochemical stain assay results showed that FaDOF2 specifically activated GUS transcription as driven by the FaSWEET9a promoter (Fig. 5 c). To confirm further the relationship between FaDOF2 and FaSWEET9a , an in vivo LUC reporter assay was performed. The luminescence intensity of N. benthamiana epidermal cells was measured. Cells co-expressing 35S:: FaDOF2 and proFaSWEET9a ::LUC exhibited enhanced luminescence than those expressing only proFaSWEET9a::LUC or the negative control (empty vector). Moreover, proFaSWEET9a exhibited the highest activity when combined with FaDOF2. The findings suggest that FaDOF2 binds to the promoter of FaSWEET9a and regulates its expression (Fig. 5 d). Transient overexpression of FaDOF2 increases sucrose content in strawberry fruits To determine whether FaDOF2 can enhance the sucrose content in strawberry fruits through the regulation of FaSWEET9a , we conducted a transient overexpression experiment in strawberry fruits. FaDOF2 was overexpressed in ‘Yanli’ fruits via A. tumefaciens -mediated infiltration. The pRI101-AN vector served as a control, and fruits infiltrated with pFaDOF2-OE exhibited a significantly higher relative expression level of FaDOF2 than that in the control (Fig. 6 a). Similarly, the relative expression level of FaSWEET9a was also up-regulated in the fruits transiently overexpressing FaDOF2 (Fig. 6 b), indicating that FaDOF2 positively regulates FaSWEET9a . The fructose, glucose, and sucrose levels were assessed in fruits exhibiting transient FaDOF2 overexpression (Fig. 6 c). The findings indicated a significant increase in sucrose content in FaDOF2 -OE fruits than that in the control. However, fructose and glucose contents in the FaDOF2 -OE fruits did not exhibit significant changes compared to the control. Overexpression of FaDOF2 enhances the sucrose content in strawberry fruits. Discussion Influence of FaSWEET9a on the development of strawberry plants FaSWEET9a , a SWEET III gene family in the octoploid strawberry genome, encodes a transporter with seven transmembrane domains (Fig. 1 c). This characteristic is consistent with its homologs in A. thaliana (Chen et al. 2010 ). We found that FaSWEET9a is highly conserved across multiple species through cross-species comparative analysis. Its homologous genes, such as AtSWEET9 and OsSWEET14 , play crucial roles in sucrose transport and accumulation (Chen et al. 2012 ; Ma et al. 2017 ), indicating that this gene has retained essential functions throughout evolution. This conservation provides a theoretical foundation for extrapolating findings from other species to strawberries (Jia et al. 2016 ; Smeekens et al. 2010 ). A phylogenetic tree constructed based on protein sequences shows that FaSWEET9a clusters with SWEET III genes from Arabidopsis (Fig. 1 e), supporting its functional specialization in sucrose transport (Lemoine et al. 2013 ). Overexpression of FaSWEET9a significantly affected the growth and morphological characteristics of strawberry plants (Supplement Fig. S4 ). In particular, plants overexpressing FaSWEET9a exhibited increased height, enlarged leaf area, and extended petiole length (Supplement Fig. S4 ). These findings suggest that FaSWEET9a may influence overall plant growth by redistributing photosynthates. Likewise, apple plants overexpressing MdSWEET9 show similar changes, with obvious alterations in nutrient transport and a notable build-up of plant material. (Braun et al. 2014 ; Ma et al. 2017 ). Influence of FaSWEET9a on fruit quality Our study confirmed the role of FaSWEET9a as a sucrose transporter, further clarifying its gene function. Heterologous expression experiments showed that FaSWEET9a could restore the ability of the mutant yeast strain SUSY7/ura to utilize sucrose (Fig. 2 ). The localization of FaSWEET9a on the plasma membrane indicates its critical role in intercellular sucrose transport (Fig. 1 d). This is consistent with the functional localization of SWEET genes in other plants. For instance, OsSWEET14 and AtSWEET11/12 are plasma membrane-localized proteins that mediate the translocation of sucrose from the photosynthetically active source tissues to the metabolically demanding sink tissues (Chen et al. 2012 ; Le et al. 2015). FaSWEET9a has been confirmed to facilitate the transport of sucrose. Thus, does it influence the sucrose content of the fruits? We investigated this question through transient overexpression and silencing of FaSWEET9a in fruits and stable genetic transformation using the vector pFaSWEET9a-OE. Transient overexpression of FaSWEET9a in fruits led to a significant increase in both FaSWEET9a expression and sucrose content than those in the control (Fig. 3 a, b). In contrast, the opposite was true in the fruits with transient silencing of FaSWEET9a (Fig. 3 c, d). After clarifying the effects of transient overexpression and silencing of FaSWEET9a on the sucrose content in fruits, we further conducted a stable genetic transformation experiment for verification. Similarly, the relative expression of FaSWEET9a increased significantly in FaSWEET9a -OE fruits during the big green, white, turning, and ripening stages (Fig. 4 b). This dynamic expression pattern aligns with the findings for OsSWEET14 and MdSWEET9b , where expression levels also increase during fruit enlargement and maturation (Eom et al. 2016 ; Zhou et al. 2014 ). Interestingly, the expression level of FaSWEET9a in FaSWEET9a -OE fruits fluctuates during the developmental process. During the early fruit developmental phase (big green stage), characterized by active cell division and expansion, the high expression of FaSWEET9a likely supplies energy substrates, such as sucrose, to fuel cellular growth, thereby promoting morphogenesis in both leaves and fruits. As the fruit transitions into the sucrose-accumulating critical phase (white to turning stages), the enhanced sucrose translocation from source organs (leaves) to sink organs (fruits) drives sustained high expression of FaSWEET9a (Fig. 4 b). This elevated expression directly correlates with increased sucrose content in fruits, with the sucrose peak synchronizing temporally with maximal FaSWEET9a transcript levels (Fig. 4 b, f). Such synchronization suggests potent sucrose-driven transcriptional regulation during this stage, which may underlie the dynamic expression fluctuations observed during fruit development. Similarly, in tomato, the sucrose content in fruits at different developmental stages also changes with the variation in the expression level of the sucrose transporter SlSWEET14 (Zhang et al. 2021 ). In grapevine, the hexose transporter VvSWEET15 initiates hexose accumulation starting at the veraison stage and peaks during fruit ripening (Lu et al. 2025 ). Sucrose, serving a dual role as both a signaling molecule and an energy source, necessitates upregulated transporter activity to meet the demands of rapid fruit expansion and carbohydrate storage (Krügel et al. 2013). This regulatory process likely operates through conserved sugar signaling pathways that induce feedback upregulation of FaSWEET9a itself or coordinate the expression of related transporters. Together, these mechanisms establish a ‘transport-accumulation-signal amplification’ positive feedback loop, ensuring sustained sucrose allocation to fruits while maintaining systemic carbon homeostasis. However, during the ripening stage, sucrose content decreases while glucose and fructose levels increase. This shift may result from reduced demand for sucrose import coupled with its subsequent breakdown into glucose and fructose, leading to decreased demand for sucrose transporters and consequent downregulation of FaSWEET9a expression (Fig. 4 b, d-f). These results demonstrate that as the expression level of FaSWEET9a increases or decreases, the sucrose content in strawberry fruits will also increase or decrease. This finding provides a potential target for the genetic improvement of strawberry fruits to enhance their sucrose content and overall quality FaSWEET9a potentially modulates sucrose accumulation in fruits through the regulation of genes associated with sucrose metabolism and transport Our experiments indicated that both FaSWEET9a expression and sucrose content peaked during the white and turning stages (Fig. 4 b, f). Therefore, we examined the relative expression levels of genes associated with sucrose metabolism and transport across these stages. Through a meticulous research process, we ascertained that during the white stage, genes implicated in sucrose synthesis, notably SUS , exhibited a marked upsurge in their relative expression levels (Fig. 4 g). SUS plays a pivotal role in sucrose metabolism, catalyzing the reversible reaction between sucrose and UDP-glucose and fructose. An up-regulation of SUS means that more substrates are converted into sucrose, directly boosting the synthesis rate. Concurrently, genes related to sucrose transport, including SPSA4 , STP7 , STP13 , STP14 , and SUC4 , also showed elevated expression (Fig. 4 g). These transporters facilitate the movement of newly synthesized sucrose from the production sites to storage compartments within the fruit cells or enable transport between different cells. This coordinated up-regulation of synthesis and transport genes forms a well-orchestrated mechanism for sucrose accumulation in the fruit during the white stage. Similar patterns have been observed in other crops. In peach fruits, the relative expression of PpSUS1 and PpSPS2 was positively correlated with sucrose content (Dai et al. 2025 ). This indicates that sucrose-related genes consistently influence sucrose levels across various fruit-bearing plants. In strawberry, virus-induced gene silencing assays for FaSS1 demonstrated that its down-regulation decreased sucrose content and inhibited ripening (Zhao et al. 2017 ). In sugarcane, overexpressing SoSPS1 in sugarcane increased SPS activity and sucrose content in transgenic lines (Anur et al. 2020 ). SPS facilitates the transformation of F6P (fructose-6-phosphate) and uridine diphosphate glucose into S6P (sucrose-6-phosphate). Subsequently, SPP then hydrolyzes the newly-formed S6P into sucrose. (Verma et al. 2011 ). During the turning stage, there were significant increases in the relative expression levels of genes associated with sucrose decomposition, including CWINV and CIN (Fig. 4 h). CWINV and CIN are key enzymes that break down sucrose into hexoses (glucose and fructose). The breakdown of sucrose into hexoses provides readily available energy for processes like color change, flavor development, and cell wall modification. Concurrently, the expression levels of sucrose transport-related genes, including STP10 , STP13 , and STP14 , showed a significant increase (Fig. 4 h). These transporters may be involved in redistributing the decomposed hexoses to different parts of the fruit where they are needed most. In tomato, increased CWINV activity is associated with elevated hexose levels (Jin et al. 2009 ; Liu et al. 2016 ). The gene expression changes related to sucrose metabolism and transport form a complex and dynamic regulatory network, indirectly affecting strawberry fruits’ overall sucrose content. FaSWEET9a , a crucial factor within this network, promotes the accumulation and maintains the dynamic balance of sucrose in fruits by regulating genes involved in sucrose metabolism and transport. This discovery offers fresh insights and new research directions for improving fruit quality. The transcription factor FaDOF regulates FaSWEET gene to enhance sucrose accumulation in strawberry fruits. Transcription factors are essential in plant development, often regulating sucrose synthesis by influencing the expression of structural genes. For example, FvSWEET9 is positively regulated by FvPHR1 and participates in sugar transport from the leaves to the fruits (Li et al. 2025 ). CitZAT5 (zinc finger of Arabidopsis thaliana 5) regulates sugar accumulation in citrus fruit by modulating the expression of CitSUS5 and CitSWEET6 (Fang et al. 2023 ). In the ‘Gala’ apple cultivar ( Malus × domestica ), MdWRKY9 interacts with MdbZIP23 and MdbZIP46, forming a protein complex that binds to the promoter region of MdSWEET9b . This binding up-regulates the expression of MdSWEET9b , thereby influencing sugar accumulation within the fruit (Zhang et al. 2023 ). This study identifies FaDOF2 as a key upstream transcription factor that regulates FaSWEET9a through bioinformatics analyses combined with experimental validation. Analysis of the promoter region of FaSWEET9a reveals a typical DOF-binding site (AAAG) (Yang et al. 2017 ). Y1H and EMSA confirmed that FaDOF2 specifically binds to this site, consequently activating FaSWEET9a transcription (Fig. 5 a, b). This binding pattern has also been observed across other plant species. For example, OsDOF11 in rice and MdDOF54 in apple regulate sugar transport and distribution via binding to SWEET gene promoters (Liu et al. 2019 ; Wang et al. 2021 ). These findings indicate the conserved nature of the DOF transcription factor family concerning their role in regulating sugar metabolism genes. To further explore the effects of FaDOF2 on FaSWEET9a expression, LUC and GUS reporter assays were conducted (Fig. 5 c, d). The results validated that FaDOF2 significantly enhances transcriptional activity associated with FaSWEET9a in vivo . Transient expression experiments additionally demonstrated that overexpression of FaDOF2 elevates transcription levels of FaSWEET9a within strawberry fruits, resulting in a substantial increase in sucrose accumulation. Thus, we infer that FaDOF2 positively regulates the transcription of FaSWEET9a and then regulates the sucrose content in strawberry fruits. Our research introduces a new regulatory mechanism for sucrose accumulation in strawberry fruits. We propose a hypothetical model to reveal this pathway (Fig. 7 ). FaSWEET9a and FaDOF2 are crucial for sucrose accumulation in strawberry fruits. Specifically, the sucrose transporter FaSWEET9a affects the phenotype of strawberry plants and promotes the accumulation of sucrose content. On the one hand, FaSWEET9a can specifically transport sucrose; on the other hand, the activity of FaSWEET9a is positively regulated by FaDOF2, and FaDOF2 then regulates sucrose content in strawberry fruits. These findings enhance our comprehension of the regulatory mechanisms controlling sucrose accumulation and provide new insights for enhancing the flavor quality of fruits. Declarations The authors declare no conflicts of interest Author contributions YX and ZHZ conceived and designed the experiments; YX, SL, HYS, JZ, CZ performed the experiments; YX analyzed the data; YX, WG, and ZHZ wrote the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (No.32130092) and the project of Liaoning Province Germplasm Innovation Grain Storage and Technology Special Program (2023020525-JH1/102-04). Data availability Source data are provided with this paper. The other data supporting this study’s fundings are available from the corresponding author on request. References Anur RM, Mufithah N, Sawitri WD, Sakakibara H, Sugiharto B (2020) Overexpression of sucrose phosphate synthase enhanced sucrose content and biomass production in transgenic sugarcane. 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Plant Cell 10(1):75-89. https://doi.org/10.1105/tpc.10.1.75 Yang B, Sugio A, White FF (2006) Os8N3 is a host disease-susceptibility gene for bacterial blight of rice. Proc Natl Acad Sci USA 103(27):10503-10508. https://doi.org/10.1073/pnas.0604088103 Yang G, Yu L, Wang Y, Wang C, Gao C (2017) The translation initiation factor 1A ( TheIF1A ) from Tamarix hispida is regulated by a Dof transcription factor and increased abiotic stress tolerance. Front Plant Sci 8:513. https://doi.org/10.3389/fpls.2017.00513 Zhang J, Liu S, Zhao S, Nie Y, Zhang Z (2024) A telomere-to-telomere haplotype-resolved genome of white-fruited strawberry reveals the complexity of fruit color formation of cultivated strawberry. Plant Biotechno J 23(1):78-80. https://doi.org/10.1111/pbi.14479 Zhang S, Wang H, Wang T, Zhang J, Liu W, Fang H, Zhang Z, Peng F, Chen X, Wang N (2023) Abscisic acid and regulation of the sugar transporter gene MdSWEET9b promote apple sugar accumulation. Plant Physiol 192(3):2081-2101. https://doi.org/10.1093/plphys/kiad119 Zhang X, Feng C, Wang M, Li T, Liu X, Jiang J (2021) Plasma membrane-localized SlSWEET7a and SlSWEET14 regulate sugar transport and storage in tomato fruits. Hortic Res 8(1):186. https://doi.org/10.1038/s41438-021-00624-w Zhao C. Hua LN, Liu XF, Li YZ, Shen YY, Guo JX (2017) Sucrose synthase FaSS1 plays an important role in the regulation of strawberry fruit ripening. Plant Growth Regul 81:175-181. https://doi.org/10.1007/s10725-016-0189-4 Zhou Y, Liu L, Huang W, Yuan M, Zhou F, Li X, Lin Y (2014) Overexpression of OsSWEET5 in rice causes growth retardation and precocious senescence by modulating sugar transport. Sci Rep 9(4):e94210. https://doi.org/10.1371/journal.pone.009421 Supplementary Files SupplementaryInformation.docx TableS1.xlsx TableS2.xlsx TableS3.xlsx supplementfigure.docx Cite Share Download PDF Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Accept 19 May, 2025 Reviewers agreed at journal 30 Apr, 2025 Reviewers invited by journal 29 Apr, 2025 Editor assigned by journal 29 Apr, 2025 First submitted to journal 25 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6529445","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449682567,"identity":"3c68bc2d-4a77-4963-80ae-8e13ff433e1b","order_by":0,"name":"Yan Xu","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Xu","suffix":""},{"id":449682568,"identity":"66f157f4-90bf-4edf-b5ce-1eab35dfa30e","order_by":1,"name":"Shuang Liu","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Liu","suffix":""},{"id":449682569,"identity":"c79e5403-ced1-4f52-8f4a-bccad714fb92","order_by":2,"name":"Hongying Sun","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hongying","middleName":"","lastName":"Sun","suffix":""},{"id":449682570,"identity":"11a2fac4-d82c-4703-9c24-1812a7724aed","order_by":3,"name":"Jian Zang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Zang","suffix":""},{"id":449682571,"identity":"06157775-7e20-4e67-a530-b525a6ae4b70","order_by":4,"name":"Chao Zhang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Zhang","suffix":""},{"id":449682572,"identity":"e0bf76a0-5e4b-41e0-bee4-5c746343d222","order_by":5,"name":"Wei Guo","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Guo","suffix":""},{"id":449682573,"identity":"6e59552d-d930-4976-9795-d229f90fd8e2","order_by":6,"name":"Zhihong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYDACCTBpA+WxEa8ljXQth0nQIj+7+dnDL3/O2xscP2PA8KHsMAP/7Ab8WhjnHDM3luG5nbjhTI4B44xzhxkk7hzAr4VZIsFMWkLidoLBgRwDZt62wwwGEgn4tbBJpH+TljA4Z29w/o0B819itPBI5JhJfkg4wLjhBtAWRmK0SEjklEkzHEhOnHnjWcHBnnPpPBI3CGiRn5G+TfLHHzt7vvPJGx/8KLOW459BQAsIMPMACYUDDAwHQC4lrB4IGH+ArGsgSu0oGAWjYBSMRAAAQxtBmVyDBqUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5935-1567","institution":"Shenyang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-04-25 13:39:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6529445/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6529445/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-025-03528-4","type":"published","date":"2025-06-02T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81933020,"identity":"558f0473-6298-4980-90f8-abf6cbac398f","added_by":"auto","created_at":"2025-05-05 05:38:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1446573,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristic analysis of \u003cem\u003eFaSWEET9a\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e Structure of the \u003cem\u003eFaSWEET\u003c/em\u003egenes. \u003cstrong\u003eb\u003c/strong\u003e The expression of \u003cem\u003eFaSWEET\u003c/em\u003e genes during fruit development (SG: small green, BG: big green, W: white, T: turning, and R: ripening). \u003cstrong\u003ec\u003c/strong\u003e Analysis of the transmembrane domains of FaSWEET9a using https://www.novopro.cn/tools/tmhmm.html. \u003cstrong\u003ed\u003c/strong\u003e Visualization of \u003cem\u003eFaSWEET9a\u003c/em\u003e-GFP subcellular localization in \u003cem\u003eNicotiana benthamiana \u003c/em\u003eleaves using a coexpressed mCherry-labeled plasma membrane marker to emphasize the plasma membrane. Scale bars represent 50 μm. \u003cstrong\u003ee \u003c/strong\u003ePhylogenetic relationship of SWEETs family proteins in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eFragaria \u003c/em\u003e× \u003cem\u003eananassa\u003c/em\u003e, \u003cem\u003eMalus\u003c/em\u003e × \u003cem\u003edomestica\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e and \u003cem\u003eSolanum lycopersicum\u003c/em\u003e. In Clade III, the FaSWEET9a marked in red font is our research object.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/bcacbdfc8b19e62b9cff2008.png"},{"id":81931585,"identity":"4fc5c74c-6f22-45a3-8de5-42b12197f1f0","added_by":"auto","created_at":"2025-05-05 05:30:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":593102,"visible":true,"origin":"","legend":"\u003cp\u003eFaSWEET9a acts as a sucrose transporter. The yeast mutant strain SUSY7/ura expressing FaSWEET9a grew in Yeast cells with pDR196-\u003cem\u003eFaSWEET9a\u003c/em\u003e or the pDR196 vector (negative control) were grown on solid Synthetic Defined Medium without uracil supplement with 2% glucose or 2% sucrose as the only carbon source. An empty vector (pDR196) was used as a negative control, the numbers on the panels indicate the dilution fold of yeast suspensions.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/618b53c679e87f632d7c7c43.png"},{"id":81931593,"identity":"0ba882c2-5e8a-4480-a4e2-c5079a6a2f8f","added_by":"auto","created_at":"2025-05-05 05:30:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":321706,"visible":true,"origin":"","legend":"\u003cp\u003eTransient overexpression or silencing of \u003cem\u003eFaSWEET9a\u003c/em\u003e alters sucrose content in strawberry fruits. \u0026nbsp;\u003cstrong\u003ea\u003c/strong\u003e Relative expression of \u003cem\u003eFaSWEET9a \u003c/em\u003ein transient overexpressing \u003cem\u003eFaSWEET9a\u003c/em\u003e plants. The empty vector pRI101-AN was set to 1 as the control. \u003cstrong\u003eb\u003c/strong\u003eSucrose, fructose, glucose contents in transient overexpressing\u003cem\u003e FaSWEET9a\u003c/em\u003eand control plants.\u003cstrong\u003e c\u003c/strong\u003e Relative expression of \u003cem\u003eFaSWEET9a \u003c/em\u003ein transient silencing \u003cem\u003eFaSWEET9a\u003c/em\u003e plants. The empty vector pRNAi-E was set to 1 as the control. \u003cstrong\u003ed\u003c/strong\u003e Sucrose, fructose, glucose contents in transient silencing\u003cem\u003e FaSWEET9a\u003c/em\u003e and control plants. Data represent mean values ± SDs from three biological replicates. Asterisks denote\u003cem\u003e P\u003c/em\u003e-values (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) based on Student’s t-test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/35d601f3448b5a8888578ac0.png"},{"id":81931599,"identity":"39189225-cd42-44c0-88ec-16088cb315d2","added_by":"auto","created_at":"2025-05-05 05:30:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":656373,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003eFaSWEET9a\u003c/em\u003e increases sucrose content in strawberry fruits. \u003cstrong\u003ea \u003c/strong\u003eThe \u003cem\u003ein vitro\u003c/em\u003e cultured \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpressing plants (OE-6, OE-7) exhibited varying soluble sugar content across different organs. \u003cstrong\u003eb \u003c/strong\u003eThe relative expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e in WT and \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpressing fruits (OE-6, OE-7) at various developmental stages. \u003cstrong\u003ec\u003c/strong\u003e Soluble sugar content in WT and \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpressing fruits (OE-6, OE-7) at variousdevelopmental stages. \u003cstrong\u003ed-f \u003c/strong\u003eThe content of fructose,\u003cstrong\u003e \u003c/strong\u003eglucose and sucrose at various developmental stages of \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpressing fruits (OE-6, OE-7). \u003cstrong\u003eg\u003c/strong\u003e Relative expression levels of sucrose metabolism and transport genes during the white stage in \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpression fruits (OE-6, OE-7). \u003cstrong\u003eh\u003c/strong\u003e Relative expression levels of sucrose metabolism and transport genes during the turning stage in \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpression fruits (OE-6, OE-7). Data from three biological replicates are presented as mean values ± SDs. Asterisks denote \u003cem\u003eP\u003c/em\u003e-values from Student’s t-test, with*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 or **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/0f7576675f912e4dddff39de.png"},{"id":81931588,"identity":"281a1c85-7960-4177-b318-647e9ff50453","added_by":"auto","created_at":"2025-05-05 05:30:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1044832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFaSWEET9a\u003c/em\u003e is regulated by FaDOF2. \u003cstrong\u003ea\u003c/strong\u003e Y1H analysis showed that FaDOF2 can bind to the\u003cem\u003e FaSWEET9a\u003c/em\u003e promoter. The empty pGADT7 vector was used as a negative control. Schematic diagram of the \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter and 2 binding sites (P1–P2) used in Y1H. And the positions relative to the full-length promoter (\u003cem\u003eproFaSWEET9a\u003c/em\u003e, -1000 bp) are indicated as follows: P1 (\u003cem\u003eproFaSWEET9a\u003c/em\u003e-P1, -270 bp), P2 (\u003cem\u003eproFaSWEET9a\u003c/em\u003e-P2, -842 bp). \u003cstrong\u003eb \u003c/strong\u003eThe binding of FaDOF2 to the \u003cem\u003eFaSWEET9a\u003c/em\u003e was verified by EMSA assay. Biotin-labeled hot probes with recognition motifs were used, and binding competition was conducted with both unlabeled and mutant probes. The symbol + and - indicate presence and absence, respectively. \u003cstrong\u003ec-d \u003c/strong\u003eGUS assay and\u003cstrong\u003e \u003c/strong\u003eLUC assay results showed that FaDOF2 can positively regulate the expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e \u003cem\u003ein vivo\u003c/em\u003e. Data from three biological replicates are presented as mean values ± SDs. Asterisks denote \u003cem\u003eP\u003c/em\u003e-values from Student’s t-test, with *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 or **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/c0329e3f2302600d0151d4c6.png"},{"id":81933026,"identity":"06657964-2e13-406c-ab6a-4f62c6d90788","added_by":"auto","created_at":"2025-05-05 05:38:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":147251,"visible":true,"origin":"","legend":"\u003cp\u003eTransient overexpression of \u003cem\u003eFaDOF2\u003c/em\u003eincreases sucrose content in strawberry fruits.\u003cstrong\u003e a \u003c/strong\u003eThe relative expression of \u003cem\u003eFaDOF2\u003c/em\u003e in the fruits transiently overexpressing \u003cem\u003eFaDOF2 \u003c/em\u003eand control. \u003cstrong\u003eb \u003c/strong\u003eThe relative expression of \u003cem\u003eFaSWEET9a \u003c/em\u003ein the fruits transiently overexpressing \u003cem\u003eFaDOF2 \u003c/em\u003eand control. \u003cstrong\u003ec\u003c/strong\u003e The content of\u003cstrong\u003e \u003c/strong\u003efructose, glucose, sucrose in the fruits transiently overexpressing \u003cem\u003eFaDOF2 \u003c/em\u003eand control. The empty vector pRI101-AN served as a control. Data from three biological replicates are presented as mean values ± SDs. Asterisks denote \u003cem\u003eP\u003c/em\u003e-values from Student’s t-test, with *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 or **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/430743c27d5f0ac723ac45f5.png"},{"id":81931603,"identity":"5d61c5de-8339-4cde-812c-adaf9a9c351e","added_by":"auto","created_at":"2025-05-05 05:30:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":147881,"visible":true,"origin":"","legend":"\u003cp\u003eModel for how FaDOF2 regulates \u003cem\u003eFaSWEET9a\u003c/em\u003e to enhance sucrose accumulation in strawberry fruits. FaDOF2 enhances the expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e by binding to its promoter, leading to increased sucrose content in strawberry fruits.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/804d138f28c2f6dc61e2a763.png"},{"id":84242729,"identity":"a98283a5-e0e0-4c20-9c4a-4e14c6195e4d","added_by":"auto","created_at":"2025-06-09 16:11:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5451043,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/0468cdc0-c0ef-40ff-9674-88275c856272.pdf"},{"id":81931584,"identity":"86ca1d15-de75-4a66-bd09-10286279ada5","added_by":"auto","created_at":"2025-05-05 05:30:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14718,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/1583ae0137afd5c9e90efaea.docx"},{"id":81934966,"identity":"c163bb42-c678-4981-ab9c-f7947479ffe9","added_by":"auto","created_at":"2025-05-05 06:00:06","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11892,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/2ab24cb57c7a384cf8843b95.xlsx"},{"id":81931594,"identity":"622209d4-16cc-47c7-80d5-3bbec5b4f9b4","added_by":"auto","created_at":"2025-05-05 05:30:18","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11306,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/619bdbd040acb79dfcf20609.xlsx"},{"id":81934964,"identity":"24d21901-f06e-4198-916e-8d4b9b36b20e","added_by":"auto","created_at":"2025-05-05 06:00:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10371,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/59d0b48a816d35ff485baf8b.xlsx"},{"id":81931768,"identity":"c0501ae5-0833-432c-88a9-1e52f05bd905","added_by":"auto","created_at":"2025-05-05 05:31:11","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6084358,"visible":true,"origin":"","legend":"","description":"","filename":"supplementfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6529445/v1/bee2a7a4e57054fe247f9b33.docx"}],"financialInterests":"","formattedTitle":"Sucrose transport gene FaSWEET9a regulated by FaDOF2 transcription factor promotes sucrose accumulation in strawberry","fulltext":[{"header":"Key Message","content":"\u003cp\u003e\u003cem\u003eFaSWEET9a,\u0026nbsp;\u003c/em\u003ean important sucrose transport gene regulated by transcription factor FaDOF2, regulates the accumulation of sucrose in strawberry fruits and affects the growth of strawberry plants.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSucrose, glucose, and fructose are key products in the carbon metabolism of plants. In the process of fruit development, an adequate supply of sucrose is essential for promoting fruit growth and maturation. Sucrose modulates gene expression related sugar transport and metabolism, improving fruit quality. Sucrose metabolism represents the most common and efficient sugar metabolic pathway. Within the cytoplasm of fruit cells, rapid and incessant repetitions of sucrose breakdown and rebuilding take place. Sucrose synthase (SUS) facilitates both the breakdown and formation of sucrose, while sucrose phosphate synthase (SPS) specifically catalyzes sucrose synthesis (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Among these enzymes, SUS plays a pivotal regulatory role in this cycle. Within the vacuole, invertase (INV) catalyzes the hydrolysis of sucrose into hexoses, although some of these hexoses are recycled back into the cytoplasm for sucrose synthesis. This cycle is thought to enhance the efficiency of sugar storage and provide sucrose equivalents (hexoses) in a stored form. In the apoplast, cell wall invertases (CWINV) facilitate the hydrolysis of sucrose, with the majority of the resulting hexoses being recycled into the cytoplasm for sucrose synthesis (Lu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The metabolic cycles of sucrose synthesis and degradation work together to regulate sugar accumulation in fruits (Nguyen et al. 2001).\u003c/p\u003e \u003cp\u003eFruits serve as vital \u0026lsquo;sink\u0026rsquo; organs within plants, and sucrose transport and metabolism are of great significance in their growth and development. The sucrose transport pathway in fruits encompasses phloem transport, unloading, intercellular transfer, and metabolic utilization. Sucrose is primarily transported from source organs such as leaves to \u0026lsquo;sink\u0026rsquo; organs like fruits through the phloem sieve tubes. Photosynthesis in leaves leads to the synthesis of sucrose. This newly-formed sucrose is then actively moved into the sieve tubes with the help of the sieve tube-companion cell complex. Subsequently, it travels to the fruits as a result of the pressure flow gradient (Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Key transporters in this process are sucrose transporters (SUT/SUC), monosaccharide transporters (MST), hexose transporters (HT), tonoplast monosaccharide transporters (TMT), tonoplast sugar transporters (TST), and sugars will eventually be exported transporters (SWEET) (Braun, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Moore et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Sucrose is transferred from the sieve tubes to fruit cells through two primary pathways. The first is the symplastic pathway, where sucrose enters fruit cells through plasmodesmata, which are cytoplasmic channels connecting adjacent plant cells. In this pathway, sucrose molecules move directly between cells via the cytoplasmic continuum, bypassing potential interference from enzymes responsible for hydrolysis within the apoplastic space (Lucas et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the second pathway, sucrose is unloaded from the sieve tubes into the apoplast. This process is facilitated by specific unloading mechanisms, likely involving certain transporter proteins that transfer sucrose into the apoplastic space (Milne et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The extracellular region called the apoplast provides an environment where acid invertase acts to split sucrose into its component sugars, glucose and fructose. Simultaneously, sucrose transporters located on the cell membranes of fruit cells utilize the proton motive force or ATP hydrolysis to actively transport sucrose into the cells against its concentration gradient. The hydrolysis-transport process differs across fruit types and is subject to varying levels of regulation (Lalonde et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Ruan et al. 2012). In strawberries, a typical non-climacteric fruit, symplastic unloading predominates during the early developmental stages. As strawberry fruits mature, apoplastic unloading gradually becomes the dominant pathway.\u003c/p\u003e \u003cp\u003eIt is well known that sucrose is formed in leaves with photosynthetic activity (source organs) by combining glucose and fructose. Subsequently, it is conveyed and loaded into the phloem\u0026rsquo;s sieve tubes via the symplastic or apoplastic route. However, the transport pathway of sucrose from mesophyll cells However, the transport pathway of sucrose from mesophyll cells to the apoplast had been unclear before the discovery of SWEET transporters. The \u003cem\u003eSWEET\u003c/em\u003e gene family was initially discovered in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, encoding a novel type of transmembrane protein. These proteins can transport both monosaccharides and disaccharides in an energy-independent manner along a concentration gradient (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xuan et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Seven transmembrane helix domains are presented in SWEET proteins (7-TMS) which mediate passive sugar transport through the unique MtN3/saliva repeat structure (Xuan et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The SWEET family is divided into four subgroups. Clade I and Clade II mainly transport hexoses; Clade III preferentially transports sucrose; and Clade IV participates in intracellular fructose transport. In plants, SWEET members from different subgroups exhibit diverse gene functions, contributing to various physiological processes. Triple mutants (\u003cem\u003eatsweet11\u003c/em\u003e;\u003cem\u003e12\u003c/em\u003e;\u003cem\u003e15\u003c/em\u003e) show increased seed coat starch, decreased seed fat, delayed embryo development, and reduced seed weight with a shriveled appearance, suggesting these proteins aid in sucrose transport from the seed coat to the embryo, supporting seed growth (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eAtSWEETs\u003c/em\u003e play roles in both above-ground and root development, and they modulate abiotic stress responses through sugar transport allocation (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). SWEET-mediated sugar efflux serves as a nutrient acquisition pathway for pathogens, first observed in rice. Rice with recessive \u003cem\u003eOsSWEET11\u003c/em\u003e/\u003cem\u003eOs8N3\u003c/em\u003e alleles shows resistance to bacterial leaf blight. In contrast, dominant allele lines allow bacterial TAL effectors to induce \u003cem\u003eOsSWEET\u003c/em\u003e expression, enhancing sugar efflux and pathogen nutrition acquisition (Yang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDOF (DNA-binding with One Finger) transcription factors, which were initially identified in maize in 1993 (Yanagisawa et al. 1993), are plant-specific zinc finger proteins. Target gene expression is regulated by transcription factors that bind to the cis-elements 5\u0026rsquo;-(T/A)/AAAG-3\u0026rsquo; (Ma et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yanagisawa, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). DOF proteins regulate carbon-nitrogen metabolism, hormone signaling, photoperiod responses, and stress adaptation (Kim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yanagisawa, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). For example, ZmDOF2 enhances DNA-binding efficiency through interactions with high-mobility group proteins, whereas \u003cem\u003eHvBPBF\u003c/em\u003e activates endosperm-specific genes by collaborating with MYB (Cai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gupta et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). DOF transcription factors play a role in plant development, influencing root growth, endosperm development, stomatal formation, and vascular differentiation. OsDOF11 in rice enhances \u003cem\u003eOsSUT1\u003c/em\u003e and \u003cem\u003eOsSWEET14\u003c/em\u003e expression (Kim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). MdDOF54 enhances drought resistance by improving photosynthesis efficiency and water transport in branches (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study identified \u003cem\u003eSWEET\u003c/em\u003e gene family members in the cultivated strawberry genome (Mao et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and discovered that \u003cem\u003eFaSWEET9a\u003c/em\u003e displays distinct expression patterns during fruit development. Transient overexpression and silencing of \u003cem\u003eFaSWEET9a\u003c/em\u003e in \u0026lsquo;Yanli\u0026rsquo; fruits significantly altered sucrose accumulation. Consistently, stable genetic transformation experiments in the strawberry cultivar \u0026lsquo;Chulian\u0026rsquo; demonstrated similar regulatory effects on sucrose metabolism (Zhang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Mechanistically, we found that the DOF transcription factor FaDOF2 bound to the \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter, leading to an upregulation of its expression. This finding holds significant implications for the study of strawberry quality and its growth and development.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eThe strawberry (\u003cem\u003eFragaria\u003c/em\u003e \u0026times; \u003cem\u003eananassa\u003c/em\u003e) cultivars \u0026lsquo;Yanli\u0026rsquo; and \u0026lsquo;Chulian\u0026rsquo; were cultivated in a greenhouse at Shenyang Agricultural University, China. \u0026lsquo;Chulian\u0026rsquo; served as the stable genetic transformation, and the non-transformed \u0026lsquo;Chulian\u0026rsquo; functioned as the wild‐type (WT) plant. \u0026lsquo;Yanli\u0026rsquo; plants were subjected to transient genetic transformation. Tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) plants were selected and then planted in a growth chamber where the temperature was kept at 25\u0026deg;C, and the lighting regime consisted of 16 hours of light followed by 8 hours of darkness. The growth medium employed was potting medium. The samples were frozen using liquid nitrogen and kept at -80\u0026deg;C until they were analyzed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA extraction and RT-qPCR analysis\u003c/h3\u003e\n\u003cp\u003eWe employed the optimized CTAB method described in the research of Wang et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The total RNA was extracted from the fruit samples. After that, the extracted RNA was subjected to reverse transcription using the PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (TaKaRa, Dalian). The cDNA was diluted to one-fourth of its original concentration and served as the template for real-time quantitative polymerase chain reaction (RT-qPCR) analysis. RT-qPCR was performed using the UltraSYBR Mixture (CWBIO, Taizhou, China). As reported in the study by Mao et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), the \u003cem\u003eGAPC2\u003c/em\u003e gene was deliberately chosen to function as the internal control for the experiment. The 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method was used to precisely estimate the relative gene expression levels. For each individual sample, the analysis was meticulously conducted with three technical replicates. Each quantitative real-time polymerase chain reaction (qPCR) assay was performed with three separate biological replicates. The Primer3 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://frodo.wi.mit.edu/\u003c/span\u003e\u003cspan address=\"http://frodo.wi.mit.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to design all primers, which are presented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of the\u003c/b\u003e \u003cb\u003eFaSWEET\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHaplotype-resolved genomic sequence and annotation information for the octoploid strawberry \u0026lsquo;Yanli\u0026rsquo; were sourced from the Rosaceae database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rosaceae.org/Analysis/147231070\u003c/span\u003e\u003cspan address=\"https://www.rosaceae.org/Analysis/147231070\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We conducted an HMMER search using the \u0026lsquo;Yanli\u0026rsquo; protein database. The Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"https://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) provided the Hidden Markov Model (HMM) profile for the SWEET domain (PF03083). BLASTp analysis was employed to look for homologous sequences between the \u003cem\u003eArabidopsis\u003c/em\u003e SWEET proteins obtained from TAIR at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and the \u0026lsquo;Yanli\u0026rsquo; protein database. An assessment of the candidate proteins was performed through the utilization of the Conserved Domain Database (CDD)(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to verify SWEET structural conservation, and non-conforming proteins were eliminated.\u003c/p\u003e\n\u003ch3\u003eChromosomal distribution and structure analysis\u003c/h3\u003e\n\u003cp\u003eChromosomal localization and gene structure analysis of the \u003cem\u003eSWEET\u003c/em\u003e family genes were performed using the GFF3 annotation file with TBtools software (v2.149) (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGene expression level analysis\u003c/h3\u003e\n\u003cp\u003eWe analyzed the transcriptome data gathered from different \u0026lsquo;Yanli\u0026rsquo; fruit development stages: small green, big green, white, turning, and ripening (Wang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The RNA-sequencing data were deposited in the NCBI database. The accession number assigned to this data set is PRJNA975298. Each experiment utilized three biological replicates, and the heatmap was generated with TBtools (v2.149).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCis-regulatory elements of the\u003c/b\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo conduct an analysis of the cis-acting elements, the PlantCARE online resource (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized. By utilizing PlantCARE, a 2000-bp sequence upstream of the coding sequence of the \u003cem\u003eFaSWEET9a\u003c/em\u003e genes was obtained. Then, the results were presented visually using TBtools (v2.149).\u003c/p\u003e \u003cp\u003e \u003cb\u003eUpstream transcription factor prediction of\u003c/b\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA comprehensive prediction of the transcription factor (TF)-binding sites in the promoter regions of the candidate genes was performed using the PlantTFDB database, which can be accessed at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://planttfdb.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://planttfdb.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The predicted TF-binding sites were presented in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e for detailed reference.\u003c/p\u003e\n\u003ch3\u003eSubcellular localization\u003c/h3\u003e\n\u003cp\u003eThe CELLO website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw/\u003c/span\u003e\u003cspan address=\"http://cello.life.nctu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to predict the intracellular localization of \u003cem\u003eFaSWEET9a\u003c/em\u003e. According to Dong et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the pRI101-\u003cem\u003eFaSWEET9a\u003c/em\u003e‐GFP fusion vector was constructed. Refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the associated primers. The bacterial suspension of \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 containing different vectors was transferred to \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. In order to observe the GFP fluorescence signals, confocal fluorescence microscopy (TCS SP8, Leica, Germany) was utilized.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe phylogenetic tree was constructed via the maximum-likelihood approach. For the calculation of phylogenetic distances, expressed as the number of amino acid substitutions per site, the Poisson correction method was employed. The entire phylogenetic analysis was carried out within the MEGA11 software.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransient expression in strawberry fruits\u003c/h3\u003e\n\u003cp\u003eThe CDS regions of \u003cem\u003eFaSWEET9a\u003c/em\u003e or \u003cem\u003eFaDOF2\u003c/em\u003e were cloned separately into the pRI101-AN vector to construct the pFaSWEET9a-OE and pFaDOF2-OE vectors for overexpression in \u0026lsquo;Yanli\u0026rsquo; fruits. To silence \u003cem\u003eFaSWEET9a\u003c/em\u003e expression in \u0026lsquo;Yanli\u0026rsquo; fruit, the forward and reverse fragments of the \u003cem\u003eFaSWEET9a\u003c/em\u003e gene were amplified and inserted into the pRNAi-E vector (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) to construct the pFaSWEET9a-RNAi vector. Refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the associated primers. These plasmids were introduced into the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etumefaciens\u003c/em\u003e strain GV3101. Then we prepared the infiltration buffer and infiltrated the fruits as per the methods of Li et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A 1 mL sterile syringe was used to inject 1 mL of infiltration buffer into large green fruit. One fruit specimen was selected and utilized for the purpose of infiltrating the target constructs into it, while another served as a control with empty vectors. Seven days subsequent to the infiltration procedure, the fruits that had undergone infiltration were harvested. A biological replicate was composed of a set of six fruits, and at least three such biological replicates were carried out in this study.\u003c/p\u003e\n\u003ch3\u003eStrawberry stable genetic transformation\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e plants of cultivated strawberry cultivar \u0026lsquo;Chulian\u0026rsquo; were subcultured on MS medium with 0.2 mg/L 6-Benzylaminopurine (6-BA), 0.1 mg/L Gibberellic Acid 3 (GA\u003csub\u003e3\u003c/sub\u003e), and 0.01 mg/L Indole-3-butyric acid (IBA). The \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpression (OE) construct was introduced into the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etumefaciens\u003c/em\u003e strain GV3101 by means of the freeze-thaw technique (Wang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). First, a single colony from the bacterial strain was selected and inoculated into the prepared liquid yeast extract peptone (YEP) medium. This medium was enhanced with 25 mg/L kanamycin and 100 mg/L spectinomycin. The medium that had been inoculated was placed on an orbital shaker set at a rotational speed of 180 rpm and incubated at 28\u0026deg;C for a duration of 16 hours. After incubation, the OD₆₀₀ of the bacterial solution was carefully adjusted to 0.1 in 50 mL of YEP medium. The solution was incubated at 28\u0026deg;C with agitation for 5 to 6 h until the OD600 of 0.5 was achieved. Next, each petiole was sectioned into 1 cm segments. These segments were then submerged in the bacterial suspension and gently swirled for 8 min. After retrieval from the suspension, the excess liquid was carefully blotted off using a sterile filter paper. Subsequently, the petiole segments were placed in a bud regeneration medium, consisting of MS medium with 2.0 mg/L thidiazuron (TDZ), and 0.2 mg/L IBA, and kept in total darkness for 3 days for co-cultivation. After the co-cultivation phase, the petiole segments were moved to a new prepared bud regeneration medium containing 250 mg/L cefotaxime and 25 mg/L kanamycin for selection. Once the selection was finalized, the petiole explants were relocated to a well-lit area for a 4 week cultivation period. When the regenerated buds attained a length of 1 cm, they were delicately excised and transferred into a development medium containing MS medium with 0.2 mg/L 6-BA, 0.1 mg/L GA3, 0.01 mg/L IBA, 250 mg/L cefotaxime, and 25 mg/L kanamycin. To sustain the selection pressure throughout the entire process, sub-culturing was performed at three week intervals.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSugar measurement\u003c/h2\u003e \u003cp\u003eFour fruit ripening stages defined as big green, white, turning, and ripening were harvested from stable transgenic strawberry plants for measuring soluble sugar content. Soluble sugar content was measured in new leaves, mature leaves, petioles, and roots collected from stable genetically transformed plants grown in MS medium, ensuring samples were taken from consistent positions on each plant. The soluble sugar contents were determined by the anthrone colorimetric method (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Each group consisted of three biological replicates.\u003c/p\u003e \u003cp\u003eFructose, glucose and sucrose were analyzed by high-performance liquid chromatography (1290 infinity II, Agilent, USA) following the method described by Li et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Each group had three biological replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHeterologous Expression of\u003c/b\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e \u003cb\u003ein Yeast Cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to Li et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the pDR196-\u003cem\u003eFaSWEET9a\u003c/em\u003e vector was constructed. The heterologous expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e in yeast was performed using the native gene sequence without codon optimization. Refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the associated primers. The constructs were transferred into the yeast mutant strain SUSY7/ura via the lithium acetate-mediated transformation technique (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Riesmeier et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Soni et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). FaSWEET9a was expressed in yeast strain SUSY7/ura. Yeast cells with pDR196-FaSWEET9a or the pDR196 vector (negative control) were grown on solid Synthetic Defined Medium without uracil using 2% (w/v) glucose or sucrose as the only carbon source.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eYeast one-hybrid (Y1H) assay\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eFaDOF2\u003c/em\u003e coding sequence was cloned into the pGADT7 vector, creating the pGADT7-FaDOF2 fusion construct. The 1000 bp promoter region of \u003cem\u003eFaSWEET9a\u003c/em\u003e containing two (A/T)AAAG elements (located at -270 bp and \u0026minus;\u0026thinsp;842 bp) was segmented to construct the reporter vectors pAbAi-\u003cem\u003eProFaSWEET9a\u003c/em\u003e-P1 (0~ -432 bp) and pAbAi-\u003cem\u003eproFaSWEET9a\u003c/em\u003e-P2 (-742 ~ -1000 bp). Refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the associated primers. According to Li et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the pGADT7-FaDOF2 and pAbAi‐\u003cem\u003eproFaSWEET9a\u003c/em\u003e fusion vectors were transformed into Y1H yeast cells for cultivation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eElectrophoretic mobility shift assay (EMSA)\u003c/h2\u003e \u003cp\u003eThe coding region of FaDOF2 was linked to the pCold vector with the His label to construct the FaDOF-pCold vector. The probe sequence (40 bp) was isolated from \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter and consisted of the (A/T)AAAG element. Refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the associated primers. \u003cem\u003eFaSWEET9a\u003c/em\u003e-probe represents the probe sequence labelled with biotin, and competitor represents the probe sequence that was not labelled by biotin. \u003cem\u003eFaSWEET9\u003c/em\u003e-probe and its competitor were synthesized by Sangon Biotechnology Co. LTD (Shanghai, China). The Chemiluminescent EMSA Kit (Beyotime, Shanghai, China) was used for performing the EMSA technique.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase (LUC) reporter assay\u003c/h2\u003e \u003cp\u003eAccording to Luo et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), the pRI101-\u003cem\u003eFaDOF2\u003c/em\u003e vector and the pro\u003cem\u003eFaSWEET9a\u003c/em\u003e::LUC vector containing a 1000-bp \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter were constructed. The bacterial suspension of \u003cem\u003eA. tumefaciens\u003c/em\u003e strains GV3101 containing the vectors mentioned above was mixed according to the requirements and then injected into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Luciferase signals were detected using the live fluorescence imager (Lb985, Berthold, Germany) after a 2-day dark incubation. The transcriptional activity was evaluated by means of the dual luciferase reporter assay kit (Beyotime, Shanghai, China). Refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the associated primers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eβ-glucuronidase (GUS) reporter assay\u003c/h2\u003e \u003cp\u003eAccording to Luo et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), the \u003cem\u003eproFaSWEET9a\u003c/em\u003e::GUS vector containing \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter (1000 bp) was constructed. Refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the associated primers. The bacterial suspension of GV3101 containing the aforementioned vectors was prepared and subsequently injected into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves as per experimental requirements. The infected tobacco leaves were subjected to GUS staining in strict accordance with the protocol stipulated by the GUS stain kit (Coolaber, Beijing, China). According to Li et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the GUS activity and GUS protein concentration of infected tobacco leaves were detected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are presented in the form of means accompanied by standard deviations (SDs). Significance analyses were conducted via the Student\u0026rsquo;s t-tests using SPSS software (version 17.0), with significance thresholds set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAccession Numbers\u003c/h2\u003e \u003cp\u003eThe sequence data presented in this article can be retrieved from the Rosaceae database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rosaceae.org/Analysis/14723107\u003c/span\u003e\u003cspan address=\"https://www.rosaceae.org/Analysis/14723107\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using the following accession numbers: \u003cem\u003eFaSWEET1a\u003c/em\u003e (FxaYL_241g0513140), \u003cem\u003eFaSWEET1b\u003c/em\u003e (FxaYL_241g0513130), \u003cem\u003eFaSWEET1c\u003c/em\u003e (FxaYL_221g0474270), \u003cem\u003eFaSWEET1d\u003c/em\u003e (FxaYL_242g0463630), \u003cem\u003eFaSWEET2a\u003c/em\u003e (FxaYL_341g0266030), \u003cem\u003eFaSWEET2b\u003c/em\u003e (FxaYL_311g0354740), \u003cem\u003eFaSWEET3\u003c/em\u003e (FxaYL_721g0968830), \u003cem\u003eFaSWEET4\u003c/em\u003e (FxaYL_511g0685950), \u003cem\u003eFaSWEET5a\u003c/em\u003e (FxaYL_511g0691570), \u003cem\u003eFaSWEET5b\u003c/em\u003e (FxaYL_611g0109740), \u003cem\u003eFaSWEET6a\u003c/em\u003e (FxaYL_611g0095590), \u003cem\u003eFaSWEET6b\u003c/em\u003e (FxaYL_611g0095800), \u003cem\u003eFaSWEET6c\u003c/em\u003e (FxaYL_612g0094050), \u003cem\u003eFaSWEET7\u003c/em\u003e (FxaYL_441g0239180), \u003cem\u003eFaSWEET9\u003c/em\u003ea (FxaYL_231g0391340), \u003cem\u003eFaSWEET9b\u003c/em\u003e (FxaYL_211g0416320), \u003cem\u003eFaSWEET9c\u003c/em\u003e (FxaYL_211g0416280), \u003cem\u003eFaSWEET9d\u003c/em\u003e (FxaYL_222g0414840), \u003cem\u003eFaSWEET10\u003c/em\u003e (FxaYL_541g0616130, \u003cem\u003eFaSWEET14a\u003c/em\u003e (FxaYL_211g0416270), \u003cem\u003eFaSWEET14b\u003c/em\u003e (FxaYL_541g0616120), \u003cem\u003eFaSWEET15a\u003c/em\u003e (FxaYL_431g0644640), \u003cem\u003eFaSWEET15b\u003c/em\u003e (FxaYL_411g0805500), \u003cem\u003eFaSWEET16\u003c/em\u003e (FxaYL_741g0928650), \u003cem\u003eFaSWEET17\u003c/em\u003e (FxaYL_411g0802810). \u003cem\u003eSPSA1\u003c/em\u003e (FxaYL_142g0861950), \u003cem\u003eSPSA3\u003c/em\u003e (FxaYL_431g0636300), \u003cem\u003eSPSA4\u003c/em\u003e (FxaYL_221g0488530), \u003cem\u003eCWIVN\u003c/em\u003e (FxaYL_612g0121980), \u003cem\u003eHXK1\u003c/em\u003e (FxaYL_112g0686890), \u003cem\u003eHXK2\u003c/em\u003e (FxaYL_221g0450840), \u003cem\u003eSUS\u003c/em\u003e (FxaYL_121g0719220), \u003cem\u003eSUS2\u003c/em\u003e (FxaYL_221g0462810), \u003cem\u003eSUS7\u003c/em\u003e (FxaYL_121g0701630), \u003cem\u003eSUC3\u003c/em\u003e (FxaYL_431g0652490), \u003cem\u003eSUC4\u003c/em\u003e (FxaYL_511g0663990), \u003cem\u003eCINV\u003c/em\u003e (FxaYL_531g0564800), \u003cem\u003eSTP1\u003c/em\u003e (FxaYL_221g0482960), \u003cem\u003eSTP13\u003c/em\u003e (FxaYL_441g0240880), \u003cem\u003eSTP14\u003c/em\u003e (FxaYL_122g0775850), \u003cem\u003eSTP7\u003c/em\u003e (FxaYL_141g0906560).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacteristic analysis of\u003c/b\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur laboratory has previously developed a high-quality haplotype-resolved genome assembly for the cultivated octoploid strawberry cultivar \u0026lsquo;Yanli\u0026rsquo;, comprising two haplotype assemblies (Hap1 and Hap2) with a total of 56 chromosomes (Mao et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Using the genome annotation, we identified \u003cem\u003eSWEET\u003c/em\u003e family members through Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"https://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) searches for the MtN3 (PF03083) domain. From the \u0026lsquo;Yanli\u0026rsquo; genome, we identified 25 \u003cem\u003eFaSWEET\u003c/em\u003e family members. These were systematically named \u003cem\u003eFaSWEET1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003e17\u003c/em\u003e based on their homology to \u003cem\u003eA. thaliana SWEET\u003c/em\u003e genes. The \u003cem\u003eFaSWEET\u003c/em\u003e genes are distributed across chromosomes 2 through 7 (Supplement Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Gene structure analysis showed that \u003cem\u003eFaSWEET\u003c/em\u003e genes contained 2\u0026ndash;6 exons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe analyzed the expression patterns of \u003cem\u003eFaSWEET\u003c/em\u003e genes across five developmental stages of \u0026lsquo;Yanli\u0026rsquo; fruits using RNA-seq.\u0026nbsp;\u003cem\u003eFaSWEET9a\u003c/em\u003e, exhibiting the highest transcript levels during the white and turning stages, was selected for further characterization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The structural analysis predicted seven transmembrane domains in FaSWEET9a (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). After conducting a comparative analysis of the amino acid sequences and stage-specific expression profiles among FaSWEET9a-d, it was found that the alleles of \u003cem\u003eFaSWEET9a\u003c/em\u003e with the highest expression levels form an allelic pair located on Hap1 and Hap2, and both possess two MtN3 domains (Supplement Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, S3). Subsequent analyses and experiments were conducted using the FaSWEET9a allele (FxaYL_231g0391340) from Hap1. Subcellular localization prediction via the CELLO database indicated potential plasma membrane (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Transiently expressing \u003cem\u003eFaSWEET9a\u003c/em\u003e-GFP fusion constructs in \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e leaves confirmed that the protein was located on the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe identified \u003cem\u003eFaSWEET\u003c/em\u003e genes from the \u0026lsquo;Yanli\u0026rsquo; genome and constructed a phylogenetic tree together with functionally characterized SWEET genes from model plant \u003cem\u003eA. thaliana\u003c/em\u003e (AtSWEETs) and important crops including rice (\u003cem\u003eOryza sativa\u003c/em\u003e, OsSWEETs), tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e, SlSWEETs) and apple (\u003cem\u003eMalus domestica\u003c/em\u003e, MdSWEETs) to comprehensively resolve the evolutionary relationships of the SWEET family in both dicot and monocot plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The maximum-likelihood phylogenetic tree revealed that SWEET family members clustered into four distinct clades (I\u0026ndash;IV) with characterized AtSWEETs. Clade III contained FaSWEET9a-d, FaSWEET10, FaSWEET14a-b, FaSWEET15a-b alongside AtSWEET9-15, OsSWEET11-15, MdSWEET9a-b, MdSWEET14, SlSWEET10a-c, SlSWEET11a-d, SlSWEET12a-d and SlSWEET14, suggesting conserved functions in sugar allocation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFaSWEET9a acts as a sucrose transporter\u003c/h2\u003e \u003cp\u003eAmong the four distinct clades within the \u003cem\u003eSWEET\u003c/em\u003e gene family, \u003cem\u003eSWEET9\u003c/em\u003e is categorized as a member of clade III. Functionally, this particular clade is predominantly responsible for facilitating sucrose transport. To assess the sucrose transport activity of FaSWEET9a, we heterologously expressed the \u003cem\u003eFaSWEET9a\u003c/em\u003e in the yeast (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e) mutant SUSY7/ura. In the experiment, yeast mutant cells transfected with the empty pDR196 vector served as negative controls. The results showed that yeast mutant cells harboring FaSWEET9a grew faster and more robustly on synthetic deficient media supplemented with sucrose than yeast cells containing the empty vector. This result demonstrates that FaSWEET9a exhibits substrate specificity for sucrose transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTransient overexpression or silencing of\u003c/b\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e \u003cb\u003ealters sucrose content in strawberry fruits\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe explored the role of \u003cem\u003eFaSWEET9a\u003c/em\u003e in strawberry fruits by overexpressing it in \u0026lsquo;Yanli\u0026rsquo; fruits through \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etumefaciens\u003c/em\u003e-mediated infiltration. The control utilized was the empty pRI101-AN vector. In the fruits infiltrated with pFaSWEET9a-OE, the relative expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e was significantly higher than that in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Moreover, the sucrose content exhibited a significant increase compared to that of the control. In contrast, there were no statistically significant differences detected in the levels of fructose and glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We used \u003cem\u003eA\u003c/em\u003e. \u003cem\u003etumefaciens\u003c/em\u003e-mediated infiltration to silence \u003cem\u003eFaSWEET9a\u003c/em\u003e expression in \u0026lsquo;Yanli\u0026rsquo; fruits. The expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e in fruits treated with pFaSWEET9a-RNAi showed a statistically significant decrease compared to the expression level in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The sucrose content was significantly reduced compared to the control. At the same time, no significant difference was detected in fructose and glucose levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The transient transformation experiments carried out on strawberry fruits showed that \u003cem\u003eFaSWEET9a\u003c/em\u003e could influence the changes in sucrose content within the strawberry fruits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e \u003cb\u003ealters morphological phenotypes and increases sucrose content in strawberries\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the impact of \u003cem\u003eFaSWEET9a\u003c/em\u003e on the phenotype of strawberry plants, we obtained two \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpressing lines (\u003cem\u003eFaSWEET9a\u003c/em\u003e-OE: OE-6 and OE-7). Morphological parameters, such as height, leaf area, the number of leaves, length and width of the third leaf, and petiole length of the third leaf were assessed during both vegetative and reproductive growth stages. During the vegetative growth stage, \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE plants exhibited significantly greater plant height than WT plants. Similarly, in terms of leaf area, length, width, and petiole length of the third leaf, \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE plants showed significant increases relative to WT plants. However, no significant differences in the number of leaves were found between \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE and WT plants (Supplement Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ea-f). In the reproductive growth stage, \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE plants were also significantly bigger than WT plants regarding plant height, leaf area, third leaf length, third leaf width, and petiole length. However, no significant differences in the number of leaves were found between \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE plants and WT plants (Supplement Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eg-l). The morphological investigations and analyses indicated that overexpression of \u003cem\u003eFaSWEET9a\u003c/em\u003e can influence various phenotype traits in strawberry plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe investigated the impact of \u003cem\u003eFaSWEET9a\u003c/em\u003e on sucrose content variations in strawberries by examining the soluble sugar levels in various organs of \u003cem\u003ein vitro\u003c/em\u003e-cultured \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE plants. The study found that soluble sugar levels in new leaves, mature leaves, petioles, and roots were markedly elevated compared to WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAfter transplanting the transgenic plants and WT, we successfully obtained fruits at various developmental stages, including big green, white, turning, and ripening. To validate the elevated transcript levels of \u003cem\u003eFaSWEET9a\u003c/em\u003e at these stages, we examined \u003cem\u003eFaSWEET9a\u003c/em\u003e expression in fruits from \u003cem\u003eFaSWEET9a\u003c/em\u003e-overexpressing (\u003cem\u003eFaSWEET9a\u003c/em\u003e-OE) plants. RT-qPCR analysis revealed that the relative expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e was significantly higher in the big green, white, turning, and ripening fruits compared to that in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). We examined the impact of \u003cem\u003eFaSWEET9a\u003c/em\u003e overexpression on sucrose levels in strawberries by analyzing soluble sugars (fructose, glucose, and sucrose) at different developmental stages in both \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE and WT fruits. The findings demonstrated that \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE fruits exhibited a significantly higher soluble sugar content compared to WT fruits at various stages of fruit development (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Further analysis of the glucose, fructose, and sucrose contents at various stages of fruit development revealed that all these sugars were present at significantly higher levels in the \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE fruits compared to WT counterparts throughout all examined stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f). Notably, the sucrose content peaked during the white and turning phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). The present findings strongly imply that \u003cem\u003eFaSWEET9a\u003c/em\u003e exerts a pivotal influence on promoting the accumulation of sucrose within the fruits of strawberry plants.\u003c/p\u003e \u003cp\u003eWe selected genes associated with sucrose metabolism and transport to analyze their relative expression in \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE fruits at the white stage and turning stage, aiming to better understand the variations in sucrose content. The results indicated a significant increase in the relative expression of genes linked to sucrose metabolism, including \u003cem\u003eSPS4\u003c/em\u003e (sucrose phosphate synthases), \u003cem\u003eCWINV\u003c/em\u003e (cell wall invertase), \u003cem\u003eHXK2\u003c/em\u003e (hexokinase), \u003cem\u003eSUS\u003c/em\u003e, \u003cem\u003eSUS7\u003c/em\u003e (sucrose synthase), during the white stage. Additionally, we observed a marked elevation in the relative expression of sucrose transport-related genes, including \u003cem\u003eSTP7\u003c/em\u003e, \u003cem\u003eSTP10\u003c/em\u003e, \u003cem\u003eSTP13\u003c/em\u003e, \u003cem\u003eSTP14\u003c/em\u003e (sugar transporter protein), \u003cem\u003eSUC4\u003c/em\u003e (sucrose carrier) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Notably, \u003cem\u003eSUS\u003c/em\u003e exhibited the highest level of relative expression among these genes. During the turning stage, we found a significant upregulation of sucrose metabolism-related genes such as \u003cem\u003eSPSA1\u003c/em\u003e, \u003cem\u003eSPSA3\u003c/em\u003e, \u003cem\u003eCWINV\u003c/em\u003e, \u003cem\u003eHXK1\u003c/em\u003e, \u003cem\u003eSUS\u003c/em\u003e, \u003cem\u003eSUS7\u003c/em\u003e, \u003cem\u003eCIN\u003c/em\u003e (cytoplasm invertase). Moreover, the relative expression of sucrose transport-related genes, including \u003cem\u003eSTP10\u003c/em\u003e, \u003cem\u003eSTP13\u003c/em\u003e, and \u003cem\u003eSTP14\u003c/em\u003e, showed a significant increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). These results indicate that \u003cem\u003eFaSWEET9a\u003c/em\u003e has the potential to enhance fruit sucrose content by modulating specific gene expression critical for sucrose degradation and resynthesis processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e \u003cb\u003eis regulated by FaDOF2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate which signals regulate the expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e, based on the analysis of the \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter cis-acting elements (Supplement Fig. S5) and the predictions from the plantTFDB website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://planttfdb.gao-lab.org/\u003c/span\u003e\u003cspan address=\"http://planttfdb.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), we selected the FaDOF2 transcription factor for further analysis, whose family members have been reported to regulate SWEET transporters (Wu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFaDOF2\u003c/em\u003e CDS was cloned into the pGADT7 vector, and we identified two (T/A)AAAG motifs at positions \u0026minus;\u0026thinsp;270 bp (P1) and \u0026minus;\u0026thinsp;842 bp (P2) within the 1000-bp promoter region of \u003cem\u003eFaSWEET9a\u003c/em\u003e and subsequently constructed two corresponding reporter vectors, pAbAi-\u003cem\u003eProFaSWEET9a\u003c/em\u003e-P1 and pAbAi-\u003cem\u003eProFaSWEET9a\u003c/em\u003e-P2. Subsequently, the recombinant plasmid pAbAi-\u003cem\u003eProFaSWEET9a\u003c/em\u003e-P1/2 was utilized in the Y1H assay. Yeast cells co-transformed with pGADT7 and pAbAi-\u003cem\u003eProFaSWEET9a\u003c/em\u003e-P1/2 failed to grow on Leu medium containing 75 ng/ml aureobasidin A (AbA). Yeast cells co-transformed with pGAD-FaDOF2 and pAbAi-\u003cem\u003eProFaSWEET9a\u003c/em\u003e-P2 grew normally on the medium, while the result of pAbAi-\u003cem\u003eProFaSWEET9a\u003c/em\u003e-P1 was consistent with that of the negative control. The result shows that FaDOF2 can bind to the promoter of \u003cem\u003eFaSWEET9a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Additionally, we analyzed the \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter and identified a\u0026thinsp;~\u0026thinsp;AAAG motif located at \u0026minus;\u0026thinsp;842 bp. To confirm the binding of FaDOF2 to the \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter, an EMSA was performed. The hot probe contains biotin-labeled P2 fragments, and EMSA was performed using the counterpart unlabeled fragments as competitors (cold probe). In the experiment, we progressively increased the amount of cold probes (10\u0026times;, and 50\u0026times; fold probe concentration). Specific binding of \u003cem\u003eFaSWEET9a\u003c/em\u003e in fragments containing FaDOF2-binding elements (TAAAG) was shown by their upward shift. The results demonstrated that competitive cold probes significantly reduced the radioactive signal, including the binding signal between FaDOF2 and the \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter. Furthermore, binding was disrupted when mutated probes were used (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eGUS assays were conducted to investigate whether FaDOF2 influences the transcriptional activity of the \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter. The GUS analysis indicated that p\u003cem\u003eProFaSWEET9a\u003c/em\u003e::GUS expression levels were significantly elevated when co-expressed with p35S::\u003cem\u003eFaDOF2\u003c/em\u003e than co-expression with pRI101-AN. Likewise, the GUS histochemical stain assay results showed that FaDOF2 specifically activated GUS transcription as driven by the \u003cem\u003eFaSWEET9a\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). To confirm further the relationship between FaDOF2 and \u003cem\u003eFaSWEET9a\u003c/em\u003e, an \u003cem\u003ein vivo\u003c/em\u003e LUC reporter assay was performed. The luminescence intensity of \u003cem\u003eN. benthamiana\u003c/em\u003e epidermal cells was measured. Cells co-expressing 35S::\u003cem\u003eFaDOF2\u003c/em\u003e and \u003cem\u003eproFaSWEET9a\u003c/em\u003e::LUC exhibited enhanced luminescence than those expressing only \u003cem\u003eproFaSWEET9a::LUC\u003c/em\u003e or the negative control (empty vector). Moreover, \u003cem\u003eproFaSWEET9a\u003c/em\u003e exhibited the highest activity when combined with FaDOF2. The findings suggest that FaDOF2 binds to the promoter of \u003cem\u003eFaSWEET9a\u003c/em\u003e and regulates its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTransient overexpression of\u003c/b\u003e \u003cb\u003eFaDOF2\u003c/b\u003e \u003cb\u003eincreases sucrose content in strawberry fruits\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether FaDOF2 can enhance the sucrose content in strawberry fruits through the regulation of \u003cem\u003eFaSWEET9a\u003c/em\u003e, we conducted a transient overexpression experiment in strawberry fruits. \u003cem\u003eFaDOF2\u003c/em\u003e was overexpressed in \u0026lsquo;Yanli\u0026rsquo; fruits via \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated infiltration. The pRI101-AN vector served as a control, and fruits infiltrated with pFaDOF2-OE exhibited a significantly higher relative expression level of \u003cem\u003eFaDOF2\u003c/em\u003e than that in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Similarly, the relative expression level of \u003cem\u003eFaSWEET9a\u003c/em\u003e was also up-regulated in the fruits transiently overexpressing \u003cem\u003eFaDOF2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), indicating that FaDOF2 positively regulates \u003cem\u003eFaSWEET9a\u003c/em\u003e. The fructose, glucose, and sucrose levels were assessed in fruits exhibiting transient \u003cem\u003eFaDOF2\u003c/em\u003e overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The findings indicated a significant increase in sucrose content in \u003cem\u003eFaDOF2\u003c/em\u003e-OE fruits than that in the control. However, fructose and glucose contents in the \u003cem\u003eFaDOF2\u003c/em\u003e-OE fruits did not exhibit significant changes compared to the control. Overexpression of \u003cem\u003eFaDOF2\u003c/em\u003e enhances the sucrose content in strawberry fruits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eInfluence of\u003c/b\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e \u003cb\u003eon the development of strawberry plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eFaSWEET9a\u003c/em\u003e, a SWEET III gene family in the octoploid strawberry genome, encodes a transporter with seven transmembrane domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). This characteristic is consistent with its homologs in \u003cem\u003eA. thaliana\u003c/em\u003e (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). We found that FaSWEET9a is highly conserved across multiple species through cross-species comparative analysis. Its homologous genes, such as \u003cem\u003eAtSWEET9\u003c/em\u003e and \u003cem\u003eOsSWEET14\u003c/em\u003e, play crucial roles in sucrose transport and accumulation (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), indicating that this gene has retained essential functions throughout evolution. This conservation provides a theoretical foundation for extrapolating findings from other species to strawberries (Jia et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Smeekens et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A phylogenetic tree constructed based on protein sequences shows that \u003cem\u003eFaSWEET9a\u003c/em\u003e clusters with \u003cem\u003eSWEET\u003c/em\u003e III genes from \u003cem\u003eArabidopsis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), supporting its functional specialization in sucrose transport (Lemoine et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Overexpression of \u003cem\u003eFaSWEET9a\u003c/em\u003e significantly affected the growth and morphological characteristics of strawberry plants (Supplement Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). In particular, plants overexpressing \u003cem\u003eFaSWEET9a\u003c/em\u003e exhibited increased height, enlarged leaf area, and extended petiole length (Supplement Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). These findings suggest that \u003cem\u003eFaSWEET9a\u003c/em\u003e may influence overall plant growth by redistributing photosynthates. Likewise, apple plants overexpressing \u003cem\u003eMdSWEET9\u003c/em\u003e show similar changes, with obvious alterations in nutrient transport and a notable build-up of plant material. (Braun et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInfluence of\u003c/b\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e \u003cb\u003eon fruit quality\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur study confirmed the role of FaSWEET9a as a sucrose transporter, further clarifying its gene function. Heterologous expression experiments showed that FaSWEET9a could restore the ability of the mutant yeast strain SUSY7/ura to utilize sucrose (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The localization of FaSWEET9a on the plasma membrane indicates its critical role in intercellular sucrose transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). This is consistent with the functional localization of \u003cem\u003eSWEET\u003c/em\u003e genes in other plants. For instance, OsSWEET14 and AtSWEET11/12 are plasma membrane-localized proteins that mediate the translocation of sucrose from the photosynthetically active source tissues to the metabolically demanding sink tissues (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Le et al. 2015). FaSWEET9a has been confirmed to facilitate the transport of sucrose. Thus, does it influence the sucrose content of the fruits? We investigated this question through transient overexpression and silencing of \u003cem\u003eFaSWEET9a\u003c/em\u003e in fruits and stable genetic transformation using the vector pFaSWEET9a-OE. Transient overexpression of \u003cem\u003eFaSWEET9a\u003c/em\u003e in fruits led to a significant increase in both \u003cem\u003eFaSWEET9a\u003c/em\u003e expression and sucrose content than those in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). In contrast, the opposite was true in the fruits with transient silencing of \u003cem\u003eFaSWEET9a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). After clarifying the effects of transient overexpression and silencing of \u003cem\u003eFaSWEET9a\u003c/em\u003e on the sucrose content in fruits, we further conducted a stable genetic transformation experiment for verification. Similarly, the relative expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e increased significantly in \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE fruits during the big green, white, turning, and ripening stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This dynamic expression pattern aligns with the findings for \u003cem\u003eOsSWEET14\u003c/em\u003e and \u003cem\u003eMdSWEET9b\u003c/em\u003e, where expression levels also increase during fruit enlargement and maturation (Eom et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, the expression level of \u003cem\u003eFaSWEET9a\u003c/em\u003e in \u003cem\u003eFaSWEET9a\u003c/em\u003e-OE fruits fluctuates during the developmental process. During the early fruit developmental phase (big green stage), characterized by active cell division and expansion, the high expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e likely supplies energy substrates, such as sucrose, to fuel cellular growth, thereby promoting morphogenesis in both leaves and fruits. As the fruit transitions into the sucrose-accumulating critical phase (white to turning stages), the enhanced sucrose translocation from source organs (leaves) to sink organs (fruits) drives sustained high expression of \u003cem\u003eFaSWEET9a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This elevated expression directly correlates with increased sucrose content in fruits, with the sucrose peak synchronizing temporally with maximal \u003cem\u003eFaSWEET9a\u003c/em\u003e transcript levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, f). Such synchronization suggests potent sucrose-driven transcriptional regulation during this stage, which may underlie the dynamic expression fluctuations observed during fruit development. Similarly, in tomato, the sucrose content in fruits at different developmental stages also changes with the variation in the expression level of the sucrose transporter \u003cem\u003eSlSWEET14\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In grapevine, the hexose transporter \u003cem\u003eVvSWEET15\u003c/em\u003e initiates hexose accumulation starting at the veraison stage and peaks during fruit ripening (Lu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Sucrose, serving a dual role as both a signaling molecule and an energy source, necessitates upregulated transporter activity to meet the demands of rapid fruit expansion and carbohydrate storage (Kr\u0026uuml;gel et al. 2013). This regulatory process likely operates through conserved sugar signaling pathways that induce feedback upregulation of \u003cem\u003eFaSWEET9a\u003c/em\u003e itself or coordinate the expression of related transporters. Together, these mechanisms establish a \u0026lsquo;transport-accumulation-signal amplification\u0026rsquo; positive feedback loop, ensuring sustained sucrose allocation to fruits while maintaining systemic carbon homeostasis. However, during the ripening stage, sucrose content decreases while glucose and fructose levels increase. This shift may result from reduced demand for sucrose import coupled with its subsequent breakdown into glucose and fructose, leading to decreased demand for sucrose transporters and consequent downregulation of \u003cem\u003eFaSWEET9a\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, d-f). These results demonstrate that as the expression level of \u003cem\u003eFaSWEET9a\u003c/em\u003e increases or decreases, the sucrose content in strawberry fruits will also increase or decrease. This finding provides a potential target for the genetic improvement of strawberry fruits to enhance their sucrose content and overall quality\u003c/p\u003e \u003cp\u003e \u003cb\u003eFaSWEET9a\u003c/b\u003e \u003cb\u003epotentially modulates sucrose accumulation in fruits through the regulation of genes associated with sucrose metabolism and transport\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur experiments indicated that both \u003cem\u003eFaSWEET9a\u003c/em\u003e expression and sucrose content peaked during the white and turning stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, f). Therefore, we examined the relative expression levels of genes associated with sucrose metabolism and transport across these stages.\u003c/p\u003e \u003cp\u003eThrough a meticulous research process, we ascertained that during the white stage, genes implicated in sucrose synthesis, notably \u003cem\u003eSUS\u003c/em\u003e, exhibited a marked upsurge in their relative expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). \u003cem\u003eSUS\u003c/em\u003e plays a pivotal role in sucrose metabolism, catalyzing the reversible reaction between sucrose and UDP-glucose and fructose. An up-regulation of \u003cem\u003eSUS\u003c/em\u003e means that more substrates are converted into sucrose, directly boosting the synthesis rate. Concurrently, genes related to sucrose transport, including \u003cem\u003eSPSA4\u003c/em\u003e, \u003cem\u003eSTP7\u003c/em\u003e, \u003cem\u003eSTP13\u003c/em\u003e, \u003cem\u003eSTP14\u003c/em\u003e, and \u003cem\u003eSUC4\u003c/em\u003e, also showed elevated expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). These transporters facilitate the movement of newly synthesized sucrose from the production sites to storage compartments within the fruit cells or enable transport between different cells. This coordinated up-regulation of synthesis and transport genes forms a well-orchestrated mechanism for sucrose accumulation in the fruit during the white stage. Similar patterns have been observed in other crops. In peach fruits, the relative expression of \u003cem\u003ePpSUS1\u003c/em\u003e and \u003cem\u003ePpSPS2\u003c/em\u003e was positively correlated with sucrose content (Dai et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This indicates that sucrose-related genes consistently influence sucrose levels across various fruit-bearing plants. In strawberry, virus-induced gene silencing assays for \u003cem\u003eFaSS1\u003c/em\u003e demonstrated that its down-regulation decreased sucrose content and inhibited ripening (Zhao et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In sugarcane, overexpressing \u003cem\u003eSoSPS1\u003c/em\u003e in sugarcane increased SPS activity and sucrose content in transgenic lines (Anur et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). SPS facilitates the transformation of F6P (fructose-6-phosphate) and uridine diphosphate glucose into S6P (sucrose-6-phosphate). Subsequently, SPP then hydrolyzes the newly-formed S6P into sucrose. (Verma et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the turning stage, there were significant increases in the relative expression levels of genes associated with sucrose decomposition, including \u003cem\u003eCWINV\u003c/em\u003e and \u003cem\u003eCIN\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). \u003cem\u003eCWINV\u003c/em\u003e and \u003cem\u003eCIN\u003c/em\u003e are key enzymes that break down sucrose into hexoses (glucose and fructose). The breakdown of sucrose into hexoses provides readily available energy for processes like color change, flavor development, and cell wall modification. Concurrently, the expression levels of sucrose transport-related genes, including \u003cem\u003eSTP10\u003c/em\u003e, \u003cem\u003eSTP13\u003c/em\u003e, and \u003cem\u003eSTP14\u003c/em\u003e, showed a significant increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). These transporters may be involved in redistributing the decomposed hexoses to different parts of the fruit where they are needed most. In tomato, increased CWINV activity is associated with elevated hexose levels (Jin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe gene expression changes related to sucrose metabolism and transport form a complex and dynamic regulatory network, indirectly affecting strawberry fruits\u0026rsquo; overall sucrose content. \u003cem\u003eFaSWEET9a\u003c/em\u003e, a crucial factor within this network, promotes the accumulation and maintains the dynamic balance of sucrose in fruits by regulating genes involved in sucrose metabolism and transport. This discovery offers fresh insights and new research directions for improving fruit quality.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe transcription factor FaDOF regulates\u003c/b\u003e \u003cb\u003eFaSWEET\u003c/b\u003e \u003cb\u003egene to enhance sucrose accumulation in strawberry fruits.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTranscription factors are essential in plant development, often regulating sucrose synthesis by influencing the expression of structural genes. For example, \u003cem\u003eFvSWEET9\u003c/em\u003e is positively regulated by FvPHR1 and participates in sugar transport from the leaves to the fruits (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). CitZAT5 (zinc finger of Arabidopsis thaliana 5) regulates sugar accumulation in citrus fruit by modulating the expression of \u003cem\u003eCitSUS5\u003c/em\u003e and \u003cem\u003eCitSWEET6\u003c/em\u003e (Fang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the \u0026lsquo;Gala\u0026rsquo; apple cultivar (\u003cem\u003eMalus\u003c/em\u003e \u0026times; \u003cem\u003edomestica\u003c/em\u003e), MdWRKY9 interacts with MdbZIP23 and MdbZIP46, forming a protein complex that binds to the promoter region of \u003cem\u003eMdSWEET9b\u003c/em\u003e. This binding up-regulates the expression of \u003cem\u003eMdSWEET9b\u003c/em\u003e, thereby influencing sugar accumulation within the fruit (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This study identifies FaDOF2 as a key upstream transcription factor that regulates \u003cem\u003eFaSWEET9a\u003c/em\u003e through bioinformatics analyses combined with experimental validation. Analysis of the promoter region of \u003cem\u003eFaSWEET9a\u003c/em\u003e reveals a typical DOF-binding site (AAAG) (Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Y1H and EMSA confirmed that FaDOF2 specifically binds to this site, consequently activating \u003cem\u003eFaSWEET9a\u003c/em\u003e transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). This binding pattern has also been observed across other plant species. For example, OsDOF11 in rice and MdDOF54 in apple regulate sugar transport and distribution via binding to \u003cem\u003eSWEET\u003c/em\u003e gene promoters (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings indicate the conserved nature of the DOF transcription factor family concerning their role in regulating sugar metabolism genes. To further explore the effects of FaDOF2 on \u003cem\u003eFaSWEET9a\u003c/em\u003e expression, LUC and GUS reporter assays were conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). The results validated that FaDOF2 significantly enhances transcriptional activity associated with \u003cem\u003eFaSWEET9a in vivo\u003c/em\u003e. Transient expression experiments additionally demonstrated that overexpression of \u003cem\u003eFaDOF2\u003c/em\u003e elevates transcription levels of \u003cem\u003eFaSWEET9a\u003c/em\u003e within strawberry fruits, resulting in a substantial increase in sucrose accumulation. Thus, we infer that FaDOF2 positively regulates the transcription of \u003cem\u003eFaSWEET9a\u003c/em\u003e and then regulates the sucrose content in strawberry fruits.\u003c/p\u003e \u003cp\u003eOur research introduces a new regulatory mechanism for sucrose accumulation in strawberry fruits. We propose a hypothetical model to reveal this pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). \u003cem\u003eFaSWEET9a\u003c/em\u003e and FaDOF2 are crucial for sucrose accumulation in strawberry fruits. Specifically, the sucrose transporter \u003cem\u003eFaSWEET9a\u003c/em\u003e affects the phenotype of strawberry plants and promotes the accumulation of sucrose content. On the one hand, \u003cem\u003eFaSWEET9a\u003c/em\u003e can specifically transport sucrose; on the other hand, the activity of \u003cem\u003eFaSWEET9a\u003c/em\u003e is positively regulated by FaDOF2, and FaDOF2 then regulates sucrose content in strawberry fruits. These findings enhance our comprehension of the regulatory mechanisms controlling sucrose accumulation and provide new insights for enhancing the flavor quality of fruits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no conflicts of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYX and ZHZ conceived and designed the experiments; YX, SL, HYS, JZ, CZ performed the experiments; YX analyzed the data; YX, WG, and ZHZ wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No.32130092) and the project of Liaoning Province Germplasm Innovation Grain Storage and Technology Special Program (2023020525-JH1/102-04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSource data are provided with this paper. The other data supporting this study\u0026rsquo;s fundings are available from the corresponding author on request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnur RM, Mufithah N, Sawitri WD, Sakakibara H, Sugiharto B (2020) Overexpression of sucrose phosphate synthase enhanced sucrose content and biomass production in transgenic sugarcane. 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Sci Rep 9(4):e94210. https://doi.org/10.1371/journal.pone.009421\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"FaSWEET9a, FaDOF2, Sucrose transport, Fruit sweetness, Gene regulation","lastPublishedDoi":"10.21203/rs.3.rs-6529445/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6529445/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study identified and characterized 25 members of the \u003cem\u003eSWEET\u003c/em\u003e gene family in the genome of cultivated strawberry (\u003cem\u003eFragaria\u003c/em\u003e × \u003cem\u003eananassa\u003c/em\u003e cv. ‘Yanli’), focusing on their potential roles in fruit development. Notably, \u003cem\u003eFaSWEET9a\u003c/em\u003e, a specific member of the SWEET family, was found to be uniquely expressed in ‘Yanli’ fruit. Functional analysis via heterologous expression in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e confirmed that FaSWEET9a acts as a sucrose transporter. To further investigate its role, we generated \u003cem\u003eFaSWEET9a\u003c/em\u003e overexpression lines and demonstrated that \u003cem\u003eFaSWEET9a\u003c/em\u003enot only enhances sucrose accumulation in strawberry fruits but also influences plant growth and development. We identified FaDOF2 that could bind to the promoter of \u003cem\u003eFaSWEET9a\u003c/em\u003e and enhance its transcription by conducting yeast one-hybrid assays, electrophoretic mobility shift assays, β-glucuronidase assays, and luciferase reporter gene assays. Moreover, transient transformation experiments revealed that FaDOF2 could elevate sucrose content in strawberry fruits by regulating \u003cem\u003eFaSWEET9a\u003c/em\u003e. This research brings new viewpoints on the molecular mechanisms that govern sucrose regulation in strawberry fruits, spotlighting the functional significance of the\u003cem\u003e FaSWEET9a\u003c/em\u003e-FaDOF2 regulatory module in the aspects of fruit quality and development.\u003c/p\u003e","manuscriptTitle":"Sucrose transport gene FaSWEET9a regulated by FaDOF2 transcription factor promotes sucrose accumulation in strawberry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 05:22:04","doi":"10.21203/rs.3.rs-6529445/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-05-19T18:54:05+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-30T06:21:36+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-29T12:12:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-29T11:43:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-04-25T09:32:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6b6e5797-c1d4-41d9-92f1-2a1e28fd4414","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:07:46+00:00","versionOfRecord":{"articleIdentity":"rs-6529445","link":"https://doi.org/10.1007/s00299-025-03528-4","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2025-06-02 15:57:48","publishedOnDateReadable":"June 2nd, 2025"},"versionCreatedAt":"2025-05-05 05:22:04","video":"","vorDoi":"10.1007/s00299-025-03528-4","vorDoiUrl":"https://doi.org/10.1007/s00299-025-03528-4","workflowStages":[]},"version":"v1","identity":"rs-6529445","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6529445","identity":"rs-6529445","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}