Wheat: Chinese Spring (CS) was used for gene amplification, Fielder was utilized for protoplast preparation and expression pattern analysis, and a transgenic wheat line overexpressing TaDOF6 specifically in the endosperm under the 1Dx5 promoter has been previously constructed (Ding et al., 2024). Tobacco: Nicotiana benthamiana was employed for transient expression assays. Arabidopsis: Columbia (Col-0) was used for target gene amplification. Wheat plants were cultivated at the transgenic plant pilot base of the Beijing Agro-Biotechnology Research Center. Arabidopsis and tobacco were grown in the Capital Normal University greenhouse under controlled conditions: 24°C/18°C with a 16 h light/8 h dark photoperiod and 50–60% relative humidity.

For metabolite extraction, 4 g of 15 days post-anthesis (DPA) wheat grains were placed in a pre-chilled mortar at –20°C and ground to a fine powder in liquid nitrogen. Subsequently, 10 mL of pre-chilled 80% methanol (Sigma-Aldrich, USA) was added, and the mixture was extracted at 4°C for 18 h. After centrifugation (6,000 rpm, 4°C, 10 min), the supernatant was collected into a fresh 50 mL tube. The precipitate was re-extracted with 8 mL of pre-chilled 80% methanol at 4°C for 10 min, followed by a second centrifugation under the same conditions. The resulting supernatants were pooled. A C18 column (Anavo, China) was activated with 4 mL of acetonitrile (Sigma-Aldrich, USA), then washed with 4 mL of distilled water. The combined supernatant was loaded onto the column, which was subsequently washed with 1 mL of distilled water and eluted with 2 mL of 45% methanol. The eluate was filtered through a 0.45 µm membrane (Millipore, USA) and used for subsequent analyses. Gibberellin A3 (GA₃), auxins, and cytokinins were quantified using ELISA kits (Biotopped, China).

Total RNA was extracted from 15 DPA grains of transgenic (TaDOF6 ^OE2) and Fielder lines using three biological replicates. cDNA synthesis, library construction, sequencing, and primary data analysis were conducted by Novogene Co., Ltd. (Beijing, China). Clean reads were aligned to the CS reference genome. Differentially expressed genes (DEGs) were identified using the R package DEGseq, with a fold change ≥ 2 and adjusted p-value (padj) < 10^-3. RNA-seq data are available at NCBI under accession number PRJNA1248066.

Total RNA was extracted using the Plant RNA Extraction Kit (TaKaRa Bio Inc., Otsu, Shiga, Japan). First-strand cDNA was synthesized with TaKaRa PrimeScript™ RT Master Mix. RT-qPCR was performed on a BioRad CFX96 real-time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR Green qPCR Master Mix (TransGen Biotech, Beijing, China). The thermal cycling conditions included an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C. The relative gene expression was calculated using the 2^-ΔΔCt method, with the Ubiquitin gene as the internal control. Primers used for RT-qPCR are listed in Supplementary Table 1 .

The coding sequence of TaDOF6 was cloned into the Sma I (TaKaRa) enzyme-linearized pGADT7-Rec vector using homologous recombination. The promoter region of TaSWEET13h was inserted between the EcoR I and Spe I sites of the pHis2.1 vector to drive LacZ reporter expression. The primers used for PCR amplification are listed in Supplementary Table 2 . The recombinant plasmid was transformed into yeast strain Y187, which was cultured on SD/-Trp/-Leu solid medium at 30°C for three days. Three randomly selected single colonies were then grown in SD/-Trp/-Leu liquid medium to an OD of 0.6–0.8. Subsequently, 5 μL of the culture were spotted onto SD/-Trp/-Leu/-His solid medium supplemented with 30 mM 3-Amino-1,2,4-triazole (3-AT) and incubated upside down at 30 °C for three days.

The TaDOF6 coding sequence was also inserted into the Xho I site of the pETMALc-H vector. The recombinant protein was expressed in E. coli BL21 (DE3) cells (TransGen Biotech). Following cell lysis, His-tagged fusion proteins in the supernatant were captured using Anti-His magnetic beads (Beyotime Biotechnology, Wuhan, China), followed by washing and elution. Protein purification was conducted according to the previously optimized protocol (Zhu et al., 2023).

The method described by Zhu et al. (2023) was followed with minor modifications. The 6×His-TaDOF6-MBP protein samples, expressed in E. coli BL21 (DE3) and purified as described above, were mixed with 6× loading buffer (Beyotime), separated by 12% SDS-PAGE, and transferred onto a PVDF membrane using the wet Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked with 5% (w/v) skim milk in TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween-20) at room temperature for 1 h, then incubated overnight at 4°C with an Anti-6×His primary antibody (Abcam, Cambridge, UK). After three washes with TBST, the membrane was incubated for 1 h at room temperature with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (TransGen Biotech). Following another three TBST washes, protein bands were visualized using the Pierce™ ECL Plus Western Blotting Substrate (Thermo Fisher).

Biotin-labeled, cold, and mutant probes were synthesized by Shanghai Sangong Bioengineering Co., Ltd. Probes were diluted to 100 μM in nuclease-free water. Forward and reverse strand probes were then mixed at a 1:1 ratio and annealed in a PCR machine (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by heating to 95°C for 5 min, followed by cooling at 0.1°C every 8 s to 25°C, then stored at 4°C. The probes were purified using the DNA Probe Purification Kit (Omega Bio-tek, Inc., Norcross, GA, USA). Finally, gel electrophoresis was performed as described previously (Zhang et al., 2016).

Genomic DNA (gDNA) was extracted from wheat leaves using the CTAB method (Zhang et al., 2013). A 1,000 bp promoter region upstream of the TaSWEET13h start codon (pTaSWEET13h) was amplified and cloned into the Kpn I and Xho I sites of the pGreenII-0800-LUC vector. The resulting pTaSWEET13h::LUC construct served as the reporter, with the Renilla luciferase (REN) gene under the 35S promoter included as an internal control. The TaDOF6 coding sequence (CDS) was inserted into the Xba I and Kpn I sites of the pGreenII-62SK vector to generate the 35S::TaDOF6 effector. Dual-luciferase assays were performed in Nicotiana benthamiana leaves following Yang et al. (2025). Agrobacterium tumefaciens cells carrying the constructs were resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES, 150 μM acetosyringone, ddH₂O) to an OD₆₀₀ of ~0.5. After 3 days, luciferase (LUC) and REN activities were measured using the Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI, USA) on a multifunctional microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The LUC/REN ratio was calculated using the empty vector (62SK) plus pTaSWEET13h as the control.

The SWEET transporter protein sequence was submitted to TMHMM (https://services.healthtech.dtu.dk/services/TMHMM-2.0/) for transmembrane domain prediction. Additionally, AlphaFold 2 (Jumper et al., 2021) was used to model the three-dimensional (3D) structure of TaSWEET13h.

The TaDOF6 CDS was cloned into the BamH I-digested 16318h-GFP vector (TaKaRa), provided by Dr. Yimiao Tang (Beijing Academy of Agriculture and Forestry Sciences). The resulting 16318h-TaDOF6-GFP and empty 16318h-GFP vectors were introduced into wheat leaf protoplasts using PEG4000, as described by Zhu et al. (2023). After 16–18 h of dark incubation at 25°C, GFP and mCherry signals were detected using an FV1000MPE confocal laser scanning microscope (Olympus, Tokyo, Japan).

AlphaFold 2 was employed to model TaSWEET13h as the receptor protein for molecular docking. Subsequently, molecular docking of small molecule ligands—sucrose, glucose, fructose, galactose, mannose, and GA₃—retrieved from https://pubchem.ncbi.nlm.nih.gov/ was conducted using AutoDockTools-1.5.7 and AutoDock Vina. The receptor protein was protonated and assigned partial charges in AutoDock, while the ligands were similarly hydrogenated and charged at the root. The prepared receptor was then docked with the ligands. Top-ranking binding conformations, based on AutoDock Vina scoring, were selected for further analysis. Finally, the resulting docked complexes were visualized in cartoon mode using PyMol (Schrödinger, Inc., New York, NY, USA).

Sucrose and hexose transport assays were carried out using sugar transporter-deficient yeast mutant strains Sey6210 and EBY.VW4000, provided by Dr. Xueli An (University of Science and Technology Beijing) and Dr. Yong Xu (Beijing Academy of Agriculture and Forestry Sciences), respectively. These strains were transformed with pDR196 (negative control), pDR196-AtSWEET13 (positive control; gift from Dr. Xueli An), or pDR196-TaSWEET13h using the standard LiAc/PEG method. Three independent colonies were used per assay. For Sey6210, colonies were grown overnight at 30°C in SD/-Ura liquid medium, serially diluted to 10⁴, 10³, 10², and 10 cells/µL, and 5 µL aliquots were spotted onto SC/-Ura plates containing 2% glucose and 2% sucrose. For EBY.VW4000, colonies were cultured overnight at 30°C in SC/-Ura medium supplemented with 2% (v/v) maltose. The cell suspensions with four concentration gradients were spotted onto SC/-Ura plates containing 2% maltose, 2% glucose, 2% fructose, 2% galactose, or 2% mannose. Plates were incubated at 30°C for three days and then photographed.

GA₃ transport assays were conducted using a modified yeast three-hybrid (Y3H) system as described previously (Kanno et al., 2016). The yeast strain Y2HGold (Weidi Biotechnology Co., Ltd., Shanghai, China) was co-transformed with pGADT7-AtGAI and pGBKT-AtGID1a, representing components of the GA signaling pathway, along with pDR196 (negative control) or pDR196-TaSWEET13h. Colonies were cultured overnight at 30°C in SD/-Trp/-Leu liquid media, serially diluted to 10⁴, 10³, 10², and 10 cells/µL, and 5 µL of each dilution from three independent colonies was spotted onto SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade/+GA₃ plates containing 30 mM 3-AT.

The sucrose transport activity of TaSWEET13h was assessed using the fluorescent sucrose analog esculin, following the method of Huai et al. (2022). Yeast mutants (Sey6210) transformed with pDR196-TaSWEET13h and control mutants carrying the empty vector pDR196 were incubated in an esculin buffer (1 mM esculin in 25 mM sodium phosphate buffer, pH 4.0) for 1 h. After washing, cells were imaged using a confocal fluorescence microscope with 405 nm excitation and 488 nm emission.

MD simulations of the TaSWEET13h–sucrose/GA₃ complex were performed using GROMACS 2022.3 (GROningen MAchine for Chemical Simulations, Department of Biochemistry, University of Groningen) on the high-performance computing platform at the Inner Mongolia High Performance Computing Public Service Platform (Huhehot, China) to obtain equilibrated conformations for subsequent MM/PBSA calculations. Trajectory analysis and image generation were conducted using PyMol (Schrödinger, Inc., New York, NY, USA) and OriginPro (OriginLab Corporation, Northampton, MA, USA).

Binding free energy and per-residue energy decomposition of the TaSWEET13h-sucrose/GA₃ complex were calculated using the gmx_mmpbsa module based on the MM/PBSA method. The binding free energy (ΔG_bind ) was calculated using the formula: ΔG_bind = ΔE_MM + ΔG_PB + ΔG_SA - TΔS, where ΔE_MM represents the electrostatic and van der Waals energy in vacuum, while ΔG_PB and ΔG_SA denote the differences in polar and nonpolar solvent solvation free energies, respectively. The entropic contribution (-TΔS) was excluded due to its computational cost and minimal variation in identical protein systems. To identify key binding residues, per-residue free energy contributions were decomposed into van der Waals and electrostatic energies (ΔG_vdw and ΔG_ele ), polar solvation free energy (ΔG_PB ), and nonpolar solvation free energy (ΔG_SA ). Residues contributing more than 1 kcal·mol^-1 to ΔG_bind were considered critical for binding affinity.

To generate the pDR196-TaSWEET13hmu plasmid, the TaSWEET13h CDS (from 16318h-TaSWEET13h) was site-specifically mutated at active amino acid residues using the KOD-Plus Mutagenesis Kit (TOYOBO, product code SMK-101).

To investigate the role of TaDOF6 in wheat grain development, we analyzed soluble sugar content and endogenous hormone levels in grains of TaDOF6 overexpression transgenic lines during the grain filling stage. Overexpression of TaDOF6 significantly increased soluble sugar and GA₃ levels in 15 DPA grains, while auxin and cytokinin levels remained unchanged ( Figures 1A–D ). To further elucidate the biological function of TaDOF6, we performed RNA-seq analysis on 15 DPA grains from overexpression lines and their transformation receptor, Fielder. The GC contents were 51.21–54.17% ( Supplementary Table S3 ). Pearson’s correlation analysis was conducted to assess inter-sample relationships ( Supplementary Figure S1 ). In total, 194 differentially expressed genes (DEGs) were identified, including 73 up-regulated and 121 down-regulated DEGs ( Figure 1E ). Gene Ontology (GO) annotation of upregulated DEGs revealed enrichment in pathways associated with carbohydrate transport, enzyme inhibitor activity, auxin response, and other biological processes ( Figure 1F ). Notably, all genes involved in the carbohydrate transport pathway belonged to the SWEET gene family. We further examined the transcriptional profiles of these SWEET genes in developing endosperm of Chinese Spring (CS) (PRJNA545291) (Gu et al., 2021) ( Figure 1G ). The results showed that TaSWEET13h expression was generally low in grains during the filling stage of wild-type wheat, but relatively high in vegetative tissues such as roots, stems, and leaves. Importantly, TaSWEET13h expression was significantly upregulated in grains of TaDOF6 overexpression lines during the filling stage, in stark contrast to wild type ( Figure 1G , Supplementary Figures S2 , S3 ), indicating that TaDOF6 overexpression significantly increased markedly enhances TaSWEET genes expression in the endosperm.

Y1H assay was conducted to determine whether the transcription factor TaDOF6 binds to the promoter region of TaSWEET13h. The coding sequence of TaDOF6 was fused with GAL4AD, while the TaSWEET13h promoter was fused with the His reporter gene. In three independent experiments, transformants co-expressing pGADT7-TaDOF6 and pHis2.1-pTaSWEET13h survived on SD/-Trp/-Leu/-His medium supplemented with 3-AT, demonstrating that TaDOF6 directly binds to the TaSWEET13h promoter ( Figure 2A ). This binding was further confirmed by EMSA, which showed interaction between TaDOF6 and the “AAAG” motif in the TaSWEET13h promoter ( Supplementary Figure S4 , Figure 2B ). In Nicotiana benthamiana leaves, co-transformation with pGreenII-62SK-TaDOF6 and pGreenII-0800-pTaSWEET13h significantly increased the LUC/REN ratio by 833.33% compared to the negative control ( Figure 2C ). Together, these results confirm that TaDOF6 directly binds to the TaSWEET13h promoter and regulates its expression.

InterProScan analysis identified two MtN3_slv domains within the TaSWEET13h protein (13–98 aa and 134–218 aa), characteristic of the plant SWEET family ( Figure 3A ), indicating that TaSWEET13h is a SWEET family member. Predictions from TMHMM and AlphaFold 2 showed that TaSWEET13h contains seven transmembrane domains (located at 12–36 aa, 48–66 aa, 72–94 aa, 106–126 aa, 132–153 aa, 165–187 aa, and 193–214 aa), with the N- and C-termini positioned on opposite sides of the membrane ( Figures 3A–C ). Upon transformation of the 16318h-TaSWEET13h-GFP construct into wheat leaf protoplasts, GFP fluorescence localized to the cytoplasmic membrane, whereas the negative control 16318h-GFP showed diffuse fluorescence throughout the cell ( Figure 3D ). These findings confirm that TaSWEET13h is a cell membrane -localized protein.

To investigate the role of TaSWEET13h in small molecule transport, we used its AlphaFold 2-predicted 3D structure as the receptor for molecular docking. Using AutoDock Vina, we calculated the binding energies of various ligands, including sucrose, glucose, fructose, galactose, mannose, and GA₃. All ligands exhibited negative binding energies and hydrogen bond distances below 4 Å ( Figure 4 ), indicating strong interactions with TaSWEET13h. These results suggest that different substrates may share the same transport channel on TaSWEET13h, potentially leading to competitive inhibition when multiple substrates are present.

The yeast mutant strain Sey6210, which lacks the extracellular invertase gene SUC2, cannot hydrolyze sucrose and thus fails to grow on media where sucrose is the sole carbon source (Milne et al., 2013). All transformants harboring pDR196 (empty vector), pDR196-AtSWEET13, or pDR196-TaSWEET13h grew well on glucose-supplemented media. However, when glucose was replaced with sucrose, Sey6210 strains expressing pDR196-TaSWEET13h exhibited significantly higher growth rates than those carrying the empty vector ( Figure 5A ). To further verify whether TaSWEET13h transports sucrose, we performed an esculin uptake assay in Sey6210. Under confocal fluorescence microscopy, fluorescence accumulated inside TaSWEET13h-expressing cells, whereas no fluorescence was detected in the negative control ( Figure 5B ), confirming that TaSWEET13h facilitates sucrose transport.

Similarly, the yeast mutant strain EBY.VW4000, which lacks all hexose transporter genes, cannot grow on media containing hexoses as the sole carbon source. While all transformants, including those carrying pDR196, pDR196-AtSWEET13, or pDR196-TaSWEET13h, grew comparably on maltose-containing media, growth differed on media containing glucose, fructose, galactose, or mannose. The EBY.VW4000 strains transformed with the pDR196 empty vector had significantly lower growth than those transformed with pDR196-TaSWEET13h ( Figure 5C ).

GID1 (gibberellin insensitive dwarf 1) and DELLA proteins are central components of the GA signaling pathway in plants, with their interaction forming its core regulatory mechanism. Under low endogenous GA levels, DELLA proteins accumulate and repress the expression of growth-related genes, thereby inhibiting plant growth. Conversely, elevated GA levels allow GA to bind its receptor, activating the GID protein complex. This complex interacts with DELLA proteins, promoting their degradation and relieving repression on growth-related genes, which facilitates plant growth and development. To investigate this interaction, we co-transformed Y2HGold yeast strains with the pDR196 empty vector or pDR196-TaSWEET13h, along with pGADT7-AtGAI and pGBKT-AtGID1a. Strains carrying the pDR196 empty vector or pDR196-TaSWEET13h failed to grow on SD/-Trp/-Leu/-His/-Ade medium without GA₃. However, those transformed with pDR196-TaSWEET13h grew on SD/-Trp/-Leu/-His/-Ade medium supplemented with 0.1 mM GA₃ ( Figure 5D ). These results indicate that TaSWEET13h is capable of transporting sucrose, glucose, fructose, galactose, mannose, and GA₃.

To investigate the transport mechanisms and key residues of the TaSWEET13h protein for sucrose and GA₃, we conducted a 1000 ns MD simulation using the optimal binding conformations of TaSWEET13h docked with sucrose and GA₃ as initial structures. Analysis of the simulation trajectories revealed that the root mean square deviation (RMSD) and radius gyration (Rg) revealed that the RMSD of the TaSWEET13h-Suc and TaSWEET13h–GA₃ complexes stabilized at 875 ns and 850 ns, respectively, with average RMSD values of 17 Å and 21 Å. The standard errors of these values remained below 1 Å post-equilibration ( Figures 6A, B ). Similarly, the Rg reached equilibrium at 750 ns and 870 ns, with respective averages of 22 Å and 23 Å, and standard errors also under 1 Å ( Figures 6C, D ). These results indicate that both complexes attained stable conformations by the end of the 1000 ns of MD simulation. To further characterize their dynamic behavior, we performed conformation sampling and clustering analysis ( Supplementary Figure S5 ). Five representative conformations of sucrose and GA₃ were tightly embedded within the hydrophobic cavity of TaSWEET13h’s active site. Minor fluctuations in ligand positioning were driven by directional shifts in the hydrophobic side chains of active site residues.

We calculated the binding free energy (ΔG_bind ) of the TaSWEET13h-Suc and TaSWEET13h-GA₃ complexes, which comprises four components: van der Waals energy (ΔG_vdw ), electrostatic Coulomb energy (ΔG_ele ), solvation free energy (ΔG_PB ), and non-polar solvation free energy (ΔG_SA ). This analysis reveals the contributions of these energy components to the binding process of TaSWEET13h with sucrose and GA₃. In both complexes, ΔG_vdw was significantly higher (TaSWEET13h-Suc: -16.54 kcal·mol^-1; TaSWEET13h-GA₃: -16.09 kcal·mol^-1) than the other components. ΔG_ele and ΔG_PB were weaker, with values for TaSWEET13h-Suc of -7.78 kcal·mol^-1 and -1.51 kcal·mol^-1, respectively, and for TaSWEET13h-GA₃ of -2.28 kcal·mol^-1 and -1.75 kcal·mol^-1, respectively. The ΔG_SA values for TaSWEET13h-Suc and TaSWEET13h-GA₃ were +14.68 kcal·mol^-1 and +12.72 kcal·mol^-1, respectively, suggesting that polar residues adversely affect binding ( Supplementary Table S4 ). These results highlight the crucial role of van der Waals interactions in the binding process of the TaSWEET13h-Suc/GA₃ complexes.

To identify the amino acid residues in TaSWEET13h essential for binding sucrose and GA₃, the MM/PBSA method was employed to decompose the binding free energy into residue-specific contributions within the TaSWEET13h-Suc and TaSWEET13h-GA₃ complexes. As shown in Figure 6E , W60, V146, and W181 contributed significantly to binding in TaSWEET13h–Suc, with ΔG_bind values of 1.97, -1.12, and -1.90 kcal·mol^-1, respectively. In the TaSWEET13h-GA₃ complex, four amino acid residues (I76, V146, F147, and W181) showed higher contributions, with ΔG_bind values of -1.22, -1.94, -0.96, and -1.49 kcal·mol^-1, respectively ( Figure 6F ). Consistent with overall binding free energy, hydrophobic interactions of each residue were critical in both complexes, particularly for W60 and W181 (ΔG_MM < -2.00 kcal·mol^-1). Notably, the indole ring of W181 engaged in both hydrophobic interactions and hydrogen bonding with sucrose and GA₃, enhancing complex stability. Other residues did not contribute to the binding affinity to sucrose and GA₃. However, S56 and G84 displayed distinct roles in the two complexes. For sucrose and GA₃, ΔG_bind of S56 were -0.54384 and 0.61992 kcal·mol^-1, respectively, while those of G84 were +0.19152 and -0.6636 kcal·mol^-1, respectively. During the 1000 ns MD simulation of the TaSWEET13h-Suc/GA₃ complexes, the root mean square fluctuation (RMSF) values of residues W60, I76, V146, F147, and W181 were all < 2.0 Å; whereas the RMSF values of residues outside the active site were generally higher ( Figures 6G, H ). Therefore, this further indicated that these key residues interact with sucrose and GA₃ to form stable and reasonable complexes.

Based on MD simulation results, three residues (W60, V146, and W181) and four residues (I76, V146, F147, and W181) were mutated to assess the affinity of TaSWEET13h for sucrose and GA₃, respectively. Compared to Sey6210 [pDR196-TaSWEET13h], strains expressing mutated TaSWEET13h [pDR196-TaSWEET13hmu] exhibited reduced growth on selection medium with sucrose as the sole carbon source ( Figure 7A ). Similarly, compared to Y2HGold [pGADT7-AtGAI; pGBKT-AtGID1a; pDR196-TaSWEET13h], the mutant strains [pGADT7-GAI; pGBKT-GID1a; pDR196-TaSWEET13hmu] showed reduced growth on medium containing 0.1 mM GA₃ ( Figure 7B ). These results indicate that the V146 and W181 residues are critical for TaSWEET13h’s affinity to sucrose and GA₃.

Grain weight is a key agronomic trait influencing crop yield and is controlled by conserved molecular pathways (Long et al., 2024). Elucidating these pathways will enhance the regulatory network of yield traits and support molecular breeding for high yield (Zhang et al., 2024). As grain size strongly correlates with grain weight, it has remained a central focus in genetic and breeding research. Our previous studies identified TaDOF6 as a DOF family TF that is specifically and highly expressed in wheat grains, regulating carbohydrate accumulation, grain size, and weight (Ding et al., 2024), although its exact regulatory mechanisms remain unclear. In rice, OsDOF11 directly regulates the sucrose transport genes OsSUT1, OsSWEET11, and OsSWEET14, thereby modulating sucrose transport (Wu et al., 2018). OsDOF11 also plays a role in seed development, and its tissue-specific overexpression increases grain weight by activating SWEET14 expression (Kim et al., 2021). This study investigates the positive regulatory role of TaDOF6 during grain filling. RNA-seq and RT-qPCR analyses confirmed that TaDOF6 overexpression significantly elevates TaSWEET13h transcript levels. Further Y1H, EMSA, and dual-luciferase assays demonstrated that TaDOF6 binds to the P-box motif. Thus, we propose that TaDOF6 enhances TaSWEET13h transcription by binding the P-box motif in the developing wheat endosperm.

SWEET family members, characterized by seven transmembrane helices and sugar transport activity, play a critical role in grain development. In maize and rice, SWEET4 transports hexoses across the endosperm cell membrane during grain filling, promoting larger grain size (Sosso et al., 2015). In Arabidopsis, AtSWEET11, AtSWEET12, and AtSWEET15 are expressed in the seed coat and endosperm to import sucrose into the embryo, supporting normal seed development. Triple mutants of these genes exhibit delayed embryo development and reduced seed weight, starch, and lipid contents (Chen et al., 2015). In soybean, overexpression of dominant alleles GmSWEET10a, GmSWEET10b, and GmSWEET39 increases seed size and oil content (Miao et al., 2020; Wang et al., 2019). Similarly, OsSWEET11, 14, and 15 contribute to carbohydrate transport into rice grains; knockout mutants of ossweet11, 14, and 15 show abnormal grain filling and reduced seed weight and starch content (Ma et al., 2017; Yang et al., 2018; Fei et al., 2021; Li et al., 2022). In lychee, spatiotemporal expression profiling suggests that LcSWEET2a and LcSWEET3b participate in seed development. In wheat, knockout of TaSWEET11 downregulates genes involved in starch biosynthesis and sucrose metabolism, resulting in impaired starch accumulation, pericarp shrinkage, and significantly reduced sucrose levels in tasweet11-ko lines (Wang et al., 2025). Collectively, these findings suggest that wheat SWEET proteins are key, functionally conserved sucrose transporters that facilitate efficient sucrose translocation during grain filling, thereby supporting grain development. Here, we show that TaSWEET13h, a SWEET family member, contains seven transmembrane helices and a conserved MtN3_slv domain, consistent with its plasma membrane localization. Previous studies have demonstrated that SWEET proteins transport not only soluble sugars but also hormones. For instance, HvSWEET11 transports both sugars and cytokinins to promote barley grain development (Radchuk et al., 2023), while AtSWEET13 and OsSWEET3a facilitate the transport of both sugars and GA (Kanno et al., 2016). In rice, OsSWEET3a exhibits dual sugar and GA transport functions, with both knockout and overexpression leading to delayed germination and slow growth (Morii et al., 2020). Consistent with these findings, we report that TaSWEET13h can transport various soluble sugars, including sucrose, glucose, and fructose, as well as gibberellins. These substrates likely bind within a common active pocket of TaSWEET13h, where hydrophobic interactions involving nonpolar amino acid residues serve as the primary driving force for its affinity to sucrose and GA₃.

However, due to the current lack of TaSWEET13h overexpression and gene-edited lines, its role in regulating wheat grain size cannot be directly validated. Based on existing findings, we propose a hypothetical model illustrating the potential mechanism by which TaDOF6 and TaSWEET13h regulate wheat grain size and weight ( Figure 8 ). During grain filling, TaDOF6 transcript levels increase sharply. TaDOF6 proteins bind to the P-box motif and activate TaSWEET13h transcription in the nucleus. The resulting TaSWEET13h protein integrates into the plasma membrane, facilitating the transport of soluble sugars and GA₃ from the extracellular space into the cytoplasm. High soluble sugar levels support starch accumulation, while gibberellins promote endosperm cell expansion. Together, these coordinated processes regulate wheat grain development. This study advances our understanding of the molecular basis of wheat grain development and offers strategies for improving yield.