Overexpression of Tonoplast Transporter FvMATE51 Simultaneously Increases Fruit Size and Sugar Accumulation in Strawberry

Fruit size and soluble sugar content are core quality traits determining consumer preference and market value. The determination of fruit size primarily depends on the precise coordination of cell division and cell expansion, under the control of a complex network involving quantitative trait loci (QTLs), plant hormone signaling, and cell wall modifying enzymes. By contrast, soluble sugar accumulation is largely attributed to the activity of specialized sugar transporters, particularly those localizing to the tonoplast which govern vacuolar sugar storage capacity (Chen et al. 2021). Nevertheless, the inherent trade-off between fruit growth and sugar accumulation constitutes one of the significant challenges in horticultural crop breeding and cultivation. Understanding the molecular basis coordinating these processes is therefore crucial for breeding high-yielding and high-quality horticultural crops. Multidrug and toxic compound extrusion (MATE) transporters, a class of secondary transporters driven by Na+ or H+ gradients, facilitate the movement of diverse substrates (e.g., organic acids and secondary metabolites) across membranes in plants (Upadhyay et al. 2019). Intriguingly, MATE members have recently been implicated in regulating the size of seeds in Arabidopsis, maize and rice (Sun et al. 2023; Suzuki et al. 2015), suggesting their roles beyond traditional substrate transport. However, the function of MATEs in regulating key quality traits of fleshy fruits, such as fruit size and sugar content, remains virtually unexplored. In a previous study, we demonstrated that FvSEP3, a key transcription factor in regulating strawberry (Fragaria vesca) fruit development and ripening, binds directly to the promoter region of FvMATE51 (FvH4_7g30023) (Figure S1) (Tang et al. 2024), a member of the MATE family, but the function of FvMATE51 remains unknown. Structurally, FvMATE51 contains 12 transmembrane helices by using transmembrane domain prediction (TMHMM) (Figure 1a). A phylogenetic analysis revealed high homology between FvMATE51 and the homologous proteins in diverse plant species (Figure S2). FvMATE51 exhibits ubiquitous transcription in vegetative and reproductive organs. Notably, FvMATE51 expression surged specifically during the white to red fruit transition (Figure 1b), strongly implicating it in the ripening process. Confocal microscopy analysis of FvMATE51-eGFP fusion protein in tobacco epidermal cells revealed a distinct continuous closed-loop fluorescence pattern that is characteristic of tonoplast localization (Figure 1c). Co-expression with a vacuolar membrane marker OsTMT1-mCherry (Cho et al. 2010) confirmed extensive signal overlap (Figure 1c). The co-localization was robustly validated in protoplasts (Figure 1d). Taken together, these results conclusively establish FvMATE51 as a vacuolar membrane protein. To investigate FvMATE51 function, we generated stable FvMATE51 knockouts (Fvmate51-1 and Fvmate51-2) using CRISPR/Cas9-mediated gene editing and overexpression lines (FvMATE51-OE-1 and FvMATE51-OE-2) in diploid strawberry (F. vesca cv. Ruegen) (Figure S3a). Genotyping analysis of the four potential off-target sites revealed no detectable mutations (Figure S3b,c). Both knockout lines harbored frameshift mutations predicted to produce nonfunctional truncated proteins (Figure S3d) and exhibited significantly reduced FvMATE51 transcript levels (Figure 1e). Conversely, the overexpression lines showed markedly elevated FvMATE51 mRNA levels (Figure 1e) and protein levels (Figure S4). During vegetative growth, FvMATE51-OE plants exhibited increased height compared to wild-type (WT), while Fvmate51 mutants showed no discernible difference (Figure S5a,b). However, Fvmate51 fruits were significantly smaller than WT at the stage of full ripening (Figure 1f), displaying reduced length and weight at 26 days post anthesis (DPA) (Figure 1g,h). Conversely, FvMATE51-OE fruits were larger (Figure 1f), with corresponding increases in both length and weight (Figure 1g,h). Notably, soluble sugar content (sucrose, glucose, and fructose) measured at 26 DPA was significantly elevated in FvMATE51-OE fruits, but remained unchanged in fruits of Fvmate51 lines (Figure 1i). Notably, the ripening progression was unaffected in all transgenic lines compared to WT (Figure S6a,b), demonstrating that FvMATE51 specifically modulates fruit size and sugar accumulation without disrupting ripening. To decipher the cellular basis of the observed fruit size phenotypes, we analysed transverse sections of 7 DPA fruits. It revealed more cells per unit area in the pith of FvMATE51-OE lines compared to WT, while Fvmate51 mutants exhibited fewer cells (Figure 1j,k). Measurements of average cell area showed an inverse pattern: cell size was smaller in fruits of FvMATE51-OE but larger in those of Fvmate51 (Figure 1k). Collectively, these changes in cell number and average size of cells demonstrate that FvMATE51 coordinately regulates both cell division and cell expansion during fruit development. We next explored the molecular mechanisms by which FvMATE51 modulates fruit size and sugar accumulation. RNA-seq analysis revealed 290 downregulated and 344 upregulated differentially expressed genes (DEGs; |Log2(fold-change)| ≥ 1 and false discovery rate (FDR) < 0.05) in the Fvmate51 mutant relative to WT (Figure S7a–c; Table S1). Subsequent Gene Ontology (GO) enrichment analysis of these common DEGs highlighted key items affected by FvMATE51 loss-of-function, such as cell wall organisation, apoplast, and primary active transmembrane transport (Figure 1l; Table S2). Among these DEGs, several functionally relevant candidates were identified: cell cycle-regulating gene FvCCA; cell wall-modifying enzyme genes FvXTH and FvXTHB (xyloglucan metabolism), FvXYL (hemicellulose degradation), and FvBGA (pectin modification); hormone signalling components FvARF23 and FvAUX28 (auxin response), FvIAA14 (auxin signalling), and FvGA3ox1 (gibberellin biosynthesis) (Figure S7d). The expression of these genes was verified by RT-qPCR (Figure 1m; Figure S7e,f). Our findings demonstrated that disrupted FvMATE51 function results in dysregulated transcription of cell wall dynamics, cell cycle progression, and hormone signalling, ultimately reducing fruit cellular division capacity. Functional analyses in yeast indicated that FvMATE51 lacked sugar transport capability (Figure S8a,b). To further examine how FvMATE51 regulates fruit size and sugar accumulation, we performed a yeast two-hybrid (Y2H) screen which identified FvTIP1;1 (FvH4_6g16770) as an interacting candidate for FvMATE51. FvTIP1;1 shared high sequence similarity (82% amino acid identity) with Arabidopsis tonoplast aquaporins AtTIP1;1 (AT2G36830), which functions as a transporter of water. The physical interaction between FvMATE51 and FvTIP1;1 was rigorously confirmed using multiple independent approaches and subcellular co-localization (Figure 1n–p; Figure S9a,b). Aquaporins facilitate transmembrane water flux, playing a crucial role in osmotic homeostasis. It has been reported that aquaporins correlate with cell size and fruit sugar accumulation (Chen et al. 2001; Zhang et al. 2019). Knockout of FvTIP1;1 via CRISPR/Cas9 drastically stunted plant growth and induced sterility, resulting in no fertile offspring (Figure S10a–c). Alternatively, transient overexpression and RNA interference (RNAi) silencing of FvTIP1;1 in fruits of the WT demonstrated its positive regulation of fruit sugar accumulation. Moreover, repression of FvTIP1;1 by RNAi in fruits of the FvMATE51-OE line partially suppressed the high sugar phenotype, while overexpression of FvTIP1;1 exhibited the opposite effect (Figure S11a–c). This suggests that FvTIP1;1 acts, at least in part, within a common pathway alongside FvMATE51 to regulate fruit sugar accumulation. We speculate that FvTIP1;1 may promote the transport activity of FvMATE51 by facilitating water transportation, which alters the concentration of Na+ or H+ and therefore affects the electrochemical gradient across the membrane, ultimately resulting in changes in fruit size and sugar accumulation. The substrates of FvMATE51 deserve further investigations. K.W., G.G., and G.Q. conceived and designed the research. K.W., C.Z., K.C., and J.L. performed the experiments. Y.W., T.C., and G.G. provided important discussions. K.W., G.G., and G.Q. analyzed the data and wrote the article. This work was supported by the CAS Project for Young Scientists in Basic Research (YSRB-093) and the National Natural Science Foundation of China (31925035). The authors declare no conflicts of interest. The data used to support the findings of this study are available in the main text and Supporting Information S1 of this article. The full legend of Figure 1 is provided in Data S1. Data S1: pbi70478-sup-0001-DataS1.zip. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.