Enhancing tomato quality, high sugar content and GABA accumulation, with mutations in ESKs and GAD3 genes

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Abdellatif, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7220491/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Tomatoes are among the most widely cultivated crops worldwide, and their nutritional value has recently gained attention in the context of functional foods. In this study, we developed tomato varieties with elevated sugar content and high gamma-aminobutyric acid (GABA) levels using a genome editing technique to introduce mutations in SlESK and SlGAD3 , aiming to enhance the nutritional value of tomatoes. Tomato plants possess three ESK homologs. We successfully generated tomatoes with mutations in all three SlESK genes and a separate line with a mutation in SlGAD3 . Compared to the wild type (WT), which exhibited a sugar content of approximately 4.26% Brix, the mutants showed a significantly higher sugar content of approximately 7.9% Brix. GABA accumulation in the mutants was approximately five times higher than in WT. Additionally, the mutants contained 1.5 times more vitamin C than WT and demonstrated enhanced drought stress tolerance. Delayed plant growth and reduced yield were also observed in the genome-edited plants. Transcriptome analysis revealed that expression of genes, which are involved in vitamin C biosynthesis, the auxin signaling pathway, and ethylene biosynthesis, were altered in the genome-edited plants. These results may contribute to cultivating high-quality tomatoes, which are expected to be in high demand. Biological sciences/Biotechnology Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Plant sciences SlESK1-3 SlGAD3 high-sugar GABA-rich high-vitamin C drought tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Tomatoes are among the most widely cultivated crops globally, with an annual production of approximately 189 million tons 1 . Additionally, research has shown that tomatoes contain numerous functional compounds, making them a vital crop for supporting and promoting health 2 . Tomatoes are also consumed as fruits, and innovative breeding techniques are being employed to develop tomato varieties with high sugar content 3 . It is expected that future advancements will lead to tomato varieties with both enhanced sugar content and enriched functional compounds. For instance, a high-GABA tomato, developed through genome editing, is already commercially available 4 . Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the mammalian central nervous system. GABA is also widely used as a dietary supplement due to its potential to lower blood pressure and reduce anxiety 5 . The genes targeted for the development of tomatoes with high GABA and sugar content are SlGAD3 ( glutamate decarboxylase 3 ) and SlESK1-3 ( Eskimo1-3; Solyc03g096030 , Solyc05g052540 , Solyc06g051350 ). SlGAD3 encodes glutamate decarboxylase 3, a key enzyme in GABA accumulation in tomato fruits. GAD3 facilitates GABA synthesis by catalyzing the removal of carboxyl group from glutamic acid. This enzyme also contains an autoinhibitory domain near its C-terminus, which stabilizes the enzyme in an inactive form under normal conditions. However, during stress responses, rising intracellular calcium ion levels trigger the formation of a calcium-calmodulin complex, which binds to the calmodulin-binding site within the autoinhibitory domain, activating GAD3 . Previous studies have shown that genome editing of the SlGAD3 autoinhibitory domain can successfully increase GABA accumulation in tomato fruits 6 7 . SlESK1-3 are homologs of the AtESK1 gene in tomato, which has been identified as a freeze tolerance-related gene in Arabidopsis thaliana . The Atesk1 mutants in Arabidopsis exhibit increased soluble sugar content and enhanced freeze tolerance under cold stress conditions 8 9 . Additionally, AtESK1 has been shown to function as a xylan acetyltransferase in Arabidopsis. Mutations in AtESK1 result in decreased secondary cell wall thickness and reduced stem strength 10 . In contrast, few studies have reported on esk mutants in other crops, particularly fruiting crops such as tomato. In this study, we aimed to generate slesk mutants in tomato and characterize the function of SlESK . Genome editing technology is anticipated to play a significant role in future breeding efforts. This technology involves inducing DNA double-strand breaks (DSBs) at specific genomic locations using artificial nucleases that recognize and cleave target sequences. Mutations arise from errors in the DNA repair process following cleavage, enabling precise modification of endogenous genes. Genome editing can be applied to a wide range of species. Prominent genome editing tools include zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 11 . In this study, we utilized CRISPR/Cas9, currently the most widely adopted genome editing tool. CRISPR/Cas9 is derived from the immune defense system widely conserved in eubacteria and archaea. It consists of the endonuclease Cas9 and a guide RNA (gRNA). During the induction of DSBs, the gRNA, containing a sequence complementary to the target site, recognizes and binds to the target DNA sequence. The Cas9 protein, which possesses two DNA cleavage domains, then cleaves the DNA at the target site, generating a DSB. Target sequence recognition by Cas9 requires the presence of a protospacer adjacent motif (PAM) sequence, such as NGG in the case of SpCas9. CRISPR/Cas9 is widely used due to its advantages, including simplicity in design and construction, as well as the ability to edit multiple genes simultaneously. Indeed, genome edited foods, such as high-GABA tomatoes, have already been commercially launched in Japan 4 . In this study, we aimed to simultaneously modify SlESK1-3 and SlGAD3 to enhance the value of the original tomato cultivar. Additionally, we investigated the phenotypes of the slesk1-3 mutants to characterize the function of SlESK . In addition to our genome editing approach, previous studies have highlighted the importance of invertase genes in sugar metabolism under stress 12 and identified gene families involved in ascorbic acid accumulation 13 , which collectively underline the complex regulation of these nutritional traits. Results Generation of esksgad3 mutants using CRISPR/Cas9 To generate tomato plants harboring T-DNA with CRISPR/Cas9, 159 regenerated plants were obtained through tissue culture of cotyledon fragments. Of these, 38 individuals were selected for sequencing after preliminary screening using MultiNA, and mutations at the target sites were confirmed in 10 individuals (Supplementary Table 3). Additionally, multiple mutations were detected in some individuals, indicating the occurrence of chimeric mutations. In the T 1 generation, the inheritance of mutations at the target sites was confirmed in five lines of esksgad3 (Supplementary Table 4). Among these individuals, homozygous mutations were successfully introduced in all target genes ( ESK1-3 and GAD3 ) in esksgad3 line #127-5 (EG Line 1) (Fig. 1 , Supplementary Table 4). Furthermore, in esksgad3 line #127-9 (EG Line 2), although mutations were observed in the ESK2 target and other target sites, several fully homozygous individuals were obtained in the subsequent T 2 generation (Supplementary table 5). Since esksgad3 #127-9-4 exhibited severe growth retardation, and no subsequent generation was obtained, it is suggested that severe mutations in three SlESK homologs may cause significant developmental damage in tomatoes. Therefore, the six-base deletion in ESK2 was selected for further characterization (Fig. 1 B). Off-target mutations were investigated in the esksgad3 #127 line, a genome-edited line, by comparing sequencing results with those of the wild type (WT). No mutations were detected at the predicted off-target candidate sites in any of the analyzed individuals (Supplementary Table 6). Additionally, amplification of the vector plasmid using 11 sets of primers revealed no residual foreign genes in esksgad3 #127-5-6 (a sibling of EG Line 1) or esksgad3 #127-9-10, #127-9-14, #127-9-18, #127-9-26, and #127-9-29 (siblings of EG Line 2) (Supplementary Fig. 1). Growth and fruit set impairment in eg mutants. In EG Line 2, ESK2target1 exhibited a heterozygous mutation in the T 1 generation. Therefore, sequence analysis of ESK2target1 was performed in the T 2 generation, with results presented in Supplementary Table 5. A delay in growth was observed in both EG Line 1 and EG Line 2 compared to the WT (Figs. 2 A, 2 B). The plant heights measured 10 weeks after the start of observations are presented as mean ± standard deviation (cm): WT = 97.0 ± 4.09, EG Line 1 = 45.3 ± 2.78, EG Line 2 = 53.8 ± 3.20. A statistically significant difference ( p < 0.05) was observed when comparing growth between WT and the EG lines (EG Line 1 and EG Line 2) across all weeks of measurement. Furthermore, fruit set rates decreased significantly. Flowers and fruits were counted 21 weeks after sowing. The fruit set rate was 43.17% in the WT, whereas it was significantly lower in EG Line 1 (4.52%) and EG Line 2 (6.56%) (Fig. 2 C). The decreased fruit set rates are results of abnormal flower developments, especially pollen development (Supplementary Fig. 2). The flowers in EG lines are smaller than in WT, and many flowers in EG lines are stopped developing and wilt (Supplementary Fig. 2A). The pollen in EG mutants also exhibited unshaped and small sized pollen (Supplementary Fig. 2B). Additionally, differences in vascular structure were observed in specific regions of the stem. In the upper part of the stem (below the third mature leaf from the top) and the middle part (above the third mature leaf from the bottom), the vessel area was significantly wider in the WT compared to the EG Line 1 and EG Line 2. However, no significant differences were observed in the lower stem region (above the boundary between the stem and root) or in the leaflets (Supplementary Fig. 3). Differences in fruit traits in EG Line1 and EG Line2 To measure fruit size, the equatorial diameter of the fruit was measured. Compared to WT, both EG Line 1 and EG Line 2 exhibited significantly smaller fruit weight and diameter, regardless of the season (Figs. 3 A, 3 C, 3 D; Supplementary Figs. 4A, 4B). In previous studies, the esk1 mutant in Arabidopsis thaliana was reported to exhibit impaired xylem structure and secondary cell wall formation 10 . Therefore, we examined the fruit structure using microscopy. The results showed no detectable differences in xylem structure among the WT and EG lines (EG Line 1 and EG Line 2) (Fig. 3 B). Regarding GABA content, a comparison of EG Line 1 and EG Line 2 with WT revealed that GABA accumulation was significantly higher in both EG Line 1 and EG Line 2 than in the WT (Fig. 3 E; Supplementary Fig. 4C), regardless of the season. For total soluble sugar content (Brix), both genome-edited lines (EG Line 1 and EG Line 2) exhibited significantly higher values compared to WT, consistently across all seasons (Fig. 3 F; Supplementary Fig. 4D). To further analyze sugar composition, sucrose, fructose, and glucose levels were measured. The results indicated that sucrose and fructose levels were significantly higher in the EG Line 1 and EG Line 2 than in WT during the spring season but less significantly elevated during the summer season (Figs. 3 G, 3 H; Supplementary Fig. 4E, 4F). In contrast, glucose content was consistently and significantly higher in the EG Line 1 and EG Line 2 across all seasons (Fig. 3 I; Supplementary Fig. 4G). From Gene Ontology (GO-) enrichment analysis and RNA-seq results, we observed the upregulation of genes associated with ascorbic acid (vitamin C) biosynthesis. Therefore, we measured ascorbic acid levels and found a significant increase in the EG Line 1 and EG Line 2 (Fig. 3 J), suggesting that these plants likely enhance antioxidant production to resist and recover from stress 16 . EG Line1 and Line2 exhibit drought stress tolerance but doesn’t exhibit cold stress tolerance. The esk1 mutant in Arabidopsis thaliana has demonstrated enhanced tolerance to various abiotic stresses, including cold, drought, and salinity, which was associated with higher abscisic acid (ABA) levels 15 . To investigate whether a similar mechanism was present in the EG Line1 and EG Line2, the expression of the ABA biosynthetic gene SlNCED1 was examined. SlNCED1 expression was found to be upregulated in the EG Line1 and EG Line2 compared to WT under normal room temperature conditions (Fig. 4 B). To assess drought stress tolerance, WT and EG lines (EG Line1 and EG Line2) were subjected to a 10-day water-starvation treatment, after which phenotypic changes, malondialdehyde (MDA) levels, and electrolyte leakage (EL) were evaluated (Figs. 4 A, 4 C, 4 D). Under normal conditions, MDA levels were higher in the EG Line1 and EG Line2 than in WT. However, after water-starvation, MDA levels increased significantly in WT but remained relatively unchanged in the EG Line1 and EG Line2 (Fig. 4 C). Although baseline levels of EL levels were higher in the EG Line1 and EG Line2, the degree of increase in EL after drought stress was significantly greater in WT compared to the EG Line1 and EG Line2 (Fig. 4 D). These results suggest that the EG Line1 and EG Line2 exhibit drought stress tolerance and sustain less cellular damage under drought conditions. However, the EG Line1 and Line2 didn’t exhibit the cold stress tolerance (Supplementary Fig. 5A). To assess cold stress tolerance, WT and EG lines were subjected to a 7-day 4°C cold stress environment. The MDA was evaluated 3-day and 7-day after the cold stress treatment. The MDA wasn’t significantly different between WT and EG lines after the cold stress treatment, while significantly different before the cold stress treatment (Supplementary Fig. 5B). RNA-seq and GO- term- enrichment- analysis in tomato fruits RNA sequencing analysis revealed that 302 genes were significantly upregulated, while 703 genes were significantly downregulated in red ripe tomato fruits of EG Line 2 compared to WT (Fig. 5 A). Among the upregulated genes identified in the GO analysis, the most notable processes were related to vitamin C biosynthesis, the auxin signaling pathway, AUX/IAA family genes, ethylene biosynthesis, and ethylene metabolism. These biological processes play critical roles in regulating the developmental responses observed in the EG Line1 and EG Line2. The highest upregulated gene associated with vitamin C regulation was Solyc05g054760.4 (dehydroascorbate reductase 1; DHAR1 ). Since the vitamin C biosynthesis pathway was upregulated, an increased concentration of ascorbic acid was also observed (Fig. 5 B). For auxin transporter and regulatory genes, key upregulated genes included Solyc01g068410.4 ( SlPIN5 ), Solyc10g080880.2 ( SlPIN7 ), Solyc09g008175.1 (SAUR-like auxin-responsive protein family), Solyc09g065850.4 ( SlIAA3 ), Solyc01g079260.4 ( SlWRKY23 ), Solyc05g047460.3 ( Sl-ARF7B ), and Solyc01g103050.3 ( Sl-ARF1 ). For ethylene biosynthesis and signaling, upregulated genes included Solyc01g095080.3 ( ACC2 ), Solyc03g118190.4 (ethylene-responsive transcription factor), Solyc07g049530.3 ( SlACO1 ), Solyc03g118190.4 (ethylene-responsive transcription factor), Solyc07g049550.3 (1-aminocyclopropane-1-carboxylate oxidase), Solyc03g044300.3 ( AP2a ) (Supplementary Fig. 4). Regarding nutrient transport and response genes, upregulated calcium-related genes included Solyc03g097100.1 (EF hand calcium-binding protein family) and Solyc01g108400.3 (calcium-dependent protein kinase-like). Additionally, the phosphate transporter Solyc02g078210.3 ( PHO2 ) and Solyc01g005000.3 ( GAD3 ) were identified as upregulated genes (Fig. 5 B; Supplementary Fig. 6). The GO analysis of downregulated genes identified 93 biological processes (BPs) significantly associated with key roles in plant growth and development. These BPs were classified into three main categories: stress response, cellular regulation, and transportation. A substantial number of downregulated genes in the fruits of EG Line1 and EG Line2 were associated with stress response processes, including responses to heat and other abiotic stimuli. The significantly downregulated genes in this category included Solyc09g065660.4 ( HsfA7 ), Solyc08g062960.4 ( HsfA2 ), and Solyc09g009100.4 ( SolycHsfA3 ), all of which are associated with heat stress response. The second category included genes involved in cellular regulation, particularly those related to cell wall biogenesis and the regulation of xyloglucan, hemicellulose, saccharide, macromolecule, and carbohydrate metabolism. The most significantly downregulated gene in this category was Solyc04g071070.2 (unknown protein, plant-type cell wall organization). The third category involved genes related to transportation, including water and lipid transport. Notable downregulated genes included Solyc01g090350.3 and Solyc01g090360.3 (non-specific lipid-transfer proteins) for lipid transport and Solyc12g044330.2 ( SlTIP2.1 ), Solyc06g074820.3 ( SlTIP1.1 ), and Solyc10g055630.2 ( SlPIP2.9 ) for water channel activity (Fig. 5 C; Supplementary Fig. 7). Discussion In this study, we demonstrated that multiple desirable traits can be improved through a single genome-editing event and investigated the characteristics of the EG Line 1 and EG Line2. Our results indicate significant differences in sugar content, GABA accumulation, and ascorbic acid levels between WT and EG lines (EG Line 1 and EG Line 2) (Fig. 3 ). Although seasonal variations were observed, the sugar content in the red ripe fruits of the EG Line 1 and EG Line 2 was consistently higher than that of the WT. Specifically, during the spring season, the average Brix value in the EG Line 1 and EG Line 2 was approximately 1.8 times higher than in the WT. Compared to the esk1 mutant in Arabidopsis thaliana , which does not produce fruits, the EG Line 1 and EG Line 2 of tomato produce fruits with elevated the sugar content, even under a greenhouse conditions without stress induction. Furthermore, both GABA and vitamin C contents were significantly increased in the EG Line 1 and EG Line 2, with GABA levels approximately 4 to 5 times higher and vitamin C levels approximately 1.5 times higher than those in WT (Fig. 3 ). These results suggest that introducing mutations into the ESKs and GAD3 genes can effectively enhance the nutritional of tomato fruits. The esks mutants in tomato could affect the stressed phenotype and GABA content. Growth retardation in the EG Line 1 and EG Line 2 was confirmed through analysis using T 2 individuals. Previous research demonstrated that esk1 mutants in Arabidopsis thaliana inhibit xylan acetylation, which promotes its degradation by endoxylanase and reduces secondary wall thickening 10 . This reduction in secondary wall thickening results in insufficient xylem formation, causing wilted leaves and dwarf traits. In tomatoes, it is also known that sugar content in tomato fruits increases when water availability is limited, which can be explained by a concentration effect due to reduced water content in the fruit 16 . Based on these findings, it is hypothesized that the growth retardation observed in the EG Line 1 and EG Line 2 results from reduced secondary wall development caused by mutations in SlESK genes and that the increased fruit sugar content is attributable to decreased water transport efficiency due to defective xylem formation. Significant differences in vessel structure were observed in the stems of the top and middle regions of the EG Line 1 and EG Line 2, with a decreased vessel area compared to the WT (Supplementary Fig. 3). RNA-seq analysis revealed upregulated expression of genes related to drought-induced stress, ABA response, and water deprivation, further supporting the high sugar phenotype observed in the EG Line 1 and EG Line 2 (Supplementary Fig. 4) Additionally, the fruit set rates are significantly decreased in the EG mutants and its results are results of stunted growth in EG mutants (Supplementary Fig. 2 and Supplementary Fig. 3). The esksgad3 #127-9-4 line (a sibling of EG Line 2) had a + 1 homozygous mutation in ESKtarget1 (Supplementary Table 5). However, this individual died shortly after genome extraction, and no other triple-frame-shift mutants were obtained from its siblings. This observation suggests that frame-shift mutations in all SlESK1-3 genes are likely lethal and that SlESK1-3 are essential for growth and efficient water transport. The esk1 mutant in Arabidopsis thaliana has exhibited tolerance to various abiotic stresses, including drought, cold, salt and osmotic stress 8 17 18 . In this study, the tomato EG Line 1 and EG Line 2 exhibited drought stress tolerance, as evidenced by reduced MDA and EL levels, including reduced damage under drought conditions. The observed drought tolerance suggests that the EG Line 1 and EG Line 2 may pre-emptively recognize stress signals and activate resistance mechanisms. Previous studies have reported that the atesk1 mutants have higher ABA levels compared to WT 15 . ABA is a key phytohormone involved in regulating plant responses to abiotic stress 19 . Consistent with these findings, we also observed increased expression of NCED1 , a key gene in ABA biosynthesis, in the EG Line 1 and EG Line 2 (Fig. 4 ) 15 . In contrast, the EG lines didn’t exhibit the cold stress tolerance not like the esk1 mutant in Arabidopsis thaliana , since tomatoes originate from tropical regions and exhibit limited cold tolerance (Supplementary Fig. 5) 20 . In conclusion, genome editing-induced mutations in tomato ESK1-3 and GAD3 genes promoted sugar content, GABA levels, and vitamin C concentration, resulting in nutrient-rich tomato fruits with increased functional value. Moreover, these results may help alleviative the high costs and labor-intensive processes associated with cultivating high-quality tomatoes. Producing high-sugar tomatoes typically requires significant amounts of fertilizers and labor. However, resources for agricultural production are limited and being depleted 21 22 , and the number of people engaged in farming is steadily decreasing 23 24 . In addition, the yield reduction commonly observed in the high-sugar tomato cultivation needs to be addressed in future studies. Materials and Methods Plant and growth conditions Tomato ( Solanum lycopersicum ) plants used in this study were provided by Sanatech Life Science Co., Ltd. The cultivation room was maintained at a constant temperature of 25°C, with a photoperiod of 16 h of light and 8 h of darkness. In the greenhouse, the temperature ranged from 11°C to 40°C during the spring season and from 20°C to 60°C during the summer season. Fertilizers included Otsuka House No. 1 and Otsuka House No. 2 (OAT Agrio Co., Ltd.). Construction of vector plasmid To create the pPcUfcoCas9-SlESKGAD3 plasmid, a reaction mixture containing 6 µl of CutSmart buffer, 2 µl of the restriction enzymes ApaI and AscI , and 50 µl of the plasmid sample pPcUbi was incubated at 37°C for 60 min. Subsequently, amplicons listed in Supplementary Table 1 (#1-#4) were amplified using KOD-Plus Neo (TOYOBO) and ligated with the digested pPcUbi plasmid. To construct pPcUfcoCas9-SlESK1,2GAD3 (Fig. 1 ), a reaction mixture containing 6 µl of CutSmart buffer, 2 µl of the restriction enzyme Xmal, and 48 µl of the pPcUfcoCas9-SlESKGAD3 plasmid sample was prepared, and the reaction was performed under the same conditions. The target sites were amplified and inserted into the restricted pPcUfcoCas9-SlESKGAD3 plasmid (#5-#6). For plasmid purification, 150 µl of Milli-Q water was added to the restriction enzyme-treated plasmid sample on ice. Then, 200 µl of the lower layer of Tris-saturated phenol- chloroform-isoamyl alcohol was added, thoroughly mixed, and centrifuged at 15,300 × g, 4°C for 5 min. The supernatant was collected, and 40 µl of 3M sodium acetate was added, followed by thorough mixing. Next, 400 µl of isopropanol was added, mixed, and centrifuged at 15,300 × g at 4°C for 20 min. The supernatant was discarded, and the pellet was washed with 500 µl of 70% ethanol, inverted five times, and centrifuged again at 15,300 × g at 4°C for 5 min. KOD-Plus Neo (TOYOBO) was used to amplify the genome-editing constructs for each target site: SlGAD3 -target, SlESK -target1 and SlESK -target2 by PCR. The reaction composition is listed Supplementary Table 1 (#1-#6), and the PCR conditions were allowed according to the manufacturer’s instructions. PCR products were extracted from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN). The ligation reaction was performed using the In-Fusion Snap Assembly Master Mix (Takara Bio). The reaction tube was kept on ice, and 25 µl of Stellar Competent Cells (Takara Bio) were added. Heat shock was applied at 42°C for 45 s to transform the plasmid into E. coli . Subsequently, 100 µl of SOC medium was added, and the culture was incubated with shaking at 180 rpm at 37°C for 60 min. The mixture was then spread on LB plates containing 50 µg/ml kanamycin and cultured at 37°C overnight. The desired vector was confirmed by sequencing the plasmid samples verify the correct insertion. Tomato transformation using the Agrobacterium method Tomato transformation was performed following the high-efficiency transformation protocol (described by Sun et al., 2006). Both the prepared vector plasmid and the Super-Agrobacterium plasmid pBBRacdSgadTAmp (Nonaka et al., 2019) were transformed into Agrobacterium tumefaciens strain GV2260. After infection of Agrobacterium into tomato cotyledons, plants were regenerated as described previously (Sun et al., 2006). Preparation of genomic DNA and amplification of target DNA regions About 5 mm 2 section of a true leaf from each plant was placed in a 1.5 ml tube and frozen with liquid nitrogen. Next, 100 µl of Buffer A (1 M Tris-HCl [pH 9.5] 30ml, KCl 23 g, 0.5 M EDTA 6 ml, distilled water to a final volume of 300 ml) was added, and the leaf section was homogenized using a hand pestle. The tube was then incubated at 95°C for 5 min. After incubation, the tube was cooled on ice and centrifuged at 17,600 × g 4°C for 5 min. The resulting supernatant was transferred to a new tube. KOD-FX Neo (TOYOBO) was used for the PCR amplification of the target DNA regions. The primers used for MultiNA sample preparation were as follows: SlGAD3-multiF and SlGAD3-multiR for the SlGAD3 target region, ESK1-target2-multinaF and ESK1-target2-multinaR for the ESK1 target region, ESK2-target2-multinaF and ESK2-target2-multinaR for the ESK2 target region, and ESK3-target2-multinaF and ESK3-target2-multinaR for the ESK3 target region (Supplementary Table 2, #1-#7). After PCR amplification, the sizes of the PCR products were confirmed by agarose gel electrophoresis. The MultiNA system (SHIMADZU) was used to identify candidate individuals with genome-editing-induced mutations. First, 4 µl of the PCR products from the target gene regions were incubated at 95°C for 5 minutes and allowed to anneal at room temperature. Size ladder preparation was performed according to the MultiNA operating protocol. When a different band pattern was detected by the MultiNA system, the PCR products were cloned into the pGEM-T Easy vector (Promega) and transformed into E. coli . After colony formation, PCR was performed using the primers M13-47 and RV-P (Supplementary Table 2, #8). The resulting PCR product was treated with Illustra ExoProStar (Cytiva) according to the manufacturer’s instructions. Finally, the samples were sequenced using the primer M13-47. Cultivation of T 1 and T 2 generations A 1% hydrochloric acid solution was poured into a beaker, fully submerging the seeds extracted from fully ripe red tomatoes. The seeds were stirred in the solution for 15 min. They were then thoroughly rinsed with water and dried. Seeds collected from individuals with confirmed mutations, along with WT seeds for comparison, were directly sown on rock wool. After root establishment, genomic DNA was extracted from the leaves of the plants to confirm the inheritance of mutations. Agrobacterium persistence test Approximately 5 mm 2 segments of plant leaves were placed in a sterile tube and homogenized using a pestle in 200 µl of sterile water. The homogenized solution was briefly centrifuged for approximately 10 s. Then, 50 µl of the supernatant was streaked onto AB medium (K₂HPO₄ 600 mg, NaH₂PO₄ 200 mg, NH₄Cl 200 mg, KCl 30 mg, CaCl₂·2H₂O 20 ml, FeSO₄·7H₂O (2.5 mg/ml) 20 ml, Agarose 3 g, 5% Glucose 20 ml, MgSO₄·7H₂O (1M) 240 µl, Kanamycin (50mg/ml) 200 µl, distilled water 200 ml), and incubated at 28°C for 5 days. As controls, streaking and culturing were also performed on the same medium using Agrobacterium tumefaciens GV2260 harboring the plasmid pPcUfcoCas9-SlESKGAD3 as a positive control and sterile water as a negative control. Evaluation of exogenous gene persistence and off-target mutations An exogenous gene residual test was performed using KOD-FX Neo (TOYOBO). The primers listed in Supplementary Table 2 (#9-#19) were used to amplify the entire plasmid region of pPcUfcocas9-SlESKGAD3 . Additionally, PCR was conducted under identical conditions using pPcUfcoCas9-SlESKGAD3 as a positive control and genomic DNA extracted from WT as a negative control. Off-target mutations were identified using Cas-OFFinder 25 . The following parameters were applied: PAM type, 5'-NGG-3'; target-genome, Solanum lycopersicum (SL4.0); maximum mismatch number, 2; DNA bulge size, 1; RNA bulge size, 1. Potential off-target mutation sites located at the target loci were selected. Among these, only sites where gene exons were registered in the Solanum lycopersicum (SL4.0) genome database were chosen for confirmation. To amplify candidate off-target mutation sites, PCR was performed using KOD-FX Neo (TOYOBO) with primers listed in Supplementary Table 2 (#20-#28). After amplification, the PCR products were sequenced for mutation verfication. Fruit measurement and analysis Fruit size and weight Fruit size was determined by measuring the maximum diameter of the fruit along the equatorial plane using a vernier caliper. Fruit weight was measured using an electronic balance. Brix Measurement The Brix value was measured using a digital refractometer (AS ONE). GABA content measurement GABA content was measured using the GABA Miel kit (Enzyme Sensor Co., Ltd.) according to the manufacturer’s instructions. Briefly, distilled water equivalent to nine times the fruit’s weight was added to the fruit sample, which was then crushed using a mortar. The resulting mixture was filtered, and 50µl of the filtrate, along with the reaction standard solution, was placed in a measurement cell at room temperature. Subsequently, 500µl of Solution A was added, and the reaction was allowed to proceed for 10 min. Then, 500 µl of Solution B was added, and the mixture was incubated for another 10 min. The GABA content (mg/100 g fresh weight) was determined using an LED colorimeter (445 nm) calibrated with distilled water and the reaction standard solution. Plant height measurement Seeds were sown and seedlings were grown in the cultivation room until they reached approximately 40cm in height. The plants were then transferred to a greenhouse, where plant height was measured weekly. Measurements were taken from the aerial part of the plant. Statistical analysis Statistical analysis was performed using R software (R Core Team, 2023). The Kruskal-Wallis test was used to compare three or more groups with different sample sizes. The Wilcoxon signed-rank test was applied to account for data asymmetry and other non-parametric characteristics, allowing for pairwise comparisons. Additionally, the Mann-Whitney U test was performed, with correction for multiple comparisons using the Holm method. For statistical analysis of plant height, the Wilcoxon signed-rank test (equivalent to the Mann-Whitney U test) was used because homoscedasticity could not be assumed, as evaluated using the F-test and Levene's test. Plant growth was calculated by subtracting the previous week's height from the current week's height, and cumulative growth values were compared. MDA measurement To measure MDA levels, 4-week-old plants were used. Prior to drought stress treatment, 100 mg of fresh leaves were collected and immediately frozen in liquid nitrogen. Under drought stress conditions, both WT and EG lines (EG Line 1 and EG Line 2) were subjected to a 10-day water starvation period, after which leaves were sampled for the same manner as the non-stressed condition. The collected leaves were finely ground in 1.5 ml of a 10% (v/v) solution of trichloroacetic acid (TCA) solution. Following centrifugation at 17,600 × g for 15 min, 1 ml of the resulting supernatant was mixed with 1 ml of a 0.6% (w/v) thiobarbituric acid (TBA) solution prepared in 10% TCA. The mixture was heated in boiling water for 20 min and then cooled to room temperature. Absorbance was measured at 450 nm, 532 nm, and 600 nm using a DU-800 spectrophotometer (Beckman Coulter). The MDA concentration was calculated using the following formula: MDA(µmol/L) = [ 6.45 × (A 532 -A 600 ) – 0.56 × A 450 ]. The MDA of the cold stress treatment also measured, after 3-days and 7-days cold stress treatment at a 4°C incubator. EL Measurement Fresh leaves from each 4-week-old plant were rinsed thoroughly with Milli-Q water (MQ) and placed in tubes filled with MQ to ensure full submersion of the leaf tissue. The tubes were maintained at room temperature for 24 h, after which EL was measured as C 1 using an ion conductivity meter (Lutron). Next, the samples were subjected to high-pressure sterilization at 121°C for 20 min and allowed to cool to room temperature. EL measurements were then recorded as C 2 . The percentage of EL was calculated using the following formula: EL (%) = (C 1 /C 2 ) × 100 RNA extraction and purification For RNA extraction, 100 mg of fresh leaves or red ripe fruits harvested from June to July were frozen in liquid nitrogen and finely ground. Total RNA was extracted using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. A total of 2µg of RNA was used for complementary DNA (cDNA) synthesis using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The primers SlNCED1 -F and SlNCED1 -R were used to amplify SlNCED1 for real-time PCR analysis (Supplementary Table 2, #29). Real-time PCR and relative abundance calculations were performed as described previously (Abellatif et al., 2002). The SlEXPRESSED gene was used as an endogenous control for gene expression analysis (Choi et al., 2018 Plant Biotechnol). RNA-seq analysis RNA sequencing analysis was outsourced to Rhelixa Co., Ltd. Quality control (QC) score were assessed using FastQC software (Version 0.11.7; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Low-quality bases (Q < 20) and adapter sequences were trimmed using Trimmomatic software (Version 0.38) with the following parameters: ILLUMINACLIP: path/to/adapter.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36. The trimmed reads were aligned to the reference genome using the RNA-seq aligner HISAT2 (Version 2.1.0). The HISAT2-generated .sam files were converted into .bam files using Samtools (Version 1.9). The resulting .bam files were used to estimate the abundance of uniquely mapped reads with featureCounts (Version 1.6.3). Raw read counts were normalized using transcripts per million (TPM). The samples were clustered using the Wald method based on the Euclidean distances of the normalized counts, employing stats (Version 3.6.1) and gplots (Version 3.0.1.1) R packages. Principal component analysis (PCA) was performed on the normalized counts, and each sample was projected onto two-dimensional plane based on the first and second PCA axes using the same R packages. Pearsonʼs correlation coefficients of the normalized counts were calculated to assess correlation between samples. Histograms and pair plots of the normalized counts were generated using the stats and gplots R packages. Heatmaps were created from Z-scores of the normalized counts using the same R packages. Raw read counts were further normalized using relative log expression normalization, and differential expression analysis was conducted with DESeq2 (Version 1.24.0). Differentially expressed genes (DEGs) were identified using the thresholds of |log 2 fold change(FC) | >1 and adjusted p-value < 0.05 (and < 0.1), calculated using the Benjamini-Hochberg (BH) method for multiple testing correction. GO enrichment analysis was performed using ShinyGO 0.80 ( http://bioinformatics.sdstate.edu/go/ ). The Slycopersicum _eg_gene Ensembl ID was employed, with the Solanum lycopersicum SL3.0 gene annotation used as the database. The false discovery rate (FDR) cutoff was set to 0.05. Ascorbic acid measurement Tomato fruit (1.0 g) was homogenized using a mortar and pestle and mixed with 2.0 ml of 5% (w/v) metaphosphoric acid. After centrifugation at 12,000 × g for 3 min, the supernatant was collected as a crude extract. The total ascorbic acid content was measured using the Ascorbic Acid Test Kit (Merck, Darmstadt, Germany) with the RQ Flex Plus 10 analyzer (Merck, Darmstadt, Germany). Microscopy analysis Microscopy analysis of stems, flowers and fruits was performed by preparing tissue sections using a Vibrating-Blade Microtome VT1200 (Leica) and staining with Toluidine Blue for xylem and with Iodine potassium for pollen. Observations were conducted using a BX50 microscope (Olympus). Declarations Data availability The transcriptome data was used Solanum lycopersicum SL3.0 gene annotation and is available in the NCBI database: the accession number is GCF_000188115.4. The sequence data used in this study can be found in the GenBank data libraries under the following accession numbers: SlESK1 (Solyc03g096030), SlESK2 (Solyc06g051350), SlESK3 (Solyc05g052540), SlGAD3 (Solyc01g005000). And the data presented in this study are available on request from the corresponding author. Acknowledgements We would like to thank Ms. Yuriko Nagai, Ms. Yumiko Iguchi, Ms. Yuri Nemoto, Ms. Kazuko Ito, and Ms. Ayako Kobayashi at T-PIRC from the University of Tsukuba, Japan for their technical support. Funding This work supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid (22H02295) and Program on Open Innovation Platform with Enterprise, Research Institute and Academia, Japan Science and Technology Agency (JST-OPERA, JPMJOP1851). Authors contributions K.M. and H.E. conceptualized the manuscript. S.C., T.I., M.K. and I.A. drafted the manuscript and prepared the figure. K.M. revised the manuscript. All authors read and approved the final manuscript. Conflict of interest statement The authors declare no conflict of interest. References FAO Statistical Yearbook. - World Food and Agriculture – World. 2023. (2023). https://reliefweb.int/report/world/fao-statistical-yearbook-2023-world-food-and-agriculture Kaboré, K. et al. Evaluation of phytonutrients composition and nutraceutical potential of tomato by-products. CyTA - J. Food . 20 (1), 404–411 (2022). Kawaguchi, K. et al. Functional disruption of cell wall invertase inhibitor by genome editing increases sugar content of tomato fruit without decrease fruit weight. Sci. Rep. 11 (1), 21534 (2021). Nagamine, A. & Ezura, H. Genome Editing for Improving Crop Nutrition. Front. Genome Ed. 4 , 850104 (2022). Boonstra, E. et al. Neurotransmitters as food supplements: the effects of GABA on brain and behavior. Front. Psychol. 6 , 1520 (2015). Takayama, M. & Ezura, H. How and why does tomato accumulate a large amount of GABA in the fruit? Front. Plant. Sci. 6 , 612 (2015). Nonaka, S. et al. Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis. Sci. Rep. 7 (1), 7057 (2017). Xin, Z. & Browse, J. eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant, Proceedings of the National Academy of Sciences, ; 95(13), pp. 7799–7804. (1998). Reyes-Díaz, M. et al. Arabidopsis thaliana avoids freezing by supercooling. J. Exp. Bot. 57 (14), 3687–3696 (2006). Yuan, Y. et al. The Arabidopsis DUF231 Domain-Containing Protein ESK1 Mediates 2-O- and 3-O-Acetylation of Xylosyl Residues in Xylan’. Plant Cell Physiol. 54 (7), 1186–1199 (2013). Jinek, M. et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337 (6096), 816–821 (2012). Ahiakpa, J. K. et al. Genome-Wide Identification and Expression Profiling of Tomato Invertase Genes Indicate Their Response to Stress and Phytohormones. J. Plant. Growth Regul. 41 , 1481–1498. https://doi.org/10.1007/s00344-021-10384-5 (2022). Munir, S. et al. Genome-wide analysis of Myo-inositol oxygenase gene family in tomato reveals their involvement in ascorbic acid accumulation. BMC Genom. 21 , 284. https://doi.org/10.1186/s12864-020-6708-8 (2020). Zhang et al. Increased Drought Tolerance through the Suppression of ESKMO1 Gene and Overexpression of CBF-Related Genes in Arabidopsis. PLOS ONE , ; 9 , e106509 (2014). Lefebvre, V. et al. ESKIMO1 Disruption in Arabidopsis Alters Vascular Tissue and Impairs Water Transport. PLoS ONE . 6 (2), e16645 (2011). Bai, S. H. et al. Combined effects of biochar and fertilizer applications on yield: A review and meta-analysis. Sci. Total Environ. 808 , 152073 (2022). Ghars, M. A. et al. Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella halophila, with special emphasis on K+/Na + selectivity and proline accumulation. J. Plant Physiol. 165 (6), 588–599 (2008). Bouchabke-Coussa, O. et al. ESKIMO1 is a key gene involved in water economy as well as cold acclimation and salt tolerance. BMC Plant Biol. 8 (1), 125 (2008). Vishwakarma, K. et al. Abscisic Acid Signaling and Abiotic Stress Tolerance in Plants: A Review on Current Knowledge and Future Prospects. Front. Plant. Sci. 8 , 161 (2017). Kong, L. et al. ‘Interactive regulation of light quality and temperature on cherry tomato growth and photosynthesis’. Environ. Exp. Bot. , ; (2021). 182. Worstall A shortage of fertilizer resources? Nature 493 (7431), 163–163 (2013). Reijnders Phosphorus resources, their depletion and conservation, a review’. Resour. Conserv. Recycl. 93 , 32–49 (2014). Mehrabi, Z. Likely decline in the number of farms globally by the middle of the century’. Nat. Sustain. 6 (8), 949–954 (2023). Keller & Kassel The number of U.S. farms continues slow deline. USDA Economic Res. Service , ; (2024). https://www.ers.usda.gov/data-products/chart-gallery/chart-detail?chartId=58268 Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30 (10), 1473–1475 (2014). Additional Declarations No competing interests reported. Supplementary Files 2.SupplementaryFile20250725.docx Cite Share Download PDF Status: Published Journal Publication published 02 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 06 Oct, 2025 Reviews received at journal 30 Sep, 2025 Reviews received at journal 20 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 11 Sep, 2025 Reviewers invited by journal 08 Aug, 2025 Editor invited by journal 31 Jul, 2025 Editor assigned by journal 30 Jul, 2025 Submission checks completed at journal 28 Jul, 2025 First submitted to journal 26 Jul, 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. 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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-7220491","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":499481499,"identity":"13f7b1bc-71f7-4984-aceb-e483d0f26e07","order_by":0,"name":"Seungje Choi","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Seungje","middleName":"","lastName":"Choi","suffix":""},{"id":499481500,"identity":"97a209ba-cbab-4625-9e53-d3381c068bbf","order_by":1,"name":"Takeru Iwama","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Takeru","middleName":"","lastName":"Iwama","suffix":""},{"id":499481502,"identity":"56795139-6ccd-497a-a285-5dbf40599cc2","order_by":2,"name":"Misaki Kobayashi","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Misaki","middleName":"","lastName":"Kobayashi","suffix":""},{"id":499481504,"identity":"a71b9a7a-a81d-4ae1-9ddb-dadfdce93968","order_by":3,"name":"Islam M.Y. Abdellatif","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Islam","middleName":"M.Y.","lastName":"Abdellatif","suffix":""},{"id":499481505,"identity":"c3db2cad-5c2f-4d99-aeb0-395a1f200115","order_by":4,"name":"Hiroshi Ezura","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Ezura","suffix":""},{"id":499481506,"identity":"463b28ea-34af-4693-997b-569868b92877","order_by":5,"name":"Kenji Miura","email":"data:image/png;base64,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","orcid":"","institution":"University of Tsukuba","correspondingAuthor":true,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Miura","suffix":""}],"badges":[],"createdAt":"2025-07-26 10:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7220491/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7220491/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-28888-5","type":"published","date":"2025-12-02T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88995816,"identity":"87beb858-dd2f-4eb1-982b-122875e3e686","added_by":"auto","created_at":"2025-08-13 14:30:33","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":137270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVector structure and mutation status of eskgad3 mutant (EG Lines)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Vector map for targeting \u003cem\u003eSlESK 1-3\u003c/em\u003e and \u003cem\u003eSlGAD3\u003c/em\u003e. RB: Right boarder, LB: Left boarder. Promoters are indicated by blue box and terminator are indicated by red box. Each target gene is marked green box and gRNA is colored by yellow box. NPT\u003cem\u003eII\u003c/em\u003e represents kanamycin resistance gene. (B) Each mutant sequence. (-) indicates deletions and red letter indicates the insertion. The number of deletions or insertion. Blued fonts indicate PAM sequences.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7220491/v1/c27132977bf211e8c800c2df.jpeg"},{"id":88995814,"identity":"a0f775da-5325-479f-8f03-9290b6436bca","added_by":"auto","created_at":"2025-08-13 14:30:33","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":179062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEG Lines exhibited plant growth retardation and reduced fruit set ratio.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Images are representatives of WT and EG Lines. Scale bars represent 5cm in length. (B) The growing height (AVE+SE) of WT and EG Lines after transfer of plants from the growth room to the greenhouse. After transfer, plant height was measured every week. (C) Fruit set rate of WT and EG Lines.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7220491/v1/68a52e06abd7106cbbbc7fee.jpeg"},{"id":88995815,"identity":"2e9a2a66-0ce1-4c79-b69e-bd34e2302925","added_by":"auto","created_at":"2025-08-13 14:30:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":734392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotype of the red ripe tomato fruits of EG Lines. The fruits of EG Lines were smaller in size than WT, but accumulated more sugar, GABA, and ascorbic acid.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Illustrates are of representative fruits of WT and EG Lines. Scale bar represents 5 mm in length. (B) Xylem structure in WT and EG Lines. Scale bar is 100 μm in length. (C-J) Parameter of fruits from WT and EG Lines during spring season. (C)Fruit weight. (D) Fruit diameter. (E) GABA concentration in fruits. (F) Brix value of fruits. (G) Sucrose concentration in fruits. (H) Fructose concentration in fruits. (I) Glucose concentration in fruits. (J) Concentration of ascorbic acid in fruits. Statistical significance: ns, no significant difference; *, p \u0026lt; 0.05; *, p \u0026lt; 0.01; ***, p \u0026lt; 0.001; ****, p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7220491/v1/3e2060a06a096bb957cf4cc1.png"},{"id":88995817,"identity":"57951b77-58f7-4558-b94c-9e610b220c91","added_by":"auto","created_at":"2025-08-13 14:30:33","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":182022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEG Lines exhibited drought stress tolerance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Illustrates are of representative plants of WT and EG Lines 10 days after water starvation. Scale bar represents 2 cm in length. (B) Relative expression of\u003cem\u003e SlNCED1\u003c/em\u003e. RNA was extracted from leaves of WT and EG Lines under normal conditions. (C) Concentration of MDA before stress (Non-stress) and after 10-days water starvation (Stress). (D) Changes of electronlyte leakage(EL). Statistical significance: ns, no significant difference; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7220491/v1/b91343093df78bfa4b1d0d7c.jpeg"},{"id":88996577,"identity":"98fc2e9f-2e75-43c3-b463-8240a057d186","added_by":"auto","created_at":"2025-08-13 14:38:33","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":351110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA-seq analysis. The red ripe tomato fruits of WT and EG Line2 were obtained and RNA was extracted from these fruits. After RNA-sequenceing, gene expression was compared between WT and EG Line2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(A) Volcano plot. Blue dots represnet down-regulated genes, red dots represent up-regulated genes (log\u003csub\u003e2\u003c/sub\u003eFC \u0026gt; 2, padj \u0026lt; 0.05). Gray dots represent genes with no significant difference. (B, C) GO enrichment analysis. (B) Results of GO enrichment analysis for up-regulated genes. (C) Results of GO enrichment analysis for down-regulated genes.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7220491/v1/aa8eeca316ca08411d510c04.jpeg"},{"id":97723794,"identity":"a91cbeee-24a2-4a78-bf63-8af1717bed1e","added_by":"auto","created_at":"2025-12-08 16:06:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2779991,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7220491/v1/ce2c6c42-9ed6-41b2-a4a9-62f587b73957.pdf"},{"id":88996579,"identity":"a9da051d-c48f-48c9-af79-deea5a8a44e0","added_by":"auto","created_at":"2025-08-13 14:38:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6607708,"visible":true,"origin":"","legend":"","description":"","filename":"2.SupplementaryFile20250725.docx","url":"https://assets-eu.researchsquare.com/files/rs-7220491/v1/bbefe86fbbdc960cf2afdae7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing tomato quality, high sugar content and GABA accumulation, with mutations in ESKs and GAD3 genes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTomatoes are among the most widely cultivated crops globally, with an annual production of approximately 189\u0026nbsp;million tons \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Additionally, research has shown that tomatoes contain numerous functional compounds, making them a vital crop for supporting and promoting health \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Tomatoes are also consumed as fruits, and innovative breeding techniques are being employed to develop tomato varieties with high sugar content \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It is expected that future advancements will lead to tomato varieties with both enhanced sugar content and enriched functional compounds. For instance, a high-GABA tomato, developed through genome editing, is already commercially available \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the mammalian central nervous system. GABA is also widely used as a dietary supplement due to its potential to lower blood pressure and reduce anxiety \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe genes targeted for the development of tomatoes with high GABA and sugar content are \u003cem\u003eSlGAD3\u003c/em\u003e (\u003cem\u003eglutamate decarboxylase 3\u003c/em\u003e) and \u003cem\u003eSlESK1-3\u003c/em\u003e (\u003cem\u003eEskimo1-3; Solyc03g096030\u003c/em\u003e, \u003cem\u003eSolyc05g052540\u003c/em\u003e, \u003cem\u003eSolyc06g051350\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eSlGAD3\u003c/em\u003e encodes glutamate decarboxylase 3, a key enzyme in GABA accumulation in tomato fruits. \u003cem\u003eGAD3\u003c/em\u003e facilitates GABA synthesis by catalyzing the removal of carboxyl group from glutamic acid. This enzyme also contains an autoinhibitory domain near its C-terminus, which stabilizes the enzyme in an inactive form under normal conditions. However, during stress responses, rising intracellular calcium ion levels trigger the formation of a calcium-calmodulin complex, which binds to the calmodulin-binding site within the autoinhibitory domain, activating \u003cem\u003eGAD3\u003c/em\u003e. Previous studies have shown that genome editing of the \u003cem\u003eSlGAD3\u003c/em\u003e autoinhibitory domain can successfully increase GABA accumulation in tomato fruits \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSlESK1-3\u003c/em\u003e are homologs of the \u003cem\u003eAtESK1\u003c/em\u003e gene in tomato, which has been identified as a freeze tolerance-related gene in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The \u003cem\u003eAtesk1\u003c/em\u003e mutants in Arabidopsis exhibit increased soluble sugar content and enhanced freeze tolerance under cold stress conditions \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eAtESK1\u003c/em\u003e has been shown to function as a xylan acetyltransferase in Arabidopsis. Mutations in \u003cem\u003eAtESK1\u003c/em\u003e result in decreased secondary cell wall thickness and reduced stem strength \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In contrast, few studies have reported on \u003cem\u003eesk\u003c/em\u003e mutants in other crops, particularly fruiting crops such as tomato. In this study, we aimed to generate \u003cem\u003eslesk\u003c/em\u003e mutants in tomato and characterize the function of \u003cem\u003eSlESK\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eGenome editing technology is anticipated to play a significant role in future breeding efforts. This technology involves inducing DNA double-strand breaks (DSBs) at specific genomic locations using artificial nucleases that recognize and cleave target sequences. Mutations arise from errors in the DNA repair process following cleavage, enabling precise modification of endogenous genes. Genome editing can be applied to a wide range of species. Prominent genome editing tools include zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 \u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we utilized CRISPR/Cas9, currently the most widely adopted genome editing tool. CRISPR/Cas9 is derived from the immune defense system widely conserved in eubacteria and archaea. It consists of the endonuclease Cas9 and a guide RNA (gRNA). During the induction of DSBs, the gRNA, containing a sequence complementary to the target site, recognizes and binds to the target DNA sequence. The Cas9 protein, which possesses two DNA cleavage domains, then cleaves the DNA at the target site, generating a DSB. Target sequence recognition by Cas9 requires the presence of a protospacer adjacent motif (PAM) sequence, such as NGG in the case of SpCas9. CRISPR/Cas9 is widely used due to its advantages, including simplicity in design and construction, as well as the ability to edit multiple genes simultaneously. Indeed, genome edited foods, such as high-GABA tomatoes, have already been commercially launched in Japan \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we aimed to simultaneously modify \u003cem\u003eSlESK1-3\u003c/em\u003e and \u003cem\u003eSlGAD3\u003c/em\u003e to enhance the value of the original tomato cultivar. Additionally, we investigated the phenotypes of the \u003cem\u003eslesk1-3\u003c/em\u003e mutants to characterize the function of \u003cem\u003eSlESK\u003c/em\u003e. In addition to our genome editing approach, previous studies have highlighted the importance of invertase genes in sugar metabolism under stress \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and identified gene families involved in ascorbic acid accumulation \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, which collectively underline the complex regulation of these nutritional traits.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eGeneration of\u003c/b\u003e \u003cb\u003eesksgad3\u003c/b\u003e \u003cb\u003emutants using CRISPR/Cas9\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo generate tomato plants harboring T-DNA with CRISPR/Cas9, 159 regenerated plants were obtained through tissue culture of cotyledon fragments. Of these, 38 individuals were selected for sequencing after preliminary screening using MultiNA, and mutations at the target sites were confirmed in 10 individuals (Supplementary Table\u0026nbsp;3). Additionally, multiple mutations were detected in some individuals, indicating the occurrence of chimeric mutations.\u003c/p\u003e\u003cp\u003eIn the T\u003csub\u003e1\u003c/sub\u003e generation, the inheritance of mutations at the target sites was confirmed in five lines of \u003cem\u003eesksgad3\u003c/em\u003e (Supplementary Table\u0026nbsp;4). Among these individuals, homozygous mutations were successfully introduced in all target genes (\u003cem\u003eESK1-3\u003c/em\u003e and \u003cem\u003eGAD3\u003c/em\u003e) in \u003cem\u003eesksgad3\u003c/em\u003e line #127-5 (EG Line 1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Table\u0026nbsp;4). Furthermore, in \u003cem\u003eesksgad3\u003c/em\u003e line #127-9 (EG Line 2), although mutations were observed in the \u003cem\u003eESK2\u003c/em\u003e target and other target sites, several fully homozygous individuals were obtained in the subsequent T\u003csub\u003e2\u003c/sub\u003e generation (Supplementary table 5). Since \u003cem\u003eesksgad3\u003c/em\u003e #127-9-4 exhibited severe growth retardation, and no subsequent generation was obtained, it is suggested that severe mutations in three \u003cem\u003eSlESK\u003c/em\u003e homologs may cause significant developmental damage in tomatoes. Therefore, the six-base deletion in \u003cem\u003eESK2\u003c/em\u003e was selected for further characterization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eOff-target mutations were investigated in the \u003cem\u003eesksgad3\u003c/em\u003e #127 line, a genome-edited line, by comparing sequencing results with those of the wild type (WT). No mutations were detected at the predicted off-target candidate sites in any of the analyzed individuals (Supplementary Table\u0026nbsp;6). Additionally, amplification of the vector plasmid using 11 sets of primers revealed no residual foreign genes in \u003cem\u003eesksgad3\u003c/em\u003e #127-5-6 (a sibling of EG Line 1) or \u003cem\u003eesksgad3\u003c/em\u003e #127-9-10, #127-9-14, #127-9-18, #127-9-26, and #127-9-29 (siblings of EG Line 2) (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth and fruit set impairment in eg mutants.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn EG Line 2, ESK2target1 exhibited a heterozygous mutation in the T\u003csub\u003e1\u003c/sub\u003e generation. Therefore, sequence analysis of ESK2target1 was performed in the T\u003csub\u003e2\u003c/sub\u003e generation, with results presented in Supplementary Table\u0026nbsp;5.\u003c/p\u003e\u003cp\u003eA delay in growth was observed in both EG Line 1 and EG Line 2 compared to the WT (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The plant heights measured 10 weeks after the start of observations are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (cm): WT\u0026thinsp;=\u0026thinsp;97.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.09, EG Line 1\u0026thinsp;=\u0026thinsp;45.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78, EG Line 2\u0026thinsp;=\u0026thinsp;53.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20. A statistically significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed when comparing growth between WT and the EG lines (EG Line 1 and EG Line 2) across all weeks of measurement.\u003c/p\u003e\u003cp\u003eFurthermore, fruit set rates decreased significantly. Flowers and fruits were counted 21 weeks after sowing. The fruit set rate was 43.17% in the WT, whereas it was significantly lower in EG Line 1 (4.52%) and EG Line 2 (6.56%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The decreased fruit set rates are results of abnormal flower developments, especially pollen development (Supplementary Fig.\u0026nbsp;2). The flowers in EG lines are smaller than in WT, and many flowers in EG lines are stopped developing and wilt (Supplementary Fig.\u0026nbsp;2A). The pollen in EG mutants also exhibited unshaped and small sized pollen (Supplementary Fig.\u0026nbsp;2B).\u003c/p\u003e\u003cp\u003eAdditionally, differences in vascular structure were observed in specific regions of the stem. In the upper part of the stem (below the third mature leaf from the top) and the middle part (above the third mature leaf from the bottom), the vessel area was significantly wider in the WT compared to the EG Line 1 and EG Line 2. However, no significant differences were observed in the lower stem region (above the boundary between the stem and root) or in the leaflets (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDifferences in fruit traits in EG Line1 and EG Line2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo measure fruit size, the equatorial diameter of the fruit was measured. Compared to WT, both EG Line 1 and EG Line 2 exhibited significantly smaller fruit weight and diameter, regardless of the season (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; Supplementary Figs.\u0026nbsp;4A, 4B).\u003c/p\u003e\u003cp\u003eIn previous studies, the \u003cem\u003eesk1\u003c/em\u003e mutant in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e was reported to exhibit impaired xylem structure and secondary cell wall formation \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, we examined the fruit structure using microscopy. The results showed no detectable differences in xylem structure among the WT and EG lines (EG Line 1 and EG Line 2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eRegarding GABA content, a comparison of EG Line 1 and EG Line 2 with WT revealed that GABA accumulation was significantly higher in both EG Line 1 and EG Line 2 than in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; Supplementary Fig.\u0026nbsp;4C), regardless of the season.\u003c/p\u003e\u003cp\u003eFor total soluble sugar content (Brix), both genome-edited lines (EG Line 1 and EG Line 2) exhibited significantly higher values compared to WT, consistently across all seasons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eF; Supplementary Fig.\u0026nbsp;4D). To further analyze sugar composition, sucrose, fructose, and glucose levels were measured. The results indicated that sucrose and fructose levels were significantly higher in the EG Line 1 and EG Line 2 than in WT during the spring season but less significantly elevated during the summer season (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eH; Supplementary Fig.\u0026nbsp;4E, 4F). In contrast, glucose content was consistently and significantly higher in the EG Line 1 and EG Line 2 across all seasons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eI; Supplementary Fig.\u0026nbsp;4G).\u003c/p\u003e\u003cp\u003eFrom Gene Ontology (GO-) enrichment analysis and RNA-seq results, we observed the upregulation of genes associated with ascorbic acid (vitamin C) biosynthesis. Therefore, we measured ascorbic acid levels and found a significant increase in the EG Line 1 and EG Line 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ), suggesting that these plants likely enhance antioxidant production to resist and recover from stress \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEG Line1 and Line2 exhibit drought stress tolerance but doesn\u0026rsquo;t exhibit cold stress tolerance.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eesk1\u003c/em\u003e mutant in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e has demonstrated enhanced tolerance to various abiotic stresses, including cold, drought, and salinity, which was associated with higher abscisic acid (ABA) levels \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. To investigate whether a similar mechanism was present in the EG Line1 and EG Line2, the expression of the ABA biosynthetic gene \u003cem\u003eSlNCED1\u003c/em\u003e was examined. \u003cem\u003eSlNCED1\u003c/em\u003e expression was found to be upregulated in the EG Line1 and EG Line2 compared to WT under normal room temperature conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To assess drought stress tolerance, WT and EG lines (EG Line1 and EG Line2) were subjected to a 10-day water-starvation treatment, after which phenotypic changes, malondialdehyde (MDA) levels, and electrolyte leakage (EL) were evaluated (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Under normal conditions, MDA levels were higher in the EG Line1 and EG Line2 than in WT. However, after water-starvation, MDA levels increased significantly in WT but remained relatively unchanged in the EG Line1 and EG Line2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Although baseline levels of EL levels were higher in the EG Line1 and EG Line2, the degree of increase in EL after drought stress was significantly greater in WT compared to the EG Line1 and EG Line2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggest that the EG Line1 and EG Line2 exhibit drought stress tolerance and sustain less cellular damage under drought conditions.\u003c/p\u003e\u003cp\u003eHowever, the EG Line1 and Line2 didn\u0026rsquo;t exhibit the cold stress tolerance (Supplementary Fig.\u0026nbsp;5A). To assess cold stress tolerance, WT and EG lines were subjected to a 7-day 4\u0026deg;C cold stress environment. The MDA was evaluated 3-day and 7-day after the cold stress treatment. The MDA wasn\u0026rsquo;t significantly different between WT and EG lines after the cold stress treatment, while significantly different before the cold stress treatment (Supplementary Fig.\u0026nbsp;5B).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA-seq and GO- term- enrichment- analysis in tomato fruits\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA sequencing analysis revealed that 302 genes were significantly upregulated, while 703 genes were significantly downregulated in red ripe tomato fruits of EG Line 2 compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eAmong the upregulated genes identified in the GO analysis, the most notable processes were related to vitamin C biosynthesis, the auxin signaling pathway, AUX/IAA family genes, ethylene biosynthesis, and ethylene metabolism. These biological processes play critical roles in regulating the developmental responses observed in the EG Line1 and EG Line2. The highest upregulated gene associated with vitamin C regulation was Solyc05g054760.4 (dehydroascorbate reductase 1; \u003cem\u003eDHAR1\u003c/em\u003e). Since the vitamin C biosynthesis pathway was upregulated, an increased concentration of ascorbic acid was also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFor auxin transporter and regulatory genes, key upregulated genes included Solyc01g068410.4 (\u003cem\u003eSlPIN5\u003c/em\u003e), Solyc10g080880.2 (\u003cem\u003eSlPIN7\u003c/em\u003e), Solyc09g008175.1 (SAUR-like auxin-responsive protein family), Solyc09g065850.4 (\u003cem\u003eSlIAA3\u003c/em\u003e), Solyc01g079260.4 (\u003cem\u003eSlWRKY23\u003c/em\u003e), Solyc05g047460.3 (\u003cem\u003eSl-ARF7B\u003c/em\u003e), and Solyc01g103050.3 (\u003cem\u003eSl-ARF1\u003c/em\u003e). For ethylene biosynthesis and signaling, upregulated genes included Solyc01g095080.3 (\u003cem\u003eACC2\u003c/em\u003e), Solyc03g118190.4 (ethylene-responsive transcription factor), Solyc07g049530.3 (\u003cem\u003eSlACO1\u003c/em\u003e), Solyc03g118190.4 (ethylene-responsive transcription factor), Solyc07g049550.3 (1-aminocyclopropane-1-carboxylate oxidase), Solyc03g044300.3 (\u003cem\u003eAP2a\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;4). Regarding nutrient transport and response genes, upregulated calcium-related genes included Solyc03g097100.1 (EF hand calcium-binding protein family) and Solyc01g108400.3 (calcium-dependent protein kinase-like). Additionally, the phosphate transporter Solyc02g078210.3 (\u003cem\u003ePHO2\u003c/em\u003e) and Solyc01g005000.3 (\u003cem\u003eGAD3\u003c/em\u003e) were identified as upregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Supplementary Fig.\u0026nbsp;6).\u003c/p\u003e\u003cp\u003eThe GO analysis of downregulated genes identified 93 biological processes (BPs) significantly associated with key roles in plant growth and development. These BPs were classified into three main categories: stress response, cellular regulation, and transportation. A substantial number of downregulated genes in the fruits of EG Line1 and EG Line2 were associated with stress response processes, including responses to heat and other abiotic stimuli. The significantly downregulated genes in this category included Solyc09g065660.4 (\u003cem\u003eHsfA7\u003c/em\u003e), Solyc08g062960.4 (\u003cem\u003eHsfA2\u003c/em\u003e), and Solyc09g009100.4 (\u003cem\u003eSolycHsfA3\u003c/em\u003e), all of which are associated with heat stress response. The second category included genes involved in cellular regulation, particularly those related to cell wall biogenesis and the regulation of xyloglucan, hemicellulose, saccharide, macromolecule, and carbohydrate metabolism. The most significantly downregulated gene in this category was Solyc04g071070.2 (unknown protein, plant-type cell wall organization). The third category involved genes related to transportation, including water and lipid transport. Notable downregulated genes included Solyc01g090350.3 and Solyc01g090360.3 (non-specific lipid-transfer proteins) for lipid transport and Solyc12g044330.2 (\u003cem\u003eSlTIP2.1\u003c/em\u003e), Solyc06g074820.3 (\u003cem\u003eSlTIP1.1\u003c/em\u003e), and Solyc10g055630.2 (\u003cem\u003eSlPIP2.9\u003c/em\u003e) for water channel activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; Supplementary Fig.\u0026nbsp;7).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that multiple desirable traits can be improved through a single genome-editing event and investigated the characteristics of the EG Line 1 and EG Line2.\u003c/p\u003e\u003cp\u003eOur results indicate significant differences in sugar content, GABA accumulation, and ascorbic acid levels between WT and EG lines (EG Line 1 and EG Line 2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although seasonal variations were observed, the sugar content in the red ripe fruits of the EG Line 1 and EG Line 2 was consistently higher than that of the WT. Specifically, during the spring season, the average Brix value in the EG Line 1 and EG Line 2 was approximately 1.8 times higher than in the WT. Compared to the \u003cem\u003eesk1\u003c/em\u003e mutant in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, which does not produce fruits, the EG Line 1 and EG Line 2 of tomato produce fruits with elevated the sugar content, even under a greenhouse conditions without stress induction.\u003c/p\u003e\u003cp\u003eFurthermore, both GABA and vitamin C contents were significantly increased in the EG Line 1 and EG Line 2, with GABA levels approximately 4 to 5 times higher and vitamin C levels approximately 1.5 times higher than those in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest that introducing mutations into the \u003cem\u003eESKs\u003c/em\u003e and \u003cem\u003eGAD3\u003c/em\u003e genes can effectively enhance the nutritional of tomato fruits. The \u003cem\u003eesks\u003c/em\u003e mutants in tomato could affect the stressed phenotype and GABA content.\u003c/p\u003e\u003cp\u003eGrowth retardation in the EG Line 1 and EG Line 2 was confirmed through analysis using T\u003csub\u003e2\u003c/sub\u003e individuals. Previous research demonstrated that \u003cem\u003eesk1\u003c/em\u003e mutants in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e inhibit xylan acetylation, which promotes its degradation by endoxylanase and reduces secondary wall thickening \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This reduction in secondary wall thickening results in insufficient xylem formation, causing wilted leaves and dwarf traits. In tomatoes, it is also known that sugar content in tomato fruits increases when water availability is limited, which can be explained by a concentration effect due to reduced water content in the fruit \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Based on these findings, it is hypothesized that the growth retardation observed in the EG Line 1 and EG Line 2 results from reduced secondary wall development caused by mutations in \u003cem\u003eSlESK\u003c/em\u003e genes and that the increased fruit sugar content is attributable to decreased water transport efficiency due to defective xylem formation. Significant differences in vessel structure were observed in the stems of the top and middle regions of the EG Line 1 and EG Line 2, with a decreased vessel area compared to the WT (Supplementary Fig.\u0026nbsp;3). RNA-seq analysis revealed upregulated expression of genes related to drought-induced stress, ABA response, and water deprivation, further supporting the high sugar phenotype observed in the EG Line 1 and EG Line 2 (Supplementary Fig.\u0026nbsp;4) Additionally, the fruit set rates are significantly decreased in the EG mutants and its results are results of stunted growth in EG mutants (Supplementary Fig.\u0026nbsp;2 and Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eesksgad3\u003c/em\u003e #127-9-4 line (a sibling of EG Line 2) had a\u0026thinsp;+\u0026thinsp;1 homozygous mutation in ESKtarget1 (Supplementary Table\u0026nbsp;5). However, this individual died shortly after genome extraction, and no other triple-frame-shift mutants were obtained from its siblings. This observation suggests that frame-shift mutations in all \u003cem\u003eSlESK1-3\u003c/em\u003e genes are likely lethal and that \u003cem\u003eSlESK1-3\u003c/em\u003e are essential for growth and efficient water transport.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eesk1\u003c/em\u003e mutant in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e has exhibited tolerance to various abiotic stresses, including drought, cold, salt and osmotic stress \u003csup\u003e8 17 18\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, the tomato EG Line 1 and EG Line 2 exhibited drought stress tolerance, as evidenced by reduced MDA and EL levels, including reduced damage under drought conditions. The observed drought tolerance suggests that the EG Line 1 and EG Line 2 may pre-emptively recognize stress signals and activate resistance mechanisms. Previous studies have reported that the \u003cem\u003eatesk1\u003c/em\u003e mutants have higher ABA levels compared to WT \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. ABA is a key phytohormone involved in regulating plant responses to abiotic stress \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, we also observed increased expression of \u003cem\u003eNCED1\u003c/em\u003e, a key gene in ABA biosynthesis, in the EG Line 1 and EG Line 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In contrast, the EG lines didn\u0026rsquo;t exhibit the cold stress tolerance not like the \u003cem\u003eesk1\u003c/em\u003e mutant in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, since tomatoes originate from tropical regions and exhibit limited cold tolerance (Supplementary Fig.\u0026nbsp;5) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn conclusion, genome editing-induced mutations in tomato \u003cem\u003eESK1-3\u003c/em\u003e and \u003cem\u003eGAD3\u003c/em\u003e genes promoted sugar content, GABA levels, and vitamin C concentration, resulting in nutrient-rich tomato fruits with increased functional value. Moreover, these results may help alleviative the high costs and labor-intensive processes associated with cultivating high-quality tomatoes. Producing high-sugar tomatoes typically requires significant amounts of fertilizers and labor. However, resources for agricultural production are limited and being depleted \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and the number of people engaged in farming is steadily decreasing \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In addition, the yield reduction commonly observed in the high-sugar tomato cultivation needs to be addressed in future studies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003ePlant and growth conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) plants used in this study were provided by Sanatech Life Science Co., Ltd. The cultivation room was maintained at a constant temperature of 25\u0026deg;C, with a photoperiod of 16 h of light and 8 h of darkness. In the greenhouse, the temperature ranged from 11\u0026deg;C to 40\u0026deg;C during the spring season and from 20\u0026deg;C to 60\u0026deg;C during the summer season. Fertilizers included Otsuka House No. 1 and Otsuka House No. 2 (OAT Agrio Co., Ltd.).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of vector plasmid\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo create the pPcUfcoCas9-SlESKGAD3 plasmid, a reaction mixture containing 6 \u0026micro;l of CutSmart buffer, 2 \u0026micro;l of the restriction enzymes \u003cem\u003eApaI\u003c/em\u003e and \u003cem\u003eAscI\u003c/em\u003e, and 50 \u0026micro;l of the plasmid sample pPcUbi was incubated at 37\u0026deg;C for 60 min. Subsequently, amplicons listed in Supplementary Table\u0026nbsp;1 (#1-#4) were amplified using KOD-Plus Neo (TOYOBO) and ligated with the digested pPcUbi plasmid.\u003c/p\u003e\u003cp\u003eTo construct pPcUfcoCas9-SlESK1,2GAD3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a reaction mixture containing 6 \u0026micro;l of CutSmart buffer, 2 \u0026micro;l of the restriction enzyme Xmal, and 48 \u0026micro;l of the pPcUfcoCas9-SlESKGAD3 plasmid sample was prepared, and the reaction was performed under the same conditions. The target sites were amplified and inserted into the restricted pPcUfcoCas9-SlESKGAD3 plasmid (#5-#6).\u003c/p\u003e\u003cp\u003eFor plasmid purification, 150 \u0026micro;l of Milli-Q water was added to the restriction enzyme-treated plasmid sample on ice. Then, 200 \u0026micro;l of the lower layer of Tris-saturated phenol- chloroform-isoamyl alcohol was added, thoroughly mixed, and centrifuged at 15,300 \u0026times; g, 4\u0026deg;C for 5 min. The supernatant was collected, and 40 \u0026micro;l of 3M sodium acetate was added, followed by thorough mixing. Next, 400 \u0026micro;l of isopropanol was added, mixed, and centrifuged at 15,300 \u0026times; g at 4\u0026deg;C for 20 min. The supernatant was discarded, and the pellet was washed with 500 \u0026micro;l of 70% ethanol, inverted five times, and centrifuged again at 15,300 \u0026times; g at 4\u0026deg;C for 5 min.\u003c/p\u003e\u003cp\u003eKOD-Plus Neo (TOYOBO) was used to amplify the genome-editing constructs for each target site: \u003cem\u003eSlGAD3\u003c/em\u003e-target, \u003cem\u003eSlESK\u003c/em\u003e-target1 and \u003cem\u003eSlESK\u003c/em\u003e-target2 by PCR. The reaction composition is listed Supplementary Table\u0026nbsp;1 (#1-#6), and the PCR conditions were allowed according to the manufacturer\u0026rsquo;s instructions. PCR products were extracted from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN).\u003c/p\u003e\u003cp\u003eThe ligation reaction was performed using the In-Fusion Snap Assembly Master Mix (Takara Bio). The reaction tube was kept on ice, and 25 \u0026micro;l of Stellar Competent Cells (Takara Bio) were added. Heat shock was applied at 42\u0026deg;C for 45 s to transform the plasmid into \u003cem\u003eE. coli\u003c/em\u003e. Subsequently, 100 \u0026micro;l of SOC medium was added, and the culture was incubated with shaking at 180 rpm at 37\u0026deg;C for 60 min. The mixture was then spread on LB plates containing 50 \u0026micro;g/ml kanamycin and cultured at 37\u0026deg;C overnight. The desired vector was confirmed by sequencing the plasmid samples verify the correct insertion.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTomato transformation using the Agrobacterium method\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTomato transformation was performed following the high-efficiency transformation protocol (described by Sun et al., 2006). Both the prepared vector plasmid and the Super-Agrobacterium plasmid pBBRacdSgadTAmp (Nonaka et al., 2019) were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV2260. After infection of \u003cem\u003eAgrobacterium\u003c/em\u003e into tomato cotyledons, plants were regenerated as described previously (Sun et al., 2006).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of genomic DNA and amplification of target DNA regions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAbout 5 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e section of a true leaf from each plant was placed in a 1.5 ml tube and frozen with liquid nitrogen. Next, 100 \u0026micro;l of Buffer A (1 M Tris-HCl [pH 9.5] 30ml, KCl 23 g, 0.5 M EDTA 6 ml, distilled water to a final volume of 300 ml) was added, and the leaf section was homogenized using a hand pestle. The tube was then incubated at 95\u0026deg;C for 5 min. After incubation, the tube was cooled on ice and centrifuged at 17,600 \u0026times; g 4\u0026deg;C for 5 min. The resulting supernatant was transferred to a new tube.\u003c/p\u003e\u003cp\u003eKOD-FX Neo (TOYOBO) was used for the PCR amplification of the target DNA regions. The primers used for MultiNA sample preparation were as follows: \u003cem\u003eSlGAD3-multiF\u003c/em\u003e and \u003cem\u003eSlGAD3-multiR\u003c/em\u003e for the \u003cem\u003eSlGAD3\u003c/em\u003e target region, \u003cem\u003eESK1-target2-multinaF\u003c/em\u003e and \u003cem\u003eESK1-target2-multinaR\u003c/em\u003e for the \u003cem\u003eESK1\u003c/em\u003e target region, \u003cem\u003eESK2-target2-multinaF\u003c/em\u003e and \u003cem\u003eESK2-target2-multinaR\u003c/em\u003e for the \u003cem\u003eESK2\u003c/em\u003e target region, and \u003cem\u003eESK3-target2-multinaF\u003c/em\u003e and \u003cem\u003eESK3-target2-multinaR\u003c/em\u003e for the \u003cem\u003eESK3\u003c/em\u003e target region (Supplementary Table\u0026nbsp;2, #1-#7). After PCR amplification, the sizes of the PCR products were confirmed by agarose gel electrophoresis.\u003c/p\u003e\u003cp\u003eThe MultiNA system (SHIMADZU) was used to identify candidate individuals with genome-editing-induced mutations. First, 4 \u0026micro;l of the PCR products from the target gene regions were incubated at 95\u0026deg;C for 5 minutes and allowed to anneal at room temperature. Size ladder preparation was performed according to the MultiNA operating protocol. When a different band pattern was detected by the MultiNA system, the PCR products were cloned into the pGEM-T \u003cem\u003eEasy\u003c/em\u003e vector (Promega) and transformed into \u003cem\u003eE. coli\u003c/em\u003e. After colony formation, PCR was performed using the primers M13-47 and RV-P (Supplementary Table\u0026nbsp;2, #8).\u003c/p\u003e\u003cp\u003eThe resulting PCR product was treated with Illustra ExoProStar (Cytiva) according to the manufacturer\u0026rsquo;s instructions. Finally, the samples were sequenced using the primer M13-47.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCultivation of T\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eand T\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003egenerations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA 1% hydrochloric acid solution was poured into a beaker, fully submerging the seeds extracted from fully ripe red tomatoes. The seeds were stirred in the solution for 15 min. They were then thoroughly rinsed with water and dried.\u003c/p\u003e\u003cp\u003eSeeds collected from individuals with confirmed mutations, along with WT seeds for comparison, were directly sown on rock wool. After root establishment, genomic DNA was extracted from the leaves of the plants to confirm the inheritance of mutations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAgrobacterium persistence test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eApproximately 5 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e segments of plant leaves were placed in a sterile tube and homogenized using a pestle in 200 \u0026micro;l of sterile water. The homogenized solution was briefly centrifuged for approximately 10 s. Then, 50 \u0026micro;l of the supernatant was streaked onto AB medium (K₂HPO₄ 600 mg, NaH₂PO₄ 200 mg, NH₄Cl 200 mg, KCl 30 mg, CaCl₂\u0026middot;2H₂O 20 ml, FeSO₄\u0026middot;7H₂O (2.5 mg/ml) 20 ml, Agarose 3 g, 5% Glucose 20 ml, MgSO₄\u0026middot;7H₂O (1M) 240 \u0026micro;l, Kanamycin (50mg/ml) 200 \u0026micro;l, distilled water 200 ml), and incubated at 28\u0026deg;C for 5 days. As controls, streaking and culturing were also performed on the same medium using \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV2260 harboring the plasmid pPcUfcoCas9-SlESKGAD3 as a positive control and sterile water as a negative control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of exogenous gene persistence and off-target mutations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAn exogenous gene residual test was performed using KOD-FX Neo (TOYOBO). The primers listed in Supplementary Table\u0026nbsp;2 (#9-#19) were used to amplify the entire plasmid region of \u003cem\u003epPcUfcocas9-SlESKGAD3\u003c/em\u003e. Additionally, PCR was conducted under identical conditions using pPcUfcoCas9-SlESKGAD3 as a positive control and genomic DNA extracted from WT as a negative control.\u003c/p\u003e\u003cp\u003eOff-target mutations were identified using Cas-OFFinder \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The following parameters were applied: PAM type, 5'-NGG-3'; target-genome, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (SL4.0); maximum mismatch number, 2; DNA bulge size, 1; RNA bulge size, 1. Potential off-target mutation sites located at the target loci were selected. Among these, only sites where gene exons were registered in the \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (SL4.0) genome database were chosen for confirmation. To amplify candidate off-target mutation sites, PCR was performed using KOD-FX Neo (TOYOBO) with primers listed in Supplementary Table\u0026nbsp;2 (#20-#28). After amplification, the PCR products were sequenced for mutation verfication.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFruit measurement and analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFruit size and weight\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFruit size was determined by measuring the maximum diameter of the fruit along the equatorial plane using a vernier caliper. Fruit weight was measured using an electronic balance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBrix Measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Brix value was measured using a digital refractometer (AS ONE).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGABA content measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGABA content was measured using the GABA Miel kit (Enzyme Sensor Co., Ltd.) according to the manufacturer\u0026rsquo;s instructions. Briefly, distilled water equivalent to nine times the fruit\u0026rsquo;s weight was added to the fruit sample, which was then crushed using a mortar. The resulting mixture was filtered, and 50\u0026micro;l of the filtrate, along with the reaction standard solution, was placed in a measurement cell at room temperature. Subsequently, 500\u0026micro;l of Solution A was added, and the reaction was allowed to proceed for 10 min. Then, 500 \u0026micro;l of Solution B was added, and the mixture was incubated for another 10 min. The GABA content (mg/100 g fresh weight) was determined using an LED colorimeter (445 nm) calibrated with distilled water and the reaction standard solution.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlant height measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeeds were sown and seedlings were grown in the cultivation room until they reached approximately 40cm in height. The plants were then transferred to a greenhouse, where plant height was measured weekly. Measurements were taken from the aerial part of the plant.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using R software (R Core Team, 2023). The Kruskal-Wallis test was used to compare three or more groups with different sample sizes. The Wilcoxon signed-rank test was applied to account for data asymmetry and other non-parametric characteristics, allowing for pairwise comparisons. Additionally, the Mann-Whitney U test was performed, with correction for multiple comparisons using the Holm method.\u003c/p\u003e\u003cp\u003eFor statistical analysis of plant height, the Wilcoxon signed-rank test (equivalent to the Mann-Whitney U test) was used because homoscedasticity could not be assumed, as evaluated using the F-test and Levene's test. Plant growth was calculated by subtracting the previous week's height from the current week's height, and cumulative growth values were compared.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMDA measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo measure MDA levels, 4-week-old plants were used. Prior to drought stress treatment, 100 mg of fresh leaves were collected and immediately frozen in liquid nitrogen. Under drought stress conditions, both WT and EG lines (EG Line 1 and EG Line 2) were subjected to a 10-day water starvation period, after which leaves were sampled for the same manner as the non-stressed condition. The collected leaves were finely ground in 1.5 ml of a 10% (v/v) solution of trichloroacetic acid (TCA) solution. Following centrifugation at 17,600 \u0026times; g for 15 min, 1 ml of the resulting supernatant was mixed with 1 ml of a 0.6% (w/v) thiobarbituric acid (TBA) solution prepared in 10% TCA. The mixture was heated in boiling water for 20 min and then cooled to room temperature. Absorbance was measured at 450 nm, 532 nm, and 600 nm using a DU-800 spectrophotometer (Beckman Coulter). The MDA concentration was calculated using the following formula: MDA(\u0026micro;mol/L) = [ 6.45 \u0026times; (A\u003csub\u003e532\u003c/sub\u003e-A\u003csub\u003e600\u003c/sub\u003e) \u0026ndash; 0.56 \u0026times; A\u003csub\u003e450\u003c/sub\u003e].\u003c/p\u003e\u003cp\u003eThe MDA of the cold stress treatment also measured, after 3-days and 7-days cold stress treatment at a 4\u0026deg;C incubator.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEL Measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFresh leaves from each 4-week-old plant were rinsed thoroughly with Milli-Q water (MQ) and placed in tubes filled with MQ to ensure full submersion of the leaf tissue. The tubes were maintained at room temperature for 24 h, after which EL was measured as C\u003csub\u003e1\u003c/sub\u003e using an ion conductivity meter (Lutron).\u003c/p\u003e\u003cp\u003eNext, the samples were subjected to high-pressure sterilization at 121\u0026deg;C for 20 min and allowed to cool to room temperature. EL measurements were then recorded as C\u003csub\u003e2\u003c/sub\u003e. The percentage of EL was calculated using the following formula: EL (%) = (C\u003csub\u003e1\u003c/sub\u003e/C\u003csub\u003e2\u003c/sub\u003e) \u0026times; 100\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA extraction and purification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor RNA extraction, 100 mg of fresh leaves or red ripe fruits harvested from June to July were frozen in liquid nitrogen and finely ground. Total RNA was extracted using TRIzol (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. A total of 2\u0026micro;g of RNA was used for complementary DNA (cDNA) synthesis using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The primers \u003cem\u003eSlNCED1\u003c/em\u003e-F and \u003cem\u003eSlNCED1\u003c/em\u003e-R were used to amplify \u003cem\u003eSlNCED1\u003c/em\u003e for real-time PCR analysis (Supplementary Table\u0026nbsp;2, #29). Real-time PCR and relative abundance calculations were performed as described previously (Abellatif et al., 2002). The \u003cem\u003eSlEXPRESSED\u003c/em\u003e gene was used as an endogenous control for gene expression analysis (Choi et al., 2018 Plant Biotechnol).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA-seq analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA sequencing analysis was outsourced to Rhelixa Co., Ltd. Quality control (QC) score were assessed using FastQC software (Version 0.11.7; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Low-quality bases (Q\u0026thinsp;\u0026lt;\u0026thinsp;20) and adapter sequences were trimmed using Trimmomatic software (Version 0.38) with the following parameters: ILLUMINACLIP: path/to/adapter.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36. The trimmed reads were aligned to the reference genome using the RNA-seq aligner HISAT2 (Version 2.1.0). The HISAT2-generated .sam files were converted into .bam files using Samtools (Version 1.9). The resulting .bam files were used to estimate the abundance of uniquely mapped reads with featureCounts (Version 1.6.3). Raw read counts were normalized using transcripts per million (TPM). The samples were clustered using the Wald method based on the Euclidean distances of the normalized counts, employing stats (Version 3.6.1) and gplots (Version 3.0.1.1) R packages. Principal component analysis (PCA) was performed on the normalized counts, and each sample was projected onto two-dimensional plane based on the first and second PCA axes using the same R packages. Pearsonʼs correlation coefficients of the normalized counts were calculated to assess correlation between samples. Histograms and pair plots of the normalized counts were generated using the stats and gplots R packages. Heatmaps were created from Z-scores of the normalized counts using the same R packages. Raw read counts were further normalized using relative log expression normalization, and differential expression analysis was conducted with DESeq2 (Version 1.24.0). Differentially expressed genes (DEGs) were identified using the thresholds of |log\u003csub\u003e2\u003c/sub\u003e fold change(FC) | \u0026gt;1 and adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (and \u0026lt;\u0026thinsp;0.1), calculated using the Benjamini-Hochberg (BH) method for multiple testing correction.\u003c/p\u003e\u003cp\u003eGO enrichment analysis was performed using ShinyGO 0.80 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The \u003cem\u003eSlycopersicum\u003c/em\u003e_eg_gene Ensembl ID was employed, with the \u003cem\u003eSolanum lycopersicum\u003c/em\u003e SL3.0 gene annotation used as the database. The false discovery rate (FDR) cutoff was set to 0.05.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAscorbic acid measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTomato fruit (1.0 g) was homogenized using a mortar and pestle and mixed with 2.0 ml of 5% (w/v) metaphosphoric acid. After centrifugation at 12,000 \u0026times; g for 3 min, the supernatant was collected as a crude extract. The total ascorbic acid content was measured using the Ascorbic Acid Test Kit (Merck, Darmstadt, Germany) with the RQ Flex Plus 10 analyzer (Merck, Darmstadt, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicroscopy analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMicroscopy analysis of stems, flowers and fruits was performed by preparing tissue sections using a Vibrating-Blade Microtome VT1200 (Leica) and staining with Toluidine Blue for xylem and with Iodine potassium for pollen. Observations were conducted using a BX50 microscope (Olympus).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptome data was used\u0026nbsp;\u003cem\u003eSolanum lycopersicum\u003c/em\u003e SL3.0 gene annotation and is available in the NCBI database: the accession number is GCF_000188115.4. The sequence data used in this study can be found in the GenBank data libraries under the following accession numbers: \u003cem\u003eSlESK1\u0026nbsp;\u003c/em\u003e(Solyc03g096030), \u003cem\u003eSlESK2\u0026nbsp;\u003c/em\u003e(Solyc06g051350), \u003cem\u003eSlESK3\u003c/em\u003e (Solyc05g052540), \u003cem\u003eSlGAD3\u003c/em\u003e (Solyc01g005000). And the data presented in this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Ms. Yuriko Nagai, Ms. Yumiko Iguchi, Ms. Yuri Nemoto, Ms. Kazuko Ito, and Ms. Ayako Kobayashi at T-PIRC from the University of Tsukuba, Japan for their technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid (22H02295) and Program on Open Innovation Platform with Enterprise, Research Institute and Academia, Japan Science and Technology Agency (JST-OPERA, JPMJOP1851).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.M. and H.E. conceptualized the manuscript. S.C., T.I., M.K. and I.A. drafted the manuscript and prepared the figure. 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Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (10), 1473\u0026ndash;1475 (2014).\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SlESK1-3, SlGAD3, high-sugar, GABA-rich, high-vitamin C, drought tolerance","lastPublishedDoi":"10.21203/rs.3.rs-7220491/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7220491/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTomatoes are among the most widely cultivated crops worldwide, and their nutritional value has recently gained attention in the context of functional foods. In this study, we developed tomato varieties with elevated sugar content and high gamma-aminobutyric acid (GABA) levels using a genome editing technique to introduce mutations in \u003cem\u003eSlESK\u003c/em\u003e and \u003cem\u003eSlGAD3\u003c/em\u003e, aiming to enhance the nutritional value of tomatoes. Tomato plants possess three \u003cem\u003eESK\u003c/em\u003e homologs. We successfully generated tomatoes with mutations in all three \u003cem\u003eSlESK\u003c/em\u003e genes and a separate line with a mutation in \u003cem\u003eSlGAD3\u003c/em\u003e. Compared to the wild type (WT), which exhibited a sugar content of approximately 4.26% Brix, the mutants showed a significantly higher sugar content of approximately 7.9% Brix. GABA accumulation in the mutants was approximately five times higher than in WT. Additionally, the mutants contained 1.5 times more vitamin C than WT and demonstrated enhanced drought stress tolerance. Delayed plant growth and reduced yield were also observed in the genome-edited plants. Transcriptome analysis revealed that expression of genes, which are involved in vitamin C biosynthesis, the auxin signaling pathway, and ethylene biosynthesis, were altered in the genome-edited plants. These results may contribute to cultivating high-quality tomatoes, which are expected to be in high demand.\u003c/p\u003e","manuscriptTitle":"Enhancing tomato quality, high sugar content and GABA accumulation, with mutations in ESKs and GAD3 genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 14:30:28","doi":"10.21203/rs.3.rs-7220491/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-06T08:38:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T06:23:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-20T09:13:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73578953668502414525108516293763815954","date":"2025-09-15T08:01:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217492819093179328628494578689348794976","date":"2025-09-11T07:33:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-08T07:43:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-31T09:28:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T05:15:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-28T12:29:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-26T10:16:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d4e780e-2646-4541-9359-97e6af8f5392","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53056717,"name":"Biological sciences/Biotechnology"},{"id":53056718,"name":"Biological sciences/Genetics"},{"id":53056719,"name":"Biological sciences/Molecular biology"},{"id":53056720,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-12-08T16:00:04+00:00","versionOfRecord":{"articleIdentity":"rs-7220491","link":"https://doi.org/10.1038/s41598-025-28888-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-02 15:57:18","publishedOnDateReadable":"December 2nd, 2025"},"versionCreatedAt":"2025-08-13 14:30:28","video":"","vorDoi":"10.1038/s41598-025-28888-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-28888-5","workflowStages":[]},"version":"v1","identity":"rs-7220491","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7220491","identity":"rs-7220491","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}