Strawberry seeds (Fragaria vesca L.) were grown in a mixture of peat, vermiculite, and perlite (volume ratio of 3:2:1) by regular fertilization. Fruits were harvested in different developmental stages and were grouped as previously reported (Jia et al., 2013); small green (about 6 days after flowering (DAF), big green (13 DAF), degreening (18 DAF), white (22 DAF), initiated redness (26 DAF), partially red (29 DAF), and fully red (33DAF); and were stored at –80°C for qPCR analyses. The vegetative tissues including root, stem, leaf bud, first leaf, second leaf, and mature leaf, as well as reproductive tissues such as flower buds, initiated opening, partial opening, and fully opened flowers, were also collected and frozen on-site in liquid nitrogen, then stored at −80°C until use.

Total RNA was extracted from specific tissues using RNAprep Pure Kit (Takara, Beijing, China) (for polysaccharide- and polyphenolic-rich plants), following the user manual from the manufacturer. Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States) and 1% agarose gel electrophoresis were used to determine RNA quality and concentration. The extracted RNA was digested with DNase I, and PrimeScript™ RT Master Mix (Takara, Beijing, China) was used to synthesize cDNA.

The open reading frame of the cDNA was amplified with gene-specific primers (Supplementary Table 2) based on FvTST1 (Accession XM_004300064) available at NCBI. The whole cDNA of FvTST1 was then amplified with mix super fidelity polymerase (Vazyme Biotech Co., Ltd.), under thermal cycling conditions of 95°C denaturation for 5 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 2 min, and a final extension of 72°C for 10 min. A single band of expected size 2217 bp was purified from gel and sub cloned into pEASY vector (TransGen Biotech, Beijing, China).

In this study, qPCR was conducted for FvTSTs in different tissues of strawberry, and for validation of some genes between transgenic and non-transgenic lines (control). We designed the primers with the Primer Premier 5.0 software (Premier Biosoft, Palo Alto, CA, United States) (Supplementary Table 2). The 18S ribosomal RNA (18S rRNA) (accession number KF668233.1) was used for transcript level normalization. PrimeScriptTM RT Kit (Perfect Real Time) (Takara, Beijing, China) was used for the preparation of the qPCR reaction, and the CFX96 platform (Bio RAD, Shanghai, China)¹ was used to perform the reaction. The 2^–ΔΔCT method was used for the calculation of the transcript levels of the genes.

The Sugar transport function was elucidated in yeast mutant strain (EBY.VW4000) (Wieczorke et al., 1999) using lithium acetate transformation with pYES2.0 vector containing the FvTST1 gene or with empty vector (Gietz et al., 1992). FvTST1 was amplified using a pair of primers with restriction sites Sac I and Xba I at their 5′ ends (Supplementary Table 3). To get pYE2.0-FvTST1, the resulting DNA fragment was cleaved with Sac I and Xba I, and was connected into pYES2.0. The pYES2.0-FvTST1-transformed yeast strain (EBY.VW4000) and transformants were cultured and selected as reported before (Li et al., 2017). For complementation assay of sugar, yeast cells were inoculated in a liquid medium with 2% maltose as a carbon source, and were incubated overnight at 30°C to an optical density (OD₆₀₀) of 1. The yeast cells were then diluted serially with (10-, 100-, and 1,000-fold), and a 4-μl dilution was cultured in the medium containing yeast nitrogen base without uracil with 2% maltose, fructose, glucose, sucrose, and mannose as carbon sources. Wild-type yeast was used as positive control, while an empty vector and EBY.VW.4000 were used as negative controls. All the plates were placed at 30°C for 2 days and were documented.

The FvTST1 gene fused with GFP was cloned into the binary vector pBI121. The CaMV35s-FvTST1-GFP fusion construct, tonoplast marker CaMV35s- γTIP-mCherry (CD3-975), and plasma membrane marker CaMV35s-PIP2A-mCherry (CD3-1007) (Nelson et al., 2007) were verified by sequencing, and then transformed into Agrobacterium. The Agrobacterium was inoculated in an LB medium and incubated overnight at 28°C in a shaker at 200 rpm. The bacterial culture was collected and then suspended in a buffer containing MES; 10 mM (pH 5.6), MgCl₂ 10 mM, and acetosyringone 200 mM to an OD₆₀₀ of 1. The mixture was kept at room temperature for 1–3 h and was infiltrated into the lower surface of Nicotiana benthamiana leaves with a 1-ml syringe. After 3 days, the infiltrated leaves were put on water glass slides for microscopic examination. Fluorescence was detected in leaf cells using a TCS-SP8 laser-scanning confocal microscope (Leica, Mannheim, Germany). The fluorescence detection wavelengths of GFP were 488 and 500–530 nm, and the fluorescence detection wavelengths of mCherry were 514 and 560–620 nm.

The CDS of FvTST1 was sub-cloned into the binary vector pBI121 employing Xba I and Sac I. The promoter of the TOMATO PROLINE RICH PROTEIN (TPRP-F1) gene (X61395.1) (Salts et al., 1991) was then placed 5′ upstream of the FvTST1 CDS in pBI121-FvTST1 using a pair of one-step recombinant cloning primers (Supplementary Table 2) employing Hind III and Xba I to get the plant expression construct pBI121-P_TPRP–F1::FvTST1.

FvTST1 and its counterpart empty vector pBI121 were infiltrated into strawberry fruits according to a previous report (Jia et al., 2013). An LB liquid medium with antibiotics was used for the inoculation of Agrobacterium and incubated overnight at 28°C to an optical density (OD₆₀₀) of 1. The culture was collected and then suspended in the buffer (10 mM MgCl₂, 10 mMMes, pH5.6, and 200 mM acetosyringone) to an OD₆₀₀ = 0.8, and placed at room temperature for 2 h before infiltration into the fruit. Fruits of strawberry attached to the plant in degreening stage (18 DAF) were injected using a sterile 1-ml hypodermic syringe.

To generate the FvTST1 transgenic tomato, Agrobacterium-mediated leaf disk transformation of tomato [Solanum lycopersicum cv. Ailsa Craig+ (AC+)] was performed as reported previously (Sun et al., 2006). Cotyledons of 2-week-old seedlings were pre-cultured in an MS medium supplemented with 0.1% Gamborg’s vitamins, 1 μg/ml 6-BA, and 40 ng/ml of IAA for 3 days. The cotyledons were then immersed for 15 min in an Agrobacterium culture (OD600 = 0.1–0.2) with the liquid MS medium. The cotyledons were dried and then co-cultured for 2 days in an MS medium containing 0.1% Gamborg’s vitamins, 0.1 μg/ml Kinetin, 0.2 μg/ml 2, 4-D, and 1.5 μg/ml acetosyringone under dark conditions. After co-cultivation, the cotyledons were transformed into a regeneration and selection MS medium (0.1% Gamborg’s vitamin, 10 μg/ml 6-BA, 0.4 μg/ml IAA, 0.5 mg/ml carbenicillin, and 50 mg/ml kanamycin). After every 3 weeks, the medium was refreshed until shooting. Two-to-three-centimeter newly developed shoots were then transformed into a rooting MS medium (0.1% Gamborg’s vitamins, 4 μg/ml IAA, 0.5 mg/ml carbenicillin, and 50 mg/ml kanamycin) until true roots developed. The presence of FvTST1 in positive regenerants from kanamycin selection was verified by PCR and sequencing.

The T1 seeds from T0 transgenic tomato plants from each independent transgenic line were geminated in a 1/2 MS medium containing kanamycin, and the non-transgenic seeds were grown in a 1/2 MS medium without kanamycin and transferred to a growth chamber, which was set at 25°C/22°C with relative humidity of 65%, a photoperiod of 16 h and a photon flux of about 200 μmoles⋅m^–2⋅s^–1. Seven-day-old (4 DAG) transgenic and control seedlings were collected, frozen in liquid nitrogen, and stored at –80°C until use. The total RNA from independent transgenic and control lines was extracted. RNA-seq was performed with a commercial RNA-seq agent (OE Biotech, Shanghai, China), and with Trimmomatic; the raw data (raw reads) were analyzed (Bolger et al., 2014). To obtain the clean reads, poly-n and low-quality reads were removed. Clean reads were mapped to reference genomes of Solanum lycopersicum cultivar “Heinz 1706,” available at the Sol Genome network ITAG v2, using HISAT2 (Kim et al., 2015). Cufflinks were used for normalization of exonic reads (Trapnell et al., 2012), which were reported as FPKM, and the number of gene reads was counted using htseq-count (Anders and Huber, 2012; Anders et al., 2015). DESeq R package functions, estimate size factors, and nbinomTest were used to find DEGs (Anders and Huber, 2012). A p value < 0.05 and a fold change > 2 or < 0.5 have been set as the significant differential expression thresholds for the selection of induced or suppressed genes. The DEGs were analyzed for GO and KEGG pathway enrichment using the R package (Kanehisa et al., 2008), which was based on hypergeometric distribution.

The contents of Fru, Glu, and Suc, in the strawberry fruit, tomato seedlings, and fruits were determined using an LC-20A high-performance liquid chromatography system (Shimadzu Company, Kyoto, Japan) with a 5-μm XB-NH2 column (4.6 mm × 250 mm; Shanghai Welch Materials, China) and a RID20A detector. To develop a standard curve, authentic standards of different sugars were purchased from Sigma (St Louis, MO, United States). Soluble sugars, Fru, Glu, and Suc were extracted according to previous reports (Castonguay et al., 1995). Plant tissues weighing 0.5 g were ground with liquid nitrogen, and soluble sugars were extracted using 10 ml of distilled water at 100°C for 1 h. The samples were centrifuged at 13,000 g for 10 min, and the supernatant was transferred to a new 10-ml tube and brought to 10 ml with distilled water. A 1-ml sample was filtered before HPLC analysis through a 0.22-μm filter membrane. The mobile phase contained acetonitrile/water with a volume-to-volume ratio of 75:25 v/v, and flow rate was adjusted to 1 ml/min with an injection volume of 10 μl.

The IAA authentic internal standard (BCCC6104) was purchased from Sigma-Aldrich (St. Louis, MO, United States. IAA was extracted following previous reports (Pan et al., 2008). Seedlings weighing 0.3 g were ground to fine powder with liquid nitrogen. Extraction buffer (isopropanol–HCl, 2: 0.002 v/v) with 2 ml was added to the powder and incubated for 30 min at 4°C and centrifuged at 13,000 g for 5 min, and repeated twice. After centrifugation, the lower organic phase was transferred to a 10-mL tube and evaporated in a constant stream of nitrogen. The samples were kept in the dark before being resuspended in a solution of 150 μl methanol and 0.1% formic acid. The samples were filtered with a 0.45-μm microfilter, and the reversed phase column (C18 ZORBAX 300SB, 3 μm; 4.6 mm × 9 mm × 150 mm; Agilent, Sta. Clara, CA, United States) was used for sample injection. The mobile phase contained methanol as solvent A, water with 0.1% formic acid was used as solvent B, and column temperature was adjusted to 30 °C. The product was purified using a solvent gradient, which began with volume adjustment to 20% methanol for 2 min, and was linearly increased to 80% methanol for 14 min, after equilibration for 5 min, and then reverted to the volume of 20% methanol for 0.1 min. A hybrid triple quadruple mass spectrometer (SCIEX 6500 QTrap; Applied Biosystems, Foster City, CA, United States) was employed for quantification analysis. System voltage was set to 4.5 kV. Source temperature was set to 500°C. Nebulizer and curtain drying gas pressure was adjusted to 75.15 and 65 psi, respectively.

The net photosynthesis rates of cotyledons of (4 DAG) seedlings were measured with CIRAS-3 (PP Systems, Amesbury, MA, United States). The analyzers act as absorption meters to measure infrared absorption. The optical bench is temperature-controlled and pressure-compensated to provide accurate CO₂ and H₂O measurement. Net photosynthesis (A) was determined from the difference between the CO₂ concentration entering (Cin) and exiting (Cout) the cuvette. IRGA CO₂ reading was corrected for water vapor, temperature, and atmospheric pressure. Since humidity dilutes the air leaving the cuvette (Cout), it was compensated using the following equation: net photosynthesis (A) = (Cin × W) – [Cout × (W+E)], where (A) is net photosynthesis, (W) is mass flow of air per unit leaf area into the cuvette, and (E) is transpiration rate.

The experiment was conducted using a completely random method. For statistical analysis, the SPSS software version 19.00 (SPSS, Chicago, IL, United States) was used. The mean values of transgenic lines and wild type lines were compared by independent t-test or one-way ANOVA test. The data was expressed as the mean ± standard deviation of three replicates.

Previously, four putative TST genes, FvTST1-4, were identified in the strawberry genome (Jiu et al., 2018; Liu et al., 2020). The phylogenetic tree revealed that FvTSTs share significant homology with ArabidopsisAtTST1, -2, -3 (Supplementary Figure 1). To understand the possible role of FvTSTs, the expression profile of these genes was analyzed in different tissues by qRT-PCR analysis (Figure 1). Expression analyses of FvTSTs indicated that they were differentially expressed in all the examined tissues in different developmental stages. Among them, FvTST1 was strongly expressed in the second leaf; however, it was weakly expressed or unnoticeable in other tissues, including young fruits, but its level was significantly increased in fully red fruits. Moreover, FvTST2 and FvTST3 had almost the same weak expression tendencies during plant growth and fruit development. On the contrary, FvTST4 expression was detected in the root, stem, and leaf, and was weakly expressed in flower buds, partially open flowers, fully open flower, and small green fruits, while higher expression level was detected in partially red fruits, and then decreased in fully red fruits. Overall, the results highlight that FvTST1 is a highly expressed TST gene during strawberry fruit development, and suggest that it might contribute to sugar accumulation in fruits.

The expression profile of genes is used as a useful tool for the selection of candidate genes; however, further evaluation is necessary. In a widely used heterologous system, the transport function of many sugar transporters has been characterized (Lemoine, 2000). The hxt-null strain EBY.VW4000 (Wieczorke et al., 1999; Conde et al., 2007) has been widely used for studying plant sugar transporters. The knockout of hexose transporters in this strain prevents it from uptaking hexoses, thus allowing the assessment of the gene introduced. FvTST1 was expressed in EBY.VW4000 hxt-null yeast mutant cells. Selection of the transformant was carried out in the medium containing maltose without uracil as a carbon source, and was confirmed by PCR (Supplementary Figure 2). The pYES2.0–FvTST1 transformant in the yeast strain was then plated in a medium containing 2% maltose, fructose, glucose, sucrose, and mannose as key carbon sources. The positive control was wild-type yeast, and the negative control was an empty vector and EBY.VW.4000. The serial dilution (10-, 100-, and 1,000-fold) of yeast strain demonstrated that the EBY.VW4000 transformant with pYES2.0–FvTST1 and wild-type yeast can grow in all the media including the sucrose-containing medium, and that the empty vector and EBY.VW.4000 can only grow in the presence of maltose (Figure 2). This result confirms that FvTST1 is a functional tonoplast sugar transporter.

For subcellular localization of FvTST1, we established a construct encoding FvTST1-GFP driven by a 35S promoter, and was co-expressed wit hCaMV35s-γTIP-mCherry (tonoplast marker, CD3-975) and CaMV35s-PIP2A-mCherry (plasma membrane marker, CD3-1007) (Nelson et al., 2007), in Nicotiana benthamiana leaves. Confocal microscopy images show that the GFP signal from FvTST1 overlapped with the mCherry signal of the tonoplast marker, and a merged yellow signal was observed in whole cells, whereas in zoom-in cells, two GFP lines and two mCherry lines from the tonoplast marker were observed and indicated by white arrows in zoom-in cells (Figure 3A). We further analyzed the co-localization of FvTST1 with the plasma membrane marker. Two linear GFP signals (white arrows), a single mCherry signal, and a weak merged yellow signal were observed in the zoom-in cells of the plasma membrane marker (Figure 3B). These data show that FvTST1 is mainly located in the tonoplast.

To elucidate the possible role of FvTST1 during fruit development, the construct with FvTST1 and pBI121 empty vectors (control) were injected in the degreening stage (18 DAF) of strawberry fruits, while the fruits were still attached to the plants. Seven days after injection, we observed that the fruits injected with FvTST1 turned red, and that the fruits with the pBI121 empty vector did not turn red (Figure 4A). We analyzed the expression level of FvTST1 in these two groups and found that FvTST1 exhibited higher expression in the FvTST1-injected fruits than in the control (Figure 4B). The sugar concentration in these two groups was assessed, and the content of Fru, Glu, and Suc was significantly induced in the FvTST1-overexpressed fruits than in the control (Figure 4C). These findings demonstrated that FvTST1 overexpression in the strawberry fruits enhanced fruit ripening and sugar accumulation.

To further elucidate the role of FvTST1 in plant growth and fruit development, the FvTST1 overexpression construct (Figure 5A) was overexpressed in the tomato plant. Transgene presence in the transgenic plants was analyzed by PCR (Figure 5B), and a high level of transgene transcript was detected in all the independent transgenic lines, but not detected in the non-transgenic (control) line (Figure 5C). In particular, the transgenic plant shows enhanced vegetative growth (Figures 5D,E), flowers, and bears fruits (Figures 5F,G) significantly earlier than control. Transgenic plant height was significantly higher than that of the control (Figure 5H). Moreover, to understand the phenotypic alteration between transgenic plants and control, the seedlings were characterized further. The root and hypocotyl length of “transgenic seedlings” was significantly longer (p < 0.05) than that of control (Figures 5I,J). Moreover, higher net photosynthetic rates were observed in some of the transgenic lines (T4, T5, and T6) than in the control (Figure 5K). Transgenic seeds were heavier than the control (Figure 5L), and they germinated 2 days earlier than the control. FvTST1 transcript level was significantly increased in the root, hypocotyl, and cotyledons of the transgenic plants, but it was not detected in the control plants (Figure 5M).

Soluble sugars (Fru, Glu, and Suc) were measured in seedlings and mature fruits of the transgenic and control plants (Figure 6). Our results showed that Suc content was significantly higher in the transgenic seedlings than in the controls, but that there were no obvious changes in Fru and Glu content (Figure 6A). However, when the transgenic plant mature fruits were compared to the controls, the Fru and Glu contents were significantly increased, but no changes in Suc content were observed (Figure 6B).

RNA-seq analysis was performed to reveal the transcriptional changes in the transgenic plants. Clean reads ranged from 46.35 to 49.29 M, total mapped reads were 97%, and Q30 of the raw bases was 94% (Supplementary Table 1), suggesting that the transcriptome data were reliable. A total of 25,934 genes were expressed in the 7-day old (4 DAG) tomato seedlings. Among the DEGs, using fold change > 2 and a Q-value < 0.05, a total of 1,142 genes were up-regulated and 746 genes were down-regulated (Supplementary Figure 3A). According to GO analysis, the down-regulated genes had 514 enriched (q < 0.05) GO terms, whereas the up-regulated genes had 427 enriched (q < 0.05) GO terms.

Gene ontology (GO) was further studied, and the top 10 GO terms of biological process (BP), cellular component (CC), and molecular function (MF) for up-regulated and down-regulated genes were analyzed (Supplementary Figures 3B,C). In the up-regulated biological process, “the auxin signaling pathway,” “auxin polar transport,” and “regulation of growth” were highly enriched terms (Supplementary Figure 3B). Apoplast, membrane system, and cell wall were enhanced, which was consistent with enhanced seedling growth (Supplementary Figure 3B). PSI was the most affected pathway in BP, CC, and MF (Supplementary Figure 3C) in the down-regulated genes. Low-affinity nitrate transmembrane transport was found to be down-regulated (Supplementary Figure 3B), suggesting alteration of nitrate/carbon balance. The genes selected during this study are available at https://solgenomics.net/ while the name and accession numbers are provided (Supplementary Table S3).

Phytochrome-interacting factors (PIFs) are a group of negative regulators of photomorphogenesis (Toledo-Ortiz et al., 2010; Kim k. et al., 2016) and are induced by sucrose (Lilley et al., 2012). In this study PIF1, -3, and -4 were significantly up-regulated, and were confirmed by qPCR analysis (Figure 7A). PHYTOCHROME B (PHYB), which is suppressed by PIFs (Jang et al., 2010), was found down-regulated with a fold change of 0.62. Many genes that were directly induced by PIFs were significantly enhanced in this study, such as BZR1, GA2ox2, TAA1, and IAA29 (Toledo-Ortiz et al., 2010), and their expression was also confirmed by qPCR (Figure 7B). PSY, another target of PIF1 (Toledo-Ortiz et al., 2010), was also found to be down-regulated. PIFs have the ability to bind to G-box, E-box, and ACE cis-elements (Zhang et al., 2013; Kim J. et al., 2016), and the existence of G-box, E-box, and ACE was checked across the 2kb segment upstream of the translation start site (ATG) for GA2ox2, BZR1, and TAA1. The results showed that 5, 8, and 18 cis-elements existed in the promoters of TAA1, BZR1, and GA2ox2, respectively (Supplementary Figure 4A). Moreover, sequences of the 2kb segment upstream of the ATG of all 1,888 DEGs from the tomato genome were examined for cis-element analysis. The results revealed that most of the promoters of the examined DEGs contained E-boxes and ACE. The promoters of the genes containing G-boxes accounted for about 1/4 to 1/3 of the total number of the affected genes (Supplementary Figure 4B).

In addition, PIFs and auxin both regulate EXPs and XTHs, which are important for hypocotyl elongation and root growth. In this study, 6 EXPs and 10 XTHs were up-regulated, except XTH8, XTH32, and EXPA1 (Figures 7C,D). Besides, the transcription of the gene SAUR19 was up-regulated (Figure 7E), which has been reported previously for hypocotyl growth stimulation indirectly regulated by PIFs (Leivar and Monte, 2014).

Interestingly, a total of 54 DEGs were obtained in the “auxin-activated signaling pathway,” with 45 up-regulated and 9 down-regulated, including SAUR9/11/20/21/24/26/32/50/63/64, and WAG2. Regarding “Auxin polar transport,” which is vital for root growth (Sabatini et al., 1999; Grieneisen et al., 2007; Gutierrez et al., 2012), linked DEGs were all up-regulated (Figure 8A). Consistently, a significantly higher auxin concentration was found in the 7-day old (4DAG) transgenic seedlings compared to the control (Figure 8B). It was worth noting that 33 DEGs out of 45 “growth regulation” genes were members of the auxin signaling pathway, indicating the pivotal role of auxin in regulating transgenic plant growth. Expression alterations of SAUR19/21/24/63/64, WAG2, were further confirmed by qPCR analysis (Figure 8C). These findings strongly suggest that PIF1, -3, -4 and/or auxin signaling pathways are involved in enhancement of root growth and hypocotyl elongation.

There are 4 TSTs in the strawberry genome. In particular, FvTST1 shares significant homology with previously identified sugar transporters such as AtTST1 (Wormit et al., 2006), VvTST1 (Afoufa-Bastien et al., 2010), and MdTST1 (Wei et al., 2014). The expression levels of these genes have been analyzed in different tissues (Figure 1). FvTST1 was highly expressed during strawberry fruit development, and its expression pattern was consistent with fruit development and sugar accumulation, warranting speculations that FvTST1 may play a role in strawberry fruit sugar accumulation. These assertions coincide with previous findings (Wei et al., 2014; Cheng J. et al., 2018).

In addition, to identify the function of FvTST1 as a sugar transporter, we overexpressed FvTST1 in hexose-deficient yeast (EBY.VW4000) (Wieczorke et al., 1999). The yeast expressing gene FvTST1 can grow in the medium containing, fructose, glucose, sucrose, and mannose, but the hexose-deficient yeast (EBY.VW4000) and empty vector did not grow (Figure 2), which revealed that FvTST1 is a functional sugar transporter. Our results are consistent with previous findings that were observed for the plasma membrane transporter and tonoplast transporter (Vignault et al., 2005; Cheng R. et al., 2018). In our study, FvTST1 transported sucrose, and our findings are concomitant with that of ArabidopsisAtTMT1,-2-transfected cells (Wormit et al., 2006; Schulz et al., 2011), which are located into the tonoplast, and have hexose and sucrose import activity. Furthermore, we have analyzed the subcellular localization of FvTST1 in N. benthamiana leaves (Figure 3), indicating that FvTST1-GFP fluorescence is mainly observed in the tonoplast, which coincides with previous studies such as on AtTST1 (Wormit et al., 2006) and CmTST2 (Cheng J. et al., 2018).

In addition, to elucidate the physiological function and substrate specificity of FvTST1 in vivo, we transiently overexpressed FvTST1 in the strawberry fruit. The fruits overexpressing FvTST1 gene matured faster, and exhibited higher sugar content than the control (Figure 4), confirming the function of FvTST1 as a sugar transporter, and promoting strawberry fruit maturation. In our study, the gene FvTST1-overexpressed fruits showed early maturity, and this alteration could be related to the involvement of sucrose signaling, because in previous studies it was documented that the content of sucrose in the cytosol has signaling functions regulating strawberry fruit maturation (Jia et al., 2013). The overexpression of FaSUT1, a sucrose transporter in the plasma membrane of strawberry, enhanced sucrose content and fruit ripening, but the down regulation of this gene reduced sucrose content and ripening of fruit (Jia et al., 2013).

To assess the role of FvTST1 in plant and fruit development, we observed the phenotypic differences between FvTST1-overexpressing tomato plants and control. The overexpression of FvTST1 exhibited visible phenotypes, such as fast vegetative growth, early flowering, and enhanced fruit ripening (Figure 5), as those observed for tomato plants overexpressing PbTMT4 (Cheng R. et al., 2018). Particularly, in FvTST1-overexpressed transgenic seedlings and fruits, the content of sucrose and hexose were significantly elevated (Figure 6), respectively. It has been reported that the overexpression of MdTST1 and MdTST2 can increase soluble sugar content in apple callus, and that MdTST1 and MdTST2 RNAi suppression significantly reduced soluble sugars (Zhu et al., 2021). Previous studies have shown that the overexpression of AtTST1, -2-altered sugar in subcellular compartments enhanced seed biomass and early plant development (Wingenter et al., 2010; Schulz et al., 2011). However, in Arabidopsis, the overexpression of AtTST1 has no effect on the abundance of soluble sugars in transgenic seedlings (Wingenter et al., 2010). Nevertheless, in this study, relative to the non-transgenic tomato seedlings, the transgenic tomato seedlings exhibited a higher level of sucrose but not hexose (Fru or Glu). These findings suggest that TST genes are conserved in many plant species, and that they are likely to have functional differences in different plants species and e1ven distinct tissues.

To understand further the underlying mechanism of FvTST1 during plant growth and development, RNA sequencing analysis was performed. Auxin activation pathway genes were found to be significantly enriched, indicating that the auxin signaling pathway is essential for seedling development. Previous research studies have indicated that Suc can induce seedling growth through auxin-mediated signaling pathways (Mishra et al., 2009; Lilley et al., 2012). The sucrose-induced auxin signaling pathway involves IAA29, YUC8 and the transcription factor phytochrome-interacting factors (PIFs) (Lilley et al., 2012). In our study, FvTST1 overexpression enhanced sucrose content in transgenic seedlings and found that PIF1, –3, and –4, were significantly up regulated, which coincides with previous findings (Lilley et al., 2012). In addition to many up-regulated genes, BZR1 and GA2ox2, TAA1, and IAA29 regulated by PIFs are crucial for the BR, GA, and auxin signaling pathways (Leivar and Monte, 2014), and sucrose-induced seedling growth (Lilley et al., 2012).