Peach fruits (Prunus persica Batsch cv. Xiatian) at 80% maturation were collected from a plantation in Hefei, China. Uniformly sized peaches, with absence of physical injury or infection, were separated into two groups. Each group was randomly assigned to three small groups. A total of 190 peaches were used for each small group.

After air-drying for approximately 1 h, all of the peaches were preserved at 4 ± 1°C with 80%–90% relative humidity for 35 days. For each small group, samples were taken at 7-day intervals. A total of 20 fruits were picked out to assess CI development and firmness. In addition, 10 fruits were picked out for the biochemical analysis, which were then stored at −80°C.

Cold-stored peaches were taken out of storage and then placed at 20°C for 3 days. The CI severity in each fruit was scored on the basis of the internal browning of peach fruits (Jia et al., 2023) and the external browning of tomato fruits (Acosta-Ramírez et al., 2024), as follows: 0, none; 1, <5%; 2, 6%–25%; 3, 26%–50%, and 4, >50%. The CI index was calculated as follows: CI index = Σ(CI scale × number of fruit within this scale)/(5 × total number of fruit).

Firmness was determined using a firmness detector. The diameters of the probe were set as 7.5 mm for peaches and 5.0 mm for tomatoes.

Liquid nitrogen-powdered fruit tissue was added to 10 mM Tris–HCl (pH 7.2) and then centrifuged at 11,000 × g at 4°C for 25 min. The obtained supernatant was diluted with 10 mM Tris–HCl (pH 7.2). Subsequently, the resulted samples were mixed with 2 mM 2′,7′-dichlorofluorescein diacetate. The reaction was carried out in the dark for 15 min. A fluorometer was used to determine the fluorescence values according to the method of Jambunathan (2010). The amount of ROS was described in arbitrary units per milligram fresh weight (FW).

Powdered fruit tissue was homogenized with 10% trichloroacetic acid. After centrifugation at 10,000 × g for 20 min, the obtained supernatant was added to 0.6% thiobarbituric acid in 10% trichloroacetic acid. The malondialdehyde (MDA) concentration was determined according to Zhang et al. (2013). The MDA content is presented in millimoles per kilogram FW.

A cork borer was used to obtain fruit disks with a thickness of 3 mm of equatorial mesocarp tissue. These disks were immersed in distilled water. Dried samples were added to 0.3 M mannitol. After 3 h, electrolyte leakage was assessed as reported by Zhang et al. (2013). The relative electrolyte leakage was calculated as a percentage of the total conductivity.

Powdered fruit samples were extracted in 0.1 M phosphate buffer (pH 6.8) and then subjected to centrifugation at 11,000 × g for 20 min at 4°C. The supernatant was used to assay the lipoxygenase (LOX) activity using the method of Liu et al. (2011). This measure is presented in units per gram FW.

Powdered fruit samples were added to 0.1 M acetic acid buffer (pH 5.6) and then centrifuged at 11,000 × g for 20 min at 4°C. The obtained supernatant was used to conduct assessment of the phospholipase D (PLD) activity based on the method of Liu et al. (2011). This measure is presented in units per gram FW.

A TRIzol kit (Tiangen Biotech, Beijing, China) was used to collect the total RNA. Subsequently, a PrimeScript™ RT Reagent Kit (Takara Bio Inc., Beijing, China) was used to obtain the first-strand cDNA. For quantitative real-time polymerase chain reaction (qRT-PCR) analysis, the primers for PpTrxh9, PpSKα, and β-actin in peach fruits are presented in Supplementary Table S1 , while those for SlTrxh9 and β-actin in tomato fruits are presented in Supplementary Table S2 . The qRT-PCR procedures were based on Jia et al. (2022). The relative expression of the target genes was determined using the 2^−ΔΔCt method.

The Matchmaker™ Gold Y2H System (Clontech, Mountain View, CA, USA) was utilized to perform the yeast two-hybrid (Y2H) assay. The coding sequences (CDSs) of PpSKα were fused with pGADT7 at the restriction sites SacI and BamHI. The CDSs of PpTrxh9 were fused with pGBKT7 at the restriction sites BamHI and PstI. Yeast cells were co-introduced with different pairs of empty or reconstructed vectors. All of the co-transformants were cultured at 30°C on a two-deficiency selection medium (SD/−Leu−Trp) and a four-deficiency selection medium (SD/−Leu−Trp−Ade−His). Subsequently, the blue color was observed after X-α-galactosidase (Gal) was added to the four-deficiency selection medium. After 3 days, the yeast cultures co-transformed with different combinations of vectors were photographed.

The CDSs of PpTrxh9 and PpSKα were inserted into pCAM35s–green fluorescent protein (GFP) at the restriction sites KpnI and XbaI, respectively. The empty pCAM35s–GFP, pCAM35s–GFP–PpTrxh9, and pCAM35s–GFP–PpSKα vectors were electroporated into Agrobacterium tumefaciens GV3101. The suspensions of A. tumefaciens (OD₆₀₀ = 0.6) containing the empty and reconstructed vectors were inoculated into the abaxial side of Nicotiana benthamiana leaves via 1-ml needleless syringes as reported in Stephan et al. (2011). mCherry was utilized to mark the cytoplasm localization. After infusion for 3 days, a confocal laser scanning microscope (Nikon A1-SHS, Tokyo, Japan) was used to observe the subcellular localizations of the target proteins.

The CDSs of PpTrxh9 and PpSKα were separately ligated into the pSPYNE and pSPYCE vectors at the restriction sites XbaI and BamHI, respectively. A. tumefaciens EHA105 was introduced with the empty or the reconstructed vectors. The various pairs of vectors were inoculated into tobacco leaves. mCherry, which was used in the subcellular localization test, was also utilized in this experiment. After cultivation of the tobacco plants for 3 days, all images of the signals in infected leaves were taken using a confocal laser scanning microscope.

The CDSs of PpTrxh9 were ligated into the pCAMBIA1300–35S–Myc vector at the restriction sites BamHI and SalI. The CDSs of PpSKα were ligated into the pCAMBIA1300–35S–3×Flag vector at the restriction sites BamHI and SalI. Tobacco leaves were injected with A. tumefaciens GV3101 harboring empty or recombinant plasmids. After 48 h, the total protein in liquid nitrogen-powdered tobacco leaves was obtained using a lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM DTT, 1 mM PMSF, 10 mM EDTA, 0.1% Triton X-100, and 1× protease inhibitor cocktail) and then incubated with anti-Flag magnetic beads for 12 h at 4°C. Western blot analysis was conducted to detect the target proteins using anti-Flag and anti-Myc antibodies.

The CDSs of PpSKα were inserted into the pGEX-4T-1 vector carrying the glutathione S-transferase (GST) tag at the restriction sites EcoRI and BamHI. The CDSs of PpTrxh9 were inserted into the pET-28a vector carrying the His tag at the restriction sites XhoI and BamHI. GST, GST–PpSKα, and His–PpTrxh9 were introduced into the Escherichia coli strain BL21. After purification, His–PpTrxh9 was incubated with GST or with GST–PpSKα. The separation of proteins was carried out with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The target proteins were analyzed using Western blot detection with anti-GST and anti-His antibodies.

The CDSs of PpSKα were inserted into the pBI121 vector at the restriction sites XbaI and SmaI. A. tumefaciens GV3101 was introduced with the empty pBI121 and the resulting pBI121–PpSKα vectors thereafter. The generation of tomato plants (Solanum lycopersicum L. cv. MicroTom) overexpressing PpSKα was conducted via infection with A. tumefaciens transformants according to the method of Hao et al. (2015). Quantitative PCR (qPCR) assay was performed to measure the PpSKα expression in the wild-type (WT) and PpSKα-overexpressed (OE) plants ( Supplementary Figure S1 ). The WT and the derived T3 generation lines were cultivated in a phytotron with a 16-h light (25°C)/8-h dark (20°C) cycle. Tomato fruits at the 80% plump stage were harvested. Subsequently, the 28-day cold storage and the collection of fruit samples proceeded as described in “Plant materials and postharvest applications.”

The tobacco rattle virus (TRV)-based vector at the restriction sites KpnI and XhoI was utilized to generate the virus-induced gene silencing (VIGS) construct of PpSKα. Subsequently, the empty and pTRV–PpSKα vectors were mobilized into A. tumefaciens GV3101. Suspensions of A. tumefaciens (OD₆₀₀ = 1.0) carrying the empty and VIGS plasmids were utilized to infect peach fruits using a 1-ml syringe according to the method of Min et al. (2018). Refrigerated storage for 2 days and the subsequent sampling were performed as detailed in “Plant materials and postharvest applications.” An infection area with a diameter of 1.5 cm and a depth of 2.0 cm in peaches was sampled.

The fruit samples were added into a radioimmunoprecipitation assay lysis buffer. Thereafter, the mixture was subjected to centrifugation at 11,000 × g for 25 min at 4°C. SDS-PAGE was used to separate the collected lysates. The subsequent Western blot analysis was performed based on the method of Jiao et al. (2016). The protein expression levels of PpTrxh9, PpSKα, and SlTrxh9 were normalized to that of RuBisCo.

All datasets were subjected to one-way analysis of variance and Duncan’s multiple range test with SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Significance of the differences among all treatments was measured at the 0.05 level.

CI symptoms in stored peach fruits became visible after 14 days. Compared with the control, cold-stored peach fruits supplemented with GB exhibited less internal browning. Moreover, GB-treated peaches showed lower CI index and higher firmness ( Figure 1 ).

As demonstrated in the transcriptomic experiments and qRT-PCR, cold stress affected the gene expression of PpTrxf, PpTrxh2, PpTrxh4-1, PpTrxh9, PpTrxm3, PpTrxo2, PpTrxx, and PpTrxy1. Among the eight PpTrxs, the enhancement of the expression of PpTrxh9 in response to cold treatment was the largest ( Supplementary Table S3 , Supplementary Figure S2 ). Therefore, PpTrxh9 was selected as the target protein for the subsequent determination of the molecular mechanisms of the GB-delayed CI progression.

The gene expression of PpTrxh9 was enhanced by GB supplementation throughout the refrigeration process. After 35 days of refrigerated storage, GB treatment improved the protein expression of PpTrxh9. Furthermore, GB treatment decreased the ROS generation. The MDA content, the electrolyte leakage, and the LOX and PLD activity also decreased with GB treatment ( Figure 2 ).

As seen from the Y2H screening, PpSKα was identified as a possible protein interacting with PpTrxh9. The Y2H assay was performed to verify the interaction between PpSKα and PpTrxh9. The transformants harboring pGBKT7–PpTrxh9 and pGADT7–PpSKα grew well on the SD/−Leu−Trp−Ade−His medium and turned blue in the presence of X-α-Gal. The transformants harboring pGBKT7–PpTrxh9 and the empty pGADT7 vector did not grow well on the SD/−Leu−Trp−Ade−His medium. These results suggest the interaction of PpSKα with PpTrxh9 ( Figure 3A ).

In the co-immunoprecipitation (Co-IP) assay, following immunoprecipitation with the anti-Flag antibody, MYC–PpTrxh9 co-precipitated with Flag–PpSKα, but not with the empty Flag. In addition, in the pull-down assay, GST–PpSKα could pull down His–PpTrxh9, while the empty GST could not. These two experiments confirmed the interaction between PpSKα and PpTrxh9 ( Figures 3B, C ).

Furthermore, as suggested in the subcellular localization assay, both PpSKα and PpTrxh9 were localized in the cell membrane and the cytoplasm. Subsequently, bimolecular fluorescence complementation (BiFC) detection was conducted to further validate the interaction. Yellow fluorescent protein (YFP) fluorescence signals were observed in the membrane and the cytoplasm in tobacco cells co-expressing PpSKα-YN plus PpTrxh9-YC and PpSKα-YC plus PpTrxh9-YN. YFP fluorescence signals were not observed when PpSKα-YN plus YC, YN plus PpTrxh9-YC, PpSKα-YC plus YN, and YC plus PpTrxh9-YN were co-transformed. Taken together, the findings corroborated the interaction of PpSKα with PpTrxh9 in the cell membrane and the cytoplasm ( Figure 4 ).

During 35 days of refrigerated storage, the gene expression of PpSKα increased with GB supplementation in peach fruits. GB treatment promoted the protein expression of PpSKα in 35-day cold-stored peaches ( Figure 5 ). The results displayed the enhancement of the gene expression of PpSKα with GB supplementation.

In comparison with the WT, the overexpression of PpSKα inhibited the visible external browning in 28-day cold-stored tomato fruits. Tomato fruits overexpressing PpSKα exhibited lower CI progression and higher firmness. The ROS production, MDA content, electrolyte leakage, and the activity of LOX and PLD decreased in the PpSKα-OE tomato fruits ( Figure 6 ). The findings illustrated that PpSKα lowered the CI degree in tomatoes.

In comparison with the WT, VIGS of PpSKα resulted in more internal browning in 2-day cold-stored peach fruits. The PpSKα-silenced peaches showed higher CI and lower firmness. Furthermore, VIGS of PpSKα led to increases in the ROS production, MDA content, electrolyte leakage, and LOX and PLD activity ( Figure 7 ). The results elucidated the positive impact of PpSKα on boosting the cold resistance in postharvest peach fruits.

A fluorometric kinase test was conducted to determine the phosphorylation of PpTrxh9 by PpSKα. After incubation of PpSKα with GST, the relative kinase activity was detectable, which showed auto-phosphorylation of PpSKα. Compared with the control (following the incubation of PpTrxh9 with GST), the relative kinase activity was higher following incubation of PpSKα with PpTrxh9. These data suggest that PpSKα phosphorylated PpTrxh9 ( Figure 8 ).

Compared with the WT, the overexpression of PpSKα upregulated the protein and gene expression of SlTrxh9 in 28-day cold-stored tomato fruits, whereas VIGS of PpSKα downregulated the protein and gene expression of PpTrxh9 in 2-day cold-stored peach fruits. This phenomenon illustrated the contribution of PpSKα to the increase in PpTrxh9 expression in peach fruits ( Figure 9 ).