The potato ‘V7’ were purchased from Xinfadi, Beijing in October 2021. About 50 kg tubers were transported to the laboratory of Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences on the same day. Tubers of uniform size and shape, free from diseases and without any visual defect were selected for the research. The tubers were carefully rinsed twice with distilled water, then immersed in 1.5% (v/v) sodium hypochlorite for 3 min to disinfect surfaces, and dried naturally. Afterward, three artificial wounds (approximately length × 1 cm, width × 1 cm, and depth × 0.5 cm) per tuber were created around the equator with a peeling knife. The wounded tubers were packed in 10-size polyethylene bags with holes (1- mm - diameter holes distributed every 3 cm). The tubers were stored at 22°C, and 4°C with 80% - 90% relative humidity in dark for healing. Wound tissues (3 mm) were collected from the wounded site using a knife after 0, 3, 5, 7, and 14 d of storage. Three biological replicates per treatment were used in all experiments. The samples were stored at -80°C.

The weight of each potato was recorded at each sampling point. Eventually following calculation formula: Weight loss (%) = (m₀ - m)/m₀ ×100%, where ‘m₀’ represents the wounded potato weight on the first day, ‘m’ represents the weight of the wounded potato at each sampling point of both control and treatments. Then take photos to record the wounded surface.

The vertical wound surface of the tuber was cut into slices (about thickness × 0.2 - 0.3 mm, width × 1 cm) with a blade. The slices were immediately rinsed with distilled water to remove starch granules and then immersed in 1% (w/v) phloroglucinol solution for 1.5 min on a glass slide with a few drops of concentrated hydrochloric acid. After 5 min, place the slide under a microscope (10×) to observe the lignin. A slice was cut in the vertical wound surface of the tuber, leaving the slices at 0.05% toluidine blue for 45 min. The residues were removed by washing with distilled water and 75% alcohol two times, respectively. Afterward, rinse twice with 95% alcohol. Finally, the tissue was stained with 1% neutral red III for 1 - 2 min and washed with distilled water and 75% alcohol. The SPA was observed by a (10×) microscope.

Lignin content was determined by Lignin Content Detection Kit (Solarbio, China). Take 3 mg of dry sample and add the reagents according to the instructions. After thorough mixing, the acetylation reaction was carried out in a water bath at 80°C for 40 min, centrifuged at 8000 g for 10 min, and the supernatant was added with glacial acetic acid. The test was conducted in a 96-well UV plate according to the manufacturer’s instructions strictly, and absorbance was read at 280 nm using a microplate reader. H₂O₂ content was determined by H₂O₂ Content Detection Kit (Solarbio, China). Weigh 0.05 g of fresh sample in 500 µL acetone ice bath homogenization, centrifuged 8000 g for 10 min at 4°C, and the reagents was added into 250 µL supernatant according to the instructions. The precipitate was dissolved after mixing and centrifuged at 4000 g for 10 min, and rested for 5 min. Absorbance values were measured at 415 nm using a 96-well plate according to the manufacturer’s instructions.

The starch, soluble sugar, and sucrose content were measured following the methods described by He (1985), and the absorbance value was measured at 620 nm. Take 0.01 g dry sample, and add 80% ethanol, 80°C water bath. After cooling, centrifuge to take the supernatant, and repeat the above operation. The supernatant was used to measure the sugar content and the precipitation was used to measure the starch. The supernatant was added 30% KOH and 300 µL anthrone reagent to measure the content of sucrose. The supernatant was added 300 µL anthrone reagent to measure the content of soluble sugar. The precipitation was diluted, add 9.2 mol L^-1 perchloric acids, and centrifuged to obtain the supernatant. Add 300 µL anthrone reagent to the supernatant to measure starch content.

The total RNA of the potato tuber was extracted using RNA prep Pure Plant Kit (TIANGEN Biotech, China) according to the manufacturer’s instructions. The RNA integrity was determined using agarose gel electrophoresis (1% agarose gel, 0.5 × TAE, 100 V, 20 min), and purity was established at an absorbance of an OD₂₆₀/OD₂₈₀ ratio. Then the RNA was reverse transcribed to cDNA by iScript™ cDNA Synthesis Kit (TRANSGEN, China). All reactions for each cDNA sample were carried out in triplicate.

Primers used in the research were included in Supplementary Table 1 . The EF1α gene was used as an internal control and was shown to be stable under the conditions used. The gene expression of Prx, PAL1 (Lulai and Neubauer, 2014), 4CL, KCS6, FAOH, GPAT5 (Lulai and Neubauer, 2014), AMY23, BAM1, AGPase and INV1 (Xie et al., 2018) were analyzed. cDNA was generated using Power SYBR Green PCR Master Mix kit (Applied Biosystems) and real-time quantitative PCR was conducted on ABI 7500 instrument (Applied Biosystems). The real-time PCR conditions were as follows: a pre-incubation at 94°C for the 30 s, then amplification of 40 cycles of 94°C for 5 s, subsequently 30 s at 60°C, 15 s at 95°C, 34 s at 60°C, and final extension step for 30 s at 95°C. The relative quantification of each gene was calculated using the 2^{− ΔCT} method and compared with the gene at the initial time.

All data were analyzed with Microsoft Excel 2019 software, which was used to calculate the mean value and standard deviation. The pictures were prepared using origin 8.5 software (Microcal Software Inc., Northampton, MA, USA). Differences between the control and treated tuber were assessed with a significant level of p < 0.05.

During healing, the wound periderm formation in tubers of all treatments continued to increase. The low-temperature treated wound periderm was reduced to a brighter color than the control within 14 days ( Figure 1A ). The accumulation of lignin has been increasing during healing in all tubers. The lignin accumulation was slower in the tubers under low-temperature treatment than in the control ( Figure 1B ). Similarly, suberin accumulation of all tubers also increased continuously during healing, however, the accumulation in the low-temperature treated tubers was significantly slower than that in the control ( Figure 1C ). The cell layers thickness of lignin and suberin increased in all tubers, and low-temperature treated tubers were lower than the control within 14 days. It indicated that the low temperature inhibited wound periderm formation and the accumulation of lignin and suberin at the wound sites.

During healing, the weight loss of all wounded tubers continued to increase. The weight loss rate of the low-temperature treated tubers became significantly slower than the control within 14 d ( Figure 1D ). The H₂O₂ content in the low-temperature treatment was significantly lower than the control, the control tubers first increased, then maintained, and finally increased to a high level. At 3 d and 14 d of healing, the H₂O₂ contents in the control tubers were 2-fold and 3.7-fold higher than that of the low-temperature treated tubers, respectively ( Figure 1E ). The lignin content in the control tubers showed a rapid increase during healing. The lignin content in the low-temperature treated tubers was significantly lower than that of the control during healing ( Figure 1F ).

During healing, starch content in all tubers decreased continuously. The content of starch in low-temperature treated tuber was significantly higher than in the control ( Figure 1G ). The content of soluble sugar initially increased and then rapidly decreased in the control tuber while that in the low-temperature treated tubers increased at 7 d of healing ( Figure 1H ). The sucrose content variation trend was similar to the soluble sugar content during healing ( Figure 1H ).

Starch and sugars are important energy supply in tuber during healing ( Figure 2A ). The expression of StAMY23 in the control increased to the maximum level at 7 d of healing and was significantly higher than that in the low temperature-treated tubers ( Figure 2B ). Expression of StBAM1 in the treated tubers was 1.7-fold and 3.2-fold higher than that in the control at 7 d and 14 d of healing, respectively ( Figure 2C ). The expression of StAGPase reached the maximum expression level at 7 d of healing, and in treated tubers was 2.0-fold higher than that in the control ( Figure 2D ). StINV1 also decreased gradually during healing. The expression of StINV1 was significantly higher in the low-temperature treated tubers than that in the control ( Figure 2E ). The results showed that low-temperature could inhibit the conversion of starch to sugar in the wound periderm of tubers, thus suppressing energy and precursor substance supply for healing.

Fatty acid metabolism reflects the accumulation of suberin in the wound periderm of potato tubers during healing ( Figure 3A ). The expression of StKCS6 and StFAOH in the controls were significantly higher than the low-temperature treated tubers during healing, while those in low-temperature treated tubers maintained at a low level ( Figures 3B, C ). The expression of StGPAT5 rapidly increased in the control at 3 d and 7 d of healing compared to the low-temperature treated tubers ( Figure 3D ). The result indicates that low temperature could inhibit the expression of StKCS6, StFAOH, and StGPAT5 to suppress suberin formation during healing.

The phenylpropane metabolic pathway in the wound tissue reflects the ability of tuber healing ( Figure 4A ). During healing, the expression of StPAL1 and St4CL increased first and then decreased in control compared to the low-temperature treated tubers, the maximum expression reached 2.7-fold and 1.4-fold at 7 d, respectively ( Figures 4B, C ). The expression of StPAL1 and St4CL in low-temperature treatments were significantly lower than that of the controls, while the StPAL1 expression in the low-temperature treatment was stable during healing. The expression of StPrx in the control was rapidly up-regulated at 3 d, and the low-temperature treatment was significantly lower than the control. However, the control tubers declined to near basal levels of expression at 7 d during healing ( Figure 4D ). The results suggested that low temperature mainly inhibited StPAL1 and St4CL expressions to suppress lignin formation. Meanwhile, StPrx suppressed further conversion of phenolics to SPP.

Low temperature inhibits the conversion of starch to sugar in the wound periderm of tubers, thus suppressing energy and precursor substance supply for healing. Starch is converted to G-6-P via sugar metabolism. On the one hand, G-6-P enters the pathway of pentose phosphate and glycolysis, and its intermediate substances are converted to shikimic acid. Shikimic acid is converted into phenylalanine through phosphorylation and other reactions to enter the phenylpropane metabolic pathway. The phenylalanine is mainly catalyzed by phenolic acids, aldehydes, and alcohols, synthesizing less lignin and SPP. On the other hand, G-6-P is converted to glycerol-3-phosphate via the glycolytic pathway, which further forms super long chain fatty acids, ultimately, form small amounts of SPA that are deposited in the wound site ( Figure 5 ).