Plant materials and growth conditions.

Commercially mature fruit of two cultivars, Mopan and Yoho (Fig. 1), of persimmon fruit were harvested on 22 Oct. 2016 at commercial maturity from a commercial orchard located in Shijiatong, Hebei, China. Standard commercial cultural practices were applied as required for the fruit trees. After harvest, the fruit were collected into bags and immediately transported to the Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Science (Shijiazhuang, Hebei, China) within 4 h in cooler boxes with ice (0 °C and 90% to 95% relative humidity) and stored at 22 to 25 °C (65% relative humidity) for the following experiments. Similar fruit without physical injuries were selected according to their uniformity of shape, color, and size. The mean sample weights of ‘Mopan’ and ‘Yoho’ persimmon fruit were (± sd) 240.1 ± 15.02 and 147 ± 11.15 g, respectively.

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Fruit storage.

After pre-cooling in air to release field heat (22 to 25 °C and 65% relative humidity), ‘Mopan’ and ‘Yoho’ persimmon fruit (900 fruit for each cultivar) were randomly divided into three groups, with 300 fruit in each group. Fruit in each group (300 fruit) were placed into commercial cardboard boxes (25 fruit/box) and transferred to each storage shelf room and stored at 22 to 25 °C and 65% relative humidity for up to 20 d. Three different shelf rooms (300 fruit for each cultivar in each room) with the same storage conditions were used. Samples were taken at 0, 4, 7, 10, 15, and 20 d of storage for measurements of various physiological properties and for selected gene analysis.

Fruit color analysis, total soluble solids measurements, and maturity assessments.

Fifteen to 20 fruit from each sample were marked at the beginning of the experiment. Samples were continuously monitored during ripening by performing optical measurements at each time point to track the progress of the color change. The fruit surface color was measured with a colorimeter (CR400; Minolta, Ramsey, NJ) at two evenly distributed equatorial sites of each fruit. L* (lightness), a* (‘− green’ to ‘+ red’), and b* (‘− blue’ to ‘+ yellow’) values were recorded and the results were expressed as L-value (lightness), chroma (C = [(a*)² + (b*)²]^0.5, color intensity), and hue angle (h° = arctan (b*/a*), actual color). Chroma and hue angle indicate the intensity of the color saturation (dull to vivid) and color changes as red–purple (0°), yellow (90°), bluish-green (180°), and blue (270′) (Amal et al., 2010).

For each sample, after skin removal, the flesh was sliced into cubes (≈1 cm³). Cubes were homogenized in a stainless-steel blender and then centrifuged for 10 min at 2500 g_n. Total soluble solids (TSS) were then determined from the juice using a digital refractometer (model 3810 PAL-1; Atago, Tokyo, Japan); 15 to 20 fruit per group were used at storage days 0, 4, 7, 10, 15, and 20, and the experiment was replicated three times.

For each cultivar, persimmon maturity was determined individually and nondestructively with a handheld delta absorbance (DA) meter (Sinteleia, Bologna, Italy), which is a portable device based on visible (vis)/near-infrared (NIR) spectroscopy (Ziosi et al., 2008). The DA meter was developed by using the absorbency properties of chlorophyll to measure the chlorophyll content in the skin of the fruit. The chlorophyll content of a fruit is a precise index of the ripeness of that fruit. The instrument provides an index of absorbance difference (I_AD = Abs₆₇₀ − Abs₇₂₀), which is calculated as the absorbance at 670 nm [Abs₆₇₀ (absorbance peak for chlorophyll)] minus the absorbance at 720 nm [Abs₇₂₀ (background of the spectrum)]. The I_AD index was measured by averaging the values recorded on the equatorial region of the two opposite sides (blushed and unblushed sides; 60 fruit from each group were marked at the beginning of the experiment).

Fruit firmness measurement.

Fifteen to 20 ‘Mopan’ and ‘Yoho’ persimmon fruit were taken at storage days 0, 4, 7, 10, 15, and 20 from each group. The firmness of persimmon flesh was measured on two equidistant peeled (≈1 cm² of the skin was removed) sites, on the blushed and unblushed side, and on the equatorial axis of each fruit using a digital hardness tester (GY-4; Handpi, Zhejiang, China) fitted with a head 7.9 mm in diameter. The hardness tester was set perpendicularly to the surface of the fruit and the indenter pressed into the pulp evenly and stopped when reaching the mark (10 mm); the results were expressed in Newtons.

Determination of soluble and total tannins.

Persimmon slices were taken on storage days 0, 4, 10, and 20 from 15 to 20 fruit in each group and ground to a fine powder using liquid nitrogen before total and soluble tannin extraction. Different solutions were used for total and soluble tannin extraction. Total tannins were extracted by 0.56 N HCl and boiled in water (100 °C) for 30 min. After cooling with running water, 2.5 N NaOH was added to neutralize the solution. For soluble tannins, 80% MeOH was used for extraction. Both total and soluble tannins in ‘Mopan’ and ‘Yoho’ persimmon fruit were determined according to the Folin-Denis method (Taira, 1996). Diluted sample solution extract (1 mL) in 6 mL of distilled water was mixed to 0.5 mL of 1 N Folin–ciocalteu reagent. After 3 min, 1 mL of saturated sodium carbonate (Na₂CO₃) was added to the mixture along with 1.5 mL of distilled water. After shaking vigorously, the mixture was kept in the dark at room temperature for 1 h. The absorbance of the resultant blue color was measured at 725 nm using a spectrophotometer (UVmini-1240; Shimadzu Corp., Kyoto, Japan). Gallic acid was used for the calibration curve. Experiments were repeated three times independently.

Measurement of ethylene production and respiration rates.

Randomly chosen ‘Mopan’ and ‘Yoho’ persimmon fruit (three replicates of 15 to 20 fruit) were sealed in a 1-L glass container for 30 min at 23 °C. Ten milliliters of headspace gas inside the glass container was collected for injection into an IR analyzer (HWF-1; Honglang Instrument Equipment Co., Henan, China) to determine the respiration rate. After being kept at 23 °C for 6 h, ethylene production was measured when 1 mL of headspace gas was taken and injected in a gas chromatographer (model GC9790II; Fuli Analysis Instrument Co., Jiangsu, China) that was equipped with a flame ionization detector (FID). Temperatures of the gasifier, FID, and oven were set as 140, 200, and 90 °C, respectively, for ethylene measurement. The carrier flow rates of the gas used were 30, 30, and 300 mL·min⁻¹ for nitrogen, hydrogen, and air, respectively. Ethylene production at storage days 0, 1, 2, 4, 7, 10, 12, 15, and 20 was calibrated with authentic ethylene gas standards, and the results were expressed as nanograms per kilogram per second. The respiration rate at storage days 0, 4, 7, 10, 15, and 20 was calculated using a CO₂ calibration curve and expressed as milligrams per kilogram per hour CO₂.

RNA preparation.

Fifteen to 20 fruit were taken after 0, 4, 10, and 20 d of fruit storage. After skin removal, the flesh was sampled by taking four wedges from each fruit, with each wedge constituting approximately one-eighth of the fruit. The flesh was quickly frozen in liquid nitrogen and ground to a fine powder; then, it was stored at −80 °C. Total RNA in persimmon fruit tissues was extracted using a modified cetyltrimethylammonium bromide (CTAB) method as described by Gasic et al. (2004). RNA extraction was conducted with three biological repeats and three technical repeats in each sample.

Real-time polymerase chain reaction.

The relative abundance of the transcripts was analyzed by real-time polymerase chain reaction (PCR). Total RNA was reverse-transcribed with the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Biomedical, Dalian, China). Quantitative real-time reverse-transcription PCR (RT-PCR) was performed using SYBR^® Premix Ex Taq™ II kit (TaKaRa Biomedical) and was performed using a CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instruction. Actin was used as an internal control (Akagi et al. 2009). The relative gene expression was calculated by the comparative Ct method (Li et al. 2017). The Dkβ-gal1, DkPG1, DkPME1, and DkPDC2 specific primers have been described by Wang et al. (2017). The DkXTH2-specific and DkERF22-specific primers have been described by Zhu et al. (2013) and Min et al. (2014), respectively. Primers specific to DkETR1, DkETR2, DkERS1, and DkCTR1 have been described by Yin et al. (2012a). Primers specific to DkADH1, DkPDC1, and DkERF19 have been described by Min et al. (2012). All primers are shown in Table 1. The RT-PCR was completed with three biological repeats and three technical repeats in each sample. All of the biological repeats showed consistent results, and the data for one of the replicates are presented in the figures. Each data point is the mean value obtained from the RT-PCR performed in triplicate on one sample, with error bars representing the sd.

Table 1.Primers and DNA sequences used for the real-time polymerase chain reaction analysis.

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Statistical analysis.

The same experiment was performed in 2016 and 2017, with consistent results, and the data of 2016 are presented in this work. All experiments were conducted with three biological repeats and three technical repeats in each sample. All data were processed using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA). Analysis of variance of data was performed using IBM SPSS Statistics (version 20; IBM Corp., Armonk, NY) and GraphPad Prism (version 7.0; GraphPad Software, San Diego, CA). Treatment means were compared using the IBM SPSS Statistics Duncan’s multiple range test (P ≤ 0.05).

Changes in fruit coloration and tss content during ‘Mopan’ and ‘Yoho’ persimmon fruit storage.

As shown in Fig. 1, ‘Mopan’ is bigger than ‘Yoho’, whereas the latter often has a more flattened fruit shape and deeper reddish-orange skin. Color is one of the major desirable characteristics that determines consumer acceptance of a product. The L, a, and b values were further evaluated regarding hue (h°) and chroma (C) color to ascertain their effects on the total color changes. As shown in Fig. 2A, the hue angle, which shows orange coloring of ‘Yoho’ and ‘Mopan’ persimmon, decreased gradually during storage. It indicates that the skin color of persimmon fruit were reddish-yellow, and ‘Yoho’ (hue = 63.83 ± 3.86) had a deeper reddish-orange skin compared with ‘Mopan’ (hue = 71.47 ± 1.67; 90° means that the persimmon is yellow and 0° indicates it is completely red). This may due to the accumulation of carotenoid contents (Khoo et al., 2011). Lee et al. (2012) reported that ethylene affected carotenoid biosynthesis and additional ripening phenotypes via SlERF6 in tomato (Solanum lycopersicum L.). Generally, fruit with high chroma (Fig. 2B) values are more vivid than those with low values, even with the same hue angle. Chroma values of ‘Yoho’ and ‘Mopan’ persimmon fruit increased, meaning that the fruit were more vivid in color, as storage time increased. Concurrently, the lightness [L-value (Fig. 2C)] decreased over ‘Yoho’ and ‘Mopan’ persimmon storage.

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Figure 2D displays that the TSS of ‘Mopan’ persimmon decreased gradually but increased in ‘Yoho’ with storage time. The TSS content of ‘Mopan’ fruit was 15.1% immediately after harvest, and it decreased to 12.2% at 20 d. This finding agreed with the information reported by Tilahun et al. (2017) regarding ‘Daebong’ persimmon. The TSS content of ‘Yoho’ fruit increased from 14% to 15.9% after 20 d. Candir et al. (2010) reported that the TSS content in nonastringent type persimmons such as ‘Fuyu’ and ‘Jiro’ increased during storage. However, ‘Yoho’ and ‘Mopan’ persimmon fruit showed different trends in TSS content changes during storage. Arnal and Del Río (2003) observed decreased TSS in ‘Rojo brillante’ persimmon (PCA) fruit, and they also found that the decrease in TSS contents in persimmon fruit was closely related to the changes in tannins from soluble to insoluble with CO₂ treatment, but the mechanism responsible for this is still unclear. The different tannin metabolisms of astringent and nonastringent persimmon fruit (Ikegami et al., 2005) may be associated with the differences in TSS. The higher weight loss rate of ‘Yoho’ fruit (15.30% loss at the end of the storage period) compared with that of ‘Mopan’ fruit (10.17% loss at the end of the storage period) during shelf life could also be related to these observed differences in TSS (Supplemental Fig. 1). It might be interesting to analyze the TSS and color effects with related gene expression changes in the current study. However, the changes do not directly lead to persimmon fruit softening and de-astringency during postharvest; they indirectly influence them through signal transmission in fruit. Only firmness, total and soluble tannin contents, ethylene production, and respiration rate–related gene expression changes were included to explore the roles of cell wall–hydrolyzing enzymes, the de-astringent process, and endogenous ethylene in postharvest ripening.

The DA meter is a new tool used to assess the maturation state of stored fruit to provide information about the shelf life that can be predicted by measuring absorbance within the chlorophyll absorption range. The instrument provides an I_AD, which is calculated as the absorbance at 670 nm minus the absorbance at 720 nm. High correlations between the I_AD index and ripeness level have been reported for apple [Malus domestica Borkh. (Nyasordzi et al., 2013)], pear [Pyrus communis L. (Wang and Sugar, 2015)], and peach [Prunus persica L. (Ziosi et al., 2008)]. This is the first report of its use to predict ripening of persimmon. In our analysis, ‘Mopan’ and ‘Yoho’ persimmon fruit showed the same trend as I_AD changes (Fig. 2E). I_AD changes correlated with changes in hue angle (R² = 0.9976 for ‘Mopan’; R² = 0.9888 for ‘Yoho’), chroma (R² = 0.8521 for ‘Mopan’; R² = 0.8289 for ‘Yoho’), lightness (R² = 0.7203 for ‘Mopan’; R² = 0.8807 for ‘Yoho’), and TSS of ‘Yoho’ (R² = 0.9175). TSS of ‘Mopan’ (R² = 0.5058) was not consistently related to measured I_AD values. This demonstrates that I_AD values that may be found in the future could provide some indication of “color” of both cultivars and “sweetness” of Yoho persimmon. The I_AD was a reliable parameter for assessing fruit maturity at harvest nondestructively and predicting persimmon fruit behavior in storage. It can be a useful tool for assessing the maturation state of the persimmon fruit during storage and providing information about the shelf life that can be expected. Furthermore, it can help select the most ripened fruit to sell at the retail stage. Measurements of the I_AD can also be used on a sorting line, which can provide an optimal distribution of persimmon fruit for storage.

Changes in fruit firmness and cell wall gene expression during ‘mopan’ and ‘yoho’ persimmon fruit storage.

Fruit firmness is one of the most important features used to assess the progress of fruit ripening after harvest because it directly affects fruit quality and storage life. Figure 3A and B showed that the firmness of ‘Mopan’ fruit was obviously lower than ‘Yoho’ and decreased rapidly after 10 d. The correlation between I_AD and firmness was R² = 0.6461 and R² = 0.7581 for ‘Mopan’ and ‘Yoho’ fruit, respectively. Farneti et al. (2015) also found that the I_AD values were significantly linked with apple firmness. Cell wall–modifying enzymes such as polygalacturonase (PG), pectin methyl esterase (PME), β-galactosidase (β-Gal), and xyloglucan endotransglycosylase/hydrolase (XTH) are closely related to fruit softening according to previous studies (Hu et al., 2009; Wang et al., 2017). In the present study, the expression of four selected genes related to fruit softening, DkPG1, DkXTH2, DkPME1, and Dkβ-GAL1, were detected up to 20 d during ‘Yoho’ and ‘Mopan’ persimmon storage, and similar trends were observed in both cultivars (Fig. 3C–F). The gene expression peaks of DkPG1, DkXTH2, and Dkβ-GAL1 appeared around day 4, and DkPME1 showed increasing expression levels as early as day 0 for ‘Mopan’ persimmon. The gene expressions of DkPG1 and DkPME1 in ‘Mopan’ fruit were higher than those in ‘Yoho’ fruit.

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Decreased fruit firmness is the result of a series of orderly adjustments applied by the related enzymes in a controlled manner (Huber, 1983). DkPG1, DkXTH2, DkPME1, and Dkβ-GAL1 genes could be considered molecular markers for firmness, and the overexpression of these genes was correlated with increased fruit softening (Wang et al., 2017). Pectin is one of the main constituents of the primary cell walls and middle lamella of higher plants. The activities of the pectin-degrading enzymes polygalacturonase (PG) and pectin methyl esterase (PME) have been found to be closely related to the degradation of pectins, which played a significant role in the softening changes in fruit and vegetable tissues (Rodoni et al., 2010). PME, the enzyme involved in pectin metabolism, catalyzes the demethylesterification of cell wall polygalacturonans to generate demethylated pectins that can be more easily hydrolyzed by PG (Micheli, 2001). PG hydrolyzes pectin acid along with the main chain of polygalacturonic acid, thereby causing cell wall dissolution and, ultimately, fruit softening. The advance work of DkPME1 may greatly affect the softening of persimmon fruit. At the same time, higher accumulation of DkPG1 and DkPME1 in ‘Mopan’ fruit may be the main reason for ‘Mopan’ fruit softening (Jiang et al., 2010; Luo, 2007).

Changes in fruit soluble tannins and total tannins content and tannins-related gene expiration during ‘Mopan’ and ‘Yoho’ persimmon fruit storage.

The total tannins and soluble tannins were used to evaluate the astringency level of persimmon fruit. The contents of the two forms of tannins of ‘Mopan’ and ‘Yoho’ fruit during storage are shown in Fig. 4A–D. The total tannin contents of both ‘Mopan’ and ‘Yoho’ fruit showed no notable changes during storage. However, values of ‘Yoho’ fruit began and remained distinctly lower than those of ‘Mopan’ fruit. Astringent-type fruit are rich in soluble tannins even at maturity, whereas the concentration of soluble tannins is significantly reduced during early development in fruit of the nonastringent type (Mo et al., 2016). Simultaneously, ‘Yoho’ fruit has a very low content of soluble tannins compared with ‘Mopan’ fruit. As shown in Fig. 4C and D, ‘Mopan’ fruit showed a dramatic decline in soluble tannins, but no significant changes were observed in the soluble tannin content of ‘Yoho’ fruit during the same period. Total tannins are composed of both soluble and insoluble tannins. The coagulation of soluble tannins to insoluble tannins most likely accounts for the natural loss of astringency (Mo et al., 2016). During this process, the total tannin barely changes. For ‘Mopan’ persimmon, the changes in I_AD were directly proportional to changes in soluble tannins concentration (R² = 0.7069). I_AD could be an effective tool for predicting the degree of de-astringency with noninvasive detection. Mo et al. (2016) reported that the soluble tannin contents of J-PCNA showed a trend similar to that of ‘Mopan’ and were always significantly lower than those of ‘Mopan’ during fruit development. Our results also showed that the two cultivars kept the same trend even after harvest.

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The loss of astringency of persimmon fruit is largely due to the reversible interconversion of ethanol and acetaldehyde catalyzed by ADH and PDC enzymes (Mo et al., 2016). The expression of de-astringency-related genes DkADH1, DkPDC1, and DkPDC2 were studied during ‘Mopan’ and ‘Yoho’ persimmon storage (Fig. 4E–G). For ‘Mopan’ and ‘Yoho’ fruit, DkADH1 showed a relatively higher expression at day 0, which decreased at day 4, and an increasing trend was observed at day 10. Similarly, the expression levels of DkPDC2 showed an obvious increase, and the peak occurred concomitantly with an advanced ripening stage at day 4. However, the DkPDC1 expression peak occurred at day 4 for ‘Yoho’ fruit, but there were no significant changes in ‘Mopan’ fruit.

Previous studies of the physiology of de-astringency of persimmons indicated that ADH and PDC were associated with persimmon de-astringency and had key roles. DkADH and DkPDC genes have been characterized from ‘Mopan’ persimmon by Min et al. (2012). The transient overexpression of the DkADH1 and DkPDC2 genes in ‘Mopan’ persimmon leaves caused a significant reduction in the levels of soluble tannins in the leaves (Mo et al., 2016). CO₂ and ethylene treatment effectively increased the transcript abundance of DkADH1, DkPDC1, and DkPDC2 (Min et al., 2012). However, under natural conditions and no treatment conditions, the expressions of DkADH1, DkPDC1, and DkPDC2 were almost constant during maturation, and this was also correlated with the slow process of natural de-astringency in ‘Mopan’ fruit without treatment. Similarly, the expression of DkADH1, DkPDC1, and DkPDC2 genes in ‘Yoho’ fruit exhibited the same patterns. Despite the high expression of de-astringency genes of ‘Yoho’ fruit after harvest, the tannin content was still low. According to Mo et al. (2016), the natural astringency loss observed in ‘Maekawa-jirou’ (J-PCNA) is unrelated to tannin coagulation, which is similar to our findings for ‘Yoho’ (J-PCNA). Using RNA sequencing, Yin et al. (2012b) isolated another three ADH genes and five PDC genes from ‘Mopan’ persimmon that were induced by the de-astringent treatment, but the roles of these genes in de-astringency need further investigation.

Changes in fruit respiration and ethylene production and ethylene-related gene expiration during ‘Mopan’ and ‘Yoho’ persimmon fruit storage.

Figure 5A and B show that the respiration rates of ‘Mopan’ and ‘Yoho’ persimmon fruit decreased during the first 4 d of storage. After that, ‘Mopan’ fruit maintained a constant respiration rate, and no significant differences were detected. ‘Yoho’ fruit experienced an increase near the end of storage. Fruit continued to respire strongly after harvesting without any treatments. The respiration process not only consumes nutrients but also produces endogenous ethylene, which may accelerate fruit ripening and senescence (Abbas and Fandi, 2002). The respiration rate (R² = 0.808 for ‘Mopan’ and R² = 0.4776 for ‘Yoho’) and ethylene production (R² = 0.7374 for ‘Mopan’ and R² = 0.9102 for ‘Yoho’) showed a clear association with I_AD values, thus indicating that I_AD values may be a reliable nondestructive ethylene estimator and respiration monitor.

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The changes in ethylene production for ‘Mopan’ and ‘Yoho’ persimmon fruit are shown in Fig. 5C and D; endogenous ethylene of persimmon was very low on the first day during storage; then, its production significantly and lineally increased. It peaked at 12 d of storage for ‘Mopan’ fruit. After 12 d, the ethylene production started to significantly decrease. For ‘Yoho’ fruit, this peak was reached at 15 d. Persimmon fruit are extremely sensitive to endogenous ethylene, although only a small amount of ethylene is generated during postharvest ripening. As previously reported, the response to endogenous ethylene was obvious during the ripening of ‘Yoho’ fruit (Yin et al., 2012a). According to our research, we found that ‘Mopan’ fruit are also sensitive to endogenous ethylene.

The natural expressions of six ethylene receptors and signal transduction genes (DkETR1, DkETR2, DkCTR1, DkERS1, DkERF19, and DkERF22) in ‘Mopan’ and ‘Yoho’ persimmon fruit have been selected and studied in the present experiments (Fig. 5E–J). ‘Mopan’ persimmon fruit were not sensitive to endogenous ethylene, and no significant differences were detected for ‘Mopan’ fruit gene expression after 20 d at 22 to 25 °C. The expression of DkETR1, DkETR2, DkCTR1, and DkERS1 increased slightly during ripening; however, for DkERF22, no significant changes were detected. DkERF19 was the gene most sensitive to endogenous ethylene and showed the largest increase in its expression at 10 d. Min et al., (2012) reported that DkERF22 rapidly responded to CO₂ treatment, whereas DkERF19 exhibited delayed the accumulation after CO₂ treatment. This indicated that ethylene has different regulatory mechanisms under natural and hypoxic conditions. However, different from ‘Mopan’ fruit, ‘Yoho’ fruit does not readily soften after harvest, but endogenous ethylene still regulates the changes inside the fruit. For ‘Yoho’ fruit, the expression levels of ethylene signal–related genes increased significantly and peaked on day 4 (DkETR1, DkETR2, DkCTR1, DkERS1, and DkERF19) or day 10 (DkERF22). The expression changes of ethylene genes did not exactly follow the ethylene level dynamics in ‘Mopan’ and ‘Yoho’ fruit, and the same conclusion was obtained by a previous study (Yin et al., 2012a). After harvest, the ethylene signal genes in ‘Yoho’ fruit were expressed at constant levels (DkETR1, DkEIL1) or had a small increase in ethylene accumulation time (DkERS1, DkETR2, DkCTR1). More diversity in expression patterns of the DkERF genes was observed. DkERF1, DkERF6, and DkERF7 increased during the early stages of storage. For ‘Mopan’ fruit, most of the ethylene signal genes were expressed at constant levels.

An analysis of the expression of ethylene receptor genes during ripening in many fruit does not follow a pattern consistent with the predicted model, indicating that more complex molecular mechanisms are operating in the regulation of ethylene perception during fruit ripening (Bapat et al., 2010). Ethylene receptors and transcription factors physically interact with the downstream signal transduction mechanisms.

Ethylene showed complex properties during fruit ripening regulation and worked through activation of the promoter to pass the signal downstream to the effector. A previous study indicated that DkERFs bind directly to the DkXTH9 and regulate this gene, which encodes an important cell wall metabolism enzyme (Wang et al., 2017). Many comprehensive studies of ‘Mopan’ were performed to investigate the transactive work of ethylene and promoters involved in softening, but little work regarding the ‘Yoho’ fruit has been performed.