Ethylene treatment of “Maekawa‐Jiro” persimmon affects peel characteristics and consequently, enables boil‐peeling

Abstract In a previous study, we reported that ethylene treatment facilitated boil‐peeling in persimmons and in several other fruits; however, the mechanism underlying the facilitating effect of ethylene was not examined in detail. Thus, in this study, we investigated the effect of ethylene treatment on the peel characteristics of persimmons, that facilitated boil‐peeling, using chemical, genomic, and histochemistry analyses. The results of the study showed that the ethylene‐related genes, DK‐ACS1 and DK‐ACO2, and the pectinase‐active gene DKPG were not expressed, even though a minor increase in ethylene generation was observed after ethylene treatment. Conversely, significant accumulation of toluidine blue O and ruthenium red dyes were observed in the sarcocarp and exocarp of the fruits, indicating an increase in the quantity of polysaccharides, including pectic substances, at the site. The results also indicate that the increased cellulase activity observed in the pericarp of the fruits may be due to the aging of the fruits, and not necessarily as a result of ethylene treatment. Furthermore, ethylene treatment increased the quantity of polysaccharides, including pectic substances, directly below the pericarp, which caused the dissolution of the site, resulting in peeling. This study provides new insights on the effect of ethylene on boil‐peeling in persimmons and provides a foundation for future research studying the effect of heat treatment in the peeling of fruits or tomato.

that ethylene produced remarkably were successful; however, in the no-ethylene treatment were unsuccessful (Murakami et al., 2019a). after ethylene treatment (Murakami et al., 2019b). However, the mechanism by which ethylene treatment facilitates boil-peeling in fruits is unclear. Hence, the purpose of this study was to clarify the mechanism by which ethylene facilitates boil-peeling in fruits.
In persimmon, successful boil-peeling without ethylene treatment has not been reported. Thus, we investigated the effect of ethylene treatment on the peel characteristics of persimmons, that facilitated boil-peeling using chemical, genomic, and histochemistry analyses.

| Plant materials
The Maekawa-Jiro persimmons used in this experiment were obtained from an orchard in Shizuoka, Japan. The persimmons were harvested on November 15 and screened to exclude fruits with physical injury, disease, and pest damage. The fruits were assigned into two groups: ethylene-treated group and the control group.
Ethylene treatment was done following the method of Murakami et al. (2019a). Immediately after harvest, the persimmon fruits were treated with 100 ppm ethylene at 25°C for 3 days, while the fruits in the control group were not treated ( Figure 1).

| Induced ethylene production and fruit quality analysis
Induced ethylene production was evaluated by incubating individual fruits in an 880 ml container for 1 hr, after which 1 ml of headspace gas was withdrawn and injected into a gas chromatograph (GC-2014). Evaluation of fruit quality was performed using 8 fruits from each treatment. Flesh firmness was determined by measuring compression using a Creep Meter (RE3305) fitted with a 5-mm diameter plunger. Each fruit was subjected to a compression speed of 1 mm/s. Soluble solid content of the fruit juice was measured using a digital refractometer (DBX-55A) and the values were expressed in percentage. Titratable acidity (TA) of the fruit juice was determined by titrating the fruit juice against 0.1 N NaOH and it was expressed as percentage citric acid equivalents (% TA).

| Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
After ethylene production and fruit quality analysis, approximately 1-mm thick peel tissue sample was collected from the fruit using a knife. The peel was frozen in liquid nitrogen and stored at −80°C for RNA extraction. Total RNA was extracted from the frozen peel samples using the method for polysaccharide-rich fruit peel tissues, as described by Ikoma et al. (1996). The samples were treated with DNase (RNeasy; QIAGEN) to eliminate any DNA contamination.
F I G U R E 1 Schematic representation of the experimental design of this study. Experimental fruits were harvested on November 15. The fruits were segregated into two treatment groups; ethylene treatment at 100 ppm for 3 days at 25°C, and the control group which were not treated with ethylene. Fruits were investigated at the points indicated by arrows First-strand cDNA was synthesized from total RNA using a reverse transcription kit (Prime Script II 1st strand cDNA synthesis kit; Takara), according to the manufacturer's instructions. The firststrand cDNA was used as a template for the amplification of cDNA fragments encoding the three ethylene-related genes in persimmon: 1-aminocyclopropane-carboxylate synthase gene (DK-ACS1) (Guinevere et al. 2006), ACC oxidase gene (DK-ACO2) (Guinevere et al. 2006), and polygalacturonase gene (DKPG1) (Liu et al., 2009).
The sequences of all primers used for qRT-PCR are listed in Table 1.
Expressed sequence tags of five ripening-related genes were obtained from the Kazusa DNA Lab. persimmon database (Kazusa DNA Research Institute) by BLAST analysis (persi mmon.kazusa. or.jp/blast.html). Oligonucleotide primers were designed according to the regions of each gene. The specificity of all the primers used was verified by melting curve analysis. qRT-PCR was performed in a PCR Thermal Cycler Dice ® Touch (Takara) using Power SYBR Green PCR (Applied Biosystems), as described in the manufacturer's protocol. Relative expression values were calculated as an average of three independent biological replications using Dk-Actin as the internal standard.

| Measurement of cellulase activity
The cellulase (β-1,4-glucanase) activity in the persimmon peel was The means of three individual experiments were determined from separate but concurrent reactions.

| Histochemical observation in peel tissue
Persimmon fruits from each treatment were used for histochemical studies. Tissue samples were obtained via horizontal sections along the equatorial axis of the outer fruit. Collected fruits, including peel tissues, were fixed with 50 mM PBS solution, including 4% paraformaldehyde, and embedded in Technovit ® 7100 (Kulzer). The embedded tissues were cut longitudinally to form sections of approximately 10 μm using a microtome. The sections were stained with 0.1% toluidine blue O and 0.01% ruthenium red. Toluidine blue O stains cell wall components, such as pectic substances (O'Brien et al., 1964).
Ruthenium red reactions were evaluated by spot testing with 57 substances and by titrating with chemically defined pectins (Luft, 1971).
Pictures of the stained sections were taken for subsequent analysis.

| Image processing and analysis
The peel region was divided into two zones: the exocarp and sarcocarp ( Figure 2). The intensity of toluidine blue O and ruthenium red staining of the exocarp and sarcocarp were quantified by analyzing images of the section. The images were processed and analyzed using ImageJ software (ImageJ software, NIH) to ascertain the polysaccharide and pectin contents of the exocarp and sarcocarp using the method of Gonzalez et al. (2010). Saturation component images were processed using different filters, and processed images were used to calculate the total cell viability from the intensity of the stained areas of the photomicrograph. The blue component of the RGB (red, green, blue) color image was selected for the toluidine blue O image, and the red component of the image was used for ruthenium red. The threshold was applied to a regular level in toluidine blue O and ruthenium red images. Some of the analyzed figures are shown in Figure 2. The analyzed area of the figure was between 115 and 783 mm 2 , excluding stone cells and tannin cells. A total of nine stained areas with toluidine blue O and ruthenium red were analyzed for staining intensity.

| Statistical analysis
The results were analyzed with BellCurve for Excel software (Social Survey Research Information Co., Ltd.) using Tukey's test (p < .05) .

| RE SULT AND D ISCUSS I ON
As compared with ethylene treatment and nonethylene treatment, In some crops, ethylene stimulates the synthesis of some enzymes, including polygalacturonase and pectin methylesterase, which results in changes in the cell wall structure (Fischer & Bennett, 1991).
Polygalacturonase is one of the hydrolases that destroys cell wall structure (Fischer & Bennett, 1991). In addition, polygalacturonase activity in several fruits increased with an increase in ethylene production, and polygalacturonase activity may be induced in harvested fruit by exogenous ethylene (Saltveit & Mcfeeters, 1980;Sawamura et al., 1978).
Therefore, we hypothesized that the increased activity of polygalacturonase by ethylene treatment facilitated boil-peeling in persimmons in our study ( Figure 2, Table 2). However, no significant increase of the pectinase activity, DKPG gene was observed in the pericarp of persimmon fruits treated with ethylene. As the reason for this, it was considered the differences analyzed region between histochemical and genomic analysis. In qRT-PCR, we sampled 1-mm thick peel tissue. On the other hand, the dyed more deeply region in histochemical analysis was approximately ~400 μm surface layer of the peel (Figure 4). It may be necessary to sample the surface layer of the peel in genetic analysis.
Any other factors, such as β-galactosidase seemed to play a major role in the ripening process of persimmon fruits (Nakamura et al., 2003).
Therefore, other factors may be responsible for facilitating boil-peeling in fruits treated with ethylene.
Although ethylene generation was induced in the Maekawa-Jiro persimmons after the ethylene treatment, the quantity generated was low (Table 1). Furthermore, no significant increase in the expres- Once persimmons receive ethylene treatment, they can be peeled with hot water. In the present study, ethylene treatment softened the fruit pulp, but the sugar and acid contents remained the same. Generally, ethylene treatment can cause fruit pulp to soften, which can negatively impact fruit processing. It is, therefore, necessary to conduct studies aimed at optimizing the ideal ethylene treatment conditions.
The use of chemicals, including lye, to peel fruits, has been widely adopted because they are cheap and easy to use (Lucas, 1967).
However, the use of lye has several disadvantages, including high peeling losses (Heaton, 1984), allergic reactions (Brenna et al., 2000), and the generation of liquid waste high in NaOH. Alternatively, there

| CON CLUS ION
In the present study, we investigated the effect of ethylene treatment on the peel characteristics of persimmons, that facili- F I G U R E 5 Quantification of stained area as a percentage of total area in "Maekawa-Jiro" persimmon exocarp and sarcocarp tissue. Vertical bars show standard errors of nine replicates. Bars bearing different letters indicate significant difference between means. Tukey's test was used for mean comparison at p < .05 significance level

ACK N OWLED G M ENT
This research was supported by grants from the Project of the Biooriented Technology Research Advancement Institution, NARO (the special scheme project on advanced research and development for next-generation technology; Tsukuba, Japan).

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from corresponding author.

F I G U R E 6
Effect of ethylene and nonethylene treatment on cellulase activity of the pericarp tissue of "Maekawa-Jiro" persimmon. Vertical bars show standard errors of three replicates. Bars bearing different letters indicate significant difference between means. Tukey's test was used for mean comparison at p < .05 significance level