Role of Brassinosteroids in Persimmon (Diospyros kaki L.) Fruit

Mar 6, 2018 - Brassinosteroids (BRs) are phytohormones that regulate numerous processes including fruit ripening. In this study, persimmon (Diospyros ...
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Cite This: J. Agric. Food Chem. 2018, 66, 2637−2644

Role of Brassinosteroids in Persimmon (Diospyros kaki L.) Fruit Ripening Yiheng He,†,‡ Jiaying Li,† Qiuyan Ban,† Shoukun Han,† and Jingping Rao*,† †

College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Hangzhou 310058, China

‡

S Supporting Information *

ABSTRACT: Brassinosteroids (BRs) are phytohormones that regulate numerous processes including fruit ripening. In this study, persimmon (Diospyros kaki L.) fruits were treated with 24-epibrassinolide (EBR) or brassinazole (Brz, a BR biosynthesis inhibitor) and then stored at ambient temperature. The results show that endogenous BR contents gradually increased during persimmon fruit ripening. EBR treatment significantly increased both the content of water-soluble pectin and the activities of polygalacturonase, pectate lyase, and endo-1,4-beta-glucanase but significantly reduced the content of acid-soluble pectin and cellulose, resulting in rapid fruit softening. The EBR treatment also promoted ethylene production and respiration rate. In contrast, Brz treatment delayed persimmon fruit ripening. qRT-PCR analysis showed that DkPG1, DkPL1, DkPE2, DkEGase1, DkACO2, DkACS1, and DkACS2 were up-regulated (especially a 38-fold increase in DkEGase1) in the fruit of the EBR-treated group. These results suggest that BRs are involved in persimmon fruit ripening by influencing cell-wall-degrading enzymes and ethylene biosynthesis. KEYWORDS: persimmon, brassinosteroids, fruit ripening, softening, ethylene

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INTRODUCTION Persimmon (Diospyros kaki L.) is a typical climacteric fruit that is highly nutritious and has a unique flavor. However, persimmon fruit softens easily during the ripening process, reducing commercial value.1 Fleshy fruit softening is characterized by polysaccharide disassembly of the cell wall.2 This cell wall degradation is catalyzed by many enzymes including polygalacturonase (PG, EC3.2.1.15), pectinesterase (PE, EC3.1.1.11), pectate lyase (PL, EC4.2.2.2), endo-1,4-betaglucanase (EGase, EC3.2.1.4), and β-galactosidase (β-gal, EC3.2.1.23).2,3 A large number of cell-wall-degrading enzyme genes related to fruit softening have been identified in several fruits including tomato, pear, and banana.3−6 In persimmon, DkPG1, DkPE1/2, DkEGase1, Dkβ-gal1/4, and DkXTH9/10 genes are involved in anaerobic-induced softening.7 Fruit ripening is a complicated process associated with a series of phytohormones such as ethylene, abscisic acid, and indole-3-acetic acid.8−10 Ethylene biosynthesis is catalyzed by two key biosynthetic enzymes: 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), which converts S-adenosyl-Lmethionine (SAM) to ACC, and ACC oxidase (ACO), which converts ACC to ethylene.11 Three ACS and two ACO genes were isolated from persimmon fruit, and marked accumulation of these mRNAs was detected with increase in ethylene production in pulp.8 Brassinosteroids (BRs) constitute a new class of steroidal phytohormones that are involved in numerous physiological processes, such as stem elongation, xylem differentiation, photosynthesis, ethylene biosynthesis, and biotic and abiotic tolerance.12−15 Among the BRs, castasterone (CS) and brassinolide (BL) are the most widely distributed, and BL is the most active compound. 6-Deoxocastasterone (6-DeoxoCS) is the precursor of CS catalyzed by brassinosteroid-6-oxidase.16 © 2018 American Chemical Society

The role of BRs in fruit ripening has been studied in recent decades. Exogenous 24-epibrassinolide (EBR) application accelerates fruit ripening in tomato and is accompanied by increases in ethylene production as well as lycopene and carbohydrate levels but also decreases in chlorophyll and ascorbic acid levels.17 Furthermore, EBR applications trigger tomato fruit ripening associated with increased transcripts of both ethylene and lycopene biosynthesis-related genes.18 The application of exogenous BRs to mango fruit accelerates fruit softening and increases ethylene production; however, no marked changes in endogenous BRs during mango fruit ripening were reported.19 Furthermore, endogenous BR levels in grape berries strongly increase at the onset of fruit ripening. In addition, exogenous application of BRs and brassinazole (Brz, a BRs biosynthesis inhibitor) to grape berries significantly promoted and inhibited grape fruit ripening, respectively.20 BRs also play a similar role in strawberry fruit ripening.21 However, no evidence of correlations between BRs and persimmon fruit ripening exists. We studied the role of BRs on persimmon fruit ripening to better understand and perhaps manipulate persimmon fruit softening and to prolong storage time. The effects of exogenous EBR and Brz treatments on persimmon fruit firmness, cell-walldegradation-related enzymatic activities, and gene expression were investigated. Moreover, endogenous BRs levels, ethylene production, and the expression pattern of ethylene biosynthesis genes were also examined. Received: Revised: Accepted: Published: 2637

December 28, 2017 March 5, 2018 March 6, 2018 March 6, 2018 DOI: 10.1021/acs.jafc.7b06117 J. Agric. Food Chem. 2018, 66, 2637−2644

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Journal of Agricultural and Food Chemistry

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Enzyme Assays. The PE activity was measured in accordance with the methods of Lin et al. with slight modifications.24 Approximately 3.0 g of frozen tissues was homogenized in 6 mL of 12% polyethylene glycol containing 0.2% sodium bisulfate at 4 °C. The mixture was centrifuged at 12 000g for 15 min at 4 °C. Afterward, the pellet was washed with 6 mL of aqueous 0.2% sodium bisulfate and then centrifuged at 12 000g for 10 min at 4 °C. The pellet was then resuspended in 8 mL of cold aqueous 7.5% NaCl containing 1% (w/v) polyvinylpyrrolidone (PVP) and maintained under agitation for 4 h at 4 °C, after which the pellet was centrifuged at 12 000g for 20 min. The supernatant was used to determined PE activity. The reaction mixture consisted of 10 mL of 1% (w/v) citrus pectin (Sigma Chemical) and 2 mL of enzyme extract, and the pH of mixture was adjusted to 7.4 with NaOH prior to the reaction. The mixture was incubated at 37 °C for 1 h and then titrated with 0.02 M NaOH to maintain a pH of 7.4. One unit of activity was defined as 1 mmol NaOH consumed h−1. The protein concentration was determined in accordance with the Bradford method, using bovine serum albumin as a standard.25 The PL activity measured in accordance with the methods of Payasi et al. with modifications.26 Frozen fruit samples (1 g) were homogenized in 7 mL of 0.02 M sodium phosphate (pH 7.0) containing cysteine-HCl (20 mM), PVP (1% w/v), and Triton X-100 (0.05%). The homogenate was then centrifuged at 12 000g for 30 min, after which the supernatant was used for PL activity assays. The reaction mixture consisted of 1 mL of enzyme extract, 0.5 mL of 0.36% (w/v) polygalacturonic acid, 1 mL of 0.1 M Tris−HCl buffer (pH 8.5), and 0.5 mL of 4 mM CaCl2 and was incubated at 37 °C for 30 min. The increase in absorbance at 235 nm was used to measure PL activity. One unit of enzyme activity was defined as the production of 1 nmol unsaturated digalacturonane h−1 under the above conditions. The PG and EGase enzymes were extracted according to the method described by Wei et al. with modifications.27 Approximately 1.0 g of frozen tissues was homogenized in 8 mL of 50 mM sodium acetate buffer (pH 5.2) containing NaCl (100 mM) and PVP (1% w/ v) at 4 °C. After rapid homogenization, the mixture was centrifuged at 12 000g for 20 min at 4 °C. The supernatant was used to determine the PG and EGase activity. The PG and EGase activities were measured in accordance with the dinitrosalicylic acid (DNS) method.4 One unit of PG activity catalyzed the liberation of 1 μg of GalA in 1 h at 37 °C. In addition, one unit of EGase activity was defined as the production of 1 mg glucose h−1. Each sample consisted of three biological replicates. Expression Analysis by qRT-PCR. The total RNA was prepared in accordance with the hot borate method.28 First-strand cDNA synthesis and qRT-PCR analysis were performed as described previously.1 One microgram of total RNA was reverse-transcribed using the Prime Script RT Reagent Kit with gDNA Eraser (Perfect Real Time, TaKaRa, Dalian, China) in accordance with the manufacturer’s protocol. The qRT-PCR analysis was performed with an iCycler iQ5 (Bio-Rad, Hercules, CA) using a SYBR PrimeScript RTPCR Kit II (TaKaRa, Kyoto, Japan). Cell-wall degradation-related and ethylene biosynthesis genes were selected according to previous reports.7,8 The primer sequences used for qPCR analysis are listed in Table S1. The relative expression levels of target genes were calculated with the formula 2−ΔΔCT.29 Each sample consisted of three biological replicates. Statistical Analysis. The data were compared using ANOVA and the least significant difference (LSD) at P < 0.05 by SPSS 22.0. The figures were prepared by Origin 2015 and the results are represented as the means ± standard errors.

MATERIALS AND METHODS

Plant Material and Treatments. Preclimacteric persimmon (D. kaki L. cv. Fuping Jianshi) fruits were harvested with 70−80% surface yellow coloration from an orchard in Fuping, Shaanxi, China (34°47′N, 109°2′E). Fruits that were of uniform maturity, size, and color and free from visual defects were selected for the experiments. Six hundred thirty fruits were randomly divided into three groups for three treatments in triplicate (70 fruits per replication). Fruit in the first group were dipped in a 10 μmol L−1 EBR (Yuanye Biotechnology, Shanghai, China) aqueous solution for 30 min at 25 °C according to our preliminary experiments. Fruit in the second group were dipped in a 5 μmol L−1 Brz (Sigma Chemical, St. Louis, MO) aqueous solution for 30 min. Fruit in the other group were treated with distilled water for 30 min and served as a control. The fruits were then air-dried for 2 h and stored at 25 ± 1 °C. In addition, fruit firmness, total soluble solids (TSS), ethylene production, and respiration rate were measured every 2 days, after which all of the samples were frozen in liquid nitrogen and stored at −80 °C for later use. Fruit Firmness and Total Soluble Solids. Fruit firmness was measured in accordance with our previous methods.1 Firmness was measured using a firmness detector (FT327, Effegi, Alfonsine, Italy) at two positions 90° apart at the fruit equator, after removal of the peel. TSS (°Brix) was measured by a digital refractometer (PAL-1, Atago, Tokyo, Japan). For each time point, six fruits were tested for replications. Each sample consisted of three biological replicates. Ethylene Production and Respiration Rate. Ethylene production and respiration rate were measured in accordance with our previous methods.1 Six persimmon fruits were sealed in a 9.17 L vessel and were incubated for 1 h at room temperature. To measure ethylene production, a 1 mL gas sample was collected from the vessel by a syringe and analyzed by injecting into GC-14A gas chromatograph (Shimadzu, Kyoto, Japan). The respiration rate was measured using a CO2 infrared gas analyzer (TEL7001; GE Telaire). Three biological replications were conducted. Endogenous Levels of BRs during Persimmon Fruit Ripening. Endogenous BRs such as BL, CS, and 6-DeoxoCS were measured using enzyme-linked immunosorbent assays (ELISA), as described by Sun et al. and Xu et al. with modifications.22,23 Approximately 0.5 g sample tissues were homogenized in 5 mL of phosphate-buffered saline (pH 7.2). The supernatant was then collected after it was centrifuged at 10 000g for 20 min, of which 10 μL was examined using ELISA Kits (Shanghai YuDuoBio, Shanghai, China) in accordance with the manufacturer’s protocol. The final absorbance was measured at 450 nm using a microplate reader (Infinite M200Pro; Tecan, Switzerland). Each sample consisted of three biological replicates. Determination of Pectin and Cellulose Contents. Pectin contents were measured as described by Bu et al. with some modifications.4 A ∼2.5 g sample of powdered tissue was dissolved using 10 mL of 95% ethanol (v/v), after which the sample was heated in a boiling water bath for 30 min while being stirred. After cooling, the sample was centrifuged at 10 000g for 10 min, and the supernatant then discarded. The pellets were washed twice with hot 95% ethanol (v/v), after which they were resuspended in 25 mL of distilled water; the mixture was then heated at 50 °C for 30 min. Afterward, the mixture was centrifuged at 10 000g. The supernatant was used to measure water-soluble pectin (WSP), and the pellets were dissolved in 25 mL of 0.5 M H2SO4 and then heated in a boiling water bath for 1 h. The supernatant was used to measure acid-soluble pectin (ASP). One mL of WSP or ASP extract was added to 6 mL of H2SO4 for 1 h in boiling water. After the mixture was cooled, 1 mL of 0.15% carbazole reagent was added, the mixture was then incubated in darkness for 30 min. Afterward, the absorbance at 530 nm was determined. A standard curve was constructed using D-(+)-galacturonic acid (GalA). The pectin content was expressed as micrograms of GalA equivalents per gram of fresh weight (FW). The cellulose contents were measured in accordance with the method described by Song et al. and were expressed as milligrams per gram of FW.5 Each sample consisted of three biological replicates.

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RESULTS Effects of EBR and Brz on Persimmon Fruit Ripening. After storage, the firmness and TSS of persimmon fruit gradually decreased, and EBR treatment significantly accelerated the reduction in fruit firmness and TSS; however, Brz treatment delayed persimmon fruit ripening and the fruit were more firm (Figure 1 and Table S2). As expected, the persimmon fruit exhibited a climacteric ethylene production 2638

DOI: 10.1021/acs.jafc.7b06117 J. Agric. Food Chem. 2018, 66, 2637−2644

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the control fruit from 0 to 8 days, and Brz reduced the contents of BL and CS during the initial storage period. Cell-Wall Composition. The content of WSP gradually increased, while the content of ASP slowly decreased during storage (Figure 3A,B). The WSP content in EBR-treated fruit was significantly higher than that in control fruit from 4 days until the end of storage; however, the WSP content was significantly lower in the Brz group than in the control group at 10 and 12 days. The ASP content was significantly lower in the EBR-treated fruit than in both the control and Brz-treated fruit after 8 days of storage. Similarly, the cellulose content slowly decreased throughout persimmon fruit ripening process (Figure 2C). However, the EBR treatment reduced the cellulose content from 8 to 14 days, and the cellulose content was significantly higher in the Brz group than in the control group at 6, 12, and 14 days. Cell-Wall-Degrading Enzyme Activities. The PG activity gradually increased in the control persimmon fruit, peaked at 12 days, and then decreased (Figure 4A). The peak activity in the EBR-treated fruit was observed at 10 days(19.62 U mg−1 protein) and was 27% higher than in the control fruit (15.33 U mg−1 protein). However, Brz treatment reduced the PG activity from 10 until 12 days. The PL activity in the fruit of all groups gradually increased during storage (Figure 4B). The PL activity was significantly higher in EBR-treated fruit than in the control fruit during storage from 4 until 10 days, and the activity was significantly lower in the Brz-treated fruit than in the control fruit from 10 to 14 days. The PE activity in the fruit of all groups increased sharply during the first 6 days, after which the activity remained relatively stable from 4 days until the end of storage (Figure 4C). However, no marked differences in PE activity were observed between the fruit in the EBR and Brz groups and that in the control group during storage. The EGase activity in the control fruit remained at a low level during the first 6 days but then increased after 6 days (Figure 4D). The EGase activity was dramatically higher in the EBRtreated fruit than in the control fruit from 10 to 12 days, which encompassed the climacteric stage; however, no distinguishable differences were observed between the EBR treatment and the control at the other time points during storage. Fruit in the Brz treatment exhibited lower EGase activity after 10 days compared with either the control or EBR-treated treatment. Expression Patterns of Cell-Wall-Degrading Enzyme Genes. The expression patterns of five cell-wall-degrading

Figure 1. Visual appearance (A), firmness (B), and ethylene production (C) of 24-epibrassinolide- (EBR) and brassinazole- (Brz) treated persimmon fruit stored at 25 ± 1 °C. Data are presented as mean ± standard error from three biological replicate assays, and different letters above the symbols indicate significant differences (LSD, P < 0.05) between three treatments at each time point. ns indicates no significant difference.

peak during ripening (Figure 1C). Compared with the control treatment, EBR treatment advanced the onset of the climacteric peak of ethylene production by 2 days, and the peak was 12.9% higher than that in control. In contrast, the Brz treatment caused a lower peak of ethylene production that occurred later. Respiration rate was stimulated by application of EBR and suppressed by Brz (Figure S1). These results suggest that the EBR treatment could accelerate persimmon fruit ripening, while the Brz treatment delayed persimmon fruit ripening. Endogenous Levels of BRs during Persimmon Fruit Ripening. To investigate the role of BRs in persimmon fruit ripening, three steroid compounds (BL, 6-DeoxoCS, and CS) in persimmon fruit were measured by ELISA (Figure 2). The results showed that BL constituted the highest content of the BRs, followed by CS and 6-DeoxoCS. The contents of BL and CS gradually increased during the first 6 days of storage and persisted at high levels during the remaining storage time (Figure 2A,B). In contrast, the levels of 6-DeoxoCS did not significantly change during the whole storage period (Figure 2C). No significant difference was found in the content of 6DeoxoCS between the control and treated fruit. The BL content was significantly higher in the EBR-treated fruit than in

Figure 2. Contents of endogenous brassinolide (A), castastarone (B), and 6-deoxocastastarone (C) of 24-epibrassinolide- (EBR) and brassinazole(Brz) treated persimmon fruit stored at 25 ± 1 °C. Data are presented as mean ± standard error from three biological replicate assays, and different letters above the columns indicate significant differences (LSD, P < 0.05) between three treatments at each time point. 2639

DOI: 10.1021/acs.jafc.7b06117 J. Agric. Food Chem. 2018, 66, 2637−2644

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Figure 3. Contents of ASP (A), WSP (B), and cellulose (C) of 24-epibrassinolide- (EBR) and brassinazole- (Brz) treated persimmon fruit stored at 25 ± 1 °C. Data are presented as mean ± standard error from three biological replicate assays, and different letters above the symbols indicate significant differences (LSD, P < 0.05) between three treatments at each time point. ns indicates no significant difference.

Figure 4. Activities of PG (A), PL (B), PE (C), and EGase (D) of 24-epibrassinolide- (EBR) and brassinazole- (Brz) treated persimmon fruit stored at 25 ± 1 °C. Data are presented as mean ± standard error from three biological replicate assays, and different letters above the symbols indicate significant differences (LSD, P < 0.05) between three treatments at each time point. ns indicates no significant difference.

enzyme genes in persimmon fruit were analyzed (Figure 5). The expression of DkPG1 in the control and EBR-treated fruits persisted at low levels from 0 to 6 days but then was sharply upregulated during the fruit dramatic softening stage (Figure 5A). At the same time, the transcript level of DkPG1 increased in response to EBR treatment from 8 until 14 days. The expression of DkPL1 in the control and EBR-treated fruit gradually increased and peaked when the ethylene production peaked (Figure 5B). The EBR treatment also significantly promoted DkPL1 mRNA abundance at most time points. The transcript level of DkPE2 was similar to that of DkPL1 (Figure 5D). The expression level of DkPE2 was up-regulated in the EBR-treated fruits, but the expression of DkPE1 was downregulated (Figure 5E). The expression level of DkEGase1 was elevated by the EBR treatment, and a 38-fold increase was observed at 12 days (Figure 5C). Moreover, the expression of DkPG1, DkPL1, DkPE2, and DkEGase1 decreased in response

to the Brz treatment. These results suggest that the BRmediated regulation of persimmon softening is related to DkPG1, DkPL1, DkPE2, and DkEGase1 expression. Expression Patterns of Ethylene Biosynthesis Genes. In this study, expression patterns of three DkACS and two DkACO genes were examined (Figure 6). The expression of DkACS1 persisted at a low level in the control fruit from 0 until 8 days, after which it increased sharply and remained at a high level (Figure 6A). In addition, the EBR treatment improved the expression of DkACS1 from 6 until 10 days; the expression increased approximately 17-fold at 8 days. DkACS2 expression levels in the EBR-treated fruit were significantly higher than those in the controls fruit from 6 to 12 days and coincided with the change in ethylene production (Figure 6B). DkACS1 and DkACS2 expression levels in the Brz-treated fruit were significantly lower than those in the control fruit from 10 to 12 days. The treated and control fruit exhibited steady-state 2640

DOI: 10.1021/acs.jafc.7b06117 J. Agric. Food Chem. 2018, 66, 2637−2644

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Figure 5. Cell-wall-degrading enzyme genes of 24-epibrassinolide- (EBR) and brassinazole- (Brz) treated persimmon fruit stored at 25 ± 1 °C. Data are presented as mean ± standard error from three biological replicate assays, and different letters above the columns indicate significant differences (LSD, P < 0.05) between three treatments at each time point.

Figure 6. Ethylene synthesis genes of 24-epibrassinolide- (EBR) and brassinazole- (Brz) treated persimmon fruit stored at 25 ± 1 °C. Data are presented as mean ± standard error from three biological replicate assays, and different letters above the columns indicate significant differences (LSD, P < 0.05) between three treatments at each time point.

levels of DkACS3 during the storage period (Figure 6C). The DkACO1 and DkACO2 genes showed similar expression patterns in the control group during persimmon fruit ripening (Figure 6D,E). However, compared with the control and Brz

treatments, the EBR treatment enhanced the expression of DkACO2 from 4 until 10 days, but no differences in the levels of the DkACO1 gene were observed between all treated fruit. These results suggest that BRs facilitate ethylene production 2641

DOI: 10.1021/acs.jafc.7b06117 J. Agric. Food Chem. 2018, 66, 2637−2644

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Journal of Agricultural and Food Chemistry during persimmon fruit ripening by promoting DkACO2, DkACS1, and DkACS2 gene expression.

study, an increase in PG, PL, and EGase activity or the expression of these corresponding genes was observed during the fruit dramatic softening stage, and the PE activity increased sharply at beginning of storage (Figures 4 and 5). These results indicated that PG, PL, and EGase play major roles in persimmon fruit softening, and PE plays an auxiliary role. Moreover, we found that the EBR treatment activated the activity of the cell-wall modification enzymes PG, PL, and EGase as well as the expression of the DkPG1, DkPL1, DkEGase1, and DkPE2 genes (Figures 4 and 5A). DkPG1, DkEGase1, and DkPE2 are correlated with persimmon fruit softening in response to high CO2 treatment.7 Also, application of EBR to Arabidopsis seedlings induces significant increases in PE activity.37 In contrast, in the present study, no effect of EBR on PE activity was observed during persimmon fruit ripening (Figure 4C). This difference may be attributed to lower levels of the DkPE1 gene in the fruits of the EBR-treated group. In addition, various BR target genes involved in different biological processes might exist. Ethylene directly regulates the transcription of various cellwall-degrading enzyme genes in many fruits.38−40 Our data showed that the timing of the EBR treatment enhances the expression of DkPG1, DkPL1, and DkPE2; however, the expression of DkEGase1 occurred prior to both ethylene production and the expression of the DkACO2, DkACS1, and DkACS2 genes (Figure 5). These results indicate that BRs regulate persimmon fruit softening by directly influencing genes related to cell-wall degradation or by crosstalk with other hormones. Additional studies are clearly required to understand how the BR response BZR transcription factors regulate genes related to persimmon fruit ripening. In summary, endogenous BR levels gradually increased during persimmon fruit ripening. Applications of EBR accelerated persimmon fruit softening and promoted ethylene production and respiration rate associated with up-regulated gene expression of DkPG1, DkPL1, DkPE2, DkEGase1, DkACO2, DkACS1, and DkACS2 (especially a 38-fold increase in DkEGase1), while Brz treatment delayed persimmon fruit ripening. These results provide new evidence of the involvement of BRs persimmon fruit ripening.

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DISCUSSION During the past two decades, abundant research has shown that BRs are involved in plant growth, development, and responses to environmental stress.30,31 However, little is known about the role of BRs in persimmon fruit ripening. In this study, the contents of BL and CS gradually increased during storage (Figure 2A,B), and this phenomenon may be involved in persimmon fruit ripening. Similarly, BRs levels significantly increase during the strawberry fruit-coloring period.21 Moreover, the contents of 6-DeoxoCS and CS increase at the onset of grape berry ripening.20 However, the contents of BRs are low and do not markedly change during mango fruit ripening.19 These results suggest that endogenous levels of BRs represent various patterns in different plant species. We also found that EBR treatment can enhance the BL content in persimmon fruit (Figure 2A), which is consistent with the reports on deseeded grape berries.23 Meanwhile, BL constituted the highest content of the BRs (Figure 2A), suggesting that BL is the most bioactive in persimmon fruit. EBR treatment can accelerated fruit coloring in nonclimacteric grape berries and strawberries,21,23 and applications of EBR promoted fruit softening and enhanced ethylene production in climacteric tomato and mango fruit.18,19 In the present study, 10 μmol L−1 EBR treatment accelerated persimmon fruit softening and markedly promoted ethylene production (Figure 1); however, the Brz treatment caused an opposite effect. These findings are inconsistent with the reports in which 5 μmol L−1 exogenous BR treatment can delay jujube fruit ripening.32 These results suggest that BRs may play a different role at different concentrations and in different fruit species. Ethylene plays an important role in persimmon fruit ripening. DkACSs and DkACOs are involved in regulating ethylene production in persimmon fruit.8 In the present study, the relative expression of DkACO2, DkACS1, and DkACS2 increased or decreased after EBR or Brz treatment (Figure 5B). Similar to results using tomato, BRs may be involved in persimmon fruit ethylene synthesis by regulating the expression of the DkACO2, DkACS1, and DkACS2 genes.18 In contrast, EBR treatment down-regulated ethylene level and ACS gene expression in pepper seedlings under chilling stress.33 Taken together, we suggest that BRs may play different roles in fruit ripening and abiotic stress. BR target genes include not only ethylene biosynthesis genes but also cell-wall-related enzyme genes.34,35 Fruit softening is closely related to the disassembly of cell-wall components, including pectin and cellulose.2 The application of EBR to persimmon fruit enhanced the content of WSP but reduced the contents of ASP and cellulose (Figure 3). These results imply that the EBR treatment promoted both the conversion of ASP to WSP and the degradation of cellulose, causing rapid softening. This implication is consistent with reports in which persimmon fruit softening is associated with pectin degradation.36 Cell-wall degradation is catalyzed by many cell-walldegrading enzymes. PG and PL are responsible for the degradation of pectic polymers homogalacturonan through hydrolysis and β-elimination reaction, respectively. PE catalyzes pectin demethylation and then promotes the action of pHdependent cell-wall hydrolases such as PG, and EGase is involved in the weakening of the primary cell wall.2 In this

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b06117. Table S1. Primes for qRT-PCR analysis used in the present study. Table S2. Contents of total soluble solids of EBR- and Brz-treated persimmon. Figure S1. Visual appearance and respiration rate of EBR- and Brz-treated persimmon. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jingping Rao: 0000-0002-8726-947X Funding

This work was supported by the National Key Research and Development Program (2016YFD0400102). Notes

The authors declare no competing financial interest. 2642

DOI: 10.1021/acs.jafc.7b06117 J. Agric. Food Chem. 2018, 66, 2637−2644

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ABBREVIATIONS USED BRs, brassinosteroids; EBR, 24-epibrassinolide; Brz, brassinazole; WSP, water-soluble pectin; ASP, acid-soluble pectin; PG, polygalacturonase (EC3.2.1.15); PE, pectinesterase (EC3.1.1.11); PL, pectate lyase (EC4.2.2.2); EGase, endo-1,4beta-glucanase (EC3.2.1.4); β-gal, β-galactosidase (EC3.2.1.23); ACC, 1-aminocyclopropane-1-carboxylate; ACS, ACC synthase; ACO, ACS oxidase; BL, brassinolide; CS, castasterone; 6-DeoxoCS, 6-deoxocastasterone; TSS, total soluble solids; ELISA, enzyme-linked immunosorbent assays

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