Near‐harvest application of methyl jasmonate affected phenolic content and antioxidant properties in “Thompson Seedless” grape

Abstract The influence of methyl jasmonate (MJ) preharvest treatment was investigated on some polyphenols and antioxidant systems in the “Thompson Seedless” table grape. The clusters were sprayed in the vineyard 2 days before harvest with 0, 1, 5, and 10 mM MJ. After picking, berries were stored for 6 days at 15°C, simulating marketing conditions. Total phenols and flavonoids were affected by MJ treatment, especially at 10 mM concentration, whereas total tannins were found to be unchanged. Antioxidant activity of the treated skin was noticeably higher compared with the control, together with PAL and POD activity. Although MJ had little effect on catechin and epicatechin, the levels of quercetin and rutin were noticeable. In addition, 5 and 10 mM MJ exerted a pronounced effect on transresveratrol content. These data showed that a single preharvest application close to the harvest time could be an effective treatment to promote the antioxidant properties of the grape.


| INTRODUC TI ON
Grapes, one of the most widely consumed fruits worldwide, are rich in phenolic compounds. These compounds in grapes are involved in qualitative and organoleptic characteristics and storability (Zhang et al., 2012). Grapes also consist of diverse kinds of phenolics contributing to improved human health (Vauzour et al., 2010;Vislocky & Fernandez, 2010;Xia et al., 2010). In view of all the aforementioned reasons, augmentation of these compounds is widely taken into consideration. In this regard, elicitation has been shown to be a strategy to stimulate the synthesis of these bioactives in fruits . Naturally occurring compounds, meanwhile, have been extensively noticed to enhance phenolic content in table grapes, such as abscisic acid (Koyama et al., 2018), salicylic acid (Champa et al., 2015;Ranjbaran et al., 2011), brassinolide (Xi et al., 2013), and methyl jasmonate (MJ; Portu et al., 2017).
Methyl jasmonate, a signal molecule applied as a chemical elicitor, induces plant defense mechanisms, leading to the synthesis of secondary metabolites (Beckers & Spoel, 2006). MJ has also been shown to affect antioxidant and defense enzymes involved in phenolic metabolism, including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) (Belhadj et al., 2006). Hence, the effect of MJ application on phenolic content and antioxidant activity to different fruits, such as raspberry , black currant (Flores & Ruiz Del Castillo, 2016), sweet cherry (Castillo et al., 2014;Saracoglu et al., 2017), kiwifruit (Öztürk & Yücedağ, 2021), medlar (Ozturk et al., 2019), plum (Karaman et al., 2013), apple , and grape wine (Portu et al., 2015) has been studied. In this context, MJ has most often been applied several times at veraison in grapes, which is considerably expensive Portu et al., 2016;. It has been shown that the content of jasmonates in grape skin decreases along with ripening (Kondo & Fukuda, 2001). Likewise, a significant decline in phenolic content of grape skin during ripening has been reported (Obreque-Slier et al., 2010), which might be associated with the mitigation of jasmonates in this process. For this reason, the application of jasmonates as a supplement near harvest time could be effective to improve berry phenolics.
To our knowledge, little information is available on a single MJ treatment as a supplement near harvest time, evaluating the residual effects on phenolic metabolism in grapes, especially table grapes, after harvest. Therefore, this work aimed to assess the impact of MJ in three different concentrations (1, 5, and 10 mM) with control applied 2 days before harvest on polyphenols and antioxidant capacity on the "Thompson Seedless" table grapes.

| Field treatments and storage
The experiment was carried out at Malayer Grape Research Station located in Hamedan, Iran (34º15′49.2′′N, 48º48′09.4′′E), using "Thompson Seedless" grapes (Vitis vinifera L.). MJ solution in different concentrations (0, 1, 5, and 10 mM) was prepared in water and Tween-80. All treatments were applied 2 days before harvest by spraying on the clusters to run-off. To determine berry maturity, the total soluble solids (TSS) of the grape juice were controlled.
Commercially matured clusters were picked and transferred immediately to the lab.
After one night precooling, intact berries with homogeneous size were selected randomly from the whole part of each bunch. About 35 clusters were allocated to each treatment. Each replicate was kept in perforated plastic clamshell containers at 15°C and 80% RH for 6 days in darkness. Twenty healthy berries were selected out of each container every day, peeled, and the skin was ground to a powder with liquid nitrogen before keeping at −80°C.

| Determination of phenolic content
Extraction of phenolic compounds was performed by the method of Li et al. (2015). Briefly, the grape powder was macerated in the extraction solvent including methanol/acetone/water (3.5:3.5: with 1% (v/v) acetic acid glacial, and centrifuged after shaking for 30 min in the dark at room temperature (RT). The supernatant was applied for phenolic and antioxidant measurements.
Total phenol content (TPC) of the extract was determined as described by Slinkard and Singleton (1977) and expressed as mg gallic acid equivalent (GAE) per gram fresh weight (fw). Shortly, an aliquot of the extract and Folin's reagent were mixed with sodium carbonate, shaken for 90 min, and then the absorbance was read at 765 nm by a spectrophotometer (Varian Cary 100 UV-Vis, US).
Total flavonoid content (TFC) of the extract was measured as developed by Yoo et al. (2008) and expressed as mg rutin equivalent (RE) per g fw. In short, an aliquot of the extract was diluted with distilled water and mixed with sodium nitrite 5%, aluminum chloride 10%, and sodium hydroxide. The absorbance of the final solution was recorded at 510 nm.
For total tannin content (TTC), the Folin-Denis method was followed (Taira, 1995) and expressed as mg tannic acid equivalent (TAE) per g fw. Briefly, diluted Folin's reagent 1 N was mixed with saturated sodium carbonate, shaken for 60 min, and read spectrophotometerically at 725 nm.

| Determination of antioxidant activity
The antioxidant activity (AA) of the extracts was determined via three different methods. DPPH radical scavenging potential was measured as proposed by Bertelli et al. (2014). An aliquot of the skin extract and/or water (as control) was mixed with DPPH radical solution, vortex-mixed well, and kept for 30 min at RT in the dark. The absorbance was read at 515 nm and the result was expressed as a percentage of inhibitory capacity of DPPH radical.
The ABTS assay was carried out following the method developed by Szymanowska et al. (2015). For making the ABTS radical solution, ABTS 7 mM was mixed with potassium persulfate 2.45 mM (1:1) and incubated at RT overnight. The working mixture was diluted to make an absorbance of 0.911 at 734 nm. The extract was then mixed with ABTS radical solution. Distilled water was considered as a control.
The ABTS radical scavenging ability of extract was expressed as a percentage of inhibitory capacity of ABTS radical.
FRAP assay was performed as followed by Benzie and Strain (1996). In brief, the working solution was prepared by mixing acetate buffer (300 mM, pH 3.6), TPTZ 10 mM solution with HCl 40 mM, and FeCl 3 -6H 2 O 20 mM (10:1:1 v/v/v). Then, the extract was mixed with the working solution and incubated at 37°C in a bain-marie for 10 min. The absorbance was recorded at 593 nm and the result was expressed as the ability to reduce 1 mM equivalent Fe(+2) to Fe(+3) per g fw.

| Determination of enzyme activity
For enzyme extraction, 400 µg of the frozen sample was suspended in extraction solution including phosphate buffer (0.05 M, pH 6.8), triton X-100 (0.05% v/v), 2-mercaptoethanol (5 mM), and PVP (0.1% w/v) (Galli et al., 2009). This mixture was centrifuged and the clear supernatant was collected as enzyme extract. The whole process was carried out at 4°C.
The PAL activity was evaluated according to Zhu et al. (2016).
Two hundred microliters of the enzyme extract was diluted by 0.2 M borate buffer. Then, 0.02 M L-PA was added, and this mixture was left to incubate at 37°C. After 30 min, 6 M HCl was added. The absorbance of the samples was recorded at 290 nm.
The activity of PPO and POD was measured as described by Christopoulos and Tsantili (2015), but with slight modifications. For the PPO activity assay, the crude extract was diluted with 0.05 M phosphate buffer (pH 6.2). Before 2-min incubation at 25°C, catechol solution (0.05 M) was added to this mixture. Finally, the changes in absorbance were followed at 420 nm.
The POD activity was analyzed by guaiacol oxidation. The reaction solution containing the extract, 0.025 M guaiacol (in 0.1 mM phosphate buffer, pH 6) and H 2 O 2 0.020 M, was incubated for 2 min at 25°C while monitoring the changes in absorbance spectrophotometerically at 470 nm.
The activity of the enzyme was expressed as an enzyme unit (the enzyme content leading to an increase of 0.001 absorbances at the relative wavelength per min under the circumstances described) per g fw.
Phenolic compounds were analyzed using an HPLC (Ultimate 3000, Dionex), equipped with a Corona ® CAD ® (Charged Aerosol Detector), an automatic sampler, and a four-channel pump. The injection volume was 10 µl. Separation of compounds was accomplished by a 250 × 4.6 mm, 5 µm, RP-18 column (Hypersil GOLD TM , Thermo Scientific, US) adjusted at 30 • C. TFA 0.1% in water (eluent A) and acetonitrile (eluent B) were used as mobile phases with a flow rate of 1 ml/min. The gradient began with 5% eluent A, reaching 30% at 20 min, 40% at 25 min, 100% at 35 min, and 5% at 37-45 min.
The compounds were identified according to the retention time and their spectra. The amount of phenolic compounds was determined by using the corresponding standard curve.

| Statistical analysis
Effects of MJ and storage time were determined by ANOVA.
Significant difference among treatments was assessed according to Duncan's multiple tests (p ≤ .05). All analyses were carried out using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). All measurements were performed in triplicate.

| Phytochemical analysis
As seen for total phenol in Figure 1. blackberries (Wang et al., 2008), and blueberries (Percival & MacKenzie, 2007). Increased phenolics during the short storage, which has previously been noticed in grapes (Maurer et al., 2017), could be a resultant organic acid decomposition providing carbons needed for phenolic formation (Kalt et al., 1999). In the present study, however, all samples subsequently experienced a reduction at the end of storage, which was slower for those of control than treated skins. The reversal of trend in TPC and TFC during storage in both treated and control berries after 5 days may be owing to the degradation and oxidation process backed by PPO and POD .
In terms of tannin, no significant differences were found when treated and control skin was evaluated during the storage time (data not shown). One of the important attributes of ripe table grapes is tannin-related astringency. Condensed tannins are commonly comprised of catechin and epicatechin monomers, and the complex structure resulted from these compounds and proanthocyanidins (Zucker, 1983). Due to their effect on the tannin content in wines, comprehensive studies have been done on wine grapes (Gawel et al., 2001). In contrast, astringency is an undesirable feature in table grapes (Dokoozlian, 2000). Therefore, our result suggested that taste of the berries might not be affected by MJ after harvest. Moreover, following a clone-dependent manner, it has been proposed that other phenolics could be favored at the expense of tannins (Portu et al., 2015). In this regard, our results showed that preharvest application of MJ increased flavonoid content, while no significant change was detected for tannin content.

| Antioxidant analysis
As presented in Figure 2. For this reason, enhanced AA of MJ-treated grapes might contribute to higher flavonoid content observed in this work.
In addition, it has already been indicated that the antioxidant potential of horticultural products is associated with phenolic content (Heredia & Cisneros-Zevallos, 2009;Saracoglu et al., 2017).
Advocating for this idea, AA in our study followed the same trend With high levels of AA, these findings in our study suggest enhanced remedial potential of MJ-treated grapes, when compared with the control.

| Enzyme analysis
Changes in PAL activity were significant over storage time  (Table 1).
Surprisingly, MJ treatment led to a noticeably higher POD activity with respect to control; however, all samples presented an increasing trend during storage (Table 1).
PAL has already been well known to be an initial enzyme in the biosynthesis of phenols (Pina & Errea, 2008). According to the literature, PAL activity can be stimulated upon different stress conditions (Dixon & Paiva, 1995). Besides grapes, increased activity of PAL after following MJ treatment has already been reported in guava, blackberry, and radish sprout ( An increment in enzyme activity during storage might be due to a de novo increase in the activity of enzyme precursors. It also can be attributed to the regeneration of enzymes over time (Christopoulos & Tsantili, 2015). Reduction in such enzymes observed at the end of storage brings about ROS accumulation which cause damage to DNA and RNA, and also peroxidation of membrane followed by early senescence (Hodges et al., 2004).
Similar to the result presented here, it has been demonstrated that JA and MJ activate the stress response paths and consequently induce PPO and POD activity (Boughton et al., 2006).

| HPLC analysis
For a better understanding of the phenolic composition and their metabolism, a detailed study was carried out through HPLC. Results of phenolic analysis are presented in Table 2.
According to ANOVA, catechin content was not affected significantly upon MJ treatment during storage (data not presented), which is inconsistent with the result of Portu et al. (2017) who found no difference between control and MJ-treated grapes in the catechin and epicatechin content even when applying a precursor, phenylalanine.
Furthermore, no response of such monomeric procyanidins to MJ treatment was detected in the "Fuji" apple (Rudell et al., 2002). As also with epicatechin, in the present study, the absence of difference between treatments and control was denoted. What is more, attenuation of the epicatechin was brought about by the highest dose of MJ (10 mM). The negative effect of MJ, as previously reported by Rudell et al. (2002), can be elucidated by MJ-induced PPO activity (Masia et al., 1998). Concomitant with this result, Portu et al. (2015) demonstrated that foliar application of MJ had no effect on flavanols in grape berries and the wine made from them. In this vein, as mentioned earlier, flavonol biosynthesis enzymes could be activated instead of those for flavanol synthesis, which is clone dependent . It should be noted that this explanation is in accordance with the result of tannin content. on rutin was kept until the end of storage. In general, higher flavonol content was obtained from grapes that had been exposed to MJ.
Preceding experiments on grapes have demonstrated that application of MJ could increase some individual flavonols; however, total flavonol was not influenced Ruiz-Garcia et al., 2012). Flores and Ruiz Del Castillo (2016), by contrast, indicated that preharvest application of 0.2 µM MJ resulted in an increase in total flavonol, although individual flavonols were not affected during storage.
It has previously been reported that spray application of MJ on raspberries and blackcurrants caused quercetin accumulation . Since flavonol synthesis follows phenylpropanoid pathway, it is deemed that MJ-induced PAL activity affects the accumulation of flavonols (Flores & del Castillo, 2014).
Results for resveratrol analysis are illustrated in Table 2.
Surprisingly, only a trace of the content of resveratrol was detected in the skin and remained unchanged during the whole storage.
This phenomenon was previously explained by Garrido and Borges (2013). These authors stated that resveratrol content is depleted along with maturity. Alleviation of resveratrol content from veraison to maturity contributed to the competition between two biosynthesis pathways triggered by CHS and STS (Jeandet et al., 1995). As can be seen, at day 1 after harvest, resveratrol content enhanced two-to threefold by 10 mM MJ treatment. Five and ten millimeter MJ showed an increasing trend during storage (1.7-and 3.7-fold for 5 and 10 mM MJ, respectively). However, the values in 10 mM MJ were much higher than those in 5 mM, indicating that 10 mM was far more effective than other doses. In contrast, resveratrol decreased in 1 mM MJ and disappeared on day 3, suggesting a short-term effect of a low dose of MJ.  , 2016). Furthermore, other results from preharvest treatment in different ways, either bunch or foliar application, also confirmed the inducing effect of MJ on stilbene content in both berries and wine (Fernandez-Marin et al., 2014;Portu et al., 2015). Based on these findings, resveratrol biosynthesis is believed to be stimulated by the MJ-activated STS gene that encodes STS, a pivotal enzyme involved in resveratrol synthesis (Xu et al., 2015). Similar results were also reported through in vitro studies under MJ application (Belhadj et al., 2008;Tassoni et al., 2005). However, further investigation into this research area is warranted.

| CON CLUS ION
The present study showed that a single MJ treatment close to the harvest increased phenolic contents during short-term storage. The effectiveness of the dose depended on the factor evaluated, but 10 mM MJ was more efficient in most factors examined. It seems that this role was accomplished by induction of antioxidant enzymes and the expression of their respective genes, therefore leading to the promoted nutritional values and the decreased oxidative detriment.
These findings suggest that the application of MJ in this way could be a useful method for enhancing health-promoting compounds.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict 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 are available within the article.