Application of Synephrine to Grape Increases Anthocyanin via Production of Hydrogen Peroxide, Not Phytohormones

Global warming has caused such problems as the poor coloration of grape skin and the decreased production of high-quality berries. We investigated the effect of synephrine (Syn) on anthocyanin accumulation. Anthocyanin accumulation in cultured grape cells treated with Syn at concentrations of 1 mM or higher showed no significant difference, indicating that the accumulation was concentration-independent. On the other hand, anthocyanin accumulation was dependent on the compound used for treatment. The sugar/acid ratio of the juice from berries treated with Syn did not differ from the control. The expression of anthocyanin-biosynthesis-related genes, but not phytohormones, was increased by the treatment with Syn at 24 h or later. The Syn treatment of cultured cells increased SOD3 expression and hydrogen peroxide (H2O2) production from 3 to 24 h after treatment. Subsequently, the expression of CAT and APX6 encoding H2O2-scavenging enzymes was also increased. Treatment of cultured cells with Syn and H2O2 increased the expression of the H2O2-responsive gene Chit4 and the anthocyanin-biosynthesis-related genes mybA1 and UFGT 4 days after the treatment and increased anthocyanin accumulation 7 days after the treatment. On the other hand, the treatment of berries with Syn and H2O2 increased anthocyanin accumulation after 9 days. These results suggest that Syn increases anthocyanin accumulation through H2O2 production without changing phytohormone biosynthesis. Syn is expected to improve grape skin coloration and contribute to high-quality berry production.


Introduction
Preventing the global-warming-induced decrease in crop quality is an urgent issue.Grapevine (Vitis spp.) is an economically important plant widely grown globally for wine production and consumption as table grapes.The increase in average temperature due to global warming has decreased grape skin coloration by inhibiting anthocyanin accumulation [1,2].It is predicted that further increases in average temperature would cause significant economic damage not only to grape growers but also the winemaking industry [3].Thus, it is necessary to prevent the decrease in grape skin coloration due to global warming.
Simple cultivation techniques for preventing grape skin coloration decrease are desired because the existing methods, such as girdling [4], leaf removal [5,6], and cluster thinning [7], require specific skills and intensive labor.The direct application of biologically active compounds that increase grape skin coloration, such as allantoin [8], amino acids [9], and vanillyl acetone [10], has stirred up interest in recent years.Therefore, we screened for biologically active compounds and found that synephrine (Syn) increases grape skin coloration (Enoki, personal communication).Syn (4-[1-hydroxy-2-(methylamino)ethyl]phenol) is an alkaloid with a phenethylamine skeleton.It is found in some orange species [11,12] and used as a dietary supplement because of its lipolytic effect [13,14].However, there are

Syn Increases Anthocyanin Accumulation in VR Cells
We used VR (Vitis Red) cells to investigate the effect of Syn on anthocyanin accumulation (Figure 1).Anthocyanin content was significantly higher in VR cells treated with Syn concentrations of 1 mM or higher than in the control (n = 3, Tukey, p < 0.01 or 0.05; Figure 1a).We found no significant difference among VR cells treated with Syn concentrations of 1 mM or higher.
Syn is biosynthesized through the phenylalanine (Phe) and tyrosine (Tyro) pathways via L-(-)-tyrosine (L-Tyro), tyramine (Tyra), and octopamine hydrochloride (Oct) using Phe as the substrate [27].Anthocyanin accumulation tended to decrease in the order of Phe, Syn, Oct, Tyra, and L-Tyro treatments.Anthocyanin contents were significantly higher in Syn-and Phe-treated VR cells than in the control (n = 3, Dunnett, p < 0.05 or 0.01; Figure 1b).The results indicate that Syn increased anthocyanin accumulation in VR cells in a concentration-independent and molecular-structure-specific manner.
orange species [11,12] and used as a dietary supplement because of its lipolytic effect [13,14].However, there are no reports on its effects on crops.To develop new technology for increasing grape skin coloration during ripening, it is necessary to examine and clarify the mechanism of the coloration effect of Syn.
In this study, we clarified the mechanism of Syn-mediated skin coloration for grape quality improvement.We performed field studies on the coloration effects of Syn and measured gene expression in berries and cultured cells.Contrary to our hypothesis, we found that Syn increases anthocyanin accumulation via the production of hydrogen peroxide (H2O2), not phytohormones.We propose a mechanism underlying Syn-mediated grape skin coloration and discuss the coloration effect of H2O2.

Syn Increases Anthocyanin Accumulation in VR Cells
We used VR (Vitis Red) cells to investigate the effect of Syn on anthocyanin accumulation (Figure 1).Anthocyanin content was significantly higher in VR cells treated with Syn concentrations of 1 mM or higher than in the control (n = 3, Tukey, p < 0.01 or 0.05; Figure 1a).We found no significant difference among VR cells treated with Syn concentrations of 1 mM or higher.
Syn is biosynthesized through the phenylalanine (Phe) and tyrosine (Tyro) pathways via L-(-)-tyrosine (L-Tyro), tyramine (Tyra), and octopamine hydrochloride (Oct) using Phe as the substrate [27].Anthocyanin accumulation tended to decrease in the order of Phe, Syn, Oct, Tyra, and L-Tyro treatments.Anthocyanin contents were significantly higher in Syn-and Phe-treated VR cells than in the control (n = 3, Dunnett, p < 0.05 or 0.01; Figure 1b).The results indicate that Syn increased anthocyanin accumulation in VR cells in a concentration-independent and molecular-structure-specific manner.

Syn Increases Anthocyanin Accumulation in Grape Skin in Field Trials
We investigated whether Syn promotes grape ripening in the field by conducting field trials in 2019 and 2021 (Figure 2a).In 2019, anthocyanin content was significantly higher in Syn-treated berries than in the control on days 10 and 20 after treatment (n = 3, t-test, p < 0.01 or 0.05; Figure 2b).Similarly, in 2021, anthocyanin content was significantly higher in Syn-treated berries than in the control on day 20 after treatment (n = 3, t-test, p < 0.01; Figure 2c).However, the sugar/acid ratio, a ripeness index, was not significantly different between Syn-treated berries and the control, even though the scale on the y-axis differed between the two years (Figure 2d,e).The results indicate that Syn increased anthocyanin accumulation in grape skin but not berry ripening. (a)

Syn Increases Anthocyanin Accumulation in Grape Skin in Field Trials
We investigated whether Syn promotes grape ripening in the field by conducting field trials in 2019 and 2021 (Figure 2a).In 2019, anthocyanin content was significantly higher in Syn-treated berries than in the control on days 10 and 20 after treatment (n = 3, t-test, p < 0.01 or 0.05; Figure 2b).Similarly, in 2021, anthocyanin content was significantly higher in Syn-treated berries than in the control on day 20 after treatment (n = 3, t-test, p < 0.01; Figure 2c).However, the sugar/acid ratio, a ripeness index, was not significantly different between Syn-treated berries and the control, even though the scale on the y-axis differed between the two years (Figure 2d,e).The results indicate that Syn increased anthocyanin accumulation in grape skin but not berry ripening.

Syn Increases Anthocyanin Accumulation in Grape Skin in Field Trials
We investigated whether Syn promotes grape ripening in the field by conducting field trials in 2019 and 2021 (Figure 2a).In 2019, anthocyanin content was significantly higher in Syn-treated berries than in the control on days 10 and 20 after treatment (n = 3, t-test, p < 0.01 or 0.05; Figure 2b).Similarly, in 2021, anthocyanin content was significantly higher in Syn-treated berries than in the control on day 20 after treatment (n = 3, t-test, p < 0.01; Figure 2c).However, the sugar/acid ratio, a ripeness index, was not significantly different between Syn-treated berries and the control, even though the scale on the y-axis differed between the two years (Figure 2d,e).The results indicate that Syn increased anthocyanin accumulation in grape skin but not berry ripening.
Upstream of the flavonoid biosynthetic pathway, the relative expression of CHS encoding chalcone synthase [EC 2.3.1.74]in Syn-treated VR cells was significantly different from that in the control only at 72 h after treatment, whereas the relative expression of CHI encoding chalcone isomerase [EC 5.5.1.6]showed a significant difference as early as 24 h after treatment (n = 4, t-test, p < 0.01).Midstream of the flavonoid biosynthetic pathway, the relative expression of F3'H encoding flavonoid 3 ′ -monooxygenase [EC 1.14.14.82] and F3'5'H encoding flavonoid 3 ′ ,5 ′ -hydroxylase [EC 1.14.14.81], which are related to red and blue anthocyanin pigment biosynthesis, differed in Syn-treated VR cells; F3'H showed a significant difference from the control at 48 h after treatment or later, whereas F3'5'H showed a significant difference at 96 h or later (n = 4, t-test, p < 0.01 or 0.05).The relative expression of F3H encoding flavanone 3-hydroxylase [EC 1.14.11.9] in Syn-treated VR cells was significantly different from that in the control at 48 h after treatment or later (n = 4, t-test, p < 0.01 or 0.05).Downstream, the relative expression of DFR encoding dihydroflavonol 4-reductase [EC 1.1.1.219]and LDOX encoding leucoanthocyanidin dioxygenase [EC 1.14.20.4] in Syntreated cells was significantly different from those in the control at 72 h after treatment or later (n = 4, t-test, p < 0.01).In the flavonoid biosynthetic pathway, significant differences in the relative expression levels of these genes were observed in the early stages of the pathway.
significant differences in the relative expression levels of these genes were observed in the early stages of the pathway.The relative expression of UFGT encoding UDP-glucose:anthocyanidin/flavonol 3-Oglucosyltransferase [EC 2.4.1.115],a key enzyme in the anthocyanin biosynthetic pathway [28], and its transcription factor mybA1 encoding Myb-related transcription factor A1 [29] was analyzed.The relative expression of mybA1 in Syn-treated VR cells was significantly higher than that in the control from 24 h after treatment, and that of UFGT from 48 h after treatment (n = 4, t-test, p < 0.01 or 0.05).Overall, the results demonstrate that Syn increased the expression of genes in the anthocyanin-biosynthesis-related pathways as early as 24 h after treatment.The relative expression of UFGT encoding UDP-glucose:anthocyanidin/flavonol 3-O-glucosyltransferase [EC 2.4.1.115],a key enzyme in the anthocyanin biosynthetic pathway [28], and its transcription factor mybA1 encoding Myb-related transcription factor A1 [29] was analyzed.The relative expression of mybA1 in Syn-treated VR cells was significantly higher than that in the control from 24 h after treatment, and that of UFGT from 48 h after treatment (n = 4, t-test, p < 0.01 or 0.05).Overall, the results demonstrate that Syn increased the expression of genes in the anthocyanin-biosynthesis-related pathways as early as 24 h after treatment.

Syn Does Not Increase the Production of Phytohormones That Promote Anthocyanin Accumulation
We measured the relative expression of NCED1 encoding 9-cis-epoxycarotenoid dioxygenase [EC 1.13.11.51] and ACS3 encoding 1-aminocyclopropane-1 carboxylate synthase [EC 4.4.1.14],the rate-limiting enzymes of ABA and ET, respectively, in VR cells.We found that Syn did not increase NECD1 expression or ABA content at 24 h after treatment (Figure 4a,b).The relative expression of ACS3 in Syn-treated VR cells was not significantly different from that in the control at 0 and 12 h after treatment but was significantly different at 24 h after treatment (n = 4, t-test, p < 0.05) (Figure 4c).Because of technical difficulties in the quantification of volatile gas ET, we measured the relative expression level of ACO2 encoding aminocyclopropanecarboxylate oxidase [EC 1.14.17.4], a key enzyme in ET biosynthesis, and found that the expression was not significantly different between Syn-treated VR cells and the control from 0 to 24 h after treatment (Figure 4d).

Syn Does Not Increase the Production of Phytohormones That Promote Anthocyanin Accumulation
We measured the relative expression of NCED1 encoding 9-cis-epoxycarotenoid dioxygenase [EC 1.13.11.51] and ACS3 encoding 1-aminocyclopropane-1 carboxylate synthase [EC 4.4.1.14],the rate-limiting enzymes of ABA and ET, respectively, in VR cells.We found that Syn did not increase NECD1 expression or ABA content at 24 h after treatment (Figure 4a,b).The relative expression of ACS3 in Syn-treated VR cells was not significantly different from that in the control at 0 and 12 h after treatment but was significantly different at 24 h after treatment (n = 4, t-test, p < 0.05) (Figure 4c).Because of technical difficulties in the quantification of volatile gas ET, we measured the relative expression level of ACO2 encoding aminocyclopropanecarboxylate oxidase [EC 1.14.17.4], a key enzyme in ET biosynthesis, and found that the expression was not significantly different between Syntreated VR cells and the control from 0 to 24 h after treatment (Figure 4d).We found that the relative expression of LOX encoding linoleate 13S-lipoxygenase [EC 1.13.11.12], the rate-limiting enzyme in the JA biosynthetic pathway, was significantly different between Syn-treated VR cells and the control at 12 h after treatment (n = 4, t-test, p < 0.05, Figure 4e).We also investigated the effect of Syn on the biosynthesis of JA, a phytohormone that increases berry skin coloration and disease resistance.Endogenous JA content in Syn-treated VR cells was not significantly different from that in the control at 24 h after treatment (Figure 4f).The results indicate that Syn is not involved in the biosynthesis of phytohormones that increase skin coloration.

Syn Increases Anthocyanin Content via H 2 O 2
The relative expression of SOD3 encoding the H 2 O 2 -generating enzyme superoxide dismutase [EC 1.15.1.1]was significantly higher in Syn-treated VR cells than in the control as early as 3 h to 12 h after treatment.H 2 O 2 content in the Syn-treated cells was significantly higher than that in the control from 3 h to 24 h (n = 4, t-test, p < 0.01 or 0.05; Figure 5a,b).We also measured the relative expression of APX6 and CAT encoding H 2 O 2 -scavenging enzymes ascorbate peroxidase [EC 1.11.1.11]and catalase [EC 1.11.1.6],respectively, as H 2 O 2 -responsive genes.The relative expression of APX6 was significantly higher (n = 4, t-test, p < 0.01 or 0.05) at 24 h, and that of CAT at 24 h and 48 h, after Syn treatment compared with the control (Figure 5c,d We found that the relative expression of LOX encoding linoleate 13S-lipoxygenase [EC 1.13.11.12], the rate-limiting enzyme in the JA biosynthetic pathway, was significantly different between Syn-treated VR cells and the control at 12 h after treatment (n = 4, t-test, p < 0.05, Figure 4e).We also investigated the effect of Syn on the biosynthesis of JA, a phytohormone that increases berry skin coloration and disease resistance.Endogenous JA content in Syn-treated VR cells was not significantly different from that in the control at 24 h after treatment (Figure 4f).The results indicate that Syn is not involved in the biosynthesis of phytohormones that increase skin coloration.

Syn Increases Anthocyanin Content via H2O2
The relative expression of SOD3 encoding the H2O2-generating enzyme superoxide dismutase [EC 1.15.1.1]was significantly higher in Syn-treated VR cells than in the control as early as 3 h to 12 h after treatment.H2O2 content in the Syn-treated cells was significantly higher than that in the control from 3 h to 24 h (n = 4, t-test, p < 0.01 or 0.05; Figure 5a,b).We also measured the relative expression of APX6 and CAT encoding H2O2-scavenging enzymes ascorbate peroxidase [EC 1.11.1.11]and catalase [EC 1.11.1.6],respectively, as H2O2-responsive genes.The relative expression of APX6 was significantly higher (n = 4, t-test, p < 0.01 or 0.05) at 24 h, and that of CAT at 24 h and 48 h, after Syn treatment compared with the control (Figure 5c,d).We measured the relative expression of H 2 O 2 -responsive gene Chit4 encoding class 4 chitinase [EC 3.2.1.14]in VR cells and found that it was significantly higher (n = 4, Dunnett, p < 0.01) 4 days after the treatment with Syn and H 2 O 2 than in the control (Figure 6a).Similarly, the relative expression of mybA1 and UFGT in VR cells showed a significant increase 4 days after the treatment with Syn and H 2 O 2 , and anthocyanin content was significantly higher 7 days after the treatment (n = 4, Dunnett, p < 0.01 or 0.05) than in the control (Figure 6b-d).The anthocyanin content in berry skin increased 9 days after the treatment with Syn and H 2 O 2 (n = 4, Dunnett, p < 0.01 or 0.05) (Figure 6e).The results show that Syn increased anthocyanin accumulation via H 2 O 2 .
We measured the relative expression of H2O2-responsive gene Chit4 encoding class 4 chitinase [EC 3.2.1.14]in VR cells and found that it was significantly higher (n = 4, Dunnett, p < 0.01) 4 days after the treatment with Syn and H2O2 than in the control (Figure 6a).Similarly, the relative expression of mybA1 and UFGT in VR cells showed a significant increase 4 days after the treatment with Syn and H2O2, and anthocyanin content was significantly higher 7 days after the treatment (n = 4, Dunnett, p < 0.01 or 0.05) than in the control (Figure 6b-d).The anthocyanin content in berry skin increased 9 days after the treatment with Syn and H2O2 (n = 4, Dunnett, p < 0.01 or 0.05) (Figure 6e).The results show that Syn increased anthocyanin accumulation via H2O2.

Discussion
We propose a mechanism by which Syn increases anthocyanin accumulation via H 2 O 2 production and not phytohormones to solve the problem of poor grape skin coloration due to global warming (Figure 7).

Discussion
We propose a mechanism by which Syn increases anthocyanin accumulation via H2O2 production and not phytohormones to solve the problem of poor grape skin coloration due to global warming (Figure 7).We showed that Syn increases SOD3 at a very early stage of treatment and generates H2O2.The Syn analog β-phenylethylethylamine promotes the immediate and transient generation of H2O2 as a product of the phenylethylamine degradation reaction by monoamine oxidase (MAO) in yeast [30,31], tobacco [32,33], and mesenchymal stem cells [34].These findings suggest that Syn is an early inducer of H2O2 in grape cells, similar to its analog β-phenylethylethylamine.
We revealed that Syn increases anthocyanin accumulation in grapes in the same manner as the treatment with H2O2.The accumulation of the antioxidant anthocyanin confers H2O2-mediated oxidative stress tolerance to plants [35].Consistent with our results, H2O2 increases anthocyanin accumulation in many plant species including grapes [36][37][38][39][40].In addition, anthocyanin from apple peel can remove H2O2 better than other phenolics [41].On the other hand, Chit4 expression can be considered a marker of H2O2-mediated oxidative stress response [42][43][44].Our finding that Syn and H2O2 upregulated the expression of Chit4 and anthocyanin-biosynthesis-related genes, which in turn increased anthocyanin accumulation in grapes, suggests that Syn increases the accumulation of the antioxidant anthocyanin by inducing H2O2-mediated oxidative stress in grape cells.However, the balance between H2O2 production and removal is strictly regulated because excess H2O2 causes oxidative injury to cells [45,46].Therefore, we assume that balancing H2O2 production and oxidative stress by regulating Syn concentration is important for H2O2-mediated anthocyanin accumulation by Syn because excess H2O2 may lead to anthocyanin degradation.
We indirectly showed that Syn did not increase ABA, ET, and JA contents, nor did it increase the sugar/acid ratio, a ripeness index related to ABA and ET.Syn only increased anthocyanin accumulation in grape berries.On the basis of these findings, we would like to emphasize that Syn-derived H2O2 is a useful coloration factor independent of phytohormones.Previous studies have focused on phytohormones to improve grape skin coloration [4,6,9,[17][18][19][20][21].H2O2 is a signaling molecule that regulates physiological processes such as plant growth and stress response, and crosstalk exists between H2O2 and phytohormones [45,47,48].This crosstalk requires further evaluation.We showed that Syn increases SOD3 at a very early stage of treatment and generates H 2 O 2 .The Syn analog β-phenylethylethylamine promotes the immediate and transient generation of H 2 O 2 as a product of the phenylethylamine degradation reaction by monoamine oxidase (MAO) in yeast [30,31], tobacco [32,33], and mesenchymal stem cells [34].These findings suggest that Syn is an early inducer of H 2 O 2 in grape cells, similar to its analog β-phenylethylethylamine.
We revealed that Syn increases anthocyanin accumulation in grapes in the same manner as the treatment with H 2 O 2 .The accumulation of the antioxidant anthocyanin confers H 2 O 2 -mediated oxidative stress tolerance to plants [35].Consistent with our results, H 2 O 2 increases anthocyanin accumulation in many plant species including grapes [36][37][38][39][40].In addition, anthocyanin from apple peel can remove H 2 O 2 better than other phenolics [41].On the other hand, Chit4 expression can be considered a marker of H 2 O 2 -mediated oxidative stress response [42][43][44].Our finding that Syn and H 2 O 2 upregulated the expression of Chit4 and anthocyanin-biosynthesis-related genes, which in turn increased anthocyanin accumulation in grapes, suggests that Syn increases the accumulation of the antioxidant anthocyanin by inducing H 2 O 2 -mediated oxidative stress in grape cells.However, the balance between H 2 O 2 production and removal is strictly regulated because excess H 2 O 2 causes oxidative injury to cells [45,46].Therefore, we assume that balancing H 2 O 2 production and oxidative stress by regulating Syn concentration is important for H 2 O 2 -mediated anthocyanin accumulation by Syn because excess H 2 O 2 may lead to anthocyanin degradation.
We indirectly showed that Syn did not increase ABA, ET, and JA contents, nor did it increase the sugar/acid ratio, a ripeness index related to ABA and ET.Syn only increased anthocyanin accumulation in grape berries.On the basis of these findings, we would like to emphasize that Syn-derived H 2 O 2 is a useful coloration factor independent of phytohormones.Previous studies have focused on phytohormones to improve grape skin coloration [4,6,9,[17][18][19][20][21].H 2 O 2 is a signaling molecule that regulates physiological processes such as plant growth and stress response, and crosstalk exists between H 2 O 2 and phytohormones [45,47,48].This crosstalk requires further evaluation.
As one of the limitations of this study, we were unable to consider alternative pathways to MAO for the generation of H 2 O 2 from Syn.This is because there are no reports of Syn analogs producing H 2 O 2 directly or indirectly via SOD except for the results of this study.The quantification of superoxide and SOD activities is needed to clarify this.He et al. (2020) reported a Syn-HCl-mediated reduction of H 2 O 2 levels in postharvest litchi, in contrast to our findings in grapes [49].These differences in results may be attributed to differences in Syn concentration, species-specific metabolic pathways, and H 2 O 2 mitigation mechanisms.Furthermore, the physiological state of the fruit before and after harvest may influence these results.Comprehensive comparative studies with different plant species and growth stages are needed to elucidate species-specific responses and verify the broad applicability of Syn.
Syn has the potential to improve grape skin coloration by increasing H 2 O 2 production without undesirable side effects such as defoliation [20] caused by ABA agents.Although there is concern about it being a health hazard because of its structural similarity to the doping agent ephedrine, Syn can be safely used as a dietary supplement [50,51].The usefulness and safety of Syn as a grape color-enhancing agent should be evaluated by further field trials and H 2 O 2 residual analysis.Syn application is expected to contribute to viticulture and the wine industry.

In Vitro Trials
Cultured grape cells (VR cells, PRC00003) were provided by the RIKEN BioResource Center Research (RIKEN BRC) through the National BioResource Project of MEXT/AMED, Japan.The cell line was derived from Vitis hybrid cv.Bailey Alicante A, which has high anthocyanin-biosynthesizing ability [52].Modified Linsmaier and Skoog (LS) medium (pH 6.1) containing 3% (w/v) sucrose, 0.05 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 g/L kinetin was used.The medium was autoclaved (1.06 kg cm −2 ) at 121 • C for 15 min, gelled with 1.2% (w/v) agar, and poured into disposable sterile plastic Petri dishes.Only white VR cells without red coloration were subcultured every week under sterile conditions and grown in a dark incubator at 27 • C.

Field Trials
Vitis vinifera cv.Syrah grapevines in the experimental vineyard (2019, 2021) of the Institute of Enology and Viticulture and an affiliated farm (2022) of the University of Yamanashi (at 35 • N, 138 • E in Yamanashi, Japan) were used.The grapevines were approximately 30 years old and grown using the double-cordon-style training method.
A solution of 1 mM Syn with 0.01% (v/v) Approach BI (Kao, Tokyo, Japan) was prepared.The grapevines were defoliated in the berry zone before veraison and sprayed with 500 mL of water (control) or Syn solution per grapevine at veraison (30 July 2019; 18 July 2021).Thereafter, grape bunches were sampled every 10 days.The bunches were photographed and stored at −80 • C until RNA analyses.
Solutions of 1 mM Syn and 300 mM H 2 O 2 with 0.01% (v/v) Approach BI (Kao, Tokyo, Japan) were prepared (10 August 2022).Nine grape bunches were randomly selected from One gram of crushed skin (or weighed VR cells) was immersed in 10 mL (or 500 µL) of 1% HCl-methanol overnight in the dark.The mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was diluted with 1% HCl-methanol to bring it within the absorbance measurement range.After mixing, absorbance was measured at 520 nm using a spectrophotometer (ASV11D-S, AS ONE, Osaka, Japan).Total anthocyanin content (malvidin-3-O-glucoside equivalent) in skin and VR cells was calculated using a published formula [58].

Sugar/Acid Ratio
Ten berries per bunch were pressed to obtain grape juice.The juice was centrifuged at 10,000 rpm for 5 min.The sugar (Brix)/acid ratio of the supernatant was measured using a pocket refractometer (PAL-BX|ACID2, ATAGO, Tokyo, Japan) following the manufacturer's instructions.Sugar content and acid content represent the percentage concentration of soluble solids and that of total acid in the juice, respectively (Brix (%) and acid (%)).

Phytohormone Contents
Each phytohormone was quantified by ELISA.JA content in VR cells was measured following the manual for plant JA using an ELISA kit (MyBioSource, San Diego, CA, USA), as reported by Tsai et al. (2019) [59].Briefly, VR cells cultured for 24 h and PBS (100 µL of PBS/10 mg of tissue) were added to a 2 mL Eppendorf tube and homogenized (30.0 Hz, 3 min) using TissueLyser II (QIAGEN).Then, 50 µL of the supernatant was centrifuged in a tabletop centrifuge and dispensed into a 96-well plate.Within 15 min after the addition of Stop Solution in the kit, absorbance was measured at 450 nm using an absorbance microplate reader, and JA content was calculated by the calibration curve method.Similarly, ABA content in VR cells cultured for 24 h was measured using a Plant Hormone ABA ELISA kit (CUSABIO, Wuhan, China) as reported by Enoki et al. (2017) [10].

H 2 O 2 Content
H 2 O 2 content was determined using a Cell Meter TM Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence* (AAT Bioquest, Sunnyvale, CA, USA) following the method of Nie et al. (2020) [60] with modifications.Briefly, VR cells and Component C assay buffer (200 mg/mL) were added to a 2 mL Eppendorf tube and the mixture was homogenized (30.0 Hz, 3 min) using a TissueLyser II (QIAGEN).The homogenate was separated using a tabletop centrifuge, and 50 µL of the supernatant was used as a test sample.After the reaction solution was added following the manufacturer's instructions, the mixtures were incubated at room temperature for 20 min, and fluorescence intensity was measured at Ex/Em = 485/538 nm using a fluorescence microplate reader.H 2 O 2 content was calculated using the calibration curve method.

Statistical Analysis
Data are presented as means ± standard error (SE) of three or four independent biological replicates.Statistical analysis was performed using BellCurve for Excel software ver.3.20.(Social Survey Research Information, Tokyo, Japan) with the Student's t-test, Tukey test, or Dunnett test.

Conclusions
We proposed a molecular mechanism for the Syn-mediated anthocyanin accumulation in grapes.Anthocyanin accumulation was increased not by phytohormones but by hydrogen peroxide and the upregulation of anthocyanin-biosynthesis-related genes in Syn-treated cells.Syn increased the expression of chitinase-encoding gene, one of stress response markers.The results suggest that Syn increases antioxidant anthocyanin accumulation by inducing oxidative stress mediated by hydrogen peroxide.The application of Syn to grape berries may be an alternative to the conventional use of phytohormone-related agents for improving grape skin coloration.

Figure 2 .
Figure 2. Effect of Syn on berry quality.Photographs of grape bunches 10 and 20 days after 1 mM Syn treatment in 2019 and 2021 (a).Anthocyanin content in berry skin (b,c).Sugar/acid ratio of juice (d,e).Data are shown as means ± SE (n = 3).* indicates significant difference at p < 0.05 and **, at 0.01 (t-test).

Figure 2 .
Figure 2. Effect of Syn on berry quality.Photographs of grape bunches 10 and 20 days after 1 mM Syn treatment in 2019 and 2021 (a).Anthocyanin content in berry skin (b,c).Sugar/acid ratio of juice (d,e).Data are shown as means ± SE (n = 3).* indicates significant difference at p < 0.05 and **, at 0.01 (t-test).

Figure 3 .
Figure 3. Expression levels of genes in anthocyanin-biosynthesis-related pathways in Syn-treated VR cells: PAL, C4H, and 4CL in the phenylpropanoid biosynthetic pathway; CHS, CHI, F3'H, F3'5'H, F3H, DFR, and LDOX in the flavonoid biosynthetic pathway; and mybA1 and UFGT in the anthocyanin biosynthetic pathway.VR cells were cultured in a medium containing 5 mM Syn for 120 h (27 °C, 54.2 μmol m −2 s −1 /16 h/day).Gene expression level was estimated by real-time RT-PCR.Data are expression levels relative to actin and are shown as means ± S.E. of four biological replicates (n = 4).* and ** indicate significant differences at p < 0.05 and 0.01, respectively (t-test).

Figure 3 .
Figure 3. Expression levels of genes in anthocyanin-biosynthesis-related pathways in Syn-treated VR cells: PAL, C4H, and 4CL in the phenylpropanoid biosynthetic pathway; CHS, CHI, F3'H, F3'5'H, F3H, DFR, and LDOX in the flavonoid biosynthetic pathway; and mybA1 and UFGT in the anthocyanin biosynthetic pathway.VR cells were cultured in a medium containing 5 mM Syn for 120 h (27 • C, 54.2 µmol m −2 s −1 /16 h/day).Gene expression level was estimated by real-time RT-PCR.Data are expression levels relative to actin and are shown as means ± S.E. of four biological replicates (n = 4).* and ** indicate significant differences at p < 0.05 and 0.01, respectively (t-test).

Figure 4 .
Figure 4. Expression levels of phytohormone biosynthesis genes and phytohormone contents in Syn-treated VR cells.Relative expression level of NECD1 encoding ABA biosynthesis rate-limiting enzyme (a) and ABA content 24 h after treatment (b).Relative expression levels of ACS3 (c) and ACO2 (d), which encode the rate-limiting enzyme of the ET biosynthetic pathway and the enzyme that biosynthesizes ET, respectively.Relative expression level of LOX encoding JA biosynthesis ratelimiting enzyme (e) and JA content 24 h after treatment (f).VR cells were cultured for 24 h (27 °C,

Figure 4 .
Figure 4. Expression levels of phytohormone biosynthesis genes and phytohormone contents in Syn-treated VR cells.Relative expression level of NECD1 encoding ABA biosynthesis rate-limiting enzyme (a) and ABA content 24 h after treatment (b).Relative expression levels of ACS3 (c) and ACO2 (d), which encode the rate-limiting enzyme of the ET biosynthetic pathway and the enzyme

Figure 5 .
Figure 5. Relative expression levels of H2O2-related genes and H2O2 contents in Syn-treated cells.SOD3 encoding H2O2-generating enzymes (a) and H2O2 content (b).Expression levels of APX6 (c) and CAT (d) encoding H2O2-scavenging enzymes.VR cells were grown in a medium containing 5 mM Syn up to 24 or 120 h (27 °C, 54.2 μmol m −2 s −1 /16 h/day).H2O2 content was measured with a fluorescence analysis kit.Gene expression levels were estimated by real-time RT-PCR.Data are expression levels relative to actin.Data are shown as means ± S.E. of four biological replicates (n = 4).* and ** indicate significant differences at p < 0.05 and 0.01, respectively (t-test).

Figure 5 .
Figure 5. Relative expression levels of H 2 O 2 -related genes and H 2 O 2 contents in Syn-treated cells.SOD3 encoding H 2 O 2 -generating enzymes (a) and H 2 O 2 content (b).Expression levels of APX6 (c) and CAT (d) encoding H 2 O 2 -scavenging enzymes.VR cells were grown in a medium containing 5 mM Syn up to 24 or 120 h (27 • C, 54.2 µmol m −2 s −1 /16 h/day).H 2 O 2 content was measured with a fluorescence analysis kit.Gene expression levels were estimated by real-time RT-PCR.Data are expression levels relative to actin.Data are shown as means ± S.E. of four biological replicates (n = 4).* and ** indicate significant differences at p < 0.05 and 0.01, respectively (t-test).

Figure 6 .
Figure 6.Effects of Syn and H2O2 treatments on VR cells and berries.Expression levels of H2O2responsive gene Chit4 (a) and anthocyanin-biosynthesis-related genes mybA1 (b) and UFGT (c).Anthocyanin content in Syn-and H2O2-treated VR cells (d).Anthocyanin content in Syn-and H2O2treated berry skin (e).VR cells were cultured in a medium containing 5 mM Syn and 10 mM H2O2 for 4 days (for gene expression levels) or 7 days (for anthocyanin content) at 27 °C, 54.2 μmol m −2 s −1 /16 h/day.Berries were harvested 9 days after treatment with 1 mM Syn and 300 mM H2O2.Gene expression levels were estimated by real-time RT-PCR.Data are expression levels relative to actin.Data are means ± S.E. for four biological replicates (n = 4).* and ** indicate significant differences at p < 0.05 and 0.01, respectively (Dunnett test).

Figure 6 .
Figure 6.Effects of Syn and H 2 O 2 treatments on VR cells and berries.Expression levels of H 2 O 2responsive gene Chit4 (a) and anthocyanin-biosynthesis-related genes mybA1 (b) and UFGT (c).Anthocyanin content in Syn-and H 2 O 2 -treated VR cells (d).Anthocyanin content in Syn-and H 2 O 2treated berry skin (e).VR cells were cultured in a medium containing 5 mM Syn and 10 mM H 2 O 2 for 4 days (for gene expression levels) or 7 days (for anthocyanin content) at 27 • C, 54.2 µmol m −2 s −1 /16 h/day.Berries were harvested 9 days after treatment with 1 mM Syn and 300 mM H 2 O 2 .Gene expression levels were estimated by real-time RT-PCR.Data are expression levels relative to actin.Data are means ± S.E. for four biological replicates (n = 4).* and ** indicate significant differences at p < 0.05 and 0.01, respectively (Dunnett test).
that biosynthesizes ET, respectively.Relative expression level of LOX encoding JA biosynthesis rate-limiting enzyme (e) and JA content 24 h after treatment (f).VR cells were cultured for 24 h (27 • C, 54.2 µmol m −2 s −1 /16 h/day) in a medium containing 5 mM Syn. Gene expression levels were estimated by real-time RT-PCR.Data are expression levels relative to actin.Phytohormone content was determined by ELISA.Data are shown as means ± S.E. of four biological replicates (n = 4).* indicates significant differences at p < 0.05 (t-test). ).