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Cluster bagging promotes melatonin biosynthesis in the berry skins of Vitis vinifera cv. Cabernet Sauvignon and Carignan during development and ripening
Food Chemistry 305 (2020) 125502
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Cluster bagging promotes melatonin biosynthesis in the berry skins of Vitis vinifera cv. Cabernet Sauvignon and Carignan during development and ripening
T
Shui-Huan Guoa,1, Teng-Fei Xua,1, Tian-Ci Shia, Xu-Qiao Jina, Ming-Xin Fenga, Xian-Hua Zhaob, ⁎ ⁎ Zhen-Wen Zhanga,c, , Jiang-Fei Menga,c, a b c
Shaanxi Engineering Research Center for Viti-Viniculture, College of Enology/College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China College of Life Sciences and Enology, Taishan University, Taian, Shandong 271021, China Heyang Experimental and Demonstrational Stations for Grape, Northwest A&F University, Heyang, Shaanxi 715300, China
A R T I C LE I N FO
A B S T R A C T
Chemical compounds studied in this article: Melatonin (PubChem CID: 896) L-tryptophan (PubChem CID: 6305) N-acetylserotonin (PubChem CID: 903) Tryptamine (PubChem CID: 1150) Serotonin (PubChem CID: 5202) 5-methoxytryptamine (PubChem CID: 1833)
Melatonin, a tryptophan derivative, is an important functional component in grape berries. We investigated the effect of cluster bagging on melatonin biosynthesis in the berries of two wine grape cultivars, Cabernet Sauvignon and Carignan, during fruit development and ripening. Cluster bagging delayed fruit coloring and ripening, and bag-treated berries of both grape cultivars synthesized more melatonin and most of the precursor compounds including L-tryptophan, N-acetylserotonin, tryptamine, and serotonin compared to those exposed to light (control) conditions. Interestingly, 5-methoxytryptamine was only detected in the berries of Carignan and not of Cabernet Sauvignon, both in the cluster bagging and control groups. In addition, melatonin and most of its precursors, decreased after veraison. VvSNAT1 and VvT5H expression levels were positively correlated with melatonin content. Our findings suggested that melatonin synthesis pathways differ among grape cultivars, and that VvSNAT1 and VvT5H may show key regulatory roles in the melatonin synthesis of grape berries.
Keywords: Melatonin Wine grape Berry skins Cluster bagging Gene expression
1. Introduction Vitis is widely cultivated around the world. According to the International Organisation of Vine and Wine (OIV) statistical report (OIV, 2017), global grape planting areas have increased rapidly in recent years, and were forecasted to reach 7.53 Mha. in 2017. The prevalence and distribution of grapes in the world is not only due to their unique sensory and excellent processing characteristics, but also due to their high nutritional value and health-promoting properties (Cho, Lee, Seo, Yokoyama, & Kim, 2018). In recent years, melatonin, resveratrol, and hydroxytyrosol have become regarded as three important bioactive compounds in grapes and wines with human health benefits (Fernández-Mar, Mateos, Garcíaparrilla, Puertas, & Cantosvillar, 2012; Iriti, Rossoni, & Faoro, 2006; Iriti & Faoro, 2009; Iriti, & Varoni, 2015). Melatonin (N-acetyl-5-methoxytryptamine), which has a low molecular weight and an indole-based structure, is ubiquitous in living
organisms. According to the summary by Fernández-Mar et al. (2012), melatonin plays significant roles in human health in terms of its antioxidant capacity, anticancer activity, immunomodulatory activity, and neuroprotective activity. Therefore, Iriti and Varoni (2015) hypothesized that melatonin in grape and wine may additively or synergistically maximize the health benefits of polyphenols. A previous study showed that melatonin can have a cardio protective effect against lethal ischemia-reperfusion injuries (Lamont et al., 2015). In addition, melatonin-rich wines protect against oxidative stress related to the central nervous system (Marhuenda et al., 2017) and can influence the generation of DNA oxidation catabolites linked to mutagenesis (Marhuenda, Medina, Martínezhernández, & Gil-Izquierdo, 2016). The melatonin content in plants is related to plant species, tissues and organs, the growth and development period and the plant growth environment. In different plant species, melatonin content varies from picograms to micrograms per gram of tissue (Tan et al., 2012). Grapes
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Corresponding authors at: College of Enology, Northwest A&F University, No. 22 Xinong Road, Yangling, Shaanxi 712100, China. E-mail addresses: [email protected] (Z.-W. Zhang), [email protected] (J.-F. Meng). 1 These authors contribute equally to this manuscript. https://doi.org/10.1016/j.foodchem.2019.125502 Received 12 June 2019; Received in revised form 13 August 2019; Accepted 7 September 2019 Available online 10 September 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Comparison of berry weight, and the levels of reducing sugar and acetic acid and between control and cluster bag-treated berries of two grape varieties: Cabernet Sauvignon or Carignan. Treatments
Control
Cluster bagging
Stages
Fruit set Expanding Veraison Maturity Fruit set Expanding Veraison Maturity
Cabernet Sauvignon
Carignan
Weight per 100 berries (g)
Reducing sugar (g/L)
Total acid (g/L)
100 grain weight (g)
Reducing sugar (g/L)
Total acid (g/L)
26.31 ± 1.23 89.56 ± 2.08 130.87 ± 6.59 133.46 ± 3.47 23.82 ± 1.25* 86.83 ± 3.23** 119.84 ± 5.35** 126.14 ± 4.51**
22.09 ± 1.06 30.24 ± 1.14 70.03 ± 3.52 213.84 ± 4.20 21.21 ± 1.00* 32.36 ± 0.94 63.98 ± 3.60** 198.14 ± 2.42**
33.06 ± 2.58 35.23 ± 1.46 12.97 ± 0.31 6.72 ± 0.39 32.76 ± 2.29** 36.24 ± 0.22 13.26 ± 0.77** 7.25 ± 0.31**
32.09 ± 0.87 149.02 ± 4.48 177.42 ± 3.35 183.16 ± 6.32 29.31 ± 1.23** 132.69 ± 3.22** 176.87 ± 5.59** 179.46 ± 5.47*
23.48 ± 2.17 28.26 ± 1.21 68.98 ± 4.12 169.36 ± 5.14 21.09 ± 1.58* 27.42 ± 1.41 57.05 ± 3.10** 158.84 ± 5.20**
24.35 ± 2.20 25.55 ± 1.72 11.15 ± 3.15 7.08 ± 0.84 24.06 ± 2.58* 24.73 ± 1.86** 14.25 ± 3.15** 7.72 ± 1.05*
Note: ‘*’ and ‘**’ indicate statistical differences according to t-examination at the 5% and 1% respectively levels between control and cluster bagged grapes of the same grape variety.
Fig. 1. Comparison of the levels melatonin and its precursors in the development of control and cluster bag-treated Cabernet Sauvignon (CS) berries. The precursors of melatonin in this study include L-tryptophan (A), tryptamine (B), serotonin (C), N-acetylserotonin (D), and melatonin (E). Data represent means ± SD (n = 3). Letters ‘a’, ‘b’, ‘c’, ‘d’, ‘e’ and ‘f’ indicate statistical differences (p < 0.05, Duncan's multiple).
content peaking at night but remaining low in the daytime. No such daily rhythmicity was observed in the organs of Lycopersicon esculentum and Pharbitis nil (Van Tassel, Roberts, Lewy, & O'Neill, 2001). UV-B radiation has been shown to increase the melatonin content in Glycyrrhiza uralensis root (Afreen, Zobayed, & Kozai, 2006). The melatonin content in Cardiandra moellendorffii leaves under natural light was higher than that under artificial light (Tan, Manchester, Helton, & Reiter, 2007). These results suggest that melatonin levels in plants may be affected by light sources. Cluster bagging is an important cultivation and management practice in viticulture to reduce the occurrence of grape fruit diseases (Xu,
are the most momentous economic crop in the word. Since the melatonin was identified in grapes in 2006 (Iriti et al., 2006), subsequent investigations have revealed that all grape berry tissues (skin, flesh, and seed) contain melatonin, and, at pre-veraison, the highest melatonin content was found in the berry skin, whereas at veraison, the highest levels were found in the seed (Vitalini et al., 2011). The endogenous melatonin content is also affected by the environment. Melatonin, as a protective molecule, accumulates in plant tissues to resist various stresses in the environment. Boccalandro, González, Wunderlin, and Silva (2011) studied Malbec grapes and found that the melatonin in grape fruits also demonstrate daily rhythmicity, with the melatonin 2
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Fig. 2. Comparison of the levels melatonin and its precursors in the development of control and cluster bag-treated Carignan (CN) berries. The precursors of melatonin in this study include L-tryptophan (A), tryptamine (B), serotonin (C), N-acetylserotonin (D), melatonin (E), and 5-methoxytryptamine (F). Data represent means ± SD (n = 3). Letters ‘a’, ‘b’, ‘c’, ‘d’, ‘e’ and ‘f’ indicate statistical differences (p < 0.05, Duncan's multiple).
Chen, & Xie, 2010), which was first used in pears and grapes in Japan in the 20th century. Pre-harvest bagging of fruit can also reduce the mechanical damage, sunburn of the skin, fruit cracking, and agrochemical residues on the fruit. Due to its many beneficial effects, fruit bagging has become an integral part of peach, apple, pear, grape, and loquat cultivation in Japan, Australia, China and the USA (Sharma, Reddy, & Jhalegar, 2014). The bagging treatment mainly changes the light intensity, temperature and humidity around the fruit, resulting in various physiological changes in the fruit (Steyn, Holcroft, Wand, & Jacobs, 2004). However, as a light-sensitive compound, it is unclear if melatonin accumulation in the grape berry could be affected by cluster bagging. Tan, Manchester, Estebanzubero, Zhou, and Reiter (2015) reported the synthetic pathway of melatonin in plants in 2015. CO2 produced tryptophan via the shikimic acid pathway, and tryptamine and serotonin were then generated in turn under the action of tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), respectively. There were two pathways for the production of melatonin from serotonin: one is through the synthesis of 5-methoxytryptamine, followed by the generation of melatonin under serotonin N-acetyl transferase (SANT); the other is via the formation of N-acetylserotonin under the action of SNAT and then synthesis of the melatonin by caffeic acid O-methyltransferase (CAMT) or N-acetylserotonin methyltransferase (ASMT). In recent years, the related enzymes in melatonin biosynthetic and metabolic pathway of plants (Supplementary Fig. 1) have been identified and their genes have also been cloned. These enzymes include TDC, T5H, SANT, ASMT etc. (Tan et al., 2015). In addition, two enzymes involved in melatonin metabolism i.e., melatonin 3-
hydroxylase and melatonin 2-hydroxylase have also been identified (Lee, Zawadzka, Czarnocki, Reiter, & Back, 2016; Byeon & Back, 2015). This study aimed to clarify the effect of cluster bagging on melatonin biosynthesis in grape berries during fruit development and ripening, compare the difference in melatonin accumulation between the two grape cultivars, and screen the key genes that affect melatonin biosynthesis in grape fruits. The present study provides experimental evidence for the further development and utilization of the bagging technique in viticulture. 2. Materials and methods 2.1. Cluster bagging treatment of field-grown berries The experimental vineyard was located at Chateau Changyu Rena (34.40°N, 108.79°E) of Shaanxi Province, China. Ten-year-old Cabernet Sauvignon (CS) and seven-year-old Carignane (CN) grapevines, on their own roots, were grown on a flat terroir with well drained sandy soil. Natural rainfall was supplemented by limited drip irrigation as required. The vines were spaced 0.8 m within rows and 3.0 m between rows, and the rows were oriented in a south-north direction. Vines were trained on a vertical single cordon positioning system with three wires. The vertical shoot-positioned canopies were uniformly managed and were trimmed twice manually, between bloom and veraison. The study vines for each variety consisted of 180 uniform vines, Half of them (three repeats, 30 vines per replicate) were bagged from fruit set until harvest with 20 × 30 cm fruit bags that contained a black double-layer inside. The black double-layer inside can block the outside sunlight, 3
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Fig. 3. Relative expression levels of melatonin-related genes in during the development of control and cluster bag-treated Cabernet Sauvignon (CS) berries. The melatonin-related genes in this study include VvSNAT1 (A), VvM3H (B), VvT5H (C), and VvTDC1 (D). Data represent means ± SD (n = 3). Letters ‘a’, ‘b’, ‘c’, ‘d’, ‘e’ and ‘f’ indicate statistical differences (p < 0.05, Duncan's multiple).
thus providing dark conditions for grape clusters. The trial was performed in triplicate with controls and cluster bagging treatments randomized over three adjacent rows. The sampling time was based on the modified E-L system (Coombe, 1995): Fruit set period (E-L number 27), expanding period (E-L number 31), veraison period (E-L number 35), and maturity period (E-L number 38).
000 rpm for 15 min. The supernatant was then collected, N2-dried at 35 °C and then added to 100 μL 30% (V/V) methanol for re-dissolution. Afterwards the sample was vortexed until complete dissolution and then centrifuged at 12, 000 rpm for 15 min, at 4 °C. Then the final supernatant was collected in the tubes which covered by aluminum foil to block the light and stored at −20 °C until use.
2.2. Berry analysis
2.3.2. Determination of melatonin and its precursors The methanol, ethanol, acetonitrile, acetic acid and formic acid (LCMS grade) were purchased from Merck (Darmstadt, Germany). The Ltryptophan, tryptamine, serotonin, N-acetylserotonin, melatonin and 5methoxytryptamine (LC-MS grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The standard was dissolved in methanol and stored at −20 °C. Before use, this standard solution was diluted to different concentrations with 70% methanol. The chromatographic analyses of melatonin and its precursors were performed using a Prominence CBM-20A UFLC system (Shimadzu, Kyoto, Japan) equipped with an Applied Biosystems 6500 QTRAP mass spectrometer (AB SCIEX LLC, Redwood, CA, USA). A Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm) was used with column oven temperature at 40 °C. Mobile phase A was consisted of 0.04% formic acid in water, and mobile phase B was acetonitrile with 0.04% formic acid. Separation was achieved using the following gradient program at a flow rate of 0.4 mL/min for 15 min: 0–11.0 min, 5–95% B; 11.0–12.0 min, 95% B; 12.0–12.1 min, 95–5% B; 12.1–15.0 min, 5% B. The injection volume was 2.0 μL. Mass spectra were acquired using electrospray ionization in positive ion mode and MRM. Ion source conditions were as follows: ion source,
Berry weight was measured for each of the three replicates (100 berries per replicate). About 200 berries were collected per replicate for each treatment at every sampling date. Grape berries were peeled in the frozen state, then the skins were frozen by liquid nitrogen and smashed into powder by a grinder. The powder was used to analyze melatonin content and extract RNA. Berry juice was collected from all sampled berries (n = 200) and used to measure the reducing sugars and total acids as the methods described by OIV (2017). 2.3. Melatonin quantification and characterization 2.3.1. Extraction of melatonin and its precursors The methods used for extraction and analysis of melatonin and its precursors were according to previous study (Huang & Mazza, 2011). Grape skin powder was frozen over 24 h under the condition of −50 °C vacuum freeze drier (FD-1C-50) until it is fully dried. The samples were ground with grinding apparatus (MM 400, Retsch) at 30 Hz for 1 min. The powder (100 mg) was extracted in 1 mL of 80% (V/V) methanol for 12 h at 4 °C and was centrifuged three times during this period at 12, 4
Food Chemistry 305 (2020) 125502
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Fig. 4. Relative expression levels of melatonin-related genes in during the development of control and cluster bag-treated Carignan (CN) berries. The melatoninrelated Genes in this study include VvSNAT1 (A), VvM3H (B), VvT5H (C), and VvTDC1 (D). Data represent means ± SD (n = 3). Letters ‘a’, ‘b’, ‘c’, ‘d’, ‘e’ and ‘f’ indicate statistical differences (p < 0.05, Duncan's multiple).
as follows: 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and then incubation at 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s, dissolution from 60 °C to 95 °C (at a rate of 0.15 °C/s), and finally, termination of the reaction. Each sample was analyzed three times. The gene VvGAPDH was used as an internal control. The data were analyzed using the 2−ΔΔCT method (Livak & Schmittgen, 2001).
Turbo Spray; electrospray capillary voltage, 5500 V; source temperature, 500 °C; curtain gas, 25 psi, ion source gas I, 50 psi; ion source gas II, 60 psi; target scan time, 0.60 s. Collision-induced dissociation employed argon as collision gas at a high manifold pressure, and collision energies and source cone potentials were optimized for each transition. Data acquisition and analysis was performed using Analyst® 1.6.3 and Multiquant® 3.0.2 software. The target mass parameters of the melatonin and its precursors were shown in Supplementary Table 2. To quantify the analytes, we constructed seven-point calibration curves, using diluted working solutions of external standards. All points on the curves represented the average of three independent determinations. The linearity of the calibration graphs was determined using regression analysis. The details about the calibration curves, including limits of detection (LOD), limits of quantitation (LOQ), linearity ranges, and correlation coefficients (R2) were shown in Supplementary Table 3.
2.5. Statistical analysis Statistical analysis of data was performed using SPSS 17.0 for Windows. One-way analysis of variance (ANOVA), independent sample t-tests and Pearson's correlation coefficient were used to determine the significance of the differences among samples. Heat maps, Euclidean's correlation analysis, and Ward clustering algorithm were performed by Metabo Analyst 3.0 (https://www.metaboanalyst.ca/).
2.4. Total RNA isolation and qRT-PCR analysis 3. Results and discussion Total RNA was extracted from the powder (1.0 g) using an RNA prep Pure Plant kit (Bai Taike, Beijing, China). First-strand 20 μL cDNA (from 500 ng total RNA) synthesis was performed using the Revert Aid First Strand cDNA Synthesis kit (Vazyme, Nanjing, China). The biosynthesis genes were measured by real-time quantitative PCR (qRT-PCR), using the IQ-SYBR Green Supermix on a MyIQTM Single Color IQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Berkeley, CA, USA). The primers were listed in Supplementary Table 1. The two-step qRT-PCR Reagent Kit (Vazyme Biotech Co., Ltd., Nanjing, China) was used according to the manufacturer's instructions. The reaction procedure was
3.1. Physiochemical parameters The basic physical and chemical characteristics of CS and CN grape berries are listed in Table 1. The maximum 100 grain weight of mature CS and CN grape fruits were 133.46 g and 183.16 g in the control group (under natural lighting conditions), respectively, but only 126.14 g and 179.46 g in the bagged group, respectively; therefore, bagging treatment was shown to inhibit the developmental growth of the fruits. The findings of the present study were almost consistent with those showed 5
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Fig. 5. Hierarchical clustering heat map and Pearson's correlation coefficient heat map of the transcript profiles of all matters and genes in Cabernet sauvignon (CS) and Carignon (CN). (A) Hierarchical clustering of the transcript profiles of all compounds and genes and a clustered heat map of melatonin, its precursors and melatonin-related genes involved in the development of Cabernet Sauvignon and Carignan grape berries. Red colors represent relatively higher relationships, and green colors represent relatively lower relationships. ‘B' and ‘NB' refer to bagging and no bagging, respectively. 1, 2, 3, and 4 represent the developmental stages: fruit set, expanding, veraison, and maturity, of grape berries, respectively. (B) Pearson's correlation coefficient heat map of correlations between the pairs of compounds and genes in Cabernet sauvignon (CS) and Carignon (CN). Red colors represent positive correlation coefficients, and blue colors represent negative correlation coefficients. ‘*’ and ‘**’ indicate statistical differences according to Pearson correlation analysis at the 5%, 1% levels of melatonin, its precursors and melatoninrelated gen. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
6
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in another recent study (Sen, Oksar, & Kesgin, 2016), which confirmed that covering materials following shading nets could delay harvest around 50 days. Bagging treatment may either enhance or inhibit the activities of key enzymes involved in fruit quality improvement (Sharma et al., 2014), and can also have multiple effects on internal fruit quality by changing the micro-environment response to fruit development (Fan & Mattheis, 1998). No significant differences in the levels of reducing sugar and total acid were observed between the two grape varieties in the bagging group, or among stages of fruit set and expanding. However, the reducing sugar content in the cluster bagging treatment was obviously lower than that of the control group, and the total acid content in the cluster bagging treatment was significantly higher than that in the control group. These findings indicated that the cluster bagging treatment slowed the deceleration of the ripening process of the grape fruits, which is consistent with the findings of most previous studies (Karajeh & Muwaffaq, 2017; Signes, Burlo, Martinrz-Snchez, & CarbonellBarrachina, 2007). The differences in results are being considered to be attributed to differences in enzyme activity and the expression levels of genes (Dai et al., 2011; Lecourieux, Lecourieux, Vignault, & Delrot, 2010; Wang, Hang, & Liu, 2010).
followed by L-tryptophan, then serotonin and tryptamine. It was an unexpected that 5-methoxytryptamine was only detected in the CN grape variety. There are two pathways for the production of melatonin from serotonin: one is through the synthesis of 5-methoxytryptamine, followed by the generation of melatonin under SANT; the other is via the formation of N-acetylserotonin under the action of SNAT and then synthesis of the melatonin by CAMT or ASMT. Considering the current understanding of the pathways involved in melatonin synthesis in plants, our findings indicated that the two varieties may utilize different synthetic pathways. The melatonin synthesis in CS may not use the pathway of the synthesis of 5-methoxytryptamine, but CN grapes might use both pathways to produce melanin. Melatonin biosynthesis appeared to be complicated throughout the different stages of ripening; therefore, future studies should be conducted to clarity the role of the precursor compounds in melatonin accumulation. Besides the direct impact of the melatonin profile on grapes and wine, the interaction among melatonin and its precursors should also be considered. Both the biosynthesis and breakdown of melatonin can affect its content during development.
3.2. Melatonin quantification and characterization
The expression patterns of genes involved in the biosynthesis of melatonin were examined by qRT-PCR for all developmental stages of grape berries from fruit set to ripeness. In Figs. 3 and 4, the expression levels of VvSNAT1, VvM3H, VvT5H and VvTDC1 for the two grape varieties (CS and CN) at different developmental stages. All target genes were expressed in the berries at some of the developmental stages analyzed or throughout the whole sampling period. The strong differences in the gene expression trends were identified between the two grape varieties. Gene VvSNAT1 showed different expression patterns in CS and CN. Basically, the expression level was higher in CS than in CN. VvSNAT1 showed different expression levels throughout the sampling period, except for the first sampling date (fruit set), and it showed a significant induction at the veraison stage in bag-treated CS (Fig. 3A), also the expression level decreased to lower than the control group before reaching full ripeness. In CN (Fig. 4A), VvSNAT1 did not show appreciable differences in expression from fruit set stage to the pre-veraison stage in either the control (light) or bagged (dark) treatment, but decreased drastically at the veraison stage in the bagged treatment. VvM3H showed two peaks of gene expression in the control (light) conditions at the veraison and ripeness stages of both grape varieties (Figs. 3B and 4B). The expression level of VvM3H in the cluster bagged (dark) treatment were lower than that in the control (light) treatment and drastically decreased as ripening progressed in both CS and CN. Similar to VvM3H, VvT5H also expressed less in bag-treatment berries, except for fruit set and veraison stages in CS grapes (Fig. 3C). In addition, VvT5H expressed during berry formation and ripening, and reached the highest value at the veraison stage. VvTDC1 showed the highest levels of gene expression in the cluster bagging treatment at the veraison stage of CS and at the early stages of development (fruit set) of CN, but was weakly expressed during the other stages (Figs. 3D and 4D). In CS, VvTDC1 showed significant differences in expression between the control and bagged treatments, and the expression level was lower at the stages of pre-veraison and after-veraison in the cluster bagging group. The expression level of VvTDC1 in CN was decreased after fruit set, and was lowest at the veraison stage, but then steadily increased. The gene expression level of VvTDC1 in bagged berries was higher than in the control group, with differences from veraison until maturity. SNAT1 and SNAT2 have been cloned from Oryza sativa (Byeon & Back, 2016) and genes MdTDC1, MdAANAT2, MdT5H4, and MdASMT1 were also already studied in Malus pumila Mill (Lei et al., 2013). TDC is deemed a rate-limiting enzyme that regulates the synthesis of tryptophan in Oryza sativa (Kang, Kang, Lee, & Back, 2007), Solanum
3.3. The expression of melatonin biosynthetic genes
Figs. 1 and 2 present the effect of cluster bagging treatment on the synthetic changes in melatonin and its precursors in CS and CN grapes. During the development and ripening of grape berries, N-acetylserotonin, L-tryptophan, tryptamine, and melatonin tends to rise, while serotonin tended to rise slowly and then decrease in CS grape berries (Fig. 1). Only the amount of serotonin in CS (Fig. 1C) and 5-methoxytryptamine in CN (Fig. 2F) were higher in the presence of light than in the cluster bagging (light avoidance) group after the berries expanded. Boccalandro et al. (2011) also reported that the synthesis of melatonin in the wine grape Malbec is affected by light and that melatonin levels peak at night but decrease during the day. L-tryptophan and tryptamine levels showed moderate increases, while the levels of N-acetylserotonin, melatonin and 5-methoxytryptamine (only detected in CN) first increased and then decreased in the CN grape berries (Fig. 2), and reached a maximum value at the expanding stage until the veraison stage. These findings were similar to those of a recent study (Murch, Krishnaraj, & Saxena, 2000), which showed that melatonin content peaked in the later period of green fruit, i.e., the veraison period, and then gradually decreased with fruit ripening. The highest melatonin counts of CS and CN grape berries were 0.118 ng/g (at the veraison stage) and 0.085 ng/g (at the expanding stage), respectively (Figs. 1E; 2E). However, serotonin content was the lowest at these stages in both varieties, thus, the regulation pattern is opposite between melatonin and serotonin. In the CS grapes the melatonin content in the veraison period was similar to that in the maturity period (Fig. 1E), but was significantly higher than that of green fruiting. In the CN grapes, the melatonin content in the expanding period was similar to that in the veraison period (Fig. 2E), but was significantly higher than that of another two periods. In 2001, a study on the melatonin content in tomato fruit at different maturity stages found that melatonin amount are the least in green fruits and the most in red fruits (Van Tassel et al., 2001). In the present study, the N-acetylserotonin levels increased in CS and decreased in CN in the maturity period led to a decrease in the serotonin content, while the levels of other compounds remained unchanged (Figs. 1D and 2D); therefore, a competitive relationship may exist between serotonin and N-acetylserotonin. Kang et al. (2011) confirmed that the first enzymatic reaction product of melatonin synthesis in rice is not serotonin but L-tryptophan. Ltryptophan can form indoleacetic acid under the action of tryptamine deaminase (TDA), which is the precursor of indoleacetic acid in plants. Considering the melatonin synthesis pathway in plants (Tan et al., 2015), N-acetylserotonin levels clearly prevailed in the present study, 7
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4. Conclusion
lycopersicum (Pang, Cheng, Pan, et al., 2018) and Paeonia lactiflora Pall flowers (Zhao et al., 2018). The results of the present study might be because of no light-dark cycle with bagging. The bagged cluster only in dark environment, resulting in increased melatonin levels.
In summary, this is the first report to compare the melatonin synthesis during berry development and ripening and the effect of cluster bagging on melatonin synthesis between two wine grape varieties, CS and CN. We found that the cluster bagging treatment decreased the weight of grape berries and delay the berry maturity, in terms of low levels of reducing sugar and high levels of total acid, in both varieties. The cluster bagging treatment induced the synthesis of melatonin and its precursors (L-tryptophan, serotonin, tryptamine, Nacetylserotonin and 5-methoxytryptamine) via changes in the expression of genes, which differed between the grape varieties. Specific differences in the synthetic pathway of melatonin were observed between the two varieties, and 5-methoxytryptamine was not detected in CS. VvSNAT1 and VvT5H expression levels showed similar tendencies with that of the melatonin content, and a high correlation was detected between them; therefore, these genes are suggested prospective to have pivotal roles in the accumulation of melatonin in grape fruits. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125502.
3.4. Correlation analysis of various substances and related genes in bagged grapes To further confirm the correlation of various substances and genes related melatonin synthesis, the key factors that affect the synthesis of melatonin in grape were screened. Fig. 5 shows the differences and connections between the gene expression levels and the synthesis of melatonin and its precursors in control or cluster bag-treated CS and CN by Hierarchical Clustering and Pearson's Correlation Coefficients respectively. As shown in Fig. 5A, significant differences in the levels of various compounds and the expression of various genes were identified between varieties and treatments, and the differences were more attributable to the difference in variety than to different light treatments. The effect of the bagging treatment on CS grapes after the veraison stage was greater than that of the green fruits, similar to those of Murch et al. (2000). Tryptamine, L-tryptophan, N-acetylserotonin and melatonin were clustered in a group with VvT5H. N-acetylserotonin was strongly correlated with melatonin in this group, which indicates that N-acetylserotonin may show an important role in melatonin synthesis. 5-methoxytryptamine, serotonin, VvSNAT1, VvM3H, and VvTDC1 formed a large category, meaning some connection between them. However, they were not directly involved in melatonin synthesis. Furthermore, we could only infer that these genes were involved in the synthesis of melatonin, but not that they were the key genes. The relationships between melatonin, its precursors and related genes were depicted by Pearson's correlation coefficient heat map (Fig. 5B). The maximum correlation coefficient between melatonin and N-acetylserotonin was 0.829 (p < 0.01). Tryptamine was highly (p < 0.01) positively correlated with both melatonin and N-acetylserotonin, and melatonin was negatively correlated with serotonin; therefore, we concluded that tryptamine was synthesized before melatonin and N-acetylserotonin, which is consistent with the synthesis pathway of plant melatonin (Tan et al., 2015). VvSNAT1 and VvT5H genes were positively correlated with melatonin synthesis but not significantly. VvT5H gene was positively correlated with melatonin and Nacetylserotonin (p < 0.05), and negatively correlated with VvTDC1. VvTDC1 was positively correlated with serotonin, but was weakly correlated with 5-methoxytryptamine, as well as negatively correlated with the remaining substances (N-acetylserotonin, L-tryptophan, tryptamine and melatonin). The prominent expression of VvT5H in the grape development cycle may indicate that this gene have a key role in bagged grape fruits. Serotonin was negatively correlated with tryptamine, N-acetylserotonin, gene VvSNAT1 and gene VvT5H, while it is positively correlated with 5-methoxytryptamine, gene VvTDC1, and gene VvM3H. What's interesting is that these substances and genes have the opposite relationship to melatonin (Fig. 5B). The expression of gene VvT5H was positively correlated with all substances and genes except serotonin. In the synthesis of melatonin, serotonin and gene VvT5H showed different roles. The expression levels of VvTDCs and VvSNAT were up-regulated, and the contents of melatonin, tryptophan, 5-Hydroxytryptophan, and N-acetyl-5-hydroxytryptophan were increased in grape seedlings under drought or salt stress (Jiao et al., 2016). In apples melatonin production was induced by up-regulating the expressions of melatonin synthesis genes MdTDC1, MdAANAT2, MdT5H4, and MdASMT1 (Lei et al., 2013), however, our results differed from this previous study. Future studies should focus on elucidating the synergistic effects of these genes in the process of melatonin synthesis, as well as other influential factors, especially regarding differences among species.
Declaration of competing interest The authors declare no competing financial interest. Acknowledgments This study received financial support from the Natural Science Foundation of China (31801811 and 31801833), China Postdoctoral Science Foundation (2018M633589 and 2019T120953), Natural Science Basic Research Plan in Shaanxi Province of China (2018JQ3018), and the Agriculture Research System of China for Grape Industry (CARS-29-zp-6). References Afreen, F., Zobayed, S. M. A., & Kozai, T. (2006). Melatonin in Glycyrrhiza uralensis: Response of plant roots to spectral quality of light and UV-B radiation. Journal of Pineal Research, 41, 108–115. https://doi.org/10.1111/j.1600-079X.2006.00337.x. Boccalandro, H. E., González, C. V., Wunderlin, D. A., & Silva, M. F. (2011). Melatonin levels, determined by LC-ESI-MS/MS, fluctuate during the day/night cycle in Vitis vinifera cv. Malbec: Evidence of its antioxidant role in fruits. Journal of Pineal Research, 51, 226–232. https://doi.org/10.1111/j.1600-079X.2011.00884.x. Byeon, Y., & Back, K. (2015). Molecular cloning of melatonin 2-hydroxylase responsible for 2-hydroxymelatonin production in rice (Oryza sativa). Journal of Pineal Research, 58(3), 343–351. https://doi.org/10.1111/jpi.12220. Byeon, Y., & Back, K. (2016). Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase. Applied Microbiology and Biotechnology, 100(15), 6683–6691. https://doi.org/10.1007/ s00253-016-7458-z. Cho, Y. J., Lee, H. G., Seo, K. H., Yokoyama, W., & Kim, H. (2018). Ant-obesity effect of prebiotic polyphenol-rich grape seed flour supplemented with probiotic kefir-derived lactic acid bacteria. Journal of Agricultural and Food Chemistry, 66(47), 12498–12511. https://doi.org/10.1021/acs.jafc.8b03720. Coombe, B. G. (1995). Growth stages of the grapevine: adoption of a system for identifying grapevine growth stages. Australian Journal of Grape and Wine Research, (pp, 1(2), 104–110). Australia: The Australian Society of Viticulture and Oenology. urn: issn:1322–7130. doi:10.1111/j.1755-0238.1995.tb00086.x. Dai, Z. W., Ollat, N., Gomes, E., Decroocq, S., Tandonnet, J. P., Bordenave, L., et al. (2011). Ecophysiological, genetic, and molecular causes of variation in grape berry weight and composition: A review. American Journal of Enology and Viticulture, 62(4), 413–425. https://doi.org/10.5344/ajev.2011.10116. Fan, X., & Mattheis, J. P. (1998). Bagging ‘fuji’ apples during fruit development affects color development and storage quality. Hortscience, 33(7), 1235–1238. Fernández-Mar, M. I., Mateos, R., Garcíaparrilla, M. C., Puertas, B., & Cantosvillar, E. (2012). Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review. Food Chemistry, 130(4), 797–813. https://doi.org/10.1016/j.foodchem.2011. 08.023. Huang, X., & Mazza, G. (2011). Simultaneous analysis of serotonin, melatonin, piceid and resveratrol in fruits using liquid chromatography tandem mass spectrometry. Journal of Chromatography A, 1218(25), 3890–3899. https://doi.org/10.1016/j.chroma. 2011.04.049. Iriti, M., & Faoro, F. (2009). Chapter 23-health-promoting effects of grape bioactive phytochemicals. Complementary & alternative therapies & the aging population, (pp 445–474). America: Academic Presshttps://doi.org/10.1016/B978-0-12-374228-5.00023-8.
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Iriti, M., Rossoni, M., & Faoro, F. (2006). Melatonin content in grape: Myth or panacea? Journal of the Science of Food and Agriculture, 86, 1432–1438. https://doi.org/10. 1002/jsfa.2537. Iriti, M., & Varoni, E. M. (2015). Melatonin in mediterranean diet, a new perspective. Journal of the Science of Food and Agriculture, 95(12), 2355–2359. https://doi.org/10. 1002/jsfa.7051. Jiao, J., Ma, Y., Chen, S., Liu, C. H., Song, Y. Y., Qin, Y., ... Liu, Y. L. (2016). Melatoninproducing endophytic bacteria from grapevine roots promote the abiotic stress-induced production of endogenous melatonin in their hosts. Frontiers in Plant Science, 7(3), 1387. https://doi.org/10.3389/fpls.2016.01387. Kang, K., Kong, K., Park, S., Natsagdorj, U., Kim, Y. S., & Back, K. (2011). Molecular cloning of a plant N-acetylserotonin methyltransferase and its expression characteristics in rice. Journal of Pineal Research, 50(3), 304–309. https://doi.org/10.1111/j. 1600-079X.2010.00841.x. Kang, S., Kang, K., Lee, K., & Back, K. (2007). Characterization of tryptamine 5-hydroxylase and serotonin synthesis in rice plants. Plant Cell Reports, 26(11), 2009–2015. https://doi.org/10.1007/s00299-007-0405-9. Karajeh, & Muwaffaq, R. (2017). Pre-harvest bagging of grape clusters as a non-chemical physical control measure against certain pests and diseases of grapevines. Organic Agriculture, 8(3), 259–264. https://doi.org/10.1007/s13165-017-0197-3. Lamont, K., Nduhirabandi, F., Adam, T., Thomas, D. P., Opie, L. H., & Lecour, S. (2015). Role of melatonin, melatonin receptors and STAT3 in the cardioprotective effect of chronic and moderate consumption of red wine. Biochemical & Biophysical Research Communications, 465(4), 719–724. https://doi.org/10.1016/j.bbrc.2015.08.064. Lecourieux, F., Lecourieux, D., Vignault, C., & Delrot, S. (2010). A sugar-inducible protein kinase, VvSK1, regulates hexose transport and sugar accumulation in grapevine cells. Plant Physiology, 152, 1096–1106. https://doi.org/10.2307/25680718. Lee, K., Zawadzka, A., Czarnocki, Z., Reiter, R. J., & Back, K. (2016). Molecular cloning of melatonin 3-hydroxylase and its production of cyclic 3-hydroxymelatonin in rice (Oryza sativa). Journal of Pineal Research, 61(4), 470–478. https://doi.org/10.1111/ jpi.12361. Lei, Q., Wang, L., Tan, D. X., Zhao, Y., Zheng, X. D., & Chen, H. (2013). Identification of genes for melatonin synthetic enzymes in ‘Red Fuji’ apple (Malus domestica Borkh. cv. Red) and their expression and melatonin production during fruit development. Journal of Pineal Research, 55, 443–451. https://doi.org/10.1111/jpi.12096. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C (T)) Method. Methods, 25, 402–408. https://doi.org/10.1006/meth.2001. Marhuenda, J., Medina, S., Martínezhernández, P., & Gil-Izquierdo, Á. (2016). Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers. Food & Function, 7(12), 4781–4796. https://doi.org/10.1039/C6FO01246A. Marhuenda, J., Medina, S., Martínez-Hernández, P., Arina, S., Zafrilla, P., Mulero, J., & Gil-Izquierdo, Á. (2017). Effect of the dietary intake of melatonin- and hydroxytyrosol-rich wines by healthy female volunteers on the systemic lipidomic-related oxylipins. Food & Function, 8(10), 3745–3757. https://doi.org/10.1039/c7fo01081h. Murch, S. J., Krishnaraj, S., & Saxena, P. D. (2000). Tryptophan is a precursor for melatonin and serotonin biosynthesis in vitro regenerated St. Jhon's wort (Hypericum
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Cluster bagging promotes melatonin biosynthesis in the berry skins of Vitis vinifera cv. Cabernet Sauvignon and Carignan during development and ripening
T
Shui-Huan Guoa,1, Teng-Fei Xua,1, Tian-Ci Shia, Xu-Qiao Jina, Ming-Xin Fenga, Xian-Hua Zhaob, ⁎ ⁎ Zhen-Wen Zhanga,c, , Jiang-Fei Menga,c, a b c
Shaanxi Engineering Research Center for Viti-Viniculture, College of Enology/College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China College of Life Sciences and Enology, Taishan University, Taian, Shandong 271021, China Heyang Experimental and Demonstrational Stations for Grape, Northwest A&F University, Heyang, Shaanxi 715300, China
A R T I C LE I N FO
A B S T R A C T
Chemical compounds studied in this article: Melatonin (PubChem CID: 896) L-tryptophan (PubChem CID: 6305) N-acetylserotonin (PubChem CID: 903) Tryptamine (PubChem CID: 1150) Serotonin (PubChem CID: 5202) 5-methoxytryptamine (PubChem CID: 1833)
Melatonin, a tryptophan derivative, is an important functional component in grape berries. We investigated the effect of cluster bagging on melatonin biosynthesis in the berries of two wine grape cultivars, Cabernet Sauvignon and Carignan, during fruit development and ripening. Cluster bagging delayed fruit coloring and ripening, and bag-treated berries of both grape cultivars synthesized more melatonin and most of the precursor compounds including L-tryptophan, N-acetylserotonin, tryptamine, and serotonin compared to those exposed to light (control) conditions. Interestingly, 5-methoxytryptamine was only detected in the berries of Carignan and not of Cabernet Sauvignon, both in the cluster bagging and control groups. In addition, melatonin and most of its precursors, decreased after veraison. VvSNAT1 and VvT5H expression levels were positively correlated with melatonin content. Our findings suggested that melatonin synthesis pathways differ among grape cultivars, and that VvSNAT1 and VvT5H may show key regulatory roles in the melatonin synthesis of grape berries.
Keywords: Melatonin Wine grape Berry skins Cluster bagging Gene expression
1. Introduction Vitis is widely cultivated around the world. According to the International Organisation of Vine and Wine (OIV) statistical report (OIV, 2017), global grape planting areas have increased rapidly in recent years, and were forecasted to reach 7.53 Mha. in 2017. The prevalence and distribution of grapes in the world is not only due to their unique sensory and excellent processing characteristics, but also due to their high nutritional value and health-promoting properties (Cho, Lee, Seo, Yokoyama, & Kim, 2018). In recent years, melatonin, resveratrol, and hydroxytyrosol have become regarded as three important bioactive compounds in grapes and wines with human health benefits (Fernández-Mar, Mateos, Garcíaparrilla, Puertas, & Cantosvillar, 2012; Iriti, Rossoni, & Faoro, 2006; Iriti & Faoro, 2009; Iriti, & Varoni, 2015). Melatonin (N-acetyl-5-methoxytryptamine), which has a low molecular weight and an indole-based structure, is ubiquitous in living
organisms. According to the summary by Fernández-Mar et al. (2012), melatonin plays significant roles in human health in terms of its antioxidant capacity, anticancer activity, immunomodulatory activity, and neuroprotective activity. Therefore, Iriti and Varoni (2015) hypothesized that melatonin in grape and wine may additively or synergistically maximize the health benefits of polyphenols. A previous study showed that melatonin can have a cardio protective effect against lethal ischemia-reperfusion injuries (Lamont et al., 2015). In addition, melatonin-rich wines protect against oxidative stress related to the central nervous system (Marhuenda et al., 2017) and can influence the generation of DNA oxidation catabolites linked to mutagenesis (Marhuenda, Medina, Martínezhernández, & Gil-Izquierdo, 2016). The melatonin content in plants is related to plant species, tissues and organs, the growth and development period and the plant growth environment. In different plant species, melatonin content varies from picograms to micrograms per gram of tissue (Tan et al., 2012). Grapes
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Corresponding authors at: College of Enology, Northwest A&F University, No. 22 Xinong Road, Yangling, Shaanxi 712100, China. E-mail addresses: [email protected] (Z.-W. Zhang), [email protected] (J.-F. Meng). 1 These authors contribute equally to this manuscript. https://doi.org/10.1016/j.foodchem.2019.125502 Received 12 June 2019; Received in revised form 13 August 2019; Accepted 7 September 2019 Available online 10 September 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Comparison of berry weight, and the levels of reducing sugar and acetic acid and between control and cluster bag-treated berries of two grape varieties: Cabernet Sauvignon or Carignan. Treatments
Control
Cluster bagging
Stages
Fruit set Expanding Veraison Maturity Fruit set Expanding Veraison Maturity
Cabernet Sauvignon
Carignan
Weight per 100 berries (g)
Reducing sugar (g/L)
Total acid (g/L)
100 grain weight (g)
Reducing sugar (g/L)
Total acid (g/L)
26.31 ± 1.23 89.56 ± 2.08 130.87 ± 6.59 133.46 ± 3.47 23.82 ± 1.25* 86.83 ± 3.23** 119.84 ± 5.35** 126.14 ± 4.51**
22.09 ± 1.06 30.24 ± 1.14 70.03 ± 3.52 213.84 ± 4.20 21.21 ± 1.00* 32.36 ± 0.94 63.98 ± 3.60** 198.14 ± 2.42**
33.06 ± 2.58 35.23 ± 1.46 12.97 ± 0.31 6.72 ± 0.39 32.76 ± 2.29** 36.24 ± 0.22 13.26 ± 0.77** 7.25 ± 0.31**
32.09 ± 0.87 149.02 ± 4.48 177.42 ± 3.35 183.16 ± 6.32 29.31 ± 1.23** 132.69 ± 3.22** 176.87 ± 5.59** 179.46 ± 5.47*
23.48 ± 2.17 28.26 ± 1.21 68.98 ± 4.12 169.36 ± 5.14 21.09 ± 1.58* 27.42 ± 1.41 57.05 ± 3.10** 158.84 ± 5.20**
24.35 ± 2.20 25.55 ± 1.72 11.15 ± 3.15 7.08 ± 0.84 24.06 ± 2.58* 24.73 ± 1.86** 14.25 ± 3.15** 7.72 ± 1.05*
Note: ‘*’ and ‘**’ indicate statistical differences according to t-examination at the 5% and 1% respectively levels between control and cluster bagged grapes of the same grape variety.
Fig. 1. Comparison of the levels melatonin and its precursors in the development of control and cluster bag-treated Cabernet Sauvignon (CS) berries. The precursors of melatonin in this study include L-tryptophan (A), tryptamine (B), serotonin (C), N-acetylserotonin (D), and melatonin (E). Data represent means ± SD (n = 3). Letters ‘a’, ‘b’, ‘c’, ‘d’, ‘e’ and ‘f’ indicate statistical differences (p < 0.05, Duncan's multiple).
content peaking at night but remaining low in the daytime. No such daily rhythmicity was observed in the organs of Lycopersicon esculentum and Pharbitis nil (Van Tassel, Roberts, Lewy, & O'Neill, 2001). UV-B radiation has been shown to increase the melatonin content in Glycyrrhiza uralensis root (Afreen, Zobayed, & Kozai, 2006). The melatonin content in Cardiandra moellendorffii leaves under natural light was higher than that under artificial light (Tan, Manchester, Helton, & Reiter, 2007). These results suggest that melatonin levels in plants may be affected by light sources. Cluster bagging is an important cultivation and management practice in viticulture to reduce the occurrence of grape fruit diseases (Xu,
are the most momentous economic crop in the word. Since the melatonin was identified in grapes in 2006 (Iriti et al., 2006), subsequent investigations have revealed that all grape berry tissues (skin, flesh, and seed) contain melatonin, and, at pre-veraison, the highest melatonin content was found in the berry skin, whereas at veraison, the highest levels were found in the seed (Vitalini et al., 2011). The endogenous melatonin content is also affected by the environment. Melatonin, as a protective molecule, accumulates in plant tissues to resist various stresses in the environment. Boccalandro, González, Wunderlin, and Silva (2011) studied Malbec grapes and found that the melatonin in grape fruits also demonstrate daily rhythmicity, with the melatonin 2
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Fig. 2. Comparison of the levels melatonin and its precursors in the development of control and cluster bag-treated Carignan (CN) berries. The precursors of melatonin in this study include L-tryptophan (A), tryptamine (B), serotonin (C), N-acetylserotonin (D), melatonin (E), and 5-methoxytryptamine (F). Data represent means ± SD (n = 3). Letters ‘a’, ‘b’, ‘c’, ‘d’, ‘e’ and ‘f’ indicate statistical differences (p < 0.05, Duncan's multiple).
Chen, & Xie, 2010), which was first used in pears and grapes in Japan in the 20th century. Pre-harvest bagging of fruit can also reduce the mechanical damage, sunburn of the skin, fruit cracking, and agrochemical residues on the fruit. Due to its many beneficial effects, fruit bagging has become an integral part of peach, apple, pear, grape, and loquat cultivation in Japan, Australia, China and the USA (Sharma, Reddy, & Jhalegar, 2014). The bagging treatment mainly changes the light intensity, temperature and humidity around the fruit, resulting in various physiological changes in the fruit (Steyn, Holcroft, Wand, & Jacobs, 2004). However, as a light-sensitive compound, it is unclear if melatonin accumulation in the grape berry could be affected by cluster bagging. Tan, Manchester, Estebanzubero, Zhou, and Reiter (2015) reported the synthetic pathway of melatonin in plants in 2015. CO2 produced tryptophan via the shikimic acid pathway, and tryptamine and serotonin were then generated in turn under the action of tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), respectively. There were two pathways for the production of melatonin from serotonin: one is through the synthesis of 5-methoxytryptamine, followed by the generation of melatonin under serotonin N-acetyl transferase (SANT); the other is via the formation of N-acetylserotonin under the action of SNAT and then synthesis of the melatonin by caffeic acid O-methyltransferase (CAMT) or N-acetylserotonin methyltransferase (ASMT). In recent years, the related enzymes in melatonin biosynthetic and metabolic pathway of plants (Supplementary Fig. 1) have been identified and their genes have also been cloned. These enzymes include TDC, T5H, SANT, ASMT etc. (Tan et al., 2015). In addition, two enzymes involved in melatonin metabolism i.e., melatonin 3-
hydroxylase and melatonin 2-hydroxylase have also been identified (Lee, Zawadzka, Czarnocki, Reiter, & Back, 2016; Byeon & Back, 2015). This study aimed to clarify the effect of cluster bagging on melatonin biosynthesis in grape berries during fruit development and ripening, compare the difference in melatonin accumulation between the two grape cultivars, and screen the key genes that affect melatonin biosynthesis in grape fruits. The present study provides experimental evidence for the further development and utilization of the bagging technique in viticulture. 2. Materials and methods 2.1. Cluster bagging treatment of field-grown berries The experimental vineyard was located at Chateau Changyu Rena (34.40°N, 108.79°E) of Shaanxi Province, China. Ten-year-old Cabernet Sauvignon (CS) and seven-year-old Carignane (CN) grapevines, on their own roots, were grown on a flat terroir with well drained sandy soil. Natural rainfall was supplemented by limited drip irrigation as required. The vines were spaced 0.8 m within rows and 3.0 m between rows, and the rows were oriented in a south-north direction. Vines were trained on a vertical single cordon positioning system with three wires. The vertical shoot-positioned canopies were uniformly managed and were trimmed twice manually, between bloom and veraison. The study vines for each variety consisted of 180 uniform vines, Half of them (three repeats, 30 vines per replicate) were bagged from fruit set until harvest with 20 × 30 cm fruit bags that contained a black double-layer inside. The black double-layer inside can block the outside sunlight, 3
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Fig. 3. Relative expression levels of melatonin-related genes in during the development of control and cluster bag-treated Cabernet Sauvignon (CS) berries. The melatonin-related genes in this study include VvSNAT1 (A), VvM3H (B), VvT5H (C), and VvTDC1 (D). Data represent means ± SD (n = 3). Letters ‘a’, ‘b’, ‘c’, ‘d’, ‘e’ and ‘f’ indicate statistical differences (p < 0.05, Duncan's multiple).
thus providing dark conditions for grape clusters. The trial was performed in triplicate with controls and cluster bagging treatments randomized over three adjacent rows. The sampling time was based on the modified E-L system (Coombe, 1995): Fruit set period (E-L number 27), expanding period (E-L number 31), veraison period (E-L number 35), and maturity period (E-L number 38).
000 rpm for 15 min. The supernatant was then collected, N2-dried at 35 °C and then added to 100 μL 30% (V/V) methanol for re-dissolution. Afterwards the sample was vortexed until complete dissolution and then centrifuged at 12, 000 rpm for 15 min, at 4 °C. Then the final supernatant was collected in the tubes which covered by aluminum foil to block the light and stored at −20 °C until use.
2.2. Berry analysis
2.3.2. Determination of melatonin and its precursors The methanol, ethanol, acetonitrile, acetic acid and formic acid (LCMS grade) were purchased from Merck (Darmstadt, Germany). The Ltryptophan, tryptamine, serotonin, N-acetylserotonin, melatonin and 5methoxytryptamine (LC-MS grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The standard was dissolved in methanol and stored at −20 °C. Before use, this standard solution was diluted to different concentrations with 70% methanol. The chromatographic analyses of melatonin and its precursors were performed using a Prominence CBM-20A UFLC system (Shimadzu, Kyoto, Japan) equipped with an Applied Biosystems 6500 QTRAP mass spectrometer (AB SCIEX LLC, Redwood, CA, USA). A Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm) was used with column oven temperature at 40 °C. Mobile phase A was consisted of 0.04% formic acid in water, and mobile phase B was acetonitrile with 0.04% formic acid. Separation was achieved using the following gradient program at a flow rate of 0.4 mL/min for 15 min: 0–11.0 min, 5–95% B; 11.0–12.0 min, 95% B; 12.0–12.1 min, 95–5% B; 12.1–15.0 min, 5% B. The injection volume was 2.0 μL. Mass spectra were acquired using electrospray ionization in positive ion mode and MRM. Ion source conditions were as follows: ion source,
Berry weight was measured for each of the three replicates (100 berries per replicate). About 200 berries were collected per replicate for each treatment at every sampling date. Grape berries were peeled in the frozen state, then the skins were frozen by liquid nitrogen and smashed into powder by a grinder. The powder was used to analyze melatonin content and extract RNA. Berry juice was collected from all sampled berries (n = 200) and used to measure the reducing sugars and total acids as the methods described by OIV (2017). 2.3. Melatonin quantification and characterization 2.3.1. Extraction of melatonin and its precursors The methods used for extraction and analysis of melatonin and its precursors were according to previous study (Huang & Mazza, 2011). Grape skin powder was frozen over 24 h under the condition of −50 °C vacuum freeze drier (FD-1C-50) until it is fully dried. The samples were ground with grinding apparatus (MM 400, Retsch) at 30 Hz for 1 min. The powder (100 mg) was extracted in 1 mL of 80% (V/V) methanol for 12 h at 4 °C and was centrifuged three times during this period at 12, 4
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Fig. 4. Relative expression levels of melatonin-related genes in during the development of control and cluster bag-treated Carignan (CN) berries. The melatoninrelated Genes in this study include VvSNAT1 (A), VvM3H (B), VvT5H (C), and VvTDC1 (D). Data represent means ± SD (n = 3). Letters ‘a’, ‘b’, ‘c’, ‘d’, ‘e’ and ‘f’ indicate statistical differences (p < 0.05, Duncan's multiple).
as follows: 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and then incubation at 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s, dissolution from 60 °C to 95 °C (at a rate of 0.15 °C/s), and finally, termination of the reaction. Each sample was analyzed three times. The gene VvGAPDH was used as an internal control. The data were analyzed using the 2−ΔΔCT method (Livak & Schmittgen, 2001).
Turbo Spray; electrospray capillary voltage, 5500 V; source temperature, 500 °C; curtain gas, 25 psi, ion source gas I, 50 psi; ion source gas II, 60 psi; target scan time, 0.60 s. Collision-induced dissociation employed argon as collision gas at a high manifold pressure, and collision energies and source cone potentials were optimized for each transition. Data acquisition and analysis was performed using Analyst® 1.6.3 and Multiquant® 3.0.2 software. The target mass parameters of the melatonin and its precursors were shown in Supplementary Table 2. To quantify the analytes, we constructed seven-point calibration curves, using diluted working solutions of external standards. All points on the curves represented the average of three independent determinations. The linearity of the calibration graphs was determined using regression analysis. The details about the calibration curves, including limits of detection (LOD), limits of quantitation (LOQ), linearity ranges, and correlation coefficients (R2) were shown in Supplementary Table 3.
2.5. Statistical analysis Statistical analysis of data was performed using SPSS 17.0 for Windows. One-way analysis of variance (ANOVA), independent sample t-tests and Pearson's correlation coefficient were used to determine the significance of the differences among samples. Heat maps, Euclidean's correlation analysis, and Ward clustering algorithm were performed by Metabo Analyst 3.0 (https://www.metaboanalyst.ca/).
2.4. Total RNA isolation and qRT-PCR analysis 3. Results and discussion Total RNA was extracted from the powder (1.0 g) using an RNA prep Pure Plant kit (Bai Taike, Beijing, China). First-strand 20 μL cDNA (from 500 ng total RNA) synthesis was performed using the Revert Aid First Strand cDNA Synthesis kit (Vazyme, Nanjing, China). The biosynthesis genes were measured by real-time quantitative PCR (qRT-PCR), using the IQ-SYBR Green Supermix on a MyIQTM Single Color IQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Berkeley, CA, USA). The primers were listed in Supplementary Table 1. The two-step qRT-PCR Reagent Kit (Vazyme Biotech Co., Ltd., Nanjing, China) was used according to the manufacturer's instructions. The reaction procedure was
3.1. Physiochemical parameters The basic physical and chemical characteristics of CS and CN grape berries are listed in Table 1. The maximum 100 grain weight of mature CS and CN grape fruits were 133.46 g and 183.16 g in the control group (under natural lighting conditions), respectively, but only 126.14 g and 179.46 g in the bagged group, respectively; therefore, bagging treatment was shown to inhibit the developmental growth of the fruits. The findings of the present study were almost consistent with those showed 5
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Fig. 5. Hierarchical clustering heat map and Pearson's correlation coefficient heat map of the transcript profiles of all matters and genes in Cabernet sauvignon (CS) and Carignon (CN). (A) Hierarchical clustering of the transcript profiles of all compounds and genes and a clustered heat map of melatonin, its precursors and melatonin-related genes involved in the development of Cabernet Sauvignon and Carignan grape berries. Red colors represent relatively higher relationships, and green colors represent relatively lower relationships. ‘B' and ‘NB' refer to bagging and no bagging, respectively. 1, 2, 3, and 4 represent the developmental stages: fruit set, expanding, veraison, and maturity, of grape berries, respectively. (B) Pearson's correlation coefficient heat map of correlations between the pairs of compounds and genes in Cabernet sauvignon (CS) and Carignon (CN). Red colors represent positive correlation coefficients, and blue colors represent negative correlation coefficients. ‘*’ and ‘**’ indicate statistical differences according to Pearson correlation analysis at the 5%, 1% levels of melatonin, its precursors and melatoninrelated gen. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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in another recent study (Sen, Oksar, & Kesgin, 2016), which confirmed that covering materials following shading nets could delay harvest around 50 days. Bagging treatment may either enhance or inhibit the activities of key enzymes involved in fruit quality improvement (Sharma et al., 2014), and can also have multiple effects on internal fruit quality by changing the micro-environment response to fruit development (Fan & Mattheis, 1998). No significant differences in the levels of reducing sugar and total acid were observed between the two grape varieties in the bagging group, or among stages of fruit set and expanding. However, the reducing sugar content in the cluster bagging treatment was obviously lower than that of the control group, and the total acid content in the cluster bagging treatment was significantly higher than that in the control group. These findings indicated that the cluster bagging treatment slowed the deceleration of the ripening process of the grape fruits, which is consistent with the findings of most previous studies (Karajeh & Muwaffaq, 2017; Signes, Burlo, Martinrz-Snchez, & CarbonellBarrachina, 2007). The differences in results are being considered to be attributed to differences in enzyme activity and the expression levels of genes (Dai et al., 2011; Lecourieux, Lecourieux, Vignault, & Delrot, 2010; Wang, Hang, & Liu, 2010).
followed by L-tryptophan, then serotonin and tryptamine. It was an unexpected that 5-methoxytryptamine was only detected in the CN grape variety. There are two pathways for the production of melatonin from serotonin: one is through the synthesis of 5-methoxytryptamine, followed by the generation of melatonin under SANT; the other is via the formation of N-acetylserotonin under the action of SNAT and then synthesis of the melatonin by CAMT or ASMT. Considering the current understanding of the pathways involved in melatonin synthesis in plants, our findings indicated that the two varieties may utilize different synthetic pathways. The melatonin synthesis in CS may not use the pathway of the synthesis of 5-methoxytryptamine, but CN grapes might use both pathways to produce melanin. Melatonin biosynthesis appeared to be complicated throughout the different stages of ripening; therefore, future studies should be conducted to clarity the role of the precursor compounds in melatonin accumulation. Besides the direct impact of the melatonin profile on grapes and wine, the interaction among melatonin and its precursors should also be considered. Both the biosynthesis and breakdown of melatonin can affect its content during development.
3.2. Melatonin quantification and characterization
The expression patterns of genes involved in the biosynthesis of melatonin were examined by qRT-PCR for all developmental stages of grape berries from fruit set to ripeness. In Figs. 3 and 4, the expression levels of VvSNAT1, VvM3H, VvT5H and VvTDC1 for the two grape varieties (CS and CN) at different developmental stages. All target genes were expressed in the berries at some of the developmental stages analyzed or throughout the whole sampling period. The strong differences in the gene expression trends were identified between the two grape varieties. Gene VvSNAT1 showed different expression patterns in CS and CN. Basically, the expression level was higher in CS than in CN. VvSNAT1 showed different expression levels throughout the sampling period, except for the first sampling date (fruit set), and it showed a significant induction at the veraison stage in bag-treated CS (Fig. 3A), also the expression level decreased to lower than the control group before reaching full ripeness. In CN (Fig. 4A), VvSNAT1 did not show appreciable differences in expression from fruit set stage to the pre-veraison stage in either the control (light) or bagged (dark) treatment, but decreased drastically at the veraison stage in the bagged treatment. VvM3H showed two peaks of gene expression in the control (light) conditions at the veraison and ripeness stages of both grape varieties (Figs. 3B and 4B). The expression level of VvM3H in the cluster bagged (dark) treatment were lower than that in the control (light) treatment and drastically decreased as ripening progressed in both CS and CN. Similar to VvM3H, VvT5H also expressed less in bag-treatment berries, except for fruit set and veraison stages in CS grapes (Fig. 3C). In addition, VvT5H expressed during berry formation and ripening, and reached the highest value at the veraison stage. VvTDC1 showed the highest levels of gene expression in the cluster bagging treatment at the veraison stage of CS and at the early stages of development (fruit set) of CN, but was weakly expressed during the other stages (Figs. 3D and 4D). In CS, VvTDC1 showed significant differences in expression between the control and bagged treatments, and the expression level was lower at the stages of pre-veraison and after-veraison in the cluster bagging group. The expression level of VvTDC1 in CN was decreased after fruit set, and was lowest at the veraison stage, but then steadily increased. The gene expression level of VvTDC1 in bagged berries was higher than in the control group, with differences from veraison until maturity. SNAT1 and SNAT2 have been cloned from Oryza sativa (Byeon & Back, 2016) and genes MdTDC1, MdAANAT2, MdT5H4, and MdASMT1 were also already studied in Malus pumila Mill (Lei et al., 2013). TDC is deemed a rate-limiting enzyme that regulates the synthesis of tryptophan in Oryza sativa (Kang, Kang, Lee, & Back, 2007), Solanum
3.3. The expression of melatonin biosynthetic genes
Figs. 1 and 2 present the effect of cluster bagging treatment on the synthetic changes in melatonin and its precursors in CS and CN grapes. During the development and ripening of grape berries, N-acetylserotonin, L-tryptophan, tryptamine, and melatonin tends to rise, while serotonin tended to rise slowly and then decrease in CS grape berries (Fig. 1). Only the amount of serotonin in CS (Fig. 1C) and 5-methoxytryptamine in CN (Fig. 2F) were higher in the presence of light than in the cluster bagging (light avoidance) group after the berries expanded. Boccalandro et al. (2011) also reported that the synthesis of melatonin in the wine grape Malbec is affected by light and that melatonin levels peak at night but decrease during the day. L-tryptophan and tryptamine levels showed moderate increases, while the levels of N-acetylserotonin, melatonin and 5-methoxytryptamine (only detected in CN) first increased and then decreased in the CN grape berries (Fig. 2), and reached a maximum value at the expanding stage until the veraison stage. These findings were similar to those of a recent study (Murch, Krishnaraj, & Saxena, 2000), which showed that melatonin content peaked in the later period of green fruit, i.e., the veraison period, and then gradually decreased with fruit ripening. The highest melatonin counts of CS and CN grape berries were 0.118 ng/g (at the veraison stage) and 0.085 ng/g (at the expanding stage), respectively (Figs. 1E; 2E). However, serotonin content was the lowest at these stages in both varieties, thus, the regulation pattern is opposite between melatonin and serotonin. In the CS grapes the melatonin content in the veraison period was similar to that in the maturity period (Fig. 1E), but was significantly higher than that of green fruiting. In the CN grapes, the melatonin content in the expanding period was similar to that in the veraison period (Fig. 2E), but was significantly higher than that of another two periods. In 2001, a study on the melatonin content in tomato fruit at different maturity stages found that melatonin amount are the least in green fruits and the most in red fruits (Van Tassel et al., 2001). In the present study, the N-acetylserotonin levels increased in CS and decreased in CN in the maturity period led to a decrease in the serotonin content, while the levels of other compounds remained unchanged (Figs. 1D and 2D); therefore, a competitive relationship may exist between serotonin and N-acetylserotonin. Kang et al. (2011) confirmed that the first enzymatic reaction product of melatonin synthesis in rice is not serotonin but L-tryptophan. Ltryptophan can form indoleacetic acid under the action of tryptamine deaminase (TDA), which is the precursor of indoleacetic acid in plants. Considering the melatonin synthesis pathway in plants (Tan et al., 2015), N-acetylserotonin levels clearly prevailed in the present study, 7
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4. Conclusion
lycopersicum (Pang, Cheng, Pan, et al., 2018) and Paeonia lactiflora Pall flowers (Zhao et al., 2018). The results of the present study might be because of no light-dark cycle with bagging. The bagged cluster only in dark environment, resulting in increased melatonin levels.
In summary, this is the first report to compare the melatonin synthesis during berry development and ripening and the effect of cluster bagging on melatonin synthesis between two wine grape varieties, CS and CN. We found that the cluster bagging treatment decreased the weight of grape berries and delay the berry maturity, in terms of low levels of reducing sugar and high levels of total acid, in both varieties. The cluster bagging treatment induced the synthesis of melatonin and its precursors (L-tryptophan, serotonin, tryptamine, Nacetylserotonin and 5-methoxytryptamine) via changes in the expression of genes, which differed between the grape varieties. Specific differences in the synthetic pathway of melatonin were observed between the two varieties, and 5-methoxytryptamine was not detected in CS. VvSNAT1 and VvT5H expression levels showed similar tendencies with that of the melatonin content, and a high correlation was detected between them; therefore, these genes are suggested prospective to have pivotal roles in the accumulation of melatonin in grape fruits. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125502.
3.4. Correlation analysis of various substances and related genes in bagged grapes To further confirm the correlation of various substances and genes related melatonin synthesis, the key factors that affect the synthesis of melatonin in grape were screened. Fig. 5 shows the differences and connections between the gene expression levels and the synthesis of melatonin and its precursors in control or cluster bag-treated CS and CN by Hierarchical Clustering and Pearson's Correlation Coefficients respectively. As shown in Fig. 5A, significant differences in the levels of various compounds and the expression of various genes were identified between varieties and treatments, and the differences were more attributable to the difference in variety than to different light treatments. The effect of the bagging treatment on CS grapes after the veraison stage was greater than that of the green fruits, similar to those of Murch et al. (2000). Tryptamine, L-tryptophan, N-acetylserotonin and melatonin were clustered in a group with VvT5H. N-acetylserotonin was strongly correlated with melatonin in this group, which indicates that N-acetylserotonin may show an important role in melatonin synthesis. 5-methoxytryptamine, serotonin, VvSNAT1, VvM3H, and VvTDC1 formed a large category, meaning some connection between them. However, they were not directly involved in melatonin synthesis. Furthermore, we could only infer that these genes were involved in the synthesis of melatonin, but not that they were the key genes. The relationships between melatonin, its precursors and related genes were depicted by Pearson's correlation coefficient heat map (Fig. 5B). The maximum correlation coefficient between melatonin and N-acetylserotonin was 0.829 (p < 0.01). Tryptamine was highly (p < 0.01) positively correlated with both melatonin and N-acetylserotonin, and melatonin was negatively correlated with serotonin; therefore, we concluded that tryptamine was synthesized before melatonin and N-acetylserotonin, which is consistent with the synthesis pathway of plant melatonin (Tan et al., 2015). VvSNAT1 and VvT5H genes were positively correlated with melatonin synthesis but not significantly. VvT5H gene was positively correlated with melatonin and Nacetylserotonin (p < 0.05), and negatively correlated with VvTDC1. VvTDC1 was positively correlated with serotonin, but was weakly correlated with 5-methoxytryptamine, as well as negatively correlated with the remaining substances (N-acetylserotonin, L-tryptophan, tryptamine and melatonin). The prominent expression of VvT5H in the grape development cycle may indicate that this gene have a key role in bagged grape fruits. Serotonin was negatively correlated with tryptamine, N-acetylserotonin, gene VvSNAT1 and gene VvT5H, while it is positively correlated with 5-methoxytryptamine, gene VvTDC1, and gene VvM3H. What's interesting is that these substances and genes have the opposite relationship to melatonin (Fig. 5B). The expression of gene VvT5H was positively correlated with all substances and genes except serotonin. In the synthesis of melatonin, serotonin and gene VvT5H showed different roles. The expression levels of VvTDCs and VvSNAT were up-regulated, and the contents of melatonin, tryptophan, 5-Hydroxytryptophan, and N-acetyl-5-hydroxytryptophan were increased in grape seedlings under drought or salt stress (Jiao et al., 2016). In apples melatonin production was induced by up-regulating the expressions of melatonin synthesis genes MdTDC1, MdAANAT2, MdT5H4, and MdASMT1 (Lei et al., 2013), however, our results differed from this previous study. Future studies should focus on elucidating the synergistic effects of these genes in the process of melatonin synthesis, as well as other influential factors, especially regarding differences among species.
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