Effects of glucose and gibberellic acid on glucosinolate content and antioxidant properties of Chinese kale sprouts

Effects of glucose and gibberellic acid on glucosinolate content and antioxidant properties of Chinese kale sprouts 葡萄糖和赤霉酸对芥蓝芽菜中芥子油苷含量及其抗氧化能力的影响 1 2 first-author 1 1 1 3 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:sunadam011@163.comqmwang@zju.edu.cn †E-mail:sunadam011@163.comqmwang@zju.edu.cn sunadam011@163.com 1 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:sunadam011@163.comqmwang@zju.edu.cn †E-mail:sunadam011@163.comqmwang@zju.edu.cn sunadam011@163.com Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Department of Horticulture, Zhejiang University, Hangzhou 310058, China Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Department of Horticulture, Zhejiang University, Hangzhou 310058, China Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Hangzhou 310058, China Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Hangzhou 310058, China College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China Glucosinolates, anthocyanins, total phenols, and vitamin C, as well as antioxidant capacity, were investigated in Chinese kale sprouts treated with both glucose and gibberellic acid (GA 3 ). The combination of 3% (0.03 g/ml) glucose and 5 μmol/L GA 3 treatment was effective in increasing glucosinolate content while glucose or GA 3 treatment alone did not influence significantly almost all individual glucosinolates or total glucosinolates. The total phenolic content and antioxidant activity of Chinese kale sprouts were enhanced by combined treatment with glucose and GA 3 , which could be useful in improving the main health-promoting compounds and antioxidant activity in Chinese kale sprouts. 目的 2 探究外源施加葡萄糖和赤霉酸对芥蓝芽菜中芥子油苷的积累，花青素、多酚和维生素C等抗氧化物的含量，以及总抗氧化活性的影响。 创新点 2 首次发现葡萄糖和赤霉酸可以协同促进芥蓝芽菜中几乎所有种类芥子油苷以及总芥子油苷的积累，并且可以大幅度提升总多酚的含量以及抗氧化能力。 方法 2 以芥蓝芽菜为材料，使用3%（0.03 g/ml）葡萄糖和5 µmo/L赤霉酸进行外源处理，以水处理作为对照组。用高效液相色谱法分析芥子油苷和维生素C的含量；采用分光光度法检测花青素的含量；用Folin-Ciocalteu试剂法测定总多酚的含量；利用亚铁还原能力实验（FRAP）法进行总抗氧化能力的评估。 结论 2 葡萄糖和赤霉酸共同处理可以有效提升芥蓝芽菜中有益健康的植物化学物质含量以及抗氧化能力。 Glucosinolate Antioxidant; Glucose Gibberellic acid Chinese kale sprouts 芥子油苷 抗氧化物 葡萄糖 赤霉酸 芥蓝芽菜 Introduction 1 Chinese kale ( Brassica oleracea var. alboglabra Bailey) is a native Chinese vegetable, which belongs to the Cruciferae family and has an extensive distribution in South China. People usually harvest stems for food when they are bolting. Nowadays, the sprouts are also consumed because of their high nutritional values and rich content of bioactive compounds, such as the anticarcinogenic glucosinolates (Cartea and Velasco, 2008 2008 ; Dinkova-Kostova and Kostov, 2012 2012 ; Kumar et al., 2015 2015 ; Mazumder et al., 2016 2016 ). The sprouts of Chinese kale and other crucifers contain at least 10 times more glucosinolates than the mature stages of the corresponding plants (Fahey et al., 1997 1997 ; Sun et al., 2011 2011 ; Yuan et al., 2010b 2010b ). Glucosinolates are a group of sulfur-and nitrogen-containing secondary metabolites which exist mainly in cruciferous plants. The glucosinolate content varies depending on genetic and environmental factors, and is modulated by external signals such as sugar (Miao et al., 2013 2013 ) and phytohormones (Yuan et al., 2009 2009 ; Sun et al., 2012 2012 ; Wang et al., 2012 2012 ; Guo et al., 2013a 2013a ; 2013b 2013b ; Huseby et al., 2013 2013 ; Kim et al., 2013 2013 ; Zang et al., 2015 2015 ). Glucose is not only the basal carbon and energy source but also a signaling molecule having regulatory effects throughout the life cycle of plant (Sheen, 2014 2014 ). Our previous studies showed that glucose dramatically increases glucosinolate content in both Arabidopsis and crop brassicas (Wei et al., 2011 2011 ; Guo et al., 2013c 2013c ; Miao et al., 2013 2013 ). Gibberellins are plant hormones participating in the regulation of plant development, which are used extensively to elevate crop yield (Naeem et al., 2001 2001 ). It has been reported that gibberellic acid (GA 3 ) increases various secondary metabolites (Abbasi et al., 2012 2012 ; Park et al., 2017 2017 ). However, studies on the effect of GA 3 on glucosinolates are limited, the only report being an observation by Huang et al. ( 2014 2014 ) that exogenous GA 3 enhanced glucosinolate content in the silique of oilseed rape remarkably. The role of GA 3 in glucosinolate accumulation in other cruciferous crops, such as Chinese kale, merits further investigation. Previous studies have demonstrated that GA 3 inhibits sucrose-induced accumulation of anthocyanin in Arabidopsis (Loreti et al., 2008 2008 ) and negatively interferes with glucose signal transduction in the regulation of cell wall invertase and sucrose synthesis in grape berry (Zhang et al., 2014 2014 ). However, there are limited reports on the relationship between GA 3 and glucose in glucosinolate biosynthesis. In the current study, we analyzed the effects of glucose and GA 3 on glucosinolate biosynthesis in Chinese kale sprouts. We also investigated whether glucose and GA 3 together have an impact on the content of other antioxidants found in Chinese kale sprouts including anthocyanins, total phenols, and vitamin C. This antioxidant activity is thought to make a great contribution to the health benefits of cruciferous vegetables (Podsędek, 2007 2007 ). Materials and methods 1 Plant materials and cultivation conditions 2 Chinese kale seeds ( Brassica oleracea var. alboglabra Bailey cv. Dasunchihua) were sterilized for 30 min in 7 ml/L sodium hypochlorite and washed with sterile water 4‒5 times. They were then soaked in sterile-distilled water for 24 h at 25 °C. Sprouts were grown in petri dishes with three pieces of wet filter paper in a plant growth chamber at 23 °C with a photoperiod of 16 h light/8 h dark. The sprouts in the treatment group were watered with 5 ml of 3% (0.03 g/ml) glucose and/or 5 μmol/L GA 3 5 d after sowing; those in the control group were watered with 5 ml sterile-distilled water. For each treatment, three replicates were taken for analysis. Three days after treatment, sprouts were harvested from the surface of the filter papers and immediately frozen in liquid nitrogen. Samples for vitamin C detection were kept in polyethylene bags at −80 °C, while the others were frozen by lyophilization with a freeze dryer (Vir Tis Inc., New York, USA), crushed into powder, and finally stored at −20 °C for further analyses of glucosinolates, anthocyanins, total phenolic content, and antioxidant capacity. Glucosinolate assay 2 Glucosinolates were extracted and analyzed as previously described with minor modifications (Sun et al., 2012 2012 ). In this study, freeze-dried samples (30 mg) were boiled in 2 ml water for 10 min. The supernatant was transferred to a new tube after centrifugation (5 min, 7000 g ), and the residues were boiled for 10 min with water (2 ml). High-performance liquid chromatography (HPLC) was performed with a Shimadzu HPLC (Shimadzu, Kyoto, Japan) with an SPD-M20A diode array detector. Data were given as nmol/mg fresh weight (FW). Anthocyanin measurement 2 Anthocyanin content was detected according to the previous method (Teng et al., 2005 2005 ). The modification is that we used freeze-dried sprouts (200 mg) which were homogenized at 4 °C in 2 ml of 1% (v/v) hydrochloric acid in methanol. Total phenolic content assay 2 Total phenolic content was determined as previously described with some modifications (Ainsworth and Gillespie, 2007 2007 ). The total phenolic compounds of Chinese kale sprouts (200 mg) were extracted with 5 ml of 30% ethanol, which were then incubated at 37 °C for 1 h and finally centrifuged at 7000 r/min for 10 min at room temperature. The supernatant was collected. Phenolic compounds were determined using Folin-Ciocalteu reagent method by reading the absorbance at 765 nm. Gallic acid was used as a standard and the results were expressed as mg gallic acid equivalent (GAE)/g dry weight (DW). Vitamin C content 2 The vitamin C content was determined as previously described with minor modifications (Yuan et al., 2010a 2010a ). Frozen sprouts (200 mg) were homogenized at 4 °C in 2 ml of 1.0% (0.01 g/ml) oxalic acid and the residues were washed twice with 1 ml 1.0% oxalic acid. The combined extract was centrifuged at 7000 r/min for 10 min. A 0.45-μm cellulose acetate filter was used to filter the supernatant. HPLC analysis was performed using the same system as in the glucosinolate assay, with a mobile phase of 0.1% oxalic acid at a flow rate of 1.0 ml/min. The vitamin C value was calculated from absorbance values at 243 nm, and mg/100 g FW was adopted to express the results. Antioxidant capacity 2 Antioxidant capacity was evaluated using the ferric reducing antioxidant power (FRAP) method of Benzie and Strain ( 1996 1996 ) with some modifications. FRAP working solution (2.7 ml) was mixed with 300 μl samples extracted from about 200 mg of freeze-dried sprouts. After 10 min incubation at 37 °C, we recorded the absorbance at 593 nm. FeSO 4 ·7H 2 O standard curves were used to calculate FRAP values which were expressed as mmol/100 g DW. Statistical analysis 2 Statistical analysis was carried out using the SPSS package program version 11.5 (SPSS Inc., Chicago, IL, USA). Differences in glucosinolate content for different treatments were tested using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) test at a 95% confidence level ( P < 0.05). Differences in the contents of total anthocyanins, phenols and vitamin C, and antioxidant activity between the treatment and control groups were analyzed using the independent-samples t -test. The values are reported as means with standard error for all results. Results 1 Effects of glucose and GA 3 treatments on glucosinolate accumulation 2 We detected 11 different kinds of glucosinolates in Chinese kale sprouts (Table 1 1 ). Seven kinds of aliphatic glucosinolate were identified, the most abundant being gluconapin at about 38%. Glucose or GA 3 treatment alone did not exert a remarkable influence on aliphatic glucosinolate content (Fig. 1 1 ), but the application of both together did. Progoitrin, sinigrin, gluconapin, gluconapoleiferin, and total aliphatic glucosinolates increased by 44%, 54%, 56%, 43%, and 45%, respectively, when compared with the control (Figs. 1b, 1c 1b, 1c , 1e, 1g 1e, 1g , and 1h 1h ). (a) Glucoiberin; (b) Progoitrin; (c) Sinigrin; (d) Glucoraphanin; (e) Gluconapin; (f) Glucoerucin; (g) Gluconapoleiferin; (h) Total aliphatic glucosinolate. Each data point represents the mean of three independent biological replicates per treatment (mean±standard error (SE)). Values not sharing a common letter are significantly different at P < 0.05 Trivial name Chemical name (side chain) Aliphatic glucosinolate  3-Carbon chain length   Glucoiberin 3-Methylsulfinylpropyl   Sinigrin 2-Propenyl  4-Carbon chain length   Glucoerucin 4-Methylthiobutyl   Glucoraphanin 4-Methylsulfinylbutyl   Gluconapin 3-Butenyl   Progoitrin 2-Hydroxy-3-butenyl  5-Carbon chain length   Gluconapoleiferin 2-Hydroxy-4-pentenyl Indole glucosinolate  Glucobrassicin 3-Indolylmethyl  Neoglucobrassicin 1-Methoxy-3-indolylmethyl  4-Hydroxyglucobrassicin 4-Hydroxy-3-indolylmethyl  4-Methoxyglucobrassicin 4-Methoxy-3-indolylmethyl There were four kinds of indolic glucosinolates in Chinese kale sprouts, with 4-methoxyglucobrassicin being the predominant one (Fig. 2 2 ). Glucose or GA 3 treatment alone remarkably elevated the glucobrassicin level (Fig. 2a 2a ). When GA 3 was applied together with glucose, significant promoting effects on 4-methoxyglucobrassicin and neoglucobrassicin were observed, which were increased by 32% and 41%, respectively, when compared with the control (Figs. 2b and 2d 2b and 2d ). (a) Glucobrassicin; (b) 4-Methoxyglucobrassicin; (c) 4-Hydroxyglucobrassicin; (d) Neoglucobrassicin; (e) Total indolic glucosinolate. Each data point represents the mean of three independent biological replicates per treatment (mean±SE). Values not sharing a common letter are significantly different at P < 0.05 As shown in Fig. 3 3 , GA 3 or glucose treatment alone did not significantly influence total glucosinolate content, but combined treatment did, raising the content by 45% compared with the control. Each data point represents the mean of three independent biological replicates per treatment (mean±SE). Values not sharing a common letter are significantly different at P < 0.05 Effects of glucose combined with GA 3 treatment on main antioxidant content and antioxidant capacity 2 As shown in Table 2 2 , GA 3 combined with glucose treatment significantly increased total phenolic content and antioxidant capacity. The antioxidant capacity was increased by approximately 150% compared with the control. However, no remarkable change in the content of anthocyanins or vitamin C was observed. Each data point represents the mean of three independent biological replicates per treatment (mean±SE). Values are significantly different for combined treatment of glucose with GA 3 when compared with water treatment ( P < 0.05)  Treatment Total anthocyanins (U/g DW) Total phenols (mg GAE/g DW) Vitamin C (mg/100 g FW) Antioxidant activity (mmol Fe 2+ /100 g DW)  Water 8.52±0.38 22.94±0.42 118.25±6.12 13.97±0.02  Glucose+GA 3 9.60±0.49 25.89±0.60 * * 121.50±11.80 34.92±0.01 * * Discussion 1 As a native Chinese vegetable, Chinese kale is widely consumed, and the relatively low seed price makes it feasible to produce sprouts commercially. Low concentrations of glucose (3%) and GA 3 (5 μmol/L), being economical and safe, were used in this study to search for a practicable way of enhancing glucosinolate and antioxidant attributes of Chinese kale sprouts. Glucosinolate has anticarcinogenic properties as well as being involved in the flavor and aroma of cruciferous crops (Dinkova-Kostova and Kostov, 2012 2012 ). A number of studies have been conducted with the aim of improving these properties. Chemical regulation was one of the most studied methods, especially with phytohormones, which have been reported to play important roles in the regulation of glucosinolate biosynthesis (Yuan et al., 2010a 2010a ; Guo et al., 2013a 2013a ; Kim et al., 2013 2013 ; Schweizer et al., 2013 2013 ; Frerigmann and Gigolashvili, 2014 2014 ; Zang et al., 2015 2015 ). Our previous research indicated that 5% (0.05 g/ml) glucose elevated glucosinolate in Chinese kale and pak choi sprouts (Wei et al., 2011 2011 ) and the study by Huang et al. ( 2014 2014 ) showed a promoting effect of 100 μmol/L GA 3 on glucosinolate content in the siliques of oilseed rape. In our present study we observed that neither glucose nor GA 3 alone affected the content or composition of individual aliphatic glucosinolates nor most of the indolic glucosinolates. This could be due to different plant species or the concentrations of glucose and GA 3 used. However, the combined treatment of glucose and GA 3 markedly boosted the accumulation of many aliphatic glucosinolate compositions and two kinds of indolic glucosinolates (4-methoxyglucobrassicin, neoglucobrassicin) (Figs. 1 1 and 2 2 ), exerting a synergistic effect on glucosinolate accumulation, although the mechanism involved remains to be elucidated. This kind of synergistic effect between glucose and a phytohormone on glucosinolate biosynthesis is not unique. We previously reported that jasmonic acid could remarkably enhance the effect of glucose on glucosinolate biosynthesis in Arabidopsis thaliana (Guo et al., 2013c 2013c ). GA 3 is much cheaper than jasmonic acid, and easy to handle making it a useful tool for improving glucosinolates in cruciferous plants. Our observation is quite different from the counteractive impact of GA 3 on sucrose-induced anthocyanin accumulation in Arabidopsis (Loreti et al., 2008 2008 ), indicating that the same phytohormone may function diversely in different signaling pathways. Phenolic compounds and vitamin C are important bioactive compounds in cruciferous vegetables for their high content and antioxidant activity, which defend cells against oxidative damage and prevent chronic disease (Podsędek, 2007 2007 ). We therefore analyzed the effects of treatment with glucose and GA 3 combined on the content of these antioxidants. Anthocyanins, a vital kind of natural pigments belonging to the phenolic compounds, are known to protect against neuronal and cardiovascular illnesses, as well as cancer and diabetes (Castañeda-Ovando et al., 2009 2009 ). Liang et al. ( 2013 2013 ) reported that GA 3 is effective in inducing phenolic production in salvia miltiorrhiza bunge hairy roots, and Loreti et al. ( 2008 2008 ) demonstrated that GA 3 has a counteractive effect on sucrose-induced anthocyanin accumulation in Arabidopsis . In this study, we found that the total phenolic content of Chinese kale is elevated when treated by glucose and GA 3 together, while no remarkable difference in anthocyanin content was observed (Table 2 2 ). Our former studies showed that Chinese kale is rich in antioxidant vitamin C (Sun et al., 2012 2012 ; Wei et al., 2011 2011 ). A similar result was observed in this survey. The content of vitamin C in Chinese kale sprout amounted to more than 100 mg per 100 g FW (Table 2 2 ). It was previously reported that GA 3 has a positive effect on maintaining vitamin C in Chinese jujube ( Zizyphus jujuba M) (Jiang et al., 2004 2004 ). In the current study, the combined application of glucose and GA 3 exerted little effect on vitamin C content in Chinese kale spouts. Our former study found that vitamin C content is dramatically reduced by 5% glucose treatment in Chinese kale sprouts (Wei et al., 2011 2011 ), and hence it is possible that glucose is counteracting the promoting effect of GA 3 on vitamin C accumulation. Treatment with glucose and GA 3 combined increased antioxidant activity by 150% compared with the control (Table 2 2 ). A high correlation had previously been found between antioxidant activity and the content of phenolic compounds in radish sprouts (Kim et al., 2006 2006 ), and a similar result was observed in the current study. The antioxidant activity of Chinese kale sprouts was boosted when the total phenolic content was increased under the combined treatment of glucose and GA 3 . Conclusions 1 The combination of 3% glucose and 5 μmol/L GA 3 treatment dramatically enhanced the accumulation of glucosinolates in Chinese kale sprouts as well as other health-promoting compounds, such as total phenols. 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The involvement of hexokinase in the coordinated regulation of glucose and gibberellin on cell wall invertase and sucrose synthesis in grape berry Compliance with ethics guidelines Hui-ying MIAO, Meng-yu WANG, Jia-qi CHANG, Han TAO, Bo SUN, and Qiao-mei WANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1700308 Project supported by the National Natural Science Foundation of China (Nos. 31270343 and 31500247) and the China Postdoctoral Science Foundation (No. 2015M581922) 2017-06-12 2017-08-14 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 18