Article pubs.acs.org/JAFC
Hydrogen-Rich Water Reestablishes ROS Homeostasis but Exerts Differential Effects on Anthocyanin Synthesis in Two Varieties of Radish Sprouts under UV‑A Irradiation Nana Su, Qi Wu, Yuanyuan Liu, Jiangtao Cai, Wenbiao Shen, Kai Xia, and Jin Cui* College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P. R. China S Supporting Information *
ABSTRACT: The aims of the study were to investigate whether hydrogen gas (H2) was involved in regulation of anthocyanin biosynthesis in two contrasting radish (Raphanus sativus L.) varieties (low [LA] and high [HA] level of anthocyanin) under UV irradiation. The results showed that hydrogen-rich water (HRW) significantly blocked the UV-A-induced increase of H2O2 and O2•− accumulation, and enhanced the UV-A-induced increase of superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities in LA and HA. Furthermore, UV-A-induced increase of anthocyanin and total phenols was further enhanced only in HA sprouts cotreated with HRW. LC-MS/MS analysis showed that five anthocyanidins existed in HA sprouts, but only two in LA sprouts. Meanwhile, the cyanidin was the most abundant anthocyanidin in HA, and the cyanidin was 2-fold higher cotreated with HRW than UV-A. Molecular analyses showed that the anthocyanin biosynthesis-related genes were upregulated significantly in both HA (in particular) and LA sprouts treated with HRW plus UV-A. These data imply that HRW reestablishes reactive oxygen species homeostasis in both LA and HA, but exerts different effects on anthocyanin accumulation between them under UV-A. KEYWORDS: hydrogen-rich water, UV-A, anthocyanin, anthocyanidin, radish, Raphanus sativus L., sprouts
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radiations alter plant morphology and architecture12 and induce lower biomass accumulation.13 However, UV rays at lowfluence rates, like UV-A, were found to induce flavonoid production14−16 and the expression of defense-related genes17 without DNA damage. Many reports have described the induction of anthocyanin biosynthesis by UV-A fluorescent lamps with peaks at 350−370 nm. For example, Hirner and Seitz (2000)18 observed UV-Adependent accumulation of anthocyanins in carrot suspension cultures. Wang et al. (2012)19 also found that UV-A (3.0 W m−2) could stimulate anthocyanin production in the hypocotyls of purple turnip. The accumulation of anthocyanins is able to form photoprotective screens to UV to reduce the absorption of UV rays.20 On the other hand, anthocyanins, possessing antioxidant activities, may scavenge UV-induced superabundant ROS.6 Hydrogen gas (H2), as an energy-storage medium that burns in a less polluting way than fossil fuels,21 also regarded as a novel antioxidant, has attracted more and more attention. The metabolism of H2 by bacteria and algae has been reported by many investigators.22−25 H2 is also found to be a potent antioxidative and antiinflammatory agent with potential for medical application.26,27 Recent results revealed that H2 could act as an important gaseous molecule with multiple biological functions in plant responses against salinity-induced and paraquat-induced oxidative stress in Arabidopsis and alfalfa seedlings cultivated with HRW.28,29
INTRODUCTION Using seed sprouts as food has spread from Far Eastern countries to parts of the Western world in the past few decades. An extraordinary variety of different types of sprouts can be found on the market in which the Cruciferae family is well represented.1 Previous studies demonstrated that cruciferous sprouts such as broccoli (Brassica oleracea L. var. italica) and radish (Raphanus sativus) contain rich health-promoting phytochemical constituents such as phenolic compounds, ascorbic acid,1,2 and glucosinolates related to cancer prevention as well as having antioxidant properties.3,4 As one of the important flavonoids in sprouts, anthocyanins generate the characteristic reddish, bluish, and purple hues and thereby contribute to the quality of fruits and vegetables.5 In addition, anthocyanins are recognized as compounds with potential health benefits for their valuable nutritional antioxidant activities.6 Beyond that, anthocyanin production is also the typical plant response to environmental stress. 7,8 The anthocyanin biosynthesis pathway is well described in plants. It is derived from phenylalanine, catalyzed by phenylalanine ammonialyase (PAL). Then it is mediated by a common step with chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H) and fluxed into anthocyanin biosynthesis by dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS).9 As one of the main light qualities, ultraviolet radiation (UV) has been recognized as a stress stimulus on plant biological processes. Responses to elevated and ambient UV include increased DNA damage, antioxidant response,10 and increased production of reactive oxygen species (ROS) such as superoxide radical (O2•−), hydroxyl radical (•OH), hydrogen peroxide (H2O2), and singlet oxygen (1O2).11 Furthermore, UV © 2014 American Chemical Society
Received: Revised: Accepted: Published: 6454
April 25, 2014 June 23, 2014 June 23, 2014 June 23, 2014 dx.doi.org/10.1021/jf5019593 | J. Agric. Food Chem. 2014, 62, 6454−6462
Journal of Agricultural and Food Chemistry
Article
as the amount of crude enzyme extract required to inhibit the reduction rate of NBT by 50%. APX activity was measured by monitoring the decrease in absorbance at 290 nm as ascorbic acid (AsA) was oxidized for at least 1 min in 3 mL of reaction mixture. CAT activity was spectrophotometrically measured by monitoring the consumption of hydrogen peroxide (H2O2) at 240 nm for at least 3 min. Real-Time RT-PCR Analysis. Total RNA was isolated from 100 mg (fresh weight) of excised radish hypocotyls by grinding with mortar and pestle in liquid nitrogen until a fine powder appeared and using TRIzol reagent (Invitrogen, Gaithersburg, MD) according to the manufacturer’s instructions. DNA-free total RNA (5 μg) from different treatments was used for first-strand cDNA synthesis in a 20 μL reaction volume containing 1 μL of RevertAid M-MuLV reverse transcriptase (Thermo Fisher Scientific Inc., USA) and 1 μL of oligo (dT)18 primer. Real-time quantification RT-PCR reactions were performed using a Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany) with Bestar SybrGreen qPCR Mastermix (DBI, Bioscience Inc., Germany) according to the manufacturer’s instructions. The cDNA was amplified using the primers as described as Table S1 in the Supporting Information. Histochemical Staining. Stress-induced generation of O2− in situ was detected by nitroblue tetrazolium (NBT) staining.32 Seedling leaves were stained with 0.1% solution of NBT in K-phosphate buffer (pH 6.4), containing 10 mM sodium azide (NaN3) until a purple-blue color became visible, and the chlorophyll of the treated leaves was removed with 95% ethanol. Hydrogen peroxide (H2O2) production was detected by 3,3′-diaminobenzidine (DAB) staining.27 Leaves were stained with 0.1% DAB solution for 6 h, and then chlorophyll was removed with 95% ethanol. After washing extensively, all the decolorized leaves were observed under a light microscope (model Stemi 2000-C; Carl Zeiss, Jena, Germany) and photographed on a color film (Powershot A620; Canon Photo Film, Tokyo, Japan). Extraction and Assay of Anthocyanins, Total Phenolic, and DPPH Radical Scavenging Activity Assay. Anthocyanin measurement was performed according to the method reported by Zhou et al. (2013)33 with some modifications. Briefly, 0.5 g samples were cut up and incubated in 10 mL of 1% HCl in methanol at room temperature in the dark for 24 h. Tubes were shaken with a vortex mixer every 6 h. The anthocyanins in the aqueous phase were then quantified by spectrophotometry (A530−A600) (UV-5200 spectrophotometer, Shanghai Metash Instruments Co., Ltd., Shanghai, China), and one unit (U) of anthocyanin was defined as the amount of 0.01 difference value. In the end, the quantity was normalized to the total fresh mass of tissue used for each sample. Determination of total phenols was performed according to the method reported by George et al. (2005)34 with some modifications. Samples (∼0.5 g) were homogenized in 50% (w/v) acetone and shaken in ultrasonic wave cleaner for 30 min, and then centrifuged at 4000g for 20 min at 4 °C. One milliliter of extract was added to 2 mL of Folin−Ciocalteu reagent. The mixture was incubated for 2 min at room temperature, and 10 mL of 10% sodium carbonate was added. The mixture was incubated for 1 h at 50 °C and finally cooled in a water−ice bath. The specific absorbance at 765 nm was immediately measured. The DPPH radical scavenging activity assay was performed according to the method reported by Chen et al. (2013).35 Briefly, standard solutions or DMSO (control) was prepared and added to a 70 μmol/L DPPH methanolic solution; the mixtures were shaken vigorously and left to stand in the dark for 30 min at room temperature, and then absorbance was read at 517 nm. Radical scavenging capacity was expressed as percentage effect (%) and calculated using the following equation:
However, whether H2 plays a specific role in the modulation of UV-A-induced anthocyanin accumulation is largely unknown, and the molecular mechanisms underlying interactions between UV and H2 signaling remain to be elucidated in anthocyanin regulation. In this paper, we report that hydrogenrich water (HRW) reestablishes reactive oxygen species homeostasis but exerts differential effects on anthocyanin synthesis in two contrasting varieties of radish sprouts under UV-A irradiation.
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MATERIALS AND METHODS
Plant Materials and Growth Conditions. Seeds of two contrasting radish (Raphanus sativus L.), i.e., cv. Qingtou (low level of anthocyanin, LA) and cv. Yanghua (high level of anthocyanin, HA), were soaked in either distilled water or hydrogen-rich water (HRW) for 12 h and then germinated for 1 d at 25 °C in darkness. Uniform gemmiparous seeds were then chosen and transferred to plastic chambers and cultured in nutrient medium (quarter-strength Hoagland’s solution) prepared with HRW or distilled water. Four treatments with 3 replicates each were established, i.e., white light (W + Con), white light plus HRW (W + HRW), UV-A (UVA + Con), and UV-A plus HRW (UVA + HRW), respectively. The nutrient medium was replaced every 12 h. Radish sprouts were grown in the incubator at 25 °C in the dark for 2 d, and then transferred to a white or UV-A illuminating incubator (Ningbo Haishu Safe Instrument Experimental Factory, Zhejiang, China) at 25 °C for 24 h. The light intensity of white light was set at 50 ± 5 μmol·m−2·s−1, while the UV-A dose was set at 5.5 W·m−2 measured with a pocket UV light meter (UV-340A, Lutron, Taiwan). Preparation of HRW. Purified hydrogen gas (99.99%, v/v) generated from a hydrogen gas generator (SHC-300; Saikesaisi Hydrogen Energy Co., Ltd., Shandong, China) was bubbled into 4000 mL of distilled water (pH 5.87, 25 °C) at a rate of 150 mL·min−1 for 1 h, a sufficient duration to saturate the solution with H2 (about 0.22 mM).28,30 Then, the corresponding HRW (pH 6.32, 25 °C) was immediately diluted to the required concentrations [1, 10, and 100% concentration (v/v)]. In our experimental conditions, the H2 concentration in freshly prepared HRW analyzed by gas chromatography (GC) was 0.22 mM, which was defined as 100% HRW. The H2 concentrations were about 22 μM in 10% HRW, 2.2 μM in 1% HRW, and maintained at a relatively constant level in 25 °C for at least 12 h. Observation of the Hypocotyl Cross Section. The hypocotyls of radish sprouts were transected with a blade, then observed under a light microscope (model Stemi 2000-C; Carl Zeiss, Germany), and photographed on color film (Powershot A620, Canon Photo Film, Japan). Determination of Thiobarbituric Acid Reactive Substances (TBARS). Lipid peroxidation was estimated by measuring the amount of TBARS as previously described.31 About 500 mg of fresh tissues was ground in 0.25% 2-thiobarbituric acid (TBA) in 10% trichloroacetic acid (TCA) using a mortar and pestle. After heating at 95 °C for 30 min, the mixture was quickly cooled in an ice bath and centrifuged at 10000g for 10 min. The absorbance of the supernatant was read at 532 nm and corrected for unspecific turbidity by subtracting the absorbance at 600 nm. The blank was 0.25% TBA in 10% TCA. The concentration of lipid peroxides, together with oxidatively modified proteins of plants, was thus quantified in terms of TBARS amount using an extinction coefficient of 155 mM−1 cm−1and expressed as nmol g−1 fresh weight (FW). Antioxidant Enzyme Assays. The activity of antioxidant enzymes was determined as described by Jin et al. (2013).28 Frozen radish plants (approximately 200 mg) were homogenized in 5 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 1% polyvinylpyrrolidone for superoxide dismutase (SOD), peroxidase (APX), and catalase (CAT) assay. The homogenate was centrifuged at 12000g for 20 min at 4 °C, and the supernatant was used as the crude enzyme extract. Total SOD activity was measured on the basis of its ability to reduce nitroblue tetrazolium (NBT) by superoxide anion generated by the riboflavin system under illumination. One unit of SOD was defined
⎛ Abscontrol − Abssample ⎞ percentage effect (%) = ⎜ ⎟ × 100 Abscontrol ⎝ ⎠ Identification and Quantification of Anthocyanidins. Sample Preparation. Fresh samples (1.0 g) were homogenized in 3 mL of 70% (w/v) ethanol containing 1% methanoic acid. The mixtures were 6455
dx.doi.org/10.1021/jf5019593 | J. Agric. Food Chem. 2014, 62, 6454−6462
Journal of Agricultural and Food Chemistry
Article
shaken in ultrasonic wave cleaner for 10 min and then centrifuged at 6000g for 20 min, and finally 2 mL of supernate was taken out. The residue was combined with 1 mL of 70% ethanol and then shaken and centrifuged as done above. Another 2 mL of supernate was taken out and merged with the supernate above. Daidzein standard samples (20 μL, 25 μM) were added into the extract and used as internal standard. After mixture and centrifugation, 5 μL was taken out for injection. The relative abundance of anthocyanidins was estimated as the ratios of peak areas of samples to peak areas of Daidzein standard samples. LC-MS/MS Analysis. The anthocyanidins were detected by a UHPLC system (Dionex, Thermo, USA) connected to a LTQOrbitrap XLhybrid mass spectrometer (Thermo Fisher Scientific). The instrument was controlled through Tune 2.6.0 and Chromeleon programs. The UHPLC column was Hypersil GOLD C18 (100 mm × 2.10 mm, 3 μm particle size, Thermo Fisher Scientific). Solvent A was 0.1% formic acid in water, B was 0.1% formic acid in acetonitrile, and separation was carried out in 30 min at a flow rate of 0.2 mL/min under the following conditions: 0−4 min, 5% B; 4−21 min, 5−95% B; 21−25 min, 95% B; 25.2−30 min, 5% B. Column oven temperature was set at 35 °C, and autosampler temperature was set at 10 °C. The capillary temperature was 300 °C, and the source voltage was set at 4 kV. Accurate mass spectra were recorded from 150 to 1200 m/z. The external mass calibration of the Orbitrap was performed once a week to ensure a working mass accuracy
