Hydrogen Sulfide Alleviates Postharvest ... - ACS Publications

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Hydrogen Sulfide Alleviates Postharvest Senescence of Broccoli by Modulating Antioxidant Defense and Senescence-Related Gene Expression Shi-Ping Li,†,§ Kang-Di Hu,†,§ Lan-Ying Hu,†,§ Yan-Hong Li,† An-Min Jiang,‡ Fang Xiao,† Yi Han,† Yong-Sheng Liu,† and Hua Zhang*,† †

School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China Nano Science and Technology Institute, University of Science and Technology of China, Suzhou Dushu Lake Higher Education Town, Suzhou 215123, China

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ABSTRACT: Accumulating evidence has shown that hydrogen sulfide (H2S) acts as a signaling regulator in plants. Here we show that H2S delays the postharvest senescence of broccoli in a dose-dependent manner. H2S maintains higher levels of metabolites, such as carotenoids, anthocyanin, and ascorbate, and reduces the accumulation of malondialdehyde, H2O2, and the superoxide anion. Further investigations showed that H2S sustained higher activities of guaiacol peroxidase, ascorbate peroxidase, catalase, and glutathione reductase and lower activities of lipoxygenase, polyphenol oxidase, phenylalanine ammonia lyase, and protease than those of water control. Moreover, the expression of the chlorophyll degradation related genes BoSGR, BoCLH2, BoPaO, BoRCCR, as well as cysteine protease BoCP1 and lipoxygenase gene BoLOX1, was down-regulated in postharvest broccoli treated with H2S. The functions of H2S on the senescence of other vegetables and fruits suggest its universal role acting as a senescence regulator. KEYWORDS: antioxidant enzymes, broccoli, hydrogen sulfide, senescence, senescence-related genes

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INTRODUCTION Fresh broccoli (Brassica oleracea var. italica) is a cruciferous vegetable with a high nutritional value due to its significant content of vitamins, antioxidant substances, and anticarcinogenic compounds.1 In recent years the demand for fresh or ready to eat broccoli has increased greatly. Floral heads of broccoli are composed of hundreds of florets with petals and chlorophyll-containing sepals. After harvest, broccoli rapidly experiences disruption in energy metabolism, nutrition, and hormone supply, which leads to rapid opening of florets and senescence, as measured by yellowing and chlorophyll degradation within the florets.2 As senescence progresses, chlorophyll and protein levels in floret tissues decline and endoprotease activity increases. In developing countries, the postharvest losses of fruits and vegetables account for as much as 50% of produce yield. Therefore, new methods should be developed to ensure the postharvest quality of broccoli and to extend its marketability at room temperature. Recently, accumulating evidence shows that hydrogen sulfide (H2S) is an endogenous signaling molecule with multifaceted physiological functions.3 Since the 1970s, the release of H2S was observed in a wide variety of plant species and is correlated with a diverse array of responses.4 H2S can be generated from cysteine or sulfite by the enzymatic actions of O-acetylserine (thiol) lyase or sulfite reductase, respectively.5 H2S has been shown to act as an endogenous gaseous regulator, participating in wheat grain germination, stomatal movement, and root organogenesis.3,6 More recently, we found that exogenous H2S is involved in preventing the senescence of cut flowers and prolonging flower vase life in a broad range of plants.7 We also investigated the role of H2S in the postharvest senescence of © 2014 American Chemical Society

fruits and found that H2S significantly prolonged the shelf life of strawberry.8 The concentration of the applied H2S required to delay senescence in strawberry is quite low, indicating that the fumigation of fruits with H2S gas released from donor solutions could be safe.8 Studies on the DES1 gene encoding L-Cys desulfhydrase (EC 4.4.1.1) unveiled the important role of sulfide in autophagy and senescence in Arabidopsis.9,10 Mutation of the DES1 gene, leads to premature leaf senescence, as demonstrated by increased expression of senescence-associated genes and transcription factors.9,10 Furthermore, exogenous H2S plays a role as a repressor of autophagy in Arabidopsis.10 Natural senescence and dark-promoted senescence are accompanied by macromolecule degradation in leaves. Whether H2S functions in alleviating the senescence of postharvest broccoli and the underlying mechanism are largely unknown. In the present study, we provide evidence that H2S fumigation effectively alleviates the postharvest senescence of broccoli, probably through decreased production of reactive oxygen species (ROS), enhanced antioxidant defense, and the modulation of senescence-related gene expressions. These data add to the notion that H2S has a widespread role in alleviating senescence in a broad range of postharvest vegetables and fruits. Received: Revised: Accepted: Published: 1119

October 19, 2013 January 12, 2014 January 14, 2014 January 14, 2014 dx.doi.org/10.1021/jf4047122 | J. Agric. Food Chem. 2014, 62, 1119−1129

Journal of Agricultural and Food Chemistry

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acid, and the homogenate was filtered through filter paper. The volume of filtrate was adjusted to 25 mL with 4% oxalic acid and titrated with 2,6-dichlorophenol-indophenol to a pink color. Determination of the Contents of Reducing Sugar, Soluble Protein, Total Soluble Solids, and Free Amino Acids. For reducing sugars, broccoli samples (1.00 ± 0.01 g) were ground with 5 mL of sodium phosphate buffer (pH 7.0, 200 mM), the homogenate was centrifuged at 10 000 rpm for 30 min, and the supernatant was collected for subsequent analysis. The determination of reducing sugar was performed spectrophotometrically according to Miller’s dinitrosalicylic acid method.17 Each analysis was repeated in triplicate, and the results were expressed as mg/g FW. Total soluble solid content was determined using a digital refractometer (Tongfang Inc., Shanghai, China) according to the method of Jiang et al.18 The content of soluble protein was determined by the method described by Bradford.19 Supernatant (0.1 mL) was mixed with 0.9 mL of distilled water and 5 mL of 0.1 mg/mL Coomassie Brilliant Blue G250. After 5 min, the OD value was recorded at 595 nm by spectrophotometer. Analyses were repeated in triplicate and the results expressed as mg/g FW. Free amino acid content was measured according to the work of Hu et al. using an automatic amino acid analyzer (model S433D, Syknm, Germany).8 Fresh-cut broccoli florets (0.50 ± 0.01 g) were ground with 5 mL of 80% ethanol, the homogenate was centrifuged at 10 000 rpm for 5 min, and the supernatant was heated at 80 °C to remove ethanol and to concentrate the sample to 1 mL. Determination of Superoxide Anion (•O2−), Hydrogen Peroxide (H2O2), and Malondialdehyde (MDA). Contents of •O2−, H2O2, and MDA were determined according to the methods described by Hu et al.8 Determination of Enzyme Activities. Activities of guaiacol peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and lipoxidase (LOX) were determined by the procedures described by Hu et al.8 Frozen broccoli floret samples (0.50 ± 0.01 g) were homogenized with 5 mL of ice-cold sodium phosphate buffer (200 mM, pH 7.8) containing 1.0 mM EDTA. The homogenate was centrifuged at 12 000 rpm (4 °C, 20 min), and the supernatant was used for activity measurement. Analysis of guaiacol POD activity was based on the oxidation of guaiacol by hydrogen peroxide. The reaction mixture contained 1.7−1.8 mL of sodium phosphate buffer (50 mM, pH 6.1), 0.1 mL of 3% H2O2, 1 mL of 1% guaiacol, and 100−200 μL of enzyme extract. The increase in absorbance at 420 nm was recorded. APX activity was determined in the presence of 0.5 mM ascorbate and 0.5 mM H2O2 by monitoring the decrease in absorbance at 290 nm. CAT activity was determined spectrophotometrically by monitoring the decrease in absorbance at 240 nm. The reaction mixture contained 2.8 mL of sodium phosphate buffer (50 mM, pH 7.0), 100 μL of 3% H2O2, and 100 μL of enzyme extract. The decrease of GR activity in absorbance at 340 nm was recorded. One unit of POD, APX, CAT, and GR activity was defined as an increase or decrease of 0.01 OD value per minute. The results were expressed as U/g FW. The activity of LOX was determined in the presence of linoleic acid by monitoring the changes in absorbance at 234 nm. One unit of LOX activity was defined as a decrease of 0.01 OD value in absorbance per minute. The results were expressed as U/g FW. The activity of polyphenol oxidase (PPO) was determined by procedures described by Benjamin and Montgomery.20 Broccoli samples (2.00 ± 0.05 g) were homogenized with 5.0 mL of sodium phosphate buffer (50 mM, pH 6.8). After centrifugation, the supernatant was used for the activity assay using catechol as substrate. One unit of PPO activity was defined as an increase of 0.01 OD value in absorbance at 410 nm per minute. The results were expressed as U/ g FW. The activity of phenylalanine ammonia lyase (PAL) was determined by the procedures described by Beaudoin-Eagan and Thorpe.21 One unit of PAL activity was defined as a change of 0.01 OD value in absorbance at 290 nm per minute. The results were expressed as U/g FW.

MATERIALS AND METHODS

Plant Materials and Treatment. Fresh broccoli (B. oleracea var. italica) that was kindly supplied by the orchard of Anhui Academy of Agricultural Sciences, Anhui, China, and selected to avoid florets showing physical damage or microbial infection was used throughout this study. Solutions of sodium hydrosulfide (NaHS·3H2O, Sigma) were used as the hydrogen sulfide (H2S) donor. Aqueous solutions (150 mL) of 0, 0.40, 0.80, 1.20, 1.60, 2.00, 2.40, 2.80, or 3.20 mM NaHS were prepared in sealed containers (volume 3 L). Six pieces of freshly cut broccoli florets (approximately 35 ± 2 g) from six different broccoli shoots were fumigated with H2S in the sealed containers at 25 °C and 85−90% relative humidity. For dark-promoted senescence, the containers were stored in darkness. Treatment solutions were renewed daily and florets were observed every 24 h. Each experiment was repeated three times. Visual Evaluation of Broccoli Senescence. The appearance of fresh-cut broccoli fumigated with H2S gas was evaluated subjectively by 20 people using five indexes: freshness, rotting rate, yellowness, flavor, texture, and floret organization.11 Each index was divided into four ranks and assigned scores of 2.0, 1.5, 1.0 and 0, with higher numbers assigned to better visual evaluation, and the sum of the five indexes is shown as a score from 0 to 10. Determination of Functional Compounds in Broccoli. Chlorophyll content of broccoli florets was determined using the colorimetric method of Lichtenthaler and Wellburn with minor modifications.12 About 0.50 ± 0.01 g of finely chopped broccoli floret samples was placed in an Erlenmeyer flask containing 5−10 mL of 80% acetone as extraction solvent. After extraction, the OD value of supernatant was recorded at 645 and 663 nm. Every analysis was repeated in triplicate, and the results were expressed as mg/g FW (fresh weight). Carotenoid content of broccoli samples was determined by the method of Nath et al.13 Fresh .50 ± 0.01 g sample was ground with 5− 10 mL of acetone by adding 1.00 g of anhydrous Na2SO4. The supernatant was transferred to a beaker and the extraction step was repeated three times. The combined supernatants were transferred to a separating funnel, and 5−10 mL of petroleum ether was added and mixed thoroughly. The upper layer of petroleum ether was collected and absorbance recorded at 452 nm, and the amount of carotenoid was expressed as mg/g FW. The following procedures were used for the assays of total phenols, flavonoids, and anthocyanin. Broccoli samples of 1.00 ± 0.01 g were homogenized with 10 mL of aqueous methanolic HCl (50% methanol, 0.05% concentrated HCl, pH 3.5), the brei was allowed to macerate for 24 h in the dark (0−4 °C), and the mixture was filtered for subsequent determination. Determination of total phenols was performed according to the method of Pirie and Mullins.14 The content of total phenols was determined spectrophotometrically at 280 nm. A calibration curve was prepared for total phenols using gallic acid as the standard. The determination of flavonoids was performed according to the work of Zhishen et al. with slight modification.15 A 1.0 mL portion of filtrate was placed in a 10 mL volumetric flask, distilled water was added to make 5 mL, 0.3 mL of 5% NaNO2 was added, and the solution was mixed thoroughly. After 5 min, 3 mL of 10% Al(NO3)3 was added, and the solution was mixed thoroughly. After 6 min, 2 mL of 1 M NaOH was added, the total was adjusted to 10 mL with distilled water, and the solution was mixed thoroughly again. The content of flavonoids was determined spectrophotometrically at 510 nm with rutin used as the standard for calibration. The determination of anthocyanin was estimated according to the method of Lee and Wicker.16 Absorption of the filtrate was recorded at 530, 620, and 650 nm. The content of anthocyanin was calculated using the formula below. One unit of anthocyanin content was expressed as a change of 0.1 OD value.

OD = (OD530 − OD620 ) − 0.1 × (OD650 − OD620 ) Ascorbate was determined by the method described by Nath et al.13 Floret samples of 2.00 ± 0.01 g were ground with 15 mL of 4% oxalic 1120

dx.doi.org/10.1021/jf4047122 | J. Agric. Food Chem. 2014, 62, 1119−1129


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Figure 1. Effects of H2S on the senescence and visual evaluation in postharvest broccoli. Broccoli florets were fumigated with different concentrations (0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, or 3.2 mM) of aqueous solutions of NaHS for 0−10 d, respectively, and the experimental treatments are shown in lower right of part A. Photographs (A) were taken from day 0 to 10, and visual evaluation (B) was recorded. The experiments and following ones were carried out at room temperature and 85−90% relative humidity. The activity of protease was measured according to the method described by Reimerdes and Klostermeyer.22 Broccoli florets (0.50 ± 0.01 g) were homogenized with 5 mL of ice-cold Tris-HCl buffer [50 mM, pH 7.5, included 1 mM ethylene diamine tetraacetic acid (EDTA), 15 mM β-mercaptoethanol, and 1% PVP]. The homogenate was centrifuged at 10 000 rpm (4 °C, 30 min), and the supernatant

was used for activity determination. One unit of protease activity was defined as an increase of 0.01 OD value at 278 nm per hour, and the results were expressed as U/g FW. RNA Extraction and Reversed Transcript Polymerase Chain Reaction (PCR). Fresh-cut broccoli florets were ground in liquid nitrogen, and RNA was extracted by the RNAiso Plus according to the 1121



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Figure 2. Effects of H2S on the contents of total chlorophyll (A), chlorophyll a (B), chlorophyll b (C), carotenoid (D), total phenols (E), flavonoid (F), anthocyanin (G), ascorbate (H), reducing sugar (I), soluble protein (J), and total soluble solids (TSS) (K) in postharvest broccoli. Broccoli florets were fumigated with 2.4 mM H2S donor NaHS aqueous solution (T) with water as the control groups (CK) for 0−5 d. Data are presented as means ± SD (n = 3). * and ** in this figure and following ones stand for a significant difference between CK and T at P < 0.05 and P < 0.01, respectively. manufacturer’s instructions (Takara, Kyoto, Japan), and total RNA was used for cDNA synthesis using a reverse transcription kit (Prime Script RT Master Mix, Takara, Kyoto, Japan). The resulting cDNA was applied as template for PCR using a PCR system with Easy Taq DNA Polymerase (TransGen, Beijing, China). The PCR primers used for detecting the genes BoSGR (stay-green protein), BoNYC (chlorophyll b reductase), BoCLH1 (chlorophyllase 1), BoCLH2 (chlorophyllase 2), BoPaO (pheophorbide a oxygenase), BoPPH (pheophytinase), BoRCCR (red catabolite chlorophyll reductase), and BoACT (actin) followed the methods of Hasperué et al.23 The following primers were utilized to assess relative expression of BoCP1 (cysteine protease) and BoLOX1 (lipoxygenase 1) by PCR: BoCP1, 5′-CGCCGATCTGACCAACGAA-3′ and 5′-CCGCTCCAATCGCAGAAAA-3′; BoLOX1, 5′-TCTCCACGAATTTCTGGGAAACAAAG-3′ and 5′AGTCCTCTGAGTGTGTAGCCTTTC-3′. Three independent extracts of RNA were done and similar results were obtained. Analysis of Rubisco. Fresh-cut broccoli florets were homogenized using a chilled mortar and pestle in sodium phosphate buffer (50 mM, pH 7.5) containing 1 mM EDTA, 15 mM 2-mercaptoethanol, and 1% (v/v) PVP, and the homogenate was centrifuged at 15 000 rpm for 10 min at 4 °C. Then the supernatant was collected for Rubisco analysis.

The Rubisco was visualized by nondenaturing PAGE with Coomassie Brilliant Blue R-250 staining according to the method of Ono et al.24 Other Vegetables and Fruits Fumigated with H2S. Fresh-cut lotus root, potato, yams, lettuce, pumpkin, and peach, kindly supplied by the orchard of Anhui Academy of Agricultural Sciences, Anhui, China, were fumigated with different concentrations of H2S donor NaHS and photographed. Statistical Analysis. Statistical significance was tested by one-way analysis of variance (ANOVA), and the results are expressed as the mean values ± standard deviation (SD) of three independent experiments. Fisher’s least significant differences (LSD) were calculated following a significant (P < 0.01 or P < 0.05) t test.

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RESULTS H 2 S Alleviates the Senescence of Postharvest Broccoli. In our experiments, broccoli florets were fumigated with H2S released by different concentrations of aqueous solutions of NaHS with water as the control. As shown in Figure 1A, H2S fumigation alleviated postharvest senescence of broccoli in a dose-dependent manner. Water control broccoli 1122


99.03 304.86 262.72 364.99 250.66

7.67 a 2.1 b 6.98 c 14 d 4.4 c ± ± ± ± ± CK0 CK1 T1 CK2 T2

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matured and yellowed quickly after 3 days of storage, while different NaHS treatment alleviated the postharvest yellowing of broccoli with an optimal concentration of 2.4 mM. Visual quality was evaluated according to the freshness, rotting rate, yellowness, flavor, texture, and organization. As shown in Figure 1B, 2.4 mM NaHS treatment showed the optimal effect on the visual quality of postharvest broccoli and thus was used in subsequent experiments. Effect of H2S on the Contents of Chlorophyll, Carotenoid, Total Phenols, Flavonoids, Anthocyanin, Ascorbate, Reducing Sugar, Soluble Proteins, and Total Soluble Solids. To further understand how H2S alleviated yellowing and browning of fumigated broccoli, we determined the contents of chlorophyll, carotenoid, phenols, flavonoids, anthocyanin, and ascorbate in broccoli. Chlorophyll contents (Figure 2A) were expressed as the sum of chlorophyll a (Figure 2B) and chlorophyll b (Figure 2C). As shown in Figure 2A−C, total chlorophyll content as well as the amounts of chlorophylls a and b decreased gradually during storage in water controls, while H2S fumigation maintained a relatively stable level of total chlorophyll and chlorophyll a until day 4, followed by a decrease on day 5, and also a higher level of chlorophyll b at the late stage of storage. Carotenoid pigments protect cellular membranes by scavenging or quenching free radicals25 acting as an essential lipid-soluble antioxidant in plants. Changes in carotenoid content, which consist of β-carotene and lutein, are shown in Figure 2D. The content of carotenoid decreased rapidly in the water control, whereas H2S fumigation significantly prevented this decrease. Regardless of treatment, total phenols increased steadily in broccoli florets with time (Figure 2E). However, broccoli fumigated with H2S maintained lower levels of total phenols compared with water controls after 2 d of storage. As shown in Figure 2F, flavonoid content increased gradually in both treatments until 3 d followed by a rapid decrease in water controls, while H2S fumigation alleviated the decrease and sustained a significantly higher level of flavonoids on days 4 and 5. The content of anthocyanin in broccoli florets increased steadily in the water control until 5 d of storage, as shown in Figure 2G, but anthocyanin content in H2S-fumigated broccoli was maintained at a significantly lower level compared with that of water controls. As shown in Figure 2H, the content of ascorbate in broccoli florets decreased during postharvest storage in both treatments; however, H2 S fumigation significantly alleviated the decrease after 2 d of storage. The contents of reducing sugars and soluble proteins are shown in Figure 2I,J. The content of reducing sugar decreased gradually in both H2S-fumigated and water control tissue throughout the storage time; however, the content of reducing sugar in postharvest broccoli fumigated with H2S was higher than that of control on days 2 and 3 of storage. As shown in Figure 2J, soluble protein levels in H2S-fumigated broccoli increased on day 1 and remained stable until day 3 followed by a drop on day 4. A similar pattern was observed in water control, except that the soluble protein level decreased sharply on day 2 and increased slightly on day 3. H2S fumigation sustained significantly higher level of soluble protein on days 2 and 3 relative to that of water control. The contents of soluble solids decreased sharply after 1 day of storage in H2S treatment and water controls; thereafter, the contents varied slightly (Figure 2K). However, H2S application

a Postharvest broccoli were fumigated with 2.4 mM H2S donor NaHS aqueous solution (T) with water as the control groups (CK) for 0−2 d (CK0, CK1, CK2, T1, and T2). Data are presented as means ± SD (n = 3). The contents of free amino acids were determined by automatic amino acid analyzer (model S433D, Syknm). Different letters indicate significant differences (p < 0.05) between the treatments.

1254.58 a 244.69 b 534.74 a 694.25 b 247.16 a ± ± ± ± ± 16276.31 20419.61 18340.6 22275.08 18994.34 223.38 a 4.96 b 24.28 b 36.36 c 16.46 d ± ± ± ± ± 424. 925.03 877.08 1342.21 1047. a b b c d 7.15 2.68 6.69 8.81 2.81 ± ± ± ± ± 84.92 229.79 212.76 326.38 257.63 15.53 a 0.1 b 4.14 b 6.1 c 1.27 d ± ± ± ± ± 186.03 275.49 273.92 350.64 311.98 8.03 a 4.16 b 7.07 b 10.25 c 0.91 c ± ± ± ± ± 67.63 198.99 188.68 318.24 326.7 6.22 a 0.79 b 8.47 b 5.69 c 18.41 b ± ± ± ± ±

83.21 a 113.87 b 56.59 b 55.1 b 34.43 b 1208.06 1892.4 1940.39 1953 1906.37 Lys 10.67 a 228.82 b 23.15 b 24.1 c 18.62 d ± ± ± ± ± 155.63 a 5.78 b 102.78 c 107.71 c 34.78 a

80.21 360.45 531.91 807.53 748.96 His ± ± ± ± ± 2403.05 3289.47 2957.29 2795.89 2328.08 Phe 289.77 a 31.23 b 150.02 c 172.31 d 60.63 c 4416.75 ± 5853.02 ± 5197.02 ± 6518.72 ± 5423.44 ± Tyr 6728.88 ± 413.05 a 5807.36 ± 39.95 b 4750.38 ± 118.91 c 5772.42 ± 210.27 b 4901.75 ± 32.34 c Leu CK0 CK1 T1 CK2 T2

23.35 91.22 86.68 137.59 104.24

122.72 ± 10.83 a 348.61 ± 2.84 b 308.27 ± 9.36 b 485.03 ± 16.73 c 402.74 ± 6.58 d total AA ± ± ± ± ± ± ± ± ± ±

431.72 842.95 753.51 1102.45 984.81 Arg

Val Ala Gly Glu Ser Asp

Table 1. Effects of H2S Fumigation on the Content of the Free Amino Acids in Postharvest Broccoli Floretsa

23.46 a 3.39 b 16.28 c 26.81 d 15.53 e

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Figure 3. Effects of H2S on the accumulation of superoxide anion (•O2−) (A), hydrogen peroxide (H2O2) (B), and malonaldehyde (MDA) (C). Broccoli florets were fumigated with 2.4 mM H2S donor NaHS aqueous solution (T) with water as the control groups (CK) for 0−5 d. Data are presented as means ± SD (n = 3).

Figure 4. Role of H2S in the regulation of the activities of guaiacol peroxidase (POD) (A), ascorbate peroxidase (APX) (B), catalase (CAT) (C), and glutathione reductase (GR) (D) and the down-regulation of lipoxygenase (LOX) (E), polyphenol oxidase (PPO) (F), phenylalanine ammonia lyase (PAL) (G), and protease (H) in postharvest broccoli florets. Postharvest broccoli florets were fumigated with 2.4 mM H2S donor NaHS aqueous solution (T) with water as the control groups (CK) for 0−5 d. Data are presented as means ± SD (n = 3).

maintained significantly higher levels of soluble solids than water controls on days 1, 3 and 4. Effect of H2S on the Contents of Free Amino Acids in Postharvest Broccoli. H2S fumigation sustained lower levels of free amino acids compared with water control (Table 1). Storage and senescence induced a rapid increase of total amino acid contents, regardless of treatment, although exposure to H2S gas alleviated the increase. With the exception of Asp, the content of free amino acids in H2S-fumigated and control broccoli was higher than that of freshly harvested broccoli. In addition, H2S did not significantly affect the levels of Gly, Ala, and His on day 1, while a significant decrease of Gly and His appeared on day 2 compared with water control. Many amino

acids, including Asn, Cys, Gln, Met, Pro, Thr, Trp, and Tyr, were not detected in broccoli during storage. H2S Decreases the Content of MDA and Reactive Oxygen Species, •O2− and H2O2. The contents of •O2−, H2O2 and MDA in broccoli fumigated with H2S and water are shown in Figure 3A−C. The overproduction of reactive oxygen species and the occurrence of oxidative damage are universal events during vegetable storage. •O2− accumulated steadily during the first 3 d of storage in water controls followed by a gradual decrease, whereas H2S fumigation significantly alleviated •O2− accumulation at 2 and 3 d of storage (Figure 3A). H2O2 production in postharvest broccoli was rapidly enhanced on 3 d of storage and thereafter was maintained at a relatively higher level than that of H2S-fumigated florets (Figure 1124



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than water control. For instance, on day 3 of storage, H2S fumigation induced a 3-fold increase of CAT activity compared with water control. Figure 4D showed similar variations in GR activities in both treatments, except that H2S fumigation induced a burst of GR activity on 3 d of storage. GR activities decreased slightly during the first day of storage in both treatments, but H2S fumigation induced a rapid increase of GR activity until day 3, followed by a sharp decline on day 4. In contrast, GR activity in water control was maintained at a relative lower and stable level compared with H2S treatment. LOXs belong to a large family of plant enzymes that catalyze the hydroperoxidation of polyunsaturated fatty acids. Figure 4E showed a similar change pattern in LOX activity between control and H2S treatment, while H2S fumigation sustained a lower level of LOX compared with water controls. LOX activity was induced greatly in the first day of storage and then fluctuated in water controls. However, LOX activity was maintained at a lower level in H2S-treated tissue than in water controls. Changes in the activities of PPO are illustrated in Figure 4F and show that H2S fumigation maintained lower PPO activities compared with controls during the entire postharvest storage period. PPO activities increased gradually in control broccoli, while those of H2S-treated florets were maintained at lower levels. Changes in PAL activity, an enzyme that catalyzes the first step in the phenylpropanoid pathway, are shown in Figure 4G. PAL activity declined during the first day of storage in both treatments. Thereafter, PAL activity increased gradually in water control broccoli, whereas the activity fluctuated at a lower level in H2S treatment. Surface browning is frequently observed in fresh-cut vegetables and is highly related to the activities of PPO and PAL. We observed lower levels of phenols and lower PAL and PPO activities in H2S-fumigated broccoli compared with water control, suggesting that H2S postponed the browning process in postharvest broccoli. The changes of protease activities are shown in Figure 4H. Consistent with the higher level of soluble protein in H2Sfumigated broccoli (Figure 2J), the activity of protease in H2Sfumigated broccoli was always lower than that of controls. On day 2 of storage, protease activity was enhanced greatly in water control broccoli. Consistent with the lower level of protease in H2S-fumigated broccoli, the total free amino acids in H2Sfumigated samples were lower than that of water controls (Table 1). Effect of H 2 S on the Relative Expressions of Senescence-Related Genes. Broccoli senescence is accompanied by chlorophyll degradation and protein degradation. Therefore, we examined the effects of H2S fumigation on the expression of the chlorophyll degradation related genes BoSGR, BoCLH2, BoPaO, BoRCCR, cysteine protease gene BoCP1, and lipoxygenase gene BoLOX1. As shown in Figure 5, the expression of BoSGR, BoCLH2, BoPaO, BoRCCR, BoCP1, and BoLOX1 were induced after 2 d of storage in water, suggesting that these genes were expressed along with senescence, while H2S fumigation showed lower expression levels of these genes on days 2 and 4 of storage. In contrast, the expression levels of BoNYC, BoCLH1, and BoPPH were relatively stable during storage and H2S fumigation showed marginal effects on the expression of these genes. Effect of H2S Fumigation on Dark-Promoted Senescence. Dark treatments have been used as a conventional method to induce plant senescence.26 Thus, we examined whether H2S fumigation could alleviate dark-promoted

Figure 5. Effect of H2S on the relative expression of BoSGR (staygreen protein), BoNYC (chlorophyll b reductase), BoCLH1 (chlorophyllase 1), BoCLH2 (chlorophyllase 2), BoPaO (pheophorbide a oxygenase), BoPPH (pheophytinase), BoRCCR (RCC reductase), BoCP1 (cysteine protease), BoLOX1 (lipoxygenase), and BoACT (actin, AF044573) in broccoli florets fumigated with 2.4 mM H2S donor NaHS aqueous solution (T) and water as the control groups (CK) for 0, 2, and 4 d, respectively. BoACT was used as a control gene.

3B). In contrast, H2O2 content of H2S-treated tissue was maintained at a low level for 3 d until a burst appeared on d 4. As shown in Figure 3C, MDA accumulated steadily in control broccoli during the first 2 d of storage and then was maintained at a stable level until 4 d followed by a slight decline. In contrast, H2S fumigation sustained a constant lower level of MDA during storage compared with water control (Figure 3C). Changes in the Activities of Guaiacol Peroxidase, Ascorbate Peroxidase, Catalase, Glutathione Reductase, Lipoxygenase, Polyphenol Oxidase, and Phenylalanine Ammonia Lyase. The activities of enzymes involved in oxidative metabolism in plants, such as guaiacol peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), lipoxygenase (LOX), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL) were measured in broccoli exposed to 2.4 mM NaHS and water (Figure 4). As shown in Figure 4A, POD activities in H2S-treated broccoli were maintained at a higher level than those of controls after 4 d of storage and then decline. In control tissue, POD increased on day 1 and decreased steadily until 4 d of storage. Figure 4B illustrates similar changes in the activity of APX in broccoli exposed to H2S or water. APX activity increased in both treatments after 1 d of storage and peaked on day 4 followed by a decline. The activity of APX in H2S-fumigated broccoli was always higher than that of control during 5 d storage. The activities of CAT in postharvest broccoli under both treatments are described in Figure 4C. CAT activity in H2S and water controls decreased gradually with the lowest level appearing on days 2 and 3, respectively. Thereafter, CAT activity increased steadily in both treatments, while H2S fumigation always maintained higher CAT activity 1125



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Figure 6. Effects of H2S on dark-induced senescence, visual evaluation, and Rubisco degradation in postharvest broccoli. Broccoli florets were fumigated with different concentrations (0, 1.2, and 2.4 mM) of aqueous solutions of NaHS for 0−5 d, respectively. Photographs (A) were taken from day 0 to 5, and the visual evaluation (B) was recorded. Effect of H2S on the degradation of Rubisco in darkness (C) in broccoli florets fumigated with 2.4 mM H2S donor NaHS aqueous solution (T) and water as the control groups (CK) for 0−5 d. Lanes 1−3 from left to right in part B represent broccoli florets in dark-induced senescence for 0 d, 5 d (CK5), and 5 d (T5), respectively. The proteins of broccoli samples were separated by nondenaturing PAGE and then stained with Coomassie Brilliant Blue R-250.

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DISCUSSION Due to the reduction in the content of carbohydrate, protein, vitamins, and antioxidants, broccoli florets are perishable and senescent after harvest.1,25 As broccoli deteriorates, it undergoes chlorophyll degradation, lipid peroxidation, cell necrosis.27 Our results show that H2S can alleviate the senescence of postharvest broccoli under natural and dark-promoted senescence conditions (Figures 1 and 6) and maintain higher contents of nutrients (Figure 2). During postharvest storage, we observed a decrease of chlorophylls, carotenoids, flavonoids, and ascorbate, while H2S application to postharvest broccoli can alleviate such decreases. The nonenzymatic antioxidant system in plants consists of ascorbate, carotenoids, and flavonoids and is capable of quenching ROS generated during postharvest storage.28,29 The yellowing of plants is positively correlated to the degradation of chlorophyll.30 The stable content of chlorophyll under H2S fumigation, as observed in the present study (Figure 1A), strongly supports the role of H2S in alleviating senescence. The content of anthocyanin accumulated highly in water control compared to H2Sfumigated broccoli during storage. Anthocyanin accumulated in leaves during autumn senescence,31 suggesting that in the present study H2S fumigation postponed senescence in broccoli florets.

senescence in broccoli. As shown in Figure 6A, 1.2 and 2.4 mM NaHS treatments effectively alleviated the dark-promoted senescence of postharvest broccoli. Visual evaluation showed that NaHS treatment maintained higher quality of postharvest broccoli than water controls (Figure 6B). During leaf senescence, Rubisco is gradually degraded, and its components are recycled within the plant; therefore, we examined whether H2S fumigation alleviated Rubisco degradation during broccoli senescence. As shown in Figure 6C, during dark-promoted senescence, H2S fumigation maintained higher level of Rubisco compared with water treatment, suggesting the important role of H2S in alleviating Rubisco degradation. H2S Alleviates the Senescence of Other Vegetables and Fruits. As shown in Figure 7, when postharvest lotus root, potato, yams, lettuce, pumpkin, and peach were fumigated with different concentrations of the H2S donor NaHS, their shelf lives were prolonged. H2S fumigation also prevented the surface browning of lotus root, potato, yams, lettuce, and peach. This survey of H2S fumigation on the senescence of these postharvest vegetables and fruits also suggests that the role of H2S in alleviating senescence might be universal in plants, probably by the activation of the antioxidant enzymes. H2S also reduced the browning process in fumigated postharvest lotus root, potato, yam, and lettuce, further confirming a role for H2S in regulating postharvest processes in these plants. 1126



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Figure 7. Effect of H2S fumigation on the senescence of other vegetables and fruits. Fresh-cut vegetables and fruits of lotus root (A), potato (B), yams (C), lettuce (D), pumpkin (E), and peach (F) were fumigated with different concentrations of NaHS as shown in the lower part of every figure. Photographs were taken at different times, as shown in each single figure.

that catalyze the hydroperoxidation of polyunsaturated fatty acids and lead to the production of MDA.27 We found that H2S sustained lower levels of LOX and lower expression level of BoLOX1 compared with water control. Correspondingly, the accumulation of MDA was also reduced by H2S, suggesting that oxidative stress in postharvest broccoli was alleviated by H2S. Surface browning is frequently observed in fresh-cut vegetables. Phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) are two key enzymes in the synthesis of free phenolics and the catalytic oxidation of phenolics to brown-colored pigments, respectively, and were found to have a strong relevance to surface browning.33,34 We observed a lower level of phenols and lower levels of PAL and PPO activities in H2S-fumigated broccoli compared with water control, implicating that H2S postponed the browning process in fresh-cut broccoli. The study of H2S on the senescence of other vegetables and fruits also suggested that the role of H2S in alleviating senescence might be universal in plants. The reduced browning process observed in H2S-fumigated fresh-cut lotus root, potato, yam, and lettuce further confirms a role of H2S in browning inhibition. H2S can be produced endogenously in plants, and H2S emission has been observed in plants attacked by pathogens or when under abiotic stresses as part of sulfur-induced resistance (SIR) response,5 suggesting a signaling role of H2S in plant growth and development. More recently, we found that

The reducing sugar, soluble protein, soluble solid, and free amino acids were also determined in broccoli during postharvest storage. H2S sustained higher level of reducing sugar, soluble protein, and soluble solid than water control. In contrast, the total content of free amino acids accumulated more in water control than in H2S-fumigated broccoli. Consistently, H2S sustained lower level of protease activity, whose primary role in plant senescence is to remobilize the nutrients out of dying cells into actively growing tissues in the plant.30 Overproduction of reactive oxygen species and oxidative damage are universal events during vegetable storage. In the present study, H2S significantly alleviated the accumulation of superoxide anion, hydrogen peroxide, and MDA. Plants have evolved the capacity to scavenge ROS by antioxidants, such as ascorbate and phenols, and antioxidative enzymes SOD, POD, APX, CAT, and GR.32 The ascorbate−glutathione cycle maintains homeostasis of ascorbate and glutathione in plants, and consequently, we measured their levels during broccoli senescence.28 In the present study, we observed decreased levels of H2O2, •O2−, and MDA and enhanced activities of POD, CAT, APX, and GR, suggesting the signaling role of H2S in activating the antioxidant enzymes. The senescence of postharvest vegetables is accompanied with lipid peroxidation mediated mainly by LOXs (lipoxygenases). LOXs belong to a large family of plant enzymes 1127



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31301820, 31300133), the Scientific Research Foundation for Returned Overseas Chinese Scholars (SRF for ROCS, SEM), the Natural Science Foundations of Anhui Province (11040606M85), and the Anhui Provincial Education Department (2012AJZR0028, ZD200910).

exogenous H2S is involved in preventing senescence of cut flowers and strawberry.7,8 Á lvarez et al. found that the DES1 mutation led to premature leaf senescence and induced autophagy phenotype in Arabidopisis, suggesting an important role of sulfide in senescence.9,10 The underlying mechanism of hydrogen sulfide in senescence alleviation is not completely known. Here we found that the expression of chlorophyll degradation related genes BoSGR, BoCLH2, BoPaO, BoRCCR, cysteine protease gene BoCP1, and lipoxygenase gene BoLOX1 was down-regulated in broccoli fumigated with H2S during storage. During chlorophyll degradation, transformation from Chl b to Chl a is catalyzed by a Chl(ide) b reductase encoded by a gene named non-yellow coloring 1 (NYC1). 35 Dephytilation of chlorophyll by chlorophyllase (CHL) is required in chlorophyll degradation. Schelbert et al. proposed that a new enzyme, pheophytinase (PPH), is involved in dephytilation of pheophytine to generate pheophorbide.36 Pheophorbide a oxygenase (PaO) catalyzes the opening of the porphyrin ring of pheophorbide and generates red chlorophyll catabolytes (RCC). This reaction is followed by the reduction of RCC by RCC reductase (RCCR). Since the photosynthetic competence of sgr leaves decreases normally during that period, it is classified as one of the nonfunctional type stay-green mutants. Then we tried to discover whether the delayed yellowing effect of H2S on broccoli is a nonfunctional stay-green type. We detected the content of Rubisco in darkpromoted senescence and found that H2S not only alleviated chlorophyll degradation but also prevented Rubisco degradation during senescence of broccoli. In the present study, H2S treatment maintained higher levels of metabolites and lower accumulation of ROS and MDA in broccoli compared with water control, suggesting that H2S treatment provides a safe way to alleviate senescence during vegetable storage. Besides, H2S could be produced endogenously in plants.5 Our previous report showed that the levels of endogenous H2S in fruits treated with exogenous H2S gas are 10−20% higher than those of the water control, further suggesting its safe usage in postharvest storage.8 The ripening and senescence of postharvest vegetables are regulated by endogenous signaling molecules and hormones, including ethylene, jasmonic acid, ABA, H2O2, and NO.8,37−40 In this work, we confirm the role of H2S in prevention of the senescence of broccoli and other vegetables and fruits, implying that H2S might be universal signal in endogenous maturation and senescence in plants. However, whether and how H2S is involved in cross-talk with other hormones and signaling molecules, such as ethylene, jasmonic acid, and NO, requires further elucidation.

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ABBREVIATIONS USED APX, ascorbate peroxidase; CAT, catalase; FW, fresh weight; GR, glutathione reductase; LOX, lipoxygenase; MDA, malondialdehyde; •O2−, superoxide anion; PAL, phenylalanine ammonia lyase; POD, guaiacol peroxidase; PPO, polyphenol oxidase; ROS, reactive oxygen species.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-18919661103. Fax: +86-551-62901043. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Russell Jones, University of California at Berkeley, for critical reading of the manuscript. This work was supported by Natural Science Foundation of China (31271803, 1128



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