Endogenous Hydrogen Sulfide Homeostasis Is Responsible for the

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Endogenous hydrogen sulfide homeostasis is responsible for the alleviation of senescence of postharvest daylily flower via increasing antioxidant capacity and maintaining energy status Dan Liu, Sheng Xu, Hua-li Hu, Jincheng Pan, Pengxia Li, and Wen-Biao Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04389 • Publication Date (Web): 06 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017

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Journal of Agricultural and Food Chemistry

Running title: Endogenous H2S delays daylily

Title: Endogenous hydrogen sulfide homeostasis is responsible for the alleviation of senescence of postharvest daylily flower via increasing antioxidant capacity and maintaining energy status

Dan Liu†,|| Sheng Xu,§,|| Huali Hu,‡ Jincheng Pan,† Pengxia Li,‡ and Wenbiao Shen,*,†

†

College of Life Sciences, Laboratory Center of Life Sciences, Nanjing Agricultural

University, Nanjing 210095, China §

Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing

210014, China ‡

Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural

Sciences, Nanjing 210014, China

1

ACS Paragon Plus Environment


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ABSTRACT: There are limited data concerning the role of endogenous H2S in

2

prolonging postharvest of vegetables and fruits. By using fluorescence microscope

3

with a specific probe, we discovered that during the senescence of postharvest daylily

4

flower, endogenous H2S homeostasis was impaired. The activities of two important

5

synthetic enzymes of H2S,

6

desulfhydrase (DCD), exhibited decreasing tendencies. By contrast, NaHS (a H2S

7

donor) not only blocked above decreased H2S production, but also extended

8

postharvest life of daylily. These beneficial roles were verified by the alleviation of

9

lipid peroxidation, and the increased activities of antioxidant enzymes. Meanwhile,

10

the energy status was sustained, and the respiration rate was decreased. In contrast to

11

NaHS, the addition of an inhibitor of H2S synthesis alone aggravated lipid

12

peroxidation, and lowered energy charge. Together, the present study implies that

13

endogenous H2S alleviates senescence of postharvest daylily via increasing

14

antioxidant capacity and maintaining energy status.

L-cysteine

desulfhydrase (LCD) and

D-cysteine

15 16

KEYWORDS: daylily, endogenous hydrogen sulfide homeostasis, senescence,

17

antioxidant capacity, energy status

18

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■ INTRODUCTION

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Flowers of daylily (Hemerocallis fulva) were widespread in China and other Asian

21

regions. Both its fresh and dried flowers have long been consumed as a nutritious food

22

in eastern Asian.1 It is rich in variety of antioxidant compounds, including β-carotene,

23

ascorbic acid, flavonoid, and (+)-catechin, etc.2 Daylily has high medicinal values,

24

such as improving sleeping,3 inhibiting cancer cell proliferation,4 restraining

25

inflammation and jaundice.5 However, the senescence of flower is rapid and highly

26

predictable, since the cell deterioration is observed just 24 h after opening.6

27

During senescence, chlorophyll and macromolecules, including proteins, membrane

28

lipids, and nucleic acids, are degraded for the redistribution of their components into

29

other organs.7 The molecule degradation partly caused the reactive oxygen species

30

(ROS) overproduction.8 In addition, energy status was an important factor for the

31

regulation of membrane integrity, and played a crucial role in regulating ripening and

32

senescence of postharvest of vegetables and fruits.9 Recently, ample evidence showed

33

that senescence-related physiological responses, such as increased membrane

34

permeability and enhanced ROS production, may be related to limited availability of

35

energy.10 Additionally, senescence usually results in programmed cell death (PCD) of

36

all the cells due to a loss of redox control.11,12

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Hydrogen sulfide (H2S) is regarded as the third endogenous signaling molecule in

38

animal systems after nitric oxide (NO) and carbon monoxide (CO).13 L-cysteine

39

desulfhydrase (LCD) and

40

cysteine-degrading enzymes for synthesis of H2S in plants.14-16 Recently, considerable

D-cysteine

desulfhydrase (DCD), are two important

3



41

studies have shown that exogenous application of H2S can delay senescence of

42

postharvest horticultural crops.17-22 The major reason of above beneficial role of H2S

43

is associated with its antioxidant behaviors via efficiently reducing ROS biosynthesis

44

and increasing antioxidant enzyme activities.23,24 However, the changes of H2S during

45

the senescence of postharvest fruits and vegetables, and corresponding roles of

46

endogenous H2S, are still elusive.

47

Thus, the aim of this work was to investigate how endogenous H2S metabolism

48

responses to the senescence of postharvest daylily flowers. Combined with the

49

fumigation, incubation test, and the application of the inhibitor of H2S synthesis, our

50

further work implies that endogenous H2S effectively alleviates senescence of

51

postharvest daylily flower probably via increasing antioxidant capacity and

52

maintaining energy status. The possibility of ascorbate peroxidase (APX) acting as a

53

direct target of H2S signaling, was preliminarily discussed. Together, this work

54

provides some further insights into the molecular mechanisms of H2S action in food

55

chemistry.

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■ MATERIALS AND METHODS

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Plant Materials and Treatments. Daylily flowers were cultivated in Suqian,

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Jiangsu province, China. Flowers of daylily with uniform size and consistent of

60

maturity, were hand-harvested in the early morning, and transported to the laboratory

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as soon as possible (within 2 h). Flowers without defects and physical damage were

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selected for experimental usage. Sodium hydrosulfide (NaHS; Sigma-Aldrich) was

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used as a hydrogen sulfide (H2S) donor, and daylily flowers were exposed to aqueous

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NaHS solutions (100 mL) of 0, 0.8, 1.6, 2.4, 3.2, 4.0, 4.8, and 5.6 mmol L-1 in sealed

65

containers (volume 21 L). Daylily fumigated with same volume of distilled water was

66

regarded as the control. Treatment solutions were renewed daily, and the storage

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temperature was 0 – 2 °C for the indicated time points.

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For another experiment, daylily were respectively immersed in plastic boxes with

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10 L of distilled water (regarded as a control), NaHS solution, or DL-propargylglycine

70

(PAG) solution for 5 min, then followed by air-drying at 20 °C for 1 h. Afterwards,

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daylily flowers were stored at 0 – 2 °C for 8 d. Each treatment was repeated at least

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three times with triplicate replicates for each.

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Endogenous H2S content assay. The content of endogenous H2S was determined

74

by using the methylene blue formation method25 with some modifications. The frozen

75

sepal tissues (1.0 g) were ground with liquid nitrogen, and extracted with 8 mL of 50

76

mmol L-1 phosphate buffered saline (PBS, pH 6.8) containing 0.2 mol L-1 ascorbic

77

acid and 0.1 mol L-1 ethylene diamine tetraacetic acid (EDTA). After centrifugation at

78

10,000 × g for 20 min (4 °C), the supernatant was transferred to a test tube, and mixed

5



79

100 mmol L-1 with PBS (pH 7.4) containing 10 mmol L-1 L-cysteine, and 2 mmol L-1

80

phosphopyridoxal. The released H2S was absorbed in a zinc acetate trap, fixed in the

81

bottom of the test tube. After the reaction for 30 min, 0.3 mL 5 mmol L-1

82

dimethyl-p-phenylenediamine dissolved in 3.5 mmol L-1 H2SO4, was added into the

83

trap. Subsequently, 0.3 mL 50 mmol L-1 ferric ammonium sulfate in 100 mmol L-1

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H2SO4 was injected into the tube, and incubated for 15 min. Finally, the H2S content

85

in the zinc acetate trap was analyzed colorimetrically at 667 nm. A calibration curve

86

was established with known concentrations of Na2S solution. The H2S content was

87

expressed as nmol g-1 fresh weight (FW).

88

Determination of L-cysteine desulfhydrase (LCD) and D-cysteine desulfhydrase

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(DCD) activities. Corresponding activities were analyzed according to the method

90

described by Hu et al.21 with some modifications. Frozen sepal tissues (1.0 g) were

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ground with a mortar and pestle in 8 mL of 20 mmol L-1 Tris-HCl (pH 8.0), and then

92

centrifuged at 12,000 × g for 20 min. For the determination of LCD activity, 1 mL of

93

supernatant was mixed with 1 mL mixture solution containing 100 mmol L-1 Tris-HCl

94

(pH 9.0), 0.8 mmol L-1 L-cysteine, and 2.5 mmol L-1 dithiothreitol (DTT). After the

95

incubation at 37 °C for 15 min, the reaction was terminated by adding 100 µL of 30

96

mmol L-1 FeCl3 dissolved in 1.2 mol L-1 HCl and 100 µL 20 mmol L-1

97

N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 mol L-1 HCl.

98

Then, the formation of the methylene blue was recorded at 670 nm. A calibration

99

curve was established with known concentrations of Na2S solution. DCD activity was

100

determined in the similar method with following modifications: D-cysteine, instead of

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L-cysteine,

was applied, and the pH of the Tris-HCl buffer was 8.0.

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Histochemical analysis. By using the fluorescent probe 7-azido-4-methylcoumarin

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(AzMC; Sigma-Aldrich), endogenous H2S was imaged as described prevously.26 The

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fresh sepals were incubated with 10 mmol L-1 degassed PBS (pH 7.4) buffer solution

105

containing 100 µmol L-1 AzMC at room temperature for 40 min. After washing with

106

distilled water for three times, the sepals were visualized immediately by using a

107

fluorescence microscope (OLYMPUS IX53). Afterwards, the relative density of the

108

fluorescent images was analyzed using Image-Pro Plus 6.0, and data are shown as the

109

mean of fluorescence intensity compared to the control samples.

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In-situ detection of hydrogen peroxide (H2O2) and superoxide anion (O2.-) was

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followed the methods reported by Yang et al.27 with some modifications. The amount

112

of O2.- in the fresh daylily were stained with a 2 mmol L-1 nitroblue tetrazolium (NBT;

113

Sigma-Aldrich) solution in 20 mmol L-1 potassium phosphate buffer (pH 6.4)

114

containing 10 mmol L-1 NaN3 under vacuum with 10 min. To visualize H2O2

115

accumulation, the daylily flowers were treated with 1 mg mL-1 3,3-diaminobenzidine

116

(DAB; Sigma-Aldrich) in 50 mM Tris-acetate buffer (pH 5.0), at room temperature

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for 12 h in the dark. In both cases, the treated flowers were boiled in 95% alcohol (v/v)

118

until the complete removal of chlorophyll, and then photographed.

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Total chlorophyll and soluble protein. Total chlorophyll was extracted using 95%

120

ethanol and quantified by UV spectrophotometer. The soluble protein content was

121

assayed by the method of Bradford28 with bovine serum albumin as standard.

122

Lipid peroxidation assay. Lipid peroxidation was assayed by measuring the

7



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contents of malondialdehyde (MDA) following the method described by Cai et al.29

124

The 2 g sepal samples were ground with 10 mL of 10% trichloroacetic acid (TCA).

125

After centrifugation for 10 min at 4000 × g, 2 mL of supernatant was added with 2 mL

126

of 0.6% 2-thiobarbituric acid (TBA) in 10% TCA, and heated in boiling water for 30

127

min, followed by cooling on ice immediately and centrifuging at 10,000 × g for 10

128

min. The MDA concentration (µmol g-1) was determined with the equation: [6.45 ×

129

(A532 – A600)] – [0.56 × A450].

130

The determinations of ATP, ADP, and AMP levels and energy charge.

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Extraction and quantification of ATP, ADP and AMP were conducted according to the

132

method described by Cao et al.9 with some modifications. The frozen sepal tissues (1

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g) were ground with 6 mL of 0.6 mol L-1 perchloric acid, followed by the

134

centrifugation at 12,000 × g for 15 min at 4 °C. The supernatant was immediately

135

neutralized to pH 6.5 – 6.8 with the addition of 1 mol L-1 KOH. After 30 min of

136

stability, it was diluted to 3 mL and passed through a 0.45 µm filter. The

137

determination of ATP, ADP and AMP contents were carried out by HPLC

138

(Waters, Alliance 2695 pump, USA) equipped with a reverse Spherisorb C-18

139

analytical column (5 µm × 250 mm × 4.6 mm) and UV detector at 254 nm. Mobile

140

phase A contained 0.06 mol L-1 disodium hydrogen phosphate and 0.04 mol L-1

141

potassium dihydrogen phosphate dissolved in deionised water and adjusted to pH 7.0

142

with the addition of 0.1 mol L-1 KOH. Mobile phase B was pure methanol. Elution

143

was performed using 85% A and 15% B for 15 min with a flow rate of 1 mL min-1.

144

Sample aliquots (20 µL) were injected into the HPLC system. Finally, the contents of

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ATP, ADP and AMP were calculated using external standards. Tissue energy status

146

was expressed by the adenine nucleotide ratio, and the energy charge was defined as

147

[ATP + 1/2ADP]/[ATP + ADP + AMP].

148

Respiratory rate assay. The respiration intensity of daylily was assayed by

149

detecting the production of carbon dioxide (CO2), following the method described by

150

Hu et al.21 Fresh daylily flower samples (about 0.3 kg) were randomly chosen and

151

sealed in gas-tight plastic boxes (21-L) for 1 h at 0 – 2 °C. A 20 mL gas sample was

152

obtained from the headspace of each box by using a gas-tight syringe, and then

153

rapidly injected into an Agilent 7820 gas chromatography (Agilent Technologies Inc.,

154

USA) for determining the CO2 concentration under the following conditions: initial

155

temperature, 70 °C; auxiliary heating temperature, 375 °C; running time, 3.5 min;

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hydrogen flow, 50 mL min-1; air flow, 400 mL min-1; carrier gas flow (nitrogen), 25

157

mL min-1.

158

Assay of antioxidant enzyme activity. Frozen sepal samples (1 g) were extracted

159

by homogenizing in 10 mL of 50 mmol L-1 potassium phosphate buffer (pH 7.0)

160

containing 1 mmol L-1 EDTA and 1 % polyvinylpyrrolidone (PVP), with the addition

161

of 1 mmol L-1 ascorbic acid in the case of ascorbate peroxidase (APX) assay. The

162

homogenate was centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatant was

163

used as the crude enzyme extract.

164

The activity of total superoxide dismutase (SOD) was determined by monitoring

165

the inhibition of the photochemical reduction of NBT.30 The amount of enzyme

166

required to cause 50% inhibition of the reduction rate of NBT was defined as one unit 9



167

(U). Catalase (CAT) activity was determined by following the decomposition of H2O2

168

at 240 nm for 3 min31 with a slight modification. The reaction mixture contained 2 mL

169

of 50 mmol L-1 sodium phosphate buffer (pH 7.0), 1 mL 0.2% H2O2, and 0.2 mL

170

enzyme extract. One unit (U) was defined as the amount of enzyme that resulted in

171

0.01 absorbance change per minute. APX activity was determined by measuring the

172

decrease at 290 nm.20 One unit (U) was also calculated as the amount of enzyme that

173

decreased 0.01 absorbance per minute.

174

Gel electrophoresis. The enzyme extract was consistent with above description.

175

Under non-denaturing conditions, different isozymes of SOD were separated on

176

discontinuous polyacrylamide gels (stacking gel 3% and separating gel 10%). For

177

each lane, 30 µl of enzyme extract was loaded. After electrophoresis, the isozymatic

178

activities of SOD on the gel were visualized.30 In order to analyze the relative activity

179

of different isozymes, gels were scanned in the transmission black-and-white mode,

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and the bands intensity was calculated by using the Quantity One v4.4.0 software

181

(Bio-Rad, Hercules, CA, USA).

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DNA laddering. DNA of daylily sepal samples was extracted as described by Cui

183

et al.32 For the DNA laddering analysis, DNA samples were incubated with RNase at

184

37 °C for 30 min. Afterwards, equal amounts of DNA were used for electrophoresis.

185

Finally, gels were stained with ethidium bromide, and then photographed under a UV

186

light.

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Statistical analysis. Results were expressed as means values ± SE of three

188

independent experiments with at least three replicates for each. Statistical analysis

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was performed by one-way ANOVA, taking P < 0.05 as significant according to

190

Duncan’s multiple comparison. Meanwhile, Fisher’s least significant differences

191

(LSD) were calculated following a significant t test (P < 0.05).

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192

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■ RESULTS AND DISCUSSION

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H2S homeostasis is impaired during storage time. To better understand the

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casual link between endogenous H2S and senescence, we firstly investigated changes

195

of endogenous H2S metabolism in postharvest daylily flower. The time-course

196

experiment (12 d; Figure 1A) showed a progressive decrease of H2S production

197

determined by spectrophotography. The changes of total activities of DCD and LCD

198

(in particular, and it exhibited a higher activity than DCD), two enzymes responsible

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for H2S synthesis in plants,14-16 approximately exhibited the similar tendencies. For

200

example, compared to the control sample at 0 d, storage time at 12 d decreased H2S

201

production, DCD and LCD activities by 48.3%, 30.3%, and 72.1%, respectively.

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These results suggested that H2S homeostasis is impaired during senescence.

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To further track endogenous H2S in situ, a commercial specific fluorescent probe

204

AzMC

was

applied.

Compared

to

the

previous

results

determined

205

spectrophotometrically (Figure 1A), AzMC-related florescent density showed the

206

similar tendencies (Figure 1C, D). Above results clearly showed a possible interaction

207

between endogenous H2S homeostasis and senescence in postharvest of daylily

208

flower.

209

NaHS is a well-known H2S donor applied in planta research, and it was previously

210

confirmed to play a vital role in extending the postharvest life of fruits and

211

vegetables.18-23 To investigate whether the maintenance of homeostatic H2S

212

concentrations is important for the delay of postharvest of daylily flower senescence,

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NaHS was applied to monitor the H2S production, with chemical-free (distilled water)

214

as a control. On the basis of phenotype and the total chlorophyll content in a pilot

215

experiment (Figure 2), 4.0 mmol L-1 NaHS fumigation was applied, and thus used in

216

the following study for investigating the roles of endogenous H2S in postharvest of

217

daylily flowers during storage (Figure 3A). As expected, further in situ analysis

218

confirmed that exogenously applied NaHS could obviously slow down the decrease in

219

endogenous H2S during storage, especially at 6 d (Figure 1C, D), which is matched

220

with beneficial phenotype triggered by NaHS (Figure 2; Figure 3A).

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H2S not only maintain high content of the total chlorophyll and soluble protein,

222

but also delay programmed cell death (PCD). Normally, senescence includes

223

cessation of photosynthesis and degeneration of cellular structures, with strong losses

224

of chlorophyll33 and proteins.34 Subsequently, we investigated the time-course

225

changes in total chlorophyll and soluble protein contents. As expected, the total

226

chlorophyll sustained decreasing tendencies during storage in control samples, while

227

H2S fumigation maintained relatively stable levels of chlorophyll content (Figure 3B).

228

Similarly, as indicated in Figure 3C, H2S-fumigated sepal remarkably reduced the loss

229

of the soluble protein contents compared with the control samples throughout the

230

storage time.

231

DNA laddering, a representative phenotype of PCD, has been observed in carpels

232

responding to senescence.35 More recently, H2S is found to delay PCD in gibberellic

233

acid-treated wheat aleurone layers.36 Zhang et al.37 also found that the H2S donor

234

NaHS significantly delayed PCD in barley aleurone layers by acting as an antioxidant. 13



235

Consistently, DNA laddering could be partially alleviated by H2S, especially in the 9

236

day of postharvest, compared with the control in daylily (Figure 3D).

237

Redox imbalance was blocked by H2S. Reactive oxygen species (ROS)

238

overproduction and oxidative damages are two universal events in postharvest

239

vegetables and fruits during storage.18,21 Increased overproduction in ROS, such as

240

H2O2 and O2.-, and afterwards lipid peroxidation, have been already well documented

241

during postharvest storage. To evaluate whether the beneficial roles of H2S were

242

associated with oxidative damages, the MDA content, a reliable marker of lipid

243

peroxidation, was measured. In this study, redox imbalance occurred during storage

244

since MDA content increased accordingly (Figure 4A). Previous results showed that

245

NaHS treatment could reduce senescence-induced oxidative stress and decreased

246

MDA accumulation in the plants.17-20 Our data further demonstrated that NaHS

247

decreased MDA levels, in comparison with the controls during storage, suggesting

248

that redox imbalance was blocked by H2S (Figure 4A).

249

This deduction was confirmed by the subsequent histochemical detection of O2.-

250

(NBT staining) and H2O2 (DAB staining) levels (Figure 4B, C). Similarly, senescence

251

alone brought about some purple-blue (NBT staining) and dark brown (DAB staining)

252

color precipitates, indicating more O2.- or H2O2 accumulation in sepals. Comparatively,

253

those pretreated with NaHS partially compromised the above-mentioned staining

254

patterns. These were highly consistent with the results of decreased MDA contents in

255

daylily (Figure 4A). Above results clearly indicated that redox imbalance was blocked

256

by H2S.

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Changes in antioxidant enzyme activities. In plants, ROS are detoxified via

258

non-enzymatic antioxidants and the antioxidant enzymes, and the latter of which

259

includes SOD, CAT, APX, and other antioxidant enzymes.12,38 We subsequently

260

investigated whether the alleviation of oxidative damage in our previous observation

261

was caused by the enhanced antioxidant enzymatic activities. Results shown in Figure

262

5A and B suggested that total activities of CAT and APX (in particularly) in controls

263

were approximately progressively decreased during senescence, both of which were

264

almost differentially blocked in H2S-fumigated daylily. By contrast, SOD activities

265

exhibited an increase in 3 d, followed by gradually decreases until 12 d of senescence,

266

respect to the control sample at 0 d (Figure 5C). However, H2S fumigation

267

significantly maintained higher SOD activities compared to corresponding control

268

samples. Furthermore, to investigate the isozymatic activities of SOD, a native

269

polyacrylamide gel electrophoresis (PAGE) was applied. Under our experimental

270

condition, at least three isozymes of SOD were detected in postharvest daylily flowers,

271

and the SOD-III isozyme contributed the most activity (Figure 5D, E). During

272

senescence, similar to the control samples, the patterns of isoforms I-III activities in

273

response to H2S were comparable to those of in-tube assay except those in 12 d. For

274

example, unlike those in in-tube assay, activities of SOD-III (the major isozyme) of

275

different treatments at 12 d were increased respect to those in 9 d. And the reason is

276

not clear. Above mentioned results are in accordance with the confirmed previous

277

reports, showing that the reduction of ROS over-accumulation by H2S-activated

278

antioxidant enzyme activities plays a vital role in prolonging postharvest storage.18-23

15



279

Together, our results indicated that redox imbalance during senescence blocked by

280

endogenous H2S was associated with the increased antioxidant enzymatic activities.

281

Changes of respiration, ATP, ADP and AMP contents, and energy charge.

282

Respiration is a good indicator of the metabolic rates of postharvested fruits and

283

vegetables. Exogenous H2S could inhibit the respiration rate in strawberry18 and water

284

spinach.21 In this study, as shown in Figure 6A, the respiration rates both in control

285

and H2S-fumigated samples were increased initially (from day 0 to day 6), followed

286

by decreasing until the end of storage. Comparatively, H2S remarkably weakened the

287

respiration rate during storage.

288

It was well-known that energy status is related to ripening and senescence of

289

postharvest vegetable.21 ATP contents in postharvest daylily were progressively

290

decreased during the entire storage periods, and H2S-fumigated plants displayed

291

significant higher levels of ATP content compared to these of controls (Figure 6B).

292

Meanwhile, ADP contents in different treatments remained stable at the beginning of

293

storage, followed decreasing after 6 days of storage, especially in H2S-treated daylily

294

(Figure 6C). By contrast, AMP content increased from day 0 to day 9, particular in

295

NaHS-fumigated samples, followed by decreasing (Figure 6D). Above results showed

296

that compared to the control samples, endogenous H2S could sustain higher levels of

297

energy charge (Figure 6E) during the major storage period in postharvest daylily,

298

which were consistent with those in water spinach21 and banana.39 Combined with the

299

contrasting observation in ATP contents triggered by H2S (Figure 6), we further

300

deduced that the respiration rates might be decreased by high ATP contents during

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301 302

senescence. Contrasting responses are observed when

DL-propargylglycine

(PAG), an H2S

303

synthetic inhibitor, and NaHS, was individually applied. To further verify the

304

possibility that endogenous H2S prevents daylily senescence, PAG, an effective H2S

305

synthetic inhibitor40 and NaHS, were applied individually, and compared. As shown in

306

Figure 7, when postharvest daylily flowers were incubated in the solutions,

307

contrasting responses in phenotypes were observed. Similar to the fumigation

308

experiments (Figures 2 and 3), NaHS solution alone could delay senescence of

309

postharvest daylily, which was supported by the increased contents of total

310

chlorophyll and energy charge (Figure 7). Meanwhile, redox homeostasis was

311

reestablished, since MDA content was decreased by 14.0% in daylily flowers,

312

compared to the control samples. Consistently, NaHS could increase activities of SOD,

313

CAT and APX by 20.3, 14.6, and 11.2%, respectively.

314

By contrast, when PAG solution was applied alone, a significant decrease in

315

chlorophyll content was observed (Figure 7), suggesting accelerating senescence

316

when endogenous H2S homeostasis was impaired. Phenotypic analysis further

317

confirmed the contrasting responses, compared to the beneficial role of NaHS. It has

318

been demonstrated that H2S functions as a signal through protein S-sulfhydration,

319

which involves the posttranslational modification of protein cysteine residues to form

320

a persulfide group (R-SSH).41,42 Recent work provided evidence, showing the

321

existence of an S-sulfhydration-modified cysteine residue on cytosolic APX in

322

Arabidopsis.43 Under our experimental condition, opposite changes of MDA content

17



323

and APX activity elicited by PAG alone were observed, in comparison with those of

324

NaHS-treated postharvest daylily. These results suggested that APX, a major

325

antioxidant enzyme mainly responsible for the scavenging of H2O2 in higher plants,38

326

might be the major target(s) of H2S. Additionally, PAG triggered a slight but no

327

significant decrease in SOD and CAT activities compared to control samples. Together,

328

these results strengthen the hypothesis that endogenous H2S homeostasis is

329

responsible for the alleviation of senescence of postharvest daylily flower by coupling

330

the increase in antioxidant capacity and maintenance of energy status. Similar

331

beneficial roles of endogenous H2S have been reported in the induction of lateral root

332

formation44 and the regulation of heavy metal tolerance.45

333

Some investigations revealed that H2S could enhance the transport of cysteine to

334

increase glutathione (GSH) production in mammalian cells.46,47 Similar to animals,

335

the cysteine and GSH contents in Arabidopsis were markedly increased in response to

336

the elevated atmospheric H2S.48 Meanwhile, water-stressed wheat plants treated with

337

NaHS displayed higher levels in ascorbate and GSH contents, as well as a higher

338

proportion of reduced forms.49 It was also reported that NaHS resulted in increases of

339

endogenous GSH contents and the ratio of GSH/oxidized GSH (GSSG) in GA-treated

340

wheat aleurone layers, and the NaHS-mediated alleviation of programmed cell death

341

(PCD) was markedly blocked by a selective inhibitor of GSH biosynthesis

342

(l-buthionine-sulfoximine, BSO).36 Since both GSH and cysteine are two reducing

343

compounds and redox buffers, the casual link between H2S and GSH/cysteine in the

344

delay of postharvest senescence in daylily flower should be further investigated.

18


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345

Together, our results revealed that the maintenance of endogenous H2S homeostasis

346

plays a vital role in delaying senescence of postharvest daylily flowers, which might

347

be, at least partly, associated with the total activities of LCD and DCD, two important

348

H2S synthetic enzymes in higher plants.14-16 Our results further provided evidence,

349

showing that the essential roles of endogenous H2S in delaying senescence of daylily

350

flowers was carried out partially via 1) the increase in antioxidant capacity to maintain

351

the redox balance; 2) the maintenance of energy status. Since recent work both in

352

animals and plants suggested that H2S exerts its functions by protein S-sulfhydration

353

under various physiological environments,41-43 further investigations are needed to

354

elucidate the potential signal transduction pathway of H2S, especially in its direct

355

target (APX, etc), in the biology of senescence.

356

19



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357

■ AUTHOR INFORMATION

358

Corresponding Author

359

*(W.S.)

360

[email protected].

361

Author Contributions

362

||

363

Funding

364

This work was supported by Jiangsu Independent Innovation Fund for Agricultural

365

Science and Technology [CX(16)1022] and the Priority Academic Program

366

Development of Jiangsu Higher Education Institutions (RAPD).

367

Notes

368

The authors declare no competing financial interest.

Phone:

+86

25

84396542.

Fax:

+86

25

84396542.

E-mail:

D.L. and S.X. contributed equally to this work.

369 370

■ ABBREVIATIONS USED

371

APX, ascorbate peroxidase; AzMC, 7-azido-4-methylcoumarin; CAT, catalase; CO,

372

carbon monoxide; CO2, carbon dioxide; DAB, 3,3-diaminobenzidine; DCD,

373

D-cysteine

374

acid; FW, fresh weight; H2O2, hydrogen peroxide; H2S, hydrogen sulfide; LCD,

375

L-cysteine

376

nitroblue

377

DL-propargylglycine;

378

PVP, polyvinylpyrrolidone; ROS, reactive oxygen species; SOD, superoxide

desulfhydrase; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic

desulfhydrase; MDA, malondialdehyde; NaHS, sodium hydrosulfide; NBT, tetrazolium;

NO,

nitric

oxide;

O2.-,

superoxide

anion;

PAG,

PBS, phosphate buffered saline; PCD, programmed cell death;

20


Page 21 of 39


379

dismutase; TBA, 2-thiobarbituric acid; TCA, trichloroacetic acid

21



380

Page 22 of 39

■ REFERENCES

381

(1) Cichewicz, R. H.; Lim, K. C.; McKerrow, J. H.; Nair, M. G. Kwanzoquinones

382

A-G and other constituents of Hemerocallis fulva ‘Kwanzo’ roots and their activity

383

against the human pathogenic trematode Schistosoma mansoni. Tetrahedron 2002, 58,

384

8597–8606.

385

(2) Fu, M.; He, Z.; Zhao, Y.; Jing, Y.; Mao, L. Antioxidant properties and involved

386

compounds of daylily flowers in relation to maturity. Food Chem. 2009, 114,

387

1192–1197.

388 389

(3) Uezu, E. Effects of hemerocallis on sleep in mice. Psychiat. Clin. Neuro. 1998, 52, 136–137.

390

(4) Cichewicz, R. H.; Zhang, Y.; Seeram, N. P.; Nair, M. G. Inhibition of human

391

tumor cell proliferation by novel anthraquinones from daylilies. Life Sci. 2004, 74,

392

1791–1799.

393 394 395 396

(5) Zhang, Y.; Cichewicz, R. H.; Nair, M. G. Lipid peroxidation inhibitory compounds from daylily (Hemerocallis fulva) leaves. Life Sci. 2004, 75, 753–763. (6) Lay-Yee, M.; Stead, A. D.; Reid, M. S. Flower senescence in daylily (Hemerocallis). Physiol. Plant. 1992, 86, 308–314.

397

(7) López-Fernández, M. P.; Burrieza, H. P.; Rizzo, A. J.; Martínez-Tosar, L. J.;

398

Maldonado, S. Cellular and molecular aspects of quinoa leaf senescence. Plant Sci.

399

2015, 238, 178–187.

400 401

(8) Bieker,

S.;

Riester,

L.;

Stahl,

M.;

Franzaring,

J.;

Zentgraf,

U.

Senescence-specific alteration of hydrogen peroxide levels in Arabidopsis thaliana

22


Page 23 of 39


402

and oilseed rape spring variety Brassica napus L. cv. mozart. J. Integr. Plant

403

Biol. 2012, 54, 540–554.

404

(9) Cao, S.; Cai, Y.; Yang, Z.; Joyce, D. C.; Zheng, Y. Effect of MeJA treatment on

405

polyamine, energy status and anthracnose rot of loquat fruit. Food Chem. 2014, 145,

406

86–89.

407

(10)

Veltman, R. H.; Lentheric, I.; Van der Plas, L. H. W.; Peppelenbos, H. W.

408

Internal browning in pear fruit (Pyrus communis L. cv Conference) may be a result of

409

a limited availability of energy and antioxidants. Postharvest Biol. Tec. 2003, 28,

410

295–302.

411 412 413

(11)

Panavas, T.; Rubinstein, B. Oxidative events during programmed cell death

of daylily (Hemerocallis hybrid) petals. Plant Sci. 1998, 133, 125–138. (12)

Rogers, H. J. ; Munné-Bosch, S. Production and scavenging of reactive

414

oxygen species and redox signaling during leaf and flower senescence: Similar but

415

different. Plant Physiol. 2016, 171, 1560–1568.

416 417 418

(13)

Wang, R. Physiological implications of hydrogen sulfide: a whiff

exploration that blossomed. Physiol. Rev. 2012, 92, 791–896. (14)

Alvarez, C.; Calo, L.; Romero, L. C.; García, I.; Gotor, C. An

419

O-acetylserine (thiol)lyase homolog with L-cysteine desulfhydrase activity regulates

420

cysteine homeostasis in Arabidopsis. Plant Physiol. 2010, 152, 656–669.

421

(15)

Romero, L. C.; García, I; Gotor, C. L-Cysteine Desulfhydrase 1 modulates

422

the generation of the signaling molecule sulfide in plant cytosol. Plant Signal Behav.

423

2013, 8, e24007.

23



424 425 426

(16)

Guo, H.; Xiao, T.; Zhou, H.; Xie, Y.; Shen, W. Hydrogen sulfide: a versatile

regulator of environmental stress in plants. Acta Physiol. Plant. 2016, 38, 1–13. (17)

Zhang, H.; Hu, S. L; Zhang, Z. J.; Hu, L. Y.; Jiang, C. X.; Wei, Z. J.; Liu, J.;

427

Wang, H. L.; Jiang, S. T. Hydrogen sulfide acts as a regulator of flower senescence in

428

plants. Postharvest Biol. Tec. 2011, 60, 251–257.

429

(18)

Hu, L. Y.; Hu, S. L.; Wu, J.; Li, Y. H.; Zheng, J. L.; Wei, Z. J.; Liu, J.; Wang,

430

H. L.; Liu, Y. S.; Zhang, H. Hydrogen sulfide prolongs postharvest shelf life of

431

strawberry and plays an antioxidative role in fruits. J. Agric. Food Chem. 2012, 60,

432

8684–8693.

433

(19)

Gao, S. P.; Hu, K. D.; Hu, L. Y.; Li, Y. H.; Han, Y.; Wang, H. L.; Lv, K.; Liu,

434

Y. S.; Zhang, H. Hydrogen sulfide delays postharvest senescence and plays an

435

antioxidative role in fresh-cut kiwifruit. HortScience 2013, 48, 1385–1392.

436

(20)

Li, S. P.; Hu, K. D.; Hu, L. Y.; Li, Y. H.; Jiang, A. M.; Xiao, F.; Han, Y.; Liu,

437

Y. S.; Zhang, H. Hydrogen sulfide alleviates postharvest senescence of broccoli by

438

modulating antioxidant defense and senescence-related gene expression. J. Agric.

439

Food Chem. 2014, 62, 1119–1129.

440

(21)

Hu, H.; Liu, D.; Li, P.; Shen, W. Hydrogen sulfide delays leaf yellowing of

441

stored water spinach (Ipomoea aquatica) during dark-induced senescence by delaying

442

chlorophyll breakdown, maintaining energy status and increasing antioxidative

443

capacity. Postharvest Biol. Tec. 2015, 108, 8–20.

444 445

(22)

Ni, Z. J.; Hu, K. D.; Song, C. B.; Ma, R. H.; Li, Z. R.; Zheng, J. L.; Fu, L.

H.; Wei, Z. J.; Zhang, H. Hydrogen sulfide alleviates postharvest senescence of grape

24


Page 24 of 39

Page 25 of 39


446

by modulating the antioxidant defenses. Oxid. Med. Cell Longev. 2016, 2016,

447

4715651.

448

(23)

Hu, K. D.; Wang, Q.; Hu, L. Y.; Gao, S. P.; Wu, J.; Li, Y. H.; Zheng, J. L.;

449

Han, Y.; Liu, Y. S.; Zhang, H. Hydrogen sulfide prolongs postharvest storage of

450

fresh-cut pears (Pyrus pyrifolia) by alleviation of oxidative damage and inhibition of

451

fungal growth. PLoS One 2014, 9, e85524.

452

(24)

Sun, Y.; Zhang, W.; Zeng, T.; Nie, Q.; Zhang, F.; Zhu, L. Hydrogen sulfide

453

inhibits enzymatic browning of fresh-cut lotus root slices by regulating phenolic

454

metabolism. Food Chem. 2015, 177, 376–381.

455 456 457

(25)

Sekiya, J.; Schmidt, A.; Wilson, L. G.; Filner, P. Emission of hydrogen

sulfide by leaf tissue in response to L-cysteine. Plant Physiol. 1982, 70, 430–436. (26)

Chen, B.; Li, W.; Lv, C.; Zhao, M.; Jin, H.; Jin, H.; Du, J.; Zhang, L.; Tang,

458

X. Fluorescent probe for highly selective and sensitive detection of hydrogen sulfide

459

in living cells and cardiac tissues. Analyst 2013, 138, 946–951.

460

(27)

Yang, L.; Tian, D.; Todd, C. D.; Luo, Y.; Hu, X. Comparative proteome

461

analyses reveal that nitric oxide is an important signal molecule in the response of rice

462

to aluminum toxicity. J. Proteome Res. 2013, 12, 1316–1330.

463

(28)

Bradford, M. M. A rapid and sensitive method for the quantitation of

464

microgram quantities of protein utilizing the principle of protein-dye binding. Anal.

465

Biochem. 1976, 72, 248–254.

466 467

(29)

Cai, C.; Chen, K. S.; Xu, W. P.; Zhang, W. S.; Li, X.; Ferguson, I. Effect of

1-MCP on postharvest quality of loquat fruit. Postharvest Biol. Tec. 2006, 40,

25



468 469 470 471

155–162. (30)

Beauchamp, C.; Fridovich, I. Superoxide dismutase: Improved assays and

an assay applicable to acrylamide gels. Anal. Biochem. 1971, 44, 276–287. (31)

Tareen, M. J.; Abbasi, N. A.; Hafiz, I. A. Postharvest application of salicylic

472

acid enhanced antioxidant enzyme activity and maintained quality of peach cv.

473

‘Flordaking’ fruit during storage. Sci. Hortic. 2012, 142, 221–228.

474

(32)

Cui, W.; Gao, C.; Fang, P.; Lin, G.; Shen, W. Alleviation of cadmium

475

toxicity in Medicago sativa by hydrogen-rich water. J. Hazard. Mater. 2013, 260,

476

715–724.

477

(33)

Ougham, H.; Hörtensteiner, S.; Armstead, I.; Donnison, I.; King, I.; Thomas,

478

H., Mur, L. The control of chlorophyll catabolism and the status of yellowing as a

479

biomarker of leaf senescence. Plant Biol. 2008, 10, 4–14.

480

(34)

Srivalli, B.; Khanna-Chopra, R. The developing reproductive ‘sink’ induces

481

oxidative stress to mediate nitrogen mobilization during monocarpic senescence in

482

wheat. Biochem. Biophys. Res. Commun. 2004, 325, 198–202.

483 484 485

(35)

Orzáez, D.; Granell, A. DNA fragmentation is regulated by ethylene during

carpel senescence in Pisum sativum. Plant J. 1997, 11, 137–144. (36)

Xie, Y.; Zhang, C.; Lai, D.; Sun, Y.; Samma, M. K.; Zhang, J.; Shen, W.

486

Hydrogen sulfide delays GA-triggered programmed cell death in wheat aleurone

487

layers by the modulation of glutathione homeostasis and heme oxygenase-1

488

expression. J. Plant Physiol. 2014, 171, 53–62.

489

(37)

Zhang, Y. X.; Hu, K. D.; Lv, K.; Li, Y. H.; Hu, L. Y.; Zhang, X. Q.; Ruan, L.;

26


Page 26 of 39

Page 27 of 39


490

Liu, Y. S.; Zhang, H. The hydrogen sulfide donor NaHS delays programmed cell

491

death in barley aleurone layers by acting as an antioxidant. Oxi. Med. Cell.

492

Longev. 2015, 2015, 1–11.

493 494 495

(38)

Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant

Sci. 2002, 7, 405–410. (39)

Li, D.; Limwachiranon, J.; Li, L.; Du, R.; Luo, Z. Involvement of energy

496

metabolism to chilling tolerance induced by hydrogen sulfide in cold-stored banana

497

fruit. Food Chem. 2016, 208, 272–278.

498 499 500

(40)

García-Mata, C.; Lamattina, L. Hydrogen sulphide, a novel gasotransmitter

involved in guard cell signalling. New Phytol. 2010, 188, 977–984. (41)

Mustafa, A. K.; Gadalla, M. M.; Sen, N.; Kim, S.; Mu, W.; Gazi, S. K.;

501

Barrow, R. K.; Yang, G.; Wang, R.; Snyder, S. H. H2S signals through protein

502

S-sulfhydration. Sci. Signal. 2009, 2, ra72.

503 504 505 506 507

(42)

Paul, B. D.; Snyder, S. H. H2S signalling through protein sulfhydration and

beyond. Nat. Rev. Mol. Cell Biol. 2012, 13, 499–507. (43)

Aroca, Á.; Serna, A.; Gotor, C.; Romero, L. C. S-sulfhydration: A cysteine

posttranslational modification in plant systems. Plant Physiol. 2015, 168, 334–342. (44)

Fang, T.; Cao, Z.; Li, J.; Shen, W.; Huang, L. Auxin-induced hydrogen

508

sulfide generation is involved in lateral root formation in tomato. Plant Physiol.

509

Biochem. 2014, 76, 44–51.

510 511

(45)

Cui, W.; Chen, H.; Zhu, K.; Jin, Q.; Xie, Y.; Cui, J.; Xia, Y.; Zhang, J.; Shen,

W. Cadmium-induced hydrogen sulfide synthesis is involved in cadmium tolerance in

27



512

Medicago sativa by reestablishment of reduced (Homo) glutathione and reactive

513

oxygen species homeostasis. PLoS One 2014, 9, e109669.

514

(46)

Kimura, Y.; Goto, Y.; Kimura, H. Hydrogen sulfide increases glutathione

515

production and suppresses oxidative stress in mitochondria. Antioxid Redox Signal.

516

2010, 12, 1–13.

517 518 519

(47)

Kimura, Y.; Kimura, H. Hydrogen sulfide protects neurons from oxidative

stress. FASEB J. 2004, 18, 1165–1167. (48)

Riemenschneider, A.; Nikiforova, V.; Hoefgen, R.; De Kok, L. J.;

520

Papenbrock, J. Impact of elevated H2S on metabolite levels, activity of enzymes and

521

expression of genes involved in cysteine metabolism. Plant Physiol. Biochem. 2005,

522

43, 473–483.

523

(49)

Shan, C.; Zhang, S.; Li, D.; Zhao, Y.; Tian, X.; Zhao, X.; Wu, Y.; Wei, X.;

524

Liu, R. Effects of exogenous hydrogen sulfide on the ascorbate and glutathione

525

metabolism in wheat seedlings leaves under water stress. Acta Physiol. Plant. 2011,

526

33, 2533–2540.

28


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Figure legends

Figure 1. Changes of endogenous H2S metabolism in postharvest daylily. H2S content (A) and activities of LCD and DCD (B) were spectrophotometrically determined in daylily flower during storage time at 0 – 2 °C. Meanwhile, samples were fumigated with 4.0 mmol L-1 NaHS during the indicated storage times at 0 – 2 °C, and corresponding sepals were loaded with AzMC (a H2S probe) for fluorescent imaging (C; bar = 1 mm), and the fluorescent density (D) was counted (n = 6). Data are the means ± SE of three independent experiments with at least three replicates for each. Bars denoted by different letters were significantly different (P < 0.05) according to Duncan’s multiple comparison.

Figure 2. Effects of H2S on the senescence in postharvest daylily. Visual appearance (A) and total chlorophyll contents (B) of daylily fumigated with different concentrations of NaHS during storage at 0 – 2 °C for 8 d. Data are means ± SE of three independent experiments with at least three replicates for each. Bars denoted by different letters were significantly different (P < 0.05) according to Duncan’s multiple comparison.

Figure 3. Changes of visual appearance (A), total chlorophyll (B), soluble protein contents (C), and DNA laddering (D). Postharvest daylily flowers were fumigated with 4.0 mmol L-1 NaHS during the indicated storage times at 0 – 2 °C. Afterwards,

29



the contents of total chlorophyll (A) and soluble protein (B) were determined. Meanwhile, the agarose gel (C) shows DNA extracted from sepals. Additionally, numbers to the right of the figure indicate DNA size of the molecular marker. The sample without chemicals was the control. Data are means ± SE of three independent experiments with at least three replicates for each. Bars denoted by different letters were significantly different (P < 0.05) according to Duncan’s multiple comparison.

Figure 4. Redox balance was reestablished by H2S. Postharvest daylily flowers were fumigated with 4.0 mmol L-1 NaHS during the indicated storage times at 0 – 2°C. Afterwards, MDA content (A) was determined. Meanwhile, corresponding sepals were stained with NBT (B) and DAB (C), respectively, and immediately photographed. Bar = 1 mm. The sample without chemicals was the control. Data are means ± SE of three independent experiments with at least three replicates for each. Bars denoted by different letters were significantly different (P < 0.05) according to Duncan’s multiple comparison.

Figure 5. Changes in total and isozymatic activities of antioxidant enzymes. Postharvest daylily flowers were fumigated with 4.0 mmol L-1 NaHS during the indicated storage times at 0 – 2 °C. Afterwards, the total activities of CAT (A), APX (B) and SOD (C) were determined. Isozymatic activity of SOD was analyzed by using electrophoresis (D), and the band intensities of the individual isozymes were expressed as % of the corresponding first isozyme of the control sample (0 d; E). The

30


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Page 31 of 39


arrows indicate the bands corresponding to various isozymes. The sample without chemicals was the control. Data are means ± SE of three independent experiments with at least three replicates for each. Bars denoted by different letters were significantly different (P < 0.05) according to Duncan’s multiple comparison.

Figure 6. Changes of respiratory rate (A), contents of ATP (B), ADP (C), AMP (D), and energy charge (E). Postharvest daylily flowers were fumigated with 4.0 mmol L-1 NaHS during the indicated storage times at 0 – 2 °C. The sample without chemicals was the control. Data are means ± SE of three independent experiments with at least three replicates for each. * in this figure indicates a significant difference between control and NaHS treatment at P < 0.05 (t test).

Figure 7. Contrasting responses in the visual appearance, contents of total chlorophyll and MDA, energy charge, and activities of SOD, CAT and APX respectively triggered by PAG and NaHS. Postharvest daylily flowers were incubated in the solution containing 2 mmol L-1 PAG, 4.0 mmol L-1 NaHS for 5 min at room temperature, then shifted to at 0 – 2 °C for 8 d. The sample without chemicals was the control. Data are means ± SE of three independent experiments with at least three replicates for each. Bars denoted by different letters were significantly different (P < 0.05) according to Duncan’s multiple comparison.

31



Figure 1 145x156mm (300 x 300 DPI)


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Figure 2 125x118mm (300 x 300 DPI)



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TOC graphic 85x51mm (300 x 300 DPI)


Endogenous Hydrogen Sulfide Homeostasis Is Responsible for the

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