Metabolomics Reveals How Cucumber (Cucumis ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

Ecotoxicology and Human Environmental Health

Metabolomics Reveals How Cucumber (Cucumis sativus) Reprograms Metabolites to Cope with Silver Ions and Silver Nanoparticle-Induced Oxidative Stress Huiling Zhang, Wenchao Du, Jose R Peralta-Videa, Jorge L. Gardea-Torresdey, Jason C. White, Arturo A. Keller, Hongyan Guo, Rong Ji, and Lijuan Zhao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02440 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

Environmental Science & Technology

1

Metabolomics Reveals How Cucumber (Cucumis

2

sativus) Reprograms Metabolites to Cope with

3

Silver Ions and Silver Nanoparticle-Induced

4

Oxidative Stress

5 6

Huiling Zhang§, Wenchao Du§, Jose R. Peralta-Videaζ, Jorge L. Gardea-Torresdeyζ,

7

Jason C. White¶, Arturo Keller⁋, Hongyan Guo§, Rong Ji§*, Lijuan Zhao§*

8 9

§

State Key Laboratory of Pollution Control and Resource Reuse, School of Environment,

10

Nanjing University, Nanjing 210023, China ζ

11 12 13

Avenue, El Paso, Texas 79968, United States ¶

Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station

14 15 16

Chemistry Department, The University of Texas at El Paso, 500 West University

(CAES), New Haven, Connecticut 06504, United States ⁋

Bren School of Environmental Science & Management, University of California, Santa Barbara, California 93106-5131, United States

17 18

*Corresponding author. Tel: +86 025-8968 0581; fax: +86 025-8968 0581.

19

Email address: [email protected]; [email protected]

1

ACS Paragon Plus Environment


20

21 22

TOC

23 24 25 26 27 28 29 30 31 32 33

2


Page 2 of 35

Page 3 of 35


34

ABSTRACT

35

Due to their well-known antifungal activity, the intentional use of Ag nanoparticle (NPs)

36

as sustainable nano-fungicides is expected to increase in agriculture. However, the

37

impacts of AgNPs on plants must be critically evaluated to guarantee their safe use in

38

food production. In this study, 4-week-old cucumber (Cucumis sativus) plants received a

39

foliar application of AgNPs (4 or 40 mg per plant) or Ag+ (0.04 or 0.4 mg per plant) for

40

seven days. Gas chromatography-mass spectrometry (GC-MS) based non-target

41

metabolomics enabled the identification and quantification of 268 metabolites in

42

cucumber leaves. Multivariate analysis revealed that all the treatments significantly

43

altered the metabolite profile. Exposure to AgNPs resulted in metabolic reprogramming,

44

including activation of antioxidant defense systems (up-regulation of phenolic

45

compounds) and down-regulation of photosynthesis (up-regulation of phytol).

46

Additionally, AgNPs enhanced respiration (up-regulation of TCA cycle intermediates),

47

inhibited photorespiration (down-regulation of glycine/serine ratio), altered membrane

48

properties (up-regulation of pentadecanoic and arachidonic acid, down-regulation of

49

linoleic and linolenic acid), and reduced of inorganic nitrogen fixation (down-regulation

50

of glutamine and asparagine). Although Ag ions induced some of the same metabolic

51

changes, alterations in the levels of carbazole, indoleactate, raffinose, adenosine,

52

lactamide, erythrose, and p-benzoquinone were AgNPs-specific. The results of this study

53

offer new insight into the molecular mechanisms by which cucumber responds to AgNPs

54

exposure and provide important information to support the sustainable use of AgNPs in

55

agriculture.

56

3



57

INTRODUCTION

58

Land degradation, declining soil organic matter, low nutrient use efficiency, and climate

59

change are all serious challenges to modern agriculture, compromising our ability to

60

produce sufficient food to support the world’s ever increasing population.1

61

Nanotechnology is emerging as a promising strategy to sustainably increase food

62

production2 and has been used to develop new agrochemical products aimed at promoting

63

plant growth and productivity3. Nano-enabled products can also help to reduce the

64

amount of applied plant protection products (PPP) and fertilizers, thereby reducing

65

environmental impacts while saving valuable water and energy inputs.

66

Given the resistance that a number of pests have exhibited toward traditional pesticides,

67

silver nanoparticles (NPs) has begun received significant attention as a novel

68

nanopesticide. AgNPs, with a broad spectrum of antimicrobial activity, have been found

69

to effectively limit a number of plant diseases. Jo et al.4 reported that Ag ions and AgNPs

70

had a significant negative impact on the colony formation of two plant pathogenic fungi

71

(Bipolaris sorokiniana and Magnaporthe grisea). Although the US EPA granted a

72

conditional registration for the first nanosilver pesticide, it should be noted that this

73

original application was for textile use and not as a plant protection product.5 Importantly,

74

significant uncertainty remains concerning the application of AgNPs in agriculture.

75

Therefore, research on the toxicity of AgNPs to non-target species such as terrestrial

76

plants is needed to ensure sustainable use in food production efforts.

77

The phytotoxicity of AgNPs to various species, including Crambe abyssinica,6

78

Arabidopsis,7 Lycopersicum esculentum, Zea mays8, Cucurbita pepo9, Phaseolus radiatus,

79

and Sorghum bicolor10 has been reported in a number of studies in recent years. Most of

4


Page 4 of 35

Page 5 of 35


80

this work has focused on the impact of exposure on physiological endpoints such as root

81

elongation, transpiration rate, and photosynthesis. A smaller number of studies have

82

sought to characterize the underlying toxicity and detoxification mechanisms of plants

83

exposed to AgNPs. Using a microarray approach, Kaveh et al.11 detected gene expression

84

changes in A. thaliana exposed to 5 mg/L AgNPs and observed that the thalianol

85

biosynthetic pathway was involved in defense against stress. In addition, a number of

86

reports have demonstrated that AgNPs trigger the overproduction of reactive oxygen

87

species (ROS), causing oxidative stress in human hepatoma cells,12 rat alveolar

88

macrophages,13 single-cell green algae,14 and plants.15 Others have addressed how plants

89

alleviate AgNPs-induced oxidative stress. Ma et al. found that glutathione (GHS) and

90

related peptides protect plants from Ag-induced nanotoxicity.6 Uncovering the

91

mechanistic strategies that plants employ to defend against AgNPs toxicity will be highly

92

informative in efforts to sustainably use these materials as plant protection products.

93

Low molecular weight metabolites are the final product of gene expression; therefore,

94

the changes in a cells metabolite profile may be a powerful strategy for assessing

95

biological activities.16 Metabolomics, defined as “the technology geared towards

96

providing an essentially unbiased, comprehensive qualitative and quantitative overview

97

of the metabolites present in an organism,”17 has been shown to be a powerful tool to

98

facilitate an understanding about how plants respond and alleviate various stressors at the

99

molecular level.18-20

100

In this study, 4-week-old cucumber plants were foliar-exposed to AgNPs for a week.

101

A control of AgNO3 was included for an ion comparison. The measured physiological

102

parameters included biomass, chlorophyll content, and lipid peroxidation. In addition,

5



103

GC-MS-based metabolomics was used to provide new insight into the metabolic response

104

of the exposed plants. A mechanistic assessment of phytotoxicity and the detoxification

105

mechanisms will not only advance understanding of environmental implications of these

106

materials but will also provide important baseline knowledge for the sustainable use of

107

AgNPs in agriculture.

108 109

EXPERIMENTAL SECTION

110

Nanoparticles and Plant Growth. Ag nanopowder was procured from Pantian nano

111

Material Co., Ltd. (Shanghai, China). The original size was 20 nm. The hydrodynamic

112

diameter of AgNPs in ultrapure water, at 10 mg/L and 100 mg/L, was 199.83 ± 10.15 nm

113

and 174.67± 4.51 nm, respectively; the ζ potential was 6.23 ± 1.45 mV, measured via

114

dynamic light scattering (Zetasizer Nano ZS, Malvern). The pH for ultrapure water, 10

115

mg/L and 100 mg/L AgNPs solution was 7.05±0.03, 5.26±0.05 and 5.67±0.04,

116

respectively. Equivalent silver salt (AgNO 3 ) was purchased from Sigma-Aldrich.

117

Cucumber (Cucumis sativus) seeds (Zhongnong No.28 F1) were purchased from

118

Hezhiyuan Seed Corporation (Shandong, China). Potting soil (Miracle-Gro, Beijing)

119

with a nutrient composition of 0.68% N, 0.27% P2O5, and 0.36% K2O was used in this

120

study. The seeds were sown at a depth of 1cm in plastic planters (14cm ×14cm ×13cm).

121

Plants were maintained in a greenhouse at a 28 °C/ 20 °C by day/night cycle. The relative

122

humidity and illumination in the greenhouse were 60% and 180 μmol·m−2·s−1,

123

respectively.

124

Exposure Assay. The foliar exposure was initiated when the cucumber seedlings were

125

four weeks old. Five treatments were established, including control (no Ag ions or

6


Page 6 of 35

Page 7 of 35


126

AgNPs), 0.04 and 0.4 mg/plant Ag ion; 4 and 40 mg/plant of AgNPs. Previous study7 and

127

our preliminary experiments showed that approximately 1% Ag ions release from AgNPs

128

over seven days; therefore, 0.04 and 0.4 mg/plant Ag ion was set up parallel to the 4 and

129

40 mg/plant treatments of AgNPs. Five replicate plants (one plant/pot) were grown for

130

each treatment. The stock solution of 10 and 100 mg/L AgNPs, 0.1 and 1 mg/L of

131

AgNO3 were prepared in nanopure water. Before application, the AgNPs suspension was

132

sonicated (KH-100DB, Hechuang Utrasonic, Jiangsu) at 45 KHz for 30 min in cool water

133

to approach a well dispersed solution. The foliar application was made 3 times per day

134

for a 7-day exposure period using a hand-held spray bottle, with total volume of 400 ml

135

per plant during 7 days applied, yielding approximate total delivered masses of 0.04 and

136

0.4 mg Ag ion per plant, and 4 and 40 mg AgNPs per plant.

137

Biomass and Ag Content Analysis. At harvest, plants were thoroughly washed with

138

deionized water to remove any residual particles. Plants were separated into leaves, stems

139

and roots. It has to be pointed that the petioles were not separated with blades and were

140

counted as leaf biomass in this study. Tissues for metal content analysis were dried at 70

141

°C for 72 h. A sample of approximately 0.02 g dried tissue was microwave digested

142

(Milestone Ethos Up, Italy) in a mixture of 8 mL H2O2 and 2 mL HNO3 (v:v=4:1) at

143

160°C for 40 min. The resulting digest was diluted to a final volume of 50 mL prior to

144

analysis. The Ag content was quantified by inductively coupled plasma-mass

145

spectrometry (ICP-MS) (NexION-300, PerkinElmer, USA).

146

Lipid Peroxidation. Lipid peroxidation in leaves was measured by the Thiobarbituric

147

Acid Reactive Substances (TBARS) assay.21 Malondialdehyde, which is the final product

148

of fatty acid degradation, is indicative of lipid peroxidation. Briefly, 0.2 g of fresh

7



149

cucumber leaves were mixed with 4 mL of 0.1% trichloroacetic acid (TCA); the mixture

150

was then centrifuged at 10,000 rpm for 15 min. A 1-ml aliquot of the supernatant was

151

mixed with 2mL of 20% TCA and 2 mL of 0.5% thiobarbituric acid (TBA); then, the

152

mixture was heated in water bath at 95 °C for 30 min. After cooling, the UV absorbance

153

was measured at 532 nm and 600 nm (UV-1800，Shimadzu Corporation, Kyoto Japan).

154

Lipid peroxidation was expressed as μmol MDA equivalent 1g-1 of fresh weight.

155

Metabolite Analysis in Cucumber Leaf Extracts. Leaf metabolites were analyzed by

156

gas chromatography-mass spectrometry (GC-MS). Details on sample preparation, GC-

157

MS analysis, and multivariate analysis are shown below.

158

Sample Preparation. At harvest, cucumber leaves were thoroughly rinsed with tap

159

water and nano pure water to remove the residual soil or particles from the surface. The

160

leaf was blotted dry with kim wipes. The fresh leaves were ground into power in liquid

161

nitrogen and 60 mg of tissue was transferred to a 1.5-mL Eppendorf tube containing two

162

small steel balls. Then 360 μL of cold methanol and 40 μL of 2-chloro-l-phenylalanine

163

(0.3 mg/mL), dissolved in methanol as internal standard, were added to each sample,

164

which were then placed at -80 °C for 2 min and sonicated at 60 HZ for 2 min. An aliquot

165

of 200 μL of chloroform was added to the samples, which were then vortexed and

166

amended with 400 μL water. The samples were vortexed again and ultrasonicated at

167

ambient temperature for 30 min. The samples were centrifuged at 13900 g for 10 min at

168

4 °C. A QC sample was prepared by mixing aliquots of all samples (a pooled sample). A

169

300 µL aliquot of supernatant was transferred to a glass sampling vial for vacuum-drying

170

at room temperature; 80 μL of 15 mg/mL methoxylamine hydrochloride in pyridine was

171

then added. The resultant mixture was vortexed vigorously for 2 min and incubated at

8


Page 8 of 35

Page 9 of 35


172

37 °C for 90 min. Eighty μL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) (with

173

1% Trimethylchlorosilane) and 20 μL n-hexane was then added, and the sample was

174

vortexed vigorously for 2 min prior to derivatization at 70 °C for 60 min. The samples

175

were placed at ambient temperature for 30 min before GC-MS analysis.

176

GC-MS Analysis. The derivatized samples were analyzed by using an Agilent 7890B

177

gas chromatography system coupled to an Agilent 5977A mass selective detector

178

(Agilent Technologies Inc., CA, USA). The column employed was a DB-5MS fused-

179

silica capillary column (30 m × 0.25 mm × 0.25 μm; Agilent J & W Scientific, Folsom,

180

CA, USA Agilent Technologies, Santa Clara, CA). Helium (> 99.999%) was used as the

181

carrier gas at a constant flow rate of 1.0 mL/min through the column. The initial oven

182

temperature was 60 °C, ramped to 125 °C at a rate of 8 °C/min, to 210 °C at a rate of 4

183

°C/min, to 270 °C at a rate of 5 °C/min, to 305 °C at a rate of 10 °C/min, and finally, held

184

at 305 °C for 3 min. The injection volume was 1 μL with the injector temperature 260 °C

185

in splitless mode. The temperature of MS quadrupole and ion source (electron impact)

186

was set to 150 and 230 °C, respectively. The collision energy was 70 eV. Mass data was

187

acquired in a full-scan mode (m/z 50-500), and the solvent delay time was set to 5 min.

188

The QC samples were injected at regular intervals (every 10 samples) throughout the

189

analytical run.

190

Multivariate Statistical Analysis. Un-supervised principal components analyses (PCA)

191

and a supervised partial least-squares discriminant analysis (PLS-DA) clustering method

192

were run based on GC-MS data via online resources (http://www.metaboanalyst.ca/).22

193

Before PCA and PLS-DA analysis, the data normalization (normalization by sum) has

194

been done for general-purpose adjustment for difference among samples, and data

9



195

transformation (log transformation) was conducted to make individual features more

196

comparable. PLS-DA uses a multiple linear regression technique to maximize the

197

separation between groups; this helps to understand which variables carry the class

198

separating information.23 Variable Importance in Projection (VIP) is the weighted sum of

199

the squares of the PLS-DA analysis, which indicates the importance of a variable to the

200

entire model.23 A variable with a VIP greater than 1 is regarded as responsible for

201

separation, defined as a discriminating metabolite in this study.24 Biological pathway

202

analysis was performed based on GC-MS data using MetaboAnalyst 2.0.25 The impact

203

value threshold calculated for pathway identification was set at 0.1.24

204 205

RESULTS AND DISCUSSION

206

Accumulation of AgNPs and Ag+ and Effect on Physiological Endpoints. Exposure to

207

AgNPs and Ag ions resulted in significant accumulation of Ag in cucumber tissues

208

(Figure S1). It must be noted that the Ag detected in the leaves is likely from the direct

209

foliar application and includes Ag both attached to the leaf surface and in the tissues.

210

However, incidental contamination of the soil during the foliar application process may

211

have occurred and as such, some fraction of the Ag in the stem and root tissues could be

212

the result of soil to plant transfer. However, such determination is out of the scope of this

213

study.

214

Visible symptoms. The 0.04 mg Ag+ treatment did not induce overt toxicity in the

215

leaves (Figure S2). However, the 0.4 mg Ag/plant treatment induced leaf yellowing by

216

day 4, which is an indicative of leaf chlorotic damage and senescence. The AgNPs at

10


Page 10 of 35

Page 11 of 35


217

both concentrations (4 and 40 mg/L) caused similar leaf damage, with the higher dose

218

also causing dehydration (Figure S2).

219

Lipid Peroxidation. The malondialdehyde (MDA) content in cucumber leaves exposed

220

to 4 and 40 mg AgNPs significantly increased (28.6% and 44.93%, respectively p ≤ 0.05)

221

as compared to the control (Figure 1). MDA is an end-product of polyunsaturated fatty

222

acid oxidation, which directly reflects the extent of lipid damage induced by oxidative

223

stress. Higher MDA levels are indicative of an increase in lipid peroxidation, especially

224

in leaves which have high levels of polyunsaturated fatty acids26. Here, the MDA

225

increase indicates potentially significant membrane damage as a function of AgNPs

226

exposure.

227

Importantly, the Ag ions treatment at both doses (0.04 and 4 mg/L) did not induce lipid

228

peroxidation relative to the controls. Navabpour et al.26 reported that silver nitrate

229

resulted in increased lipid peroxidation and cellular damage in A. thaliana. Although this

230

differs from our results, it should be noted that their dosing was 1 mM AgNO3 (169

231

mg/L), which was 16.9 times higher than the 10 mg/L concentration used in the current

232

study. However, this does suggest that the damage noted in the AgNP treatment may be a

233

result of ion release and may not be the result of nanoparticle exposure.

234

Biomass. Figure S3 A and B presents the average fresh biomass of root, stem and leaf

235

and total biomass of cucumber plants exposed to AgNPs or Ag+ for seven days. Although

236

lipid peroxidation and visible toxicity symptoms were observed with the 4 and 40 mg

237

AgNPs treatments, there were generally no significant changes in biomass of root, stem

238

and leaf compared with the controls and Ag+/AgNPs treated plants, except that 100 mg/L

239

AgNPs significantly (p1. The 40 metabolites that lead to the separation of the control and all Ag-

265

treated groups are shown in Figure S4. In order to further discriminate Ag ion and NPs

266

response, a metabolic dataset of Ag+ groups vs. control and AgNPs vs. control PLS-DA

267

analyses were conducted separately. In addition, Ag ions-specific and AgNPs-specific

268

metabolites were screened out separately (Figure S5 and S6). The univariate statistical

269

analysis (ANOVA) was performed to detect metabolites significantly changed by AgNPs

270

and Ag+, which may be omitted by a multivariate analysis. The combined multivariate

271

and univariate results for the differentially regulated metabolites as a function of Ag ions

272

or AgNPs are listed in a visualized square (Figure S7). A significant overlap of

273

differentially impacted metabolites was observed in response to AgNPs and Ag+ (76

274

significant metabolite changes, 21 down-regulated and 55 up-regulated), suggesting that a

275

significant fraction of AgNP-induced stress may originate predominantly from toxicity of

276

the released ion. That being said, there were some metabolite changes that were NPs

277

specific, which does indicates a nanoscale particle effect. These findings are consistent

278

with Kavel et al.,11 who note that both Ag ion and the NP contribute to the observed

279

toxicity of AgNPs to Arabidopsis thaliana. The metabolites that were significantly

280

changed according to the PLS-DA and one way ANOVA (Figure S7) were then

281

classified into different groups based on their metabolic functions and pathway; a

282

discussion of this data follows below.

283

Metabolites Changed by both Ag+ and AgNPs. Among the 93 significantly changed

284

metabolites, 76 or nearly 82% were due to both Ag+ and AgNPs exposure, highlighting

285

the importance of ion release to plant response.

13



286

Phytol. Phytol, a degradation product of chlorophyll, was significantly increased (1.5-

287

2.2 -fold) by exposure to both Ag+ and AgNPs in a dose-dependent manner (Figure 3).

288

The increase in phytol accumulation is indicative of chlorophyll degradation. As noted

289

above, the increased MDA (Figure 1) suggests oxidative stress and lipid peroxidation.

290

Plants have evolved convergent mechanisms to cope with stress that are, in general,

291

energetically demanding.30 Such energetic demands may require the disassembly of

292

organelles or organelle components to overcome stress-induced damage and to

293

adequately distribute cellular resources. The degradation of chloroplasts and the recycling

294

of their nutrients is known to be important under stress conditions and during leaf

295

senescence.30 Mach28 reported that phytol from chlorophyll degradation is used in the

296

biosynthesis of tocopherol, which is important lipid-soluble antioxidant to protect lipids

297

from oxidative damage. The degradation of chlorophyll to phytol could be an active

298

protective mechanism for cucumber to combat oxidative stress. Phytol response to the Cu

299

induced stress has been previously reported.20 In addition, phytol has antimicrobial

300

properties and is also induced by water stress,31 which makes them a general biomarker

301

of plant defense against stress.

302

Antioxidants (β-glucoside and phenolic acids). Notably, arbutin and salicin are the

303

metabolites with the highest VIP scores (Figure S4), indicating that both compounds

304

contribute significantly to the separation between the control and the Ag NPs/Ag+ groups.

305

Arbutin and salicin were not detected in un-exposed control and 0.04 mg Ag+ treatment.

306

However, 0.4 mg Ag ions and both AgNPs treatments triggered significantly (p

Metabolomics Reveals How Cucumber (Cucumis ... - ACS Publications

Recommend Documents