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Ecotoxicology and Human Environmental Health

Metabolomics Reveals How Cucumber (Cucumissativus) Reprograms Metabolites to Cope with Silver

Ions and Silver Nanoparticle-Induced Oxidative StressHuiling Zhang, Wenchao Du, Jose R Peralta-Videa, Jorge L. Gardea-Torresdey,

Jason C. White, Arturo A. Keller, Hongyan Guo, Rong Ji, and Lijuan ZhaoEnviron. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02440 • Publication Date (Web): 14 Jun 2018

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1

Metabolomics Reveals How Cucumber (Cucumis 1

sativus) Reprograms Metabolites to Cope with 2

Silver Ions and Silver Nanoparticle-Induced 3

Oxidative Stress 4

5

Huiling Zhang§, Wenchao Du§, Jose R. Peralta-Videaζ, Jorge L. Gardea-Torresdeyζ, 6

Jason C. White¶, Arturo Keller⁋, Hongyan Guo§, Rong Ji§*, Lijuan Zhao§* 7

8

§State Key Laboratory of Pollution Control and Resource Reuse, School of Environment, 9

Nanjing University, Nanjing 210023, China 10

ζ Chemistry Department, The University of Texas at El Paso, 500 West University 11

Avenue, El Paso, Texas 79968, United States 12

¶Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station 13

(CAES), New Haven, Connecticut 06504, United States 14

⁋Bren School of Environmental Science & Management, University of California, Santa 15

Barbara, California 93106-5131, United States 16

17

*Corresponding author. Tel: +86 025-8968 0581; fax: +86 025-8968 0581. 18

Email address: [email protected]; [email protected] 19

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ABSTRACT 34

Due to their well-known antifungal activity, the intentional use of Ag nanoparticle (NPs) 35

as sustainable nano-fungicides is expected to increase in agriculture. However, the 36

impacts of AgNPs on plants must be critically evaluated to guarantee their safe use in 37

food production. In this study, 4-week-old cucumber (Cucumis sativus) plants received a 38

foliar application of AgNPs (4 or 40 mg per plant) or Ag+ (0.04 or 0.4 mg per plant) for 39

seven days. Gas chromatography-mass spectrometry (GC-MS) based non-target 40

metabolomics enabled the identification and quantification of 268 metabolites in 41

cucumber leaves. Multivariate analysis revealed that all the treatments significantly 42

altered the metabolite profile. Exposure to AgNPs resulted in metabolic reprogramming, 43

including activation of antioxidant defense systems (up-regulation of phenolic 44

compounds) and down-regulation of photosynthesis (up-regulation of phytol). 45

Additionally, AgNPs enhanced respiration (up-regulation of TCA cycle intermediates), 46

inhibited photorespiration (down-regulation of glycine/serine ratio), altered membrane 47

properties (up-regulation of pentadecanoic and arachidonic acid, down-regulation of 48

linoleic and linolenic acid), and reduced of inorganic nitrogen fixation (down-regulation 49

of glutamine and asparagine). Although Ag ions induced some of the same metabolic 50

changes, alterations in the levels of carbazole, indoleactate, raffinose, adenosine, 51

lactamide, erythrose, and p-benzoquinone were AgNPs-specific. The results of this study 52

offer new insight into the molecular mechanisms by which cucumber responds to AgNPs 53

exposure and provide important information to support the sustainable use of AgNPs in 54

agriculture. 55

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INTRODUCTION 57

Land degradation, declining soil organic matter, low nutrient use efficiency, and climate 58

change are all serious challenges to modern agriculture, compromising our ability to 59

produce sufficient food to support the world’s ever increasing population.1 60

Nanotechnology is emerging as a promising strategy to sustainably increase food 61

production2 and has been used to develop new agrochemical products aimed at promoting 62

plant growth and productivity3. Nano-enabled products can also help to reduce the 63

amount of applied plant protection products (PPP) and fertilizers, thereby reducing 64

environmental impacts while saving valuable water and energy inputs. 65

Given the resistance that a number of pests have exhibited toward traditional pesticides, 66

silver nanoparticles (NPs) has begun received significant attention as a novel 67

nanopesticide. AgNPs, with a broad spectrum of antimicrobial activity, have been found 68

to effectively limit a number of plant diseases. Jo et al.4 reported that Ag ions and AgNPs 69

had a significant negative impact on the colony formation of two plant pathogenic fungi 70

(Bipolaris sorokiniana and Magnaporthe grisea). Although the US EPA granted a 71

conditional registration for the first nanosilver pesticide, it should be noted that this 72

original application was for textile use and not as a plant protection product.5 Importantly, 73

significant uncertainty remains concerning the application of AgNPs in agriculture. 74

Therefore, research on the toxicity of AgNPs to non-target species such as terrestrial 75

plants is needed to ensure sustainable use in food production efforts. 76

The phytotoxicity of AgNPs to various species, including Crambe abyssinica,6 77

Arabidopsis,7 Lycopersicum esculentum, Zea mays8, Cucurbita pepo9, Phaseolus radiatus, 78

and Sorghum bicolor10 has been reported in a number of studies in recent years. Most of 79

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this work has focused on the impact of exposure on physiological endpoints such as root 80

elongation, transpiration rate, and photosynthesis. A smaller number of studies have 81

sought to characterize the underlying toxicity and detoxification mechanisms of plants 82

exposed to AgNPs. Using a microarray approach, Kaveh et al.11 detected gene expression 83

changes in A. thaliana exposed to 5 mg/L AgNPs and observed that the thalianol 84

biosynthetic pathway was involved in defense against stress. In addition, a number of 85

reports have demonstrated that AgNPs trigger the overproduction of reactive oxygen 86

species (ROS), causing oxidative stress in human hepatoma cells,12 rat alveolar 87

macrophages,13 single-cell green algae,14 and plants.15 Others have addressed how plants 88

alleviate AgNPs-induced oxidative stress. Ma et al. found that glutathione (GHS) and 89

related peptides protect plants from Ag-induced nanotoxicity.6 Uncovering the 90

mechanistic strategies that plants employ to defend against AgNPs toxicity will be highly 91

informative in efforts to sustainably use these materials as plant protection products. 92

Low molecular weight metabolites are the final product of gene expression; therefore, 93

the changes in a cells metabolite profile may be a powerful strategy for assessing 94

biological activities.16 Metabolomics, defined as “the technology geared towards 95

providing an essentially unbiased, comprehensive qualitative and quantitative overview 96

of the metabolites present in an organism,”17 has been shown to be a powerful tool to 97

facilitate an understanding about how plants respond and alleviate various stressors at the 98

molecular level.18-20 99

In this study, 4-week-old cucumber plants were foliar-exposed to AgNPs for a week. 100

A control of AgNO3 was included for an ion comparison. The measured physiological 101

parameters included biomass, chlorophyll content, and lipid peroxidation. In addition, 102

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GC-MS-based metabolomics was used to provide new insight into the metabolic response 103

of the exposed plants. A mechanistic assessment of phytotoxicity and the detoxification 104

mechanisms will not only advance understanding of environmental implications of these 105

materials but will also provide important baseline knowledge for the sustainable use of 106

AgNPs in agriculture. 107

108

EXPERIMENTAL SECTION 109

Nanoparticles and Plant Growth. Ag nanopowder was procured from Pantian nano 110

Material Co., Ltd. (Shanghai, China). The original size was 20 nm. The hydrodynamic 111

diameter of AgNPs in ultrapure water, at 10 mg/L and 100 mg/L, was 199.83 ± 10.15 nm 112

and 174.67± 4.51 nm, respectively; the ζ potential was 6.23 ± 1.45 mV, measured via 113

dynamic light scattering (Zetasizer Nano ZS, Malvern). The pH for ultrapure water, 10 114

mg/L and 100 mg/L AgNPs solution was 7.05±0.03, 5.26±0.05 and 5.67±0.04, 115

respectively. Equivalent silver salt (AgNO3) was purchased from Sigma-Aldrich. 116

Cucumber (Cucumis sativus) seeds (Zhongnong No.28 F1) were purchased from 117

Hezhiyuan Seed Corporation (Shandong, China). Potting soil (Miracle-Gro, Beijing) 118

with a nutrient composition of 0.68% N, 0.27% P2O5, and 0.36% K2O was used in this 119

study. The seeds were sown at a depth of 1cm in plastic planters (14cm ×14cm ×13cm). 120

Plants were maintained in a greenhouse at a 28 °C/ 20 °C by day/night cycle. The relative 121

humidity and illumination in the greenhouse were 60% and 180 μmol·m−2·s−1, 122

respectively. 123

Exposure Assay. The foliar exposure was initiated when the cucumber seedlings were 124

four weeks old. Five treatments were established, including control (no Ag ions or 125

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AgNPs), 0.04 and 0.4 mg/plant Ag ion; 4 and 40 mg/plant of AgNPs. Previous study7 and 126

our preliminary experiments showed that approximately 1% Ag ions release from AgNPs 127

over seven days; therefore, 0.04 and 0.4 mg/plant Ag ion was set up parallel to the 4 and 128

40 mg/plant treatments of AgNPs. Five replicate plants (one plant/pot) were grown for 129

each treatment. The stock solution of 10 and 100 mg/L AgNPs, 0.1 and 1 mg/L of 130

AgNO3 were prepared in nanopure water. Before application, the AgNPs suspension was 131

sonicated (KH-100DB, Hechuang Utrasonic, Jiangsu) at 45 KHz for 30 min in cool water 132

to approach a well dispersed solution. The foliar application was made 3 times per day 133

for a 7-day exposure period using a hand-held spray bottle, with total volume of 400 ml 134

per plant during 7 days applied, yielding approximate total delivered masses of 0.04 and 135

0.4 mg Ag ion per plant, and 4 and 40 mg AgNPs per plant. 136

Biomass and Ag Content Analysis. At harvest, plants were thoroughly washed with 137

deionized water to remove any residual particles. Plants were separated into leaves, stems 138

and roots. It has to be pointed that the petioles were not separated with blades and were 139

counted as leaf biomass in this study. Tissues for metal content analysis were dried at 70 140

°C for 72 h. A sample of approximately 0.02 g dried tissue was microwave digested 141

(Milestone Ethos Up, Italy) in a mixture of 8 mL H2O2 and 2 mL HNO3 (v:v=4:1) at 142

160°C for 40 min. The resulting digest was diluted to a final volume of 50 mL prior to 143

analysis. The Ag content was quantified by inductively coupled plasma-mass 144

spectrometry (ICP-MS) (NexION-300, PerkinElmer, USA). 145

Lipid Peroxidation. Lipid peroxidation in leaves was measured by the Thiobarbituric 146

Acid Reactive Substances (TBARS) assay.21 Malondialdehyde, which is the final product 147

of fatty acid degradation, is indicative of lipid peroxidation. Briefly, 0.2 g of fresh 148

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cucumber leaves were mixed with 4 mL of 0.1% trichloroacetic acid (TCA); the mixture 149

was then centrifuged at 10,000 rpm for 15 min. A 1-ml aliquot of the supernatant was 150

mixed with 2mL of 20% TCA and 2 mL of 0.5% thiobarbituric acid (TBA); then, the 151

mixture was heated in water bath at 95 °C for 30 min. After cooling, the UV absorbance 152

was measured at 532 nm and 600 nm (UV-1800，Shimadzu Corporation, Kyoto Japan). 153

Lipid peroxidation was expressed as μmol MDA equivalent 1g-1 of fresh weight. 154

Metabolite Analysis in Cucumber Leaf Extracts. Leaf metabolites were analyzed by 155

gas chromatography-mass spectrometry (GC-MS). Details on sample preparation, GC-156

MS analysis, and multivariate analysis are shown below. 157

Sample Preparation. At harvest, cucumber leaves were thoroughly rinsed with tap 158

water and nano pure water to remove the residual soil or particles from the surface. The 159

leaf was blotted dry with kim wipes. The fresh leaves were ground into power in liquid 160

nitrogen and 60 mg of tissue was transferred to a 1.5-mL Eppendorf tube containing two 161

small steel balls. Then 360 μL of cold methanol and 40 μL of 2-chloro-l-phenylalanine 162

(0.3 mg/mL), dissolved in methanol as internal standard, were added to each sample, 163

which were then placed at -80 °C for 2 min and sonicated at 60 HZ for 2 min. An aliquot 164

of 200 μL of chloroform was added to the samples, which were then vortexed and 165

amended with 400 μL water. The samples were vortexed again and ultrasonicated at 166

ambient temperature for 30 min. The samples were centrifuged at 13900 g for 10 min at 167

4 °C. A QC sample was prepared by mixing aliquots of all samples (a pooled sample). A 168

300 µL aliquot of supernatant was transferred to a glass sampling vial for vacuum-drying 169

at room temperature; 80 μL of 15 mg/mL methoxylamine hydrochloride in pyridine was 170

then added. The resultant mixture was vortexed vigorously for 2 min and incubated at 171

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37 °C for 90 min. Eighty μL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) (with 172

1% Trimethylchlorosilane) and 20 μL n-hexane was then added, and the sample was 173

vortexed vigorously for 2 min prior to derivatization at 70 °C for 60 min. The samples 174

were placed at ambient temperature for 30 min before GC-MS analysis. 175

GC-MS Analysis. The derivatized samples were analyzed by using an Agilent 7890B 176

gas chromatography system coupled to an Agilent 5977A mass selective detector 177

(Agilent Technologies Inc., CA, USA). The column employed was a DB-5MS fused-178

silica capillary column (30 m × 0.25 mm × 0.25 μm; Agilent J & W Scientific, Folsom, 179

CA, USA Agilent Technologies, Santa Clara, CA). Helium (> 99.999%) was used as the 180

carrier gas at a constant flow rate of 1.0 mL/min through the column. The initial oven 181

temperature was 60 °C, ramped to 125 °C at a rate of 8 °C/min, to 210 °C at a rate of 4 182

°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 183

at 305 °C for 3 min. The injection volume was 1 μL with the injector temperature 260 °C 184

in splitless mode. The temperature of MS quadrupole and ion source (electron impact) 185

was set to 150 and 230 °C, respectively. The collision energy was 70 eV. Mass data was 186

acquired in a full-scan mode (m/z 50-500), and the solvent delay time was set to 5 min. 187

The QC samples were injected at regular intervals (every 10 samples) throughout the 188

analytical run. 189

Multivariate Statistical Analysis. Un-supervised principal components analyses (PCA) 190

and a supervised partial least-squares discriminant analysis (PLS-DA) clustering method 191

were run based on GC-MS data via online resources (http://www.metaboanalyst.ca/).22 192

Before PCA and PLS-DA analysis, the data normalization (normalization by sum) has 193

been done for general-purpose adjustment for difference among samples, and data 194

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http://www.metaboanalyst.ca/

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transformation (log transformation) was conducted to make individual features more 195

comparable. PLS-DA uses a multiple linear regression technique to maximize the 196

separation between groups; this helps to understand which variables carry the class 197

separating information.23 Variable Importance in Projection (VIP) is the weighted sum of 198

the squares of the PLS-DA analysis, which indicates the importance of a variable to the 199

entire model.23 A variable with a VIP greater than 1 is regarded as responsible for 200

separation, defined as a discriminating metabolite in this study.24 Biological pathway 201

analysis was performed based on GC-MS data using MetaboAnalyst 2.0.25 The impact 202

value threshold calculated for pathway identification was set at 0.1.24 203

204

RESULTS AND DISCUSSION 205

Accumulation of AgNPs and Ag+ and Effect on Physiological Endpoints. Exposure to 206

AgNPs and Ag ions resulted in significant accumulation of Ag in cucumber tissues 207

(Figure S1). It must be noted that the Ag detected in the leaves is likely from the direct 208

foliar application and includes Ag both attached to the leaf surface and in the tissues. 209

However, incidental contamination of the soil during the foliar application process may 210

have occurred and as such, some fraction of the Ag in the stem and root tissues could be 211

the result of soil to plant transfer. However, such determination is out of the scope of this 212

study. 213

Visible symptoms. The 0.04 mg Ag+ treatment did not induce overt toxicity in the 214

leaves (Figure S2). However, the 0.4 mg Ag/plant treatment induced leaf yellowing by 215

day 4, which is an indicative of leaf chlorotic damage and senescence. The AgNPs at 216

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both concentrations (4 and 40 mg/L) caused similar leaf damage, with the higher dose 217

also causing dehydration (Figure S2). 218

Lipid Peroxidation. The malondialdehyde (MDA) content in cucumber leaves exposed 219

to 4 and 40 mg AgNPs significantly increased (28.6% and 44.93%, respectively p ≤ 0.05) 220

as compared to the control (Figure 1). MDA is an end-product of polyunsaturated fatty 221

acid oxidation, which directly reflects the extent of lipid damage induced by oxidative 222

stress. Higher MDA levels are indicative of an increase in lipid peroxidation, especially 223

in leaves which have high levels of polyunsaturated fatty acids26. Here, the MDA 224

increase indicates potentially significant membrane damage as a function of AgNPs 225

exposure. 226

Importantly, the Ag ions treatment at both doses (0.04 and 4 mg/L) did not induce lipid 227

peroxidation relative to the controls. Navabpour et al.26 reported that silver nitrate 228

resulted in increased lipid peroxidation and cellular damage in A. thaliana. Although this 229

differs from our results, it should be noted that their dosing was 1 mM AgNO3 (169 230

mg/L), which was 16.9 times higher than the 10 mg/L concentration used in the current 231

study. However, this does suggest that the damage noted in the AgNP treatment may be a 232

result of ion release and may not be the result of nanoparticle exposure. 233

Biomass. Figure S3 A and B presents the average fresh biomass of root, stem and leaf 234

and total biomass of cucumber plants exposed to AgNPs or Ag+ for seven days. Although 235

lipid peroxidation and visible toxicity symptoms were observed with the 4 and 40 mg 236

AgNPs treatments, there were generally no significant changes in biomass of root, stem 237

and leaf compared with the controls and Ag+/AgNPs treated plants, except that 100 mg/L 238

AgNPs significantly (p<0.05) decreased root biomass compared to other treatments 239

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(Figure S3 A). In addition, there were no difference between control and Ag groups in 240

regarding with total biomass (Figure S3 B). This lack of correlation may highlights the 241

more sensitive nature of endpoints that focus on molecular changes within the plant 242

tissues as compared to more gross measures such as biomass. 243

Metabolic Response of Cucumber to AgNPs and Ag ions. The physiological 244

alterations noted above are clearly the result of intracellular metabolic changes. A total 245

of 268 metabolites were identified and semi-quantified on the basis of their mass spectral 246

fingerprints and retention-index matches. First, an un-supervised clustering method - 247

principal component analysis (PCA) was performed on the GC-MS data using 248

MetaboAnalyst, which provides a general overview of the clustering information between 249

groups. This model was used without any sample designation.27 The grouping of the 250

samples in a PCA scores plot is based on the similarities between sample or treatment 251

metabolic profiles.28 The PCA scores plot (Figure 2 A) shows that there was no 252

noticeable separation between 0.04 mg Ag ion group and the control; however, the other 253

treatments (0.4 mg Ag+, 4 and 40 mg AgNPs) were clearly separated from control group 254

along the first principal component (PC1), which explained 25% of the total variance. 255

Importantly, no clear separation was observed between 0.4 mg Ag+ group and 4 mg 256

AgNPs group using PCA model. PLS-DA, a supervised clustering method, generally 257

provides greater discrimination power compared to PCA.29 To maximize the separation 258

between groups, the PLS-DA multivariate analysis based on GC-MS dataset was 259

performed. The score plot (Figure 2 B) shows that all AgNPs and Ag+ groups were 260

clearly separate from the controls, and that this separation along PC1 was generally dose-261

dependent. These results indicate that both AgNPs and Ag+ altered the metabolic profile 262

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of cucumber leaves. The responsive metabolites were subsequently isolated on the basis 263

of VIP score >1. The 40 metabolites that lead to the separation of the control and all Ag-264

treated groups are shown in Figure S4. In order to further discriminate Ag ion and NPs 265

response, a metabolic dataset of Ag+ groups vs. control and AgNPs vs. control PLS-DA 266

analyses were conducted separately. In addition, Ag ions-specific and AgNPs-specific 267

metabolites were screened out separately (Figure S5 and S6). The univariate statistical 268

analysis (ANOVA) was performed to detect metabolites significantly changed by AgNPs 269

and Ag+, which may be omitted by a multivariate analysis. The combined multivariate 270

and univariate results for the differentially regulated metabolites as a function of Ag ions 271

or AgNPs are listed in a visualized square (Figure S7). A significant overlap of 272

differentially impacted metabolites was observed in response to AgNPs and Ag+ (76 273

significant metabolite changes, 21 down-regulated and 55 up-regulated), suggesting that a 274

significant fraction of AgNP-induced stress may originate predominantly from toxicity of 275

the released ion. That being said, there were some metabolite changes that were NPs 276

specific, which does indicates a nanoscale particle effect. These findings are consistent 277

with Kavel et al.,11 who note that both Ag ion and the NP contribute to the observed 278

toxicity of AgNPs to Arabidopsis thaliana. The metabolites that were significantly 279

changed according to the PLS-DA and one way ANOVA (Figure S7) were then 280

classified into different groups based on their metabolic functions and pathway; a 281

discussion of this data follows below. 282

Metabolites Changed by both Ag+ and AgNPs. Among the 93 significantly changed 283

metabolites, 76 or nearly 82% were due to both Ag+ and AgNPs exposure, highlighting 284

the importance of ion release to plant response. 285

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Phytol. Phytol, a degradation product of chlorophyll, was significantly increased (1.5-286

2.2 -fold) by exposure to both Ag+ and AgNPs in a dose-dependent manner (Figure 3). 287

The increase in phytol accumulation is indicative of chlorophyll degradation. As noted 288

above, the increased MDA (Figure 1) suggests oxidative stress and lipid peroxidation. 289

Plants have evolved convergent mechanisms to cope with stress that are, in general, 290

energetically demanding.30 Such energetic demands may require the disassembly of 291

organelles or organelle components to overcome stress-induced damage and to 292

adequately distribute cellular resources. The degradation of chloroplasts and the recycling 293

of their nutrients is known to be important under stress conditions and during leaf 294

senescence.30 Mach28 reported that phytol from chlorophyll degradation is used in the 295

biosynthesis of tocopherol, which is important lipid-soluble antioxidant to protect lipids 296

from oxidative damage. The degradation of chlorophyll to phytol could be an active 297

protective mechanism for cucumber to combat oxidative stress. Phytol response to the Cu 298

induced stress has been previously reported.20 In addition, phytol has antimicrobial 299

properties and is also induced by water stress,31 which makes them a general biomarker 300

of plant defense against stress. 301

Antioxidants (β-glucoside and phenolic acids). Notably, arbutin and salicin are the 302

metabolites with the highest VIP scores (Figure S4), indicating that both compounds 303

contribute significantly to the separation between the control and the Ag NPs/Ag+ groups. 304

Arbutin and salicin were not detected in un-exposed control and 0.04 mg Ag+ treatment. 305

However, 0.4 mg Ag ions and both AgNPs treatments triggered significantly (p<0.01) 306

up-regulation in the production of these two compounds (Figure 4). Salicin and arbutin 307

are alcoholic and phenolic glycosides, respectively, and they may participate in the 308

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defense of cucumber plant against Ag-induced stress, either as free radical scavengers or 309

as signaling molecules. 310

In addition to glucosides, the presence of number of aromatic compounds was 311

increased by exposure to AgNPs and Ag ions (Figure 4). The aromatic compounds 4-312

Hydroxyquinazoline (4-HQ), 3-hydroxybenzoic acid (phenolic acid), 1,2,4-benzenetriol 313

(BT) and pyrogallol were significantly (p < 0.05) increased by all Ag/AgNP treatments, 314

indicating they are very sensitive to the presence of this metal. 4-HQ has been found to 315

limit the increase of ROS produced by plasma membrane NADH oxidase.32 1,2,4-316

benzenetriol (BT) is a polyphenolic metabolite of benzene, and has been shown to 317

significantly reduce the generation of ROS in H2O2-induced BV-2 cells.33 Pyrogallol has 318

also been reported to function as an antioxidant.34 Taguri et al. found that polyphenols 319

having pyrogallol groups also have strong antibacterial activity.35 Thus, significant 320

increases in the accumulation of these compounds are likely related to the quenching of 321

oxidative stress and to general defense. Somewhat differently, Cis-2-hydroxycinnamic 322

acid, ferulic acid and ethyl cinnamate (Esters of coumaric acid) were unchanged by 0.04 323

mg Ag+, but were significantly increased by the other Ag treatments (Figure 4). The up-324

regulation of these aromatic compounds may be another active protective mechanism 325

employed by cucumber upon Ag stress. 326

The biosynthesis of antioxidant compounds is believed to result in the scavenging of 327

excess ROS. Biological pathway analysis also reveals that phenylalanine metabolism, 328

which is a stress response-related biological pathway, was disturbed at 40 mg AgNPs, 329

(Figure S8). The results clearly indicate that the antioxidant defense system was 330

activated by AgNPs; this may probably lead to a switching of cellular energy metabolism 331

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from growth to defense, thereby resulting in the accumulation of defense related 332

metabolites.36 333

Tricarboxylic acid (TCA) cycle. Succinic acid, a TCA cycle intermediate, was 334

significantly increased in a dose-dependent manner with Ag exposure (Figure 5). Except 335

0.04 mg Ag+, all other treatments significantly (p<0.05) increased succinic levels (1.6-336

1.7-fold) in cucumber leaves. Isocitric acid, another TCA cycle intermediate, increased 2-337

fold upon exposure to 40 mg AgNPs. The up-regulation of TCA cycle intermediates may 338

indicate activation of the TCA pathway; biological pathway analysis confirms that the 339

TCA cycle was altered by the 40 mg AgNPs treatment (Figure S8). The TCA cycle is the 340

core of the cell’s respiratory machinery; it is likely that plants up-regulate respiration to 341

produce energy for the manufacture of defense compounds needed to address oxidative 342

stress. 343

Sugar and sugar alcohol (polyols). Sugars are important signaling molecules in the 344

regulation of plant metabolism, and they are known to accumulate during stress, as well 345

as in senescing leaves.37 A significant increase (p ≤ 0.05) in the accumulation of lyxose, 346

levoglucosan, and lentiobiose was identified upon exposure to 0.4 mg Ag+, as well as 4 347

and 40 mg AgNPs (Figure 5). In addition, several sugar alcohols, including threitol, D-348

arabitol, and sorbitol, which were unaffected at 0.04 mg Ag+ but were significantly (p ≤ 349

0.05) increased in a dose-dependent fashion with the other Ag treatments (Figure 5). The 350

accumulation of sugar polyols is known to help maintain cellular hydration and functions 351

during leaf senescence;38 increased polyol content could possibly function in the stress 352

tolerance of plants. 353

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Nitrogen Metabolism (Amino acids). Amino acids are essential components of plant 354

primary metabolism and play an important role in plant physiological process, such as 355

acting as osmolytes, modulating stomatal opening, serving as precursors for the synthesis 356

of defense-related metabolites and signaling metabolites.39 Results showed that a number 357

of amino acids, namely ornithine, cycloleucine, norleucine, isoleucine, threonine, 358

methionine, lysine, and beta-alanine; were dramatically increased after Ag exposure 359

(Figure 6). For example, cycloleucine was not detected in the control leaves; but levels 360

were significantly increased in a dose-dependent fashion with Ag exposure. The 361

increased accumulation several amino acids suggests that nitrogen metabolism was 362

disturbed by AgNPs or that significant protein degradation has occurred. One exception 363

is N-Methyl-DL-alanine, which was unchanged by exposure to 0.04 mg Ag ions, but then 364

decreased in a dose-dependent manner with Ag ions and AgNPs. The reason for the 365

observed changes in those amino acid content are unknown. 366

Glutamine (Gln) and asparagine (Asn) are two important amides and were found 367

unchanged by 0.04 mg Ag ions; however, their content decreased in a dose-dependent 368

manner with Ag treatment. Specifically, exposure to 40 mg AgNPs resulted in the 369

significant (p ≤ 0.05) 4- and 15-fold decrease of Gln and Asn, respectively, compared to 370

control. Gln and Asn are the main N-rich amino acids in leaves, and are involved in 371

fixing inorganic nitrogen; 40 the reduction in these two amino acids suggests that this 372

important process may be disturbed. 373

Glycine content was decreased with treatment but serine levels were increased in a 374

dose-dependent manner in the Ag exposed plants (Figure 3). Glycine (Gly) and serine 375

(Ser) are two essential amino acids formed during photorespiration, and the Gly/Ser ratio 376

Page 17 of 35



18

is commonly used as an indicator of photorespiratory activity.40 It is interesting to note 377

that Gly/Ser ratio decreased 3-fold under exposure to 40 mg AgNPs, suggesting that 378

photorespiration was inhibited under this condition. Wingler et al.37 suggested that the 379

photorespiration pathway may provide additional protection against oxidative damage in 380

under high light induced stress by supplying glycine, which can be used for the synthesis 381

of the broad defense molecule glutathione. Earlier reports also demonstrated that the 382

Gly/Ser ratio is a sensitive biomarker of leaf senescence, changing significantly even 383

prior to senescence symptoms.40 In summary, AgNPs and Ag ions induced significant 384

changes in the amino acid composition of cucumber leaves (Figure 3). 385

Fatty acids. Pentadecanoic acid, a saturated phospholipid fatty acid, was significantly 386

(p ≤ 0.05) increased in a dose-dependent manner with both Ag ions and AgNPs (Figure 387

7). Pentadecanoic acid is a component of the phospholipid bilayer and increases in its 388

content may be indicative of membrane remodeling or synthesis to adapt to the adverse 389

condition. Given the increase in MDA content, the up-regulation of pentadecanoic acid 390

was possibly for repairing the damaged membrane. The unsaturated fatty acids linoleic 391

and linolenic acids were unchanged by the 0.04 mg Ag+ treatment; however, levels were 392

significantly (p ≤ 0.05) decreased by the other Ag exposures with the 40 mg AgNPs 393

decrease being approximately 1-fold. It is known that as plants acclimate to biotic and 394

abiotic stress, the response may include a remodeling membrane fluidity by releasing α-395

linolenic (18:3) from membrane lipids.41 This flexible adjustment of membrane fluidity 396

maintains an environment suitable for the function of critical integral proteins during 397

stress. Arachidonic acid, an unsaturated phospholipid fatty acid, was unaffected by 0.04 398

mg Ag+ but was significantly (p ≤ 0.05) increased by the high ion and the AgNPs 399

Page 18 of 35



19

treatments from 1~6 fold. All of these metabolite changes are indicative of Ag-induced 400

disruption to the composition and integrity of lipid membranes. Lipid peroxidation is a 401

chain reaction and is created by free radicals influencing unsaturated fatty acids in cell 402

membranes, leading to their damage. Clearly one potential reason for the observed up- or 403

down regulation of fatty acid metabolites is the result of lipid peroxidation. Another 404

possibility is that plants adjust the membrane composition to rebuild the membrane 405

integrity and to restrict Ag ions permeation into cells. Phospholipids are comprised of 406

polar head groups, glycerides, and two fatty acyl chains. We found that diglycerol, the 407

polar head group component of phospholipid, was significantly (p ≤ 0.05) increased 2.3-408

fold under exposure to 40 mg AgNPs (Figure 7). These findings support the hypothesis 409

that cucumber leaves altered intracellular metabolism in order to rebuild the membrane 410

integrity. 411

Signaling Molecule and plant hormone. Unlike most metabolites which were 412

unaffected by 0.04 mg Ag+, salicylic acid (SA) was significantly (p ≤ 0.05) increased by 413

at this lower ion dose and was affected in a dose-dependent manner with Ag content in 414

tissues (Figure 3). Notably, a significant (p ≤ 0.01) 8-fold increase in the accumulation of 415

SA was noted upon exposure to 40 mg AgNPs. SA is a plant hormone that plays an 416

important role in plant defense. Previous evidence indicates that SA is a signaling 417

molecule for the activation of plant defense response, including systemic acquired 418

resistance (SAR).42 Similarly, Shulaev et al. 29 demonstrated that SA produced as a result 419

of stress can serve as signaling molecules activating systemic defense and acclimation 420

responses. In addition to serving as a signaling molecule, SA has been reported to 421

mediate the phenylpropanoid pathway and to play an important role against pathogens, 422

Page 19 of 35



20

some insect pests, and other abiotic stresses.43 It has been reported that SA is synthesized 423

at the site of wounding or pathogen infection in tobacco leaves, possibly serving as an 424

endogenous signal for disease resistance44. Taken together, the up-regulation of SA is 425

clearly a broad defense-related behavior of cucumber in response to Ag induced stress. In 426

addition to signaling molecules, AgNPs also induced significant changes in defense-427

related secondary metabolites; some discussion of this can be found in the Supporting 428

Information. 429

AgNPs Specific Response. Importantly, some metabolites were up- or downregulated 430

in response to AgNPs only, indicating a nanoscale size-specific effect on the plant. The 431

metabolites upregulated specifically by AgNPs include carbazole, raffinose, lactulose, 432

citraconic acid, aspartic acid, dithioerythritol, D-erythronolactone, and N-Methyl-L-433

glutamic acid. Among them, arbazole, raffinose, lactulose and citraconic acid were not 434

detected in control and Ag ion treated plants, but were present at significant levels in 435

AgNPs treated plants (Figure S10). Notably, N-Methyl-L-glutamic acid increased 116-436

fold upon exposure to 40 mg AgNPs as compared to unexposed control. Carbazoles are 437

bioactive compounds with known antioxidative activity.45 The polysaccharides raffinose 438

and lactulose were not detected in the leaves of unexposed plants and Ag ions treated 439

plants; however, their levels were significantly increased at 40 mg AgNPs. Raffinose is a 440

sugar with no known energetic role; however, it has been reported to accumulate in 441

response to a broad range of abiotic stressors such as drought, extreme temperatures, and 442

salinity.46 Nishizawa et al. speculated that raffinose is possibly involved in membrane 443

protection and radical scavenging in response to oxidative damage caused by salinity or 444

chilling.47 Raffinose have been associated with reduction processes in oxidative 445

Page 20 of 35



21

membrane damage and ROS scavenging,48 which may explain its increase concurrent 446

with MDA upon exposure to 40 mg Ag NPs. 447

In addition, a number of metabolites were notably downregulated (8-17-fold change) 448

specifically by AgNPs, including acetanilide, p-benzoquinone, 5,6-dihydrouracil, 449

dibenzofuran, oxalic acid, oxamic acid, lactamide (Figure S11). Remarkably, adrenaline 450

became undetectable in response to AgNP exposure. Kaveh et al. also reported that 451

exposure of Arabidopsis thaliana to AgNPs induced some nanoparticle-specific gene 452

responses;11 the authors reported that specific responses to AgNPs including the 453

induction of genes involved in defense against insects and pathogens (AT1G52040) and 454

wounding (AT2G01520), which are believed to be related to mechanical damage to plant 455

tissues caused by AgNPs. The reason for down-regulation of those compounds in our 456

current study is unknown. 457

Environmental Implications. This work has provided insight into the metabolic 458

response of cucumber to exposure to AgNPs and Ag ions. Our data suggest that AgNPs at 459

4 and 40 mg/plant induced overt toxicity symptoms and significant lipid peroxidation in 460

cucumber. Additionally, both doses resulted in alteration of the leaf metabolite profiles. 461

A mechanistic understanding of AgNPs toxicity is very important for future AgNPs 462

applications in agriculture. Metabolomic analysis proved to be a powerful tool and 463

revealed that plants employ multiple strategies for increasing their tolerance of AgNPs or 464

Ag ion induced oxidative stress, including: adjustment of membrane phospholipid 465

composition, increasing the sugar and sugar alcohol levels, and activation of antioxidant 466

defense pathways. These findings provide very valuable information for understanding 467

the molecular mechanisms involved in plant response to AgNP induced stress, and will 468

Page 21 of 35



22

be useful to efforts at seeking to accurately characterize the risk of these materials in the 469

environment, as well as to the development of sustainable nano-enabled plant protection 470

strategies. 471

472

Supporting Information 473

Ag concentrations in cucumber root, stem, and leaf (Figure S1); Photograph of cucumber 474

leaves with phytotoxicity symptoms (Figure S2); Biomass of cucumber (Figure S3); VIP 475

scores from PLS-DA analysis of cucumber leaves (Figure S4, S5 and S6); Significantly 476

changed metabolites of Ag ions group, AgNPs group and their overlap (Figure S7); 477

Biological pathway analysis (Figure S8); significantly changed secondary metabolites 478

(Figure S9); significantly increased (Figure S10) and decreased (Figure S11) metabolites 479

specific to AgNPs exposure. Discussion regarding other significantly changed secondary 480

metabolites are available as Supporting Information. This material is available free of 481

charge via the Internet at http://pubs.acs.org. 482

483

ACKNOWLEDGMENTS 484

This work was supported by the National Key Research and Development Program of 485

China under 2016YFD0800207. We also acknowledge the National Natural Science 486

Foundation of China under 21607071. Any opinions, finding, and conclusions or 487

recommendations expressed in this material are those of authors and do not necessarily 488

reflect the views of National Science Foundation of China. Authors also acknowledge the 489

National Science Foundation and the Environmental Protection Agency under 490

Cooperative Agreement Number DBI-1266377, the USDA grant 2016-67021-24985. 491

Page 22 of 35



https://webmail.utep.edu/owa/redir.aspx?C=5b01ec6f63f3430fa3cec20cb128d864&URL=http%3a%2f%2fpubs.acs.org

23

References: 492

1. Shahid, S. A.; Al-Shankiti, A., Sustainable food production in marginal lands—493 Case of GDLA member countries. International Soil and Water Conservation Research 494 2013, 1, (1), 24-38. 495 2. Gogos, A.; Knauer, K.; Bucheli, T. D., Nanomaterials in Plant Protection and 496 Fertilization: Current State, Foreseen Applications, and Research Priorities. Journal of 497 Agricultural and Food Chemistry 2012, 60, (39), 9781-9792. 498 3. Walker, G. W.; Kookana, R. S.; Smith, N. E.; Kah, M.; Doolette, C. L.; Reeves, 499 P. T.; Lovell, W.; Anderson, D. J.; Turney, T. W.; Navarro, D. A., Ecological Risk 500 Assessment of Nano-enabled Pesticides: A Perspective on Problem Formulation. Journal 501 of Agricultural and Food Chemistry 2017. 502 4. Jo, Y.-K.; Kim, B.; Jung, G., Antifungal Activity of Silver Ions and Nanoparticles 503

on Phytopathogenic Fungi. 2009; Vol. 93, p 1037-1043. 504 5. Kah, M.; Hofmann, T., Nanopesticide research: Current trends and future 505 priorities. Environment International 2014, 63, 224-235. 506 6. Ma, C.; Chhikara, S.; Minocha, R.; Long, S.; Musante, C.; White, J. C.; Xing, B.; 507 Dhankher, O. P., Reduced Silver Nanoparticle Phytotoxicity in Crambe abyssinica with 508 Enhanced Glutathione Production by Overexpressing Bacterial γ-Glutamylcysteine 509 Synthase. Environmental Science & Technology 2015, 49, (16), 10117-10126. 510

7. Wang, J.; Koo, Y.; Alexander, A.; Yang, Y.; Westerhof, S.; Zhang, Q.; Schnoor, 511 J. L.; Colvin, V. L.; Braam, J.; Alvarez, P. J. J., Phytostimulation of Poplars and 512 Arabidopsis Exposed to Silver Nanoparticles and Ag+ at Sublethal Concentrations. 513 Environmental Science & Technology 2013, 47, (10), 5442-5449. 514 8. Ravindran, A.; Prathna, T. C.; Verma, V. K.; Chandrasekaran, N.; Mukherjee, A., 515

Bovine serum albumin mediated decrease in silver nanoparticle phytotoxicity: root 516 elongation and seed germination assay. Toxicological & Environmental Chemistry 2012, 517 94, (1), 91-98. 518 9. Stampoulis, D.; Sinha, S. K.; White, J. C., Assay-dependent phytotoxicity of 519 nanoparticles to plants. Environmental science & technology 2009, 43, (24), 9473-9479. 520 10. Lee, W.-M.; Kwak, J. I.; An, Y.-J., Effect of silver nanoparticles in crop plants 521 Phaseolus radiatus and Sorghum bicolor: Media effect on phytotoxicity. Chemosphere 522 2012, 86, (5), 491-499. 523

11. Kaveh, R.; Li, Y.-S.; Ranjbar, S.; Tehrani, R.; Brueck, C. L.; Van Aken, B., 524 Changes in Arabidopsis thaliana Gene Expression in Response to Silver Nanoparticles 525 and Silver Ions. Environmental Science & Technology 2013, 47, (18), 10637-10644. 526 12. Kim, S.; Choi, J. E.; Choi, J.; Chung, K.-H.; Park, K.; Yi, J.; Ryu, D.-Y., 527 Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells. 528

Toxicology in Vitro 2009, 23, (6), 1076-1084. 529 13. Carlson, C.; Hussain, S. M.; Schrand, A. M.; K. Braydich-Stolle, L.; Hess, K. L.; 530

Jones, R. L.; Schlager, J. J., Unique Cellular Interaction of Silver Nanoparticles: Size-531 Dependent Generation of Reactive Oxygen Species. The Journal of Physical Chemistry B 532 2008, 112, (43), 13608-13619. 533 14. Panda, K. K.; Achary, V. M. M.; Krishnaveni, R.; Padhi, B. K.; Sarangi, S. N.; 534 Sahu, S. N.; Panda, B. B., In vitro biosynthesis and genotoxicity bioassay of silver 535 nanoparticles using plants. Toxicology in Vitro 2011, 25, (5), 1097-1105. 536

Page 23 of 35



24

15. Yuan, L.; Richardson, C. J.; Ho, M.; Willis, C. W.; Colman, B. P.; Wiesner, M. 537

R., Stress Responses of Aquatic Plants to Silver Nanoparticles. Environmental Science & 538 Technology 2018, 52, (5), 2558-2565. 539 16. Hong, J.; Yang, L.; Zhang, D.; Shi, J., Plant Metabolomics: An Indispensable 540 System Biology Tool for Plant Science. International Journal of Molecular Sciences 541 2016, 17, (6), 767. 542 17. Hall, R. D., Plant metabolomics: from holistic hope, to hype, to hot topic. New 543 Phytologist 2006, 169, (3), 453-468. 544 18. Zhao, L.; Huang, Y.; Hu, J.; Zhou, H.; Adeleye, A. S.; Keller, A. A., 1H NMR 545 and GC-MS Based Metabolomics Reveal Defense and Detoxification Mechanism of 546 Cucumber Plant under Nano-Cu Stress. Environmental Science & Technology 2016, 50, 547 (4), 2000-2010. 548 19. Zhao, L.; Ortiz, C.; Adeleye, A. S.; Hu, Q.; Zhou, H.; Huang, Y.; Keller, A. A., 549

Metabolomics to Detect Response of Lettuce (Lactuca sativa) to Cu(OH)2 550 Nanopesticides: Oxidative Stress Response and Detoxification Mechanisms. 551 Environmental Science & Technology 2016, 50, (17), 9697-9707. 552 20. Zhao, L.; Huang, Y.; Adeleye, A. S.; Keller, A. A., Metabolomics Reveals 553 Cu(OH)2 Nanopesticide-Activated Anti-oxidative Pathways and Decreased Beneficial 554 Antioxidants in Spinach Leaves. Environmental Science & Technology 2017, 51, (17), 555 10184-10194. 556

21. Jambunathan, N., Determination and Detection of Reactive Oxygen Species 557 (ROS), Lipid Peroxidation, and Electrolyte Leakage in Plants. In Plant Stress Tolerance: 558 Methods and Protocols, Sunkar, R., Ed. Humana Press: Totowa, NJ, 2010; pp 291-297. 559 22. Xia, J.; Sinelnikov, I. V.; Han, B.; Wishart, D. S., MetaboAnalyst 3.0—making 560 metabolomics more meaningful. Nucleic Acids Res. 2015, 43, (W1), W251-W257. 561

23. Jung, Y.; Ahn, Y. G.; Kim, H. K.; Moon, B. C.; Lee, A. Y.; Hwang, G.-S., 562 Characterization of dandelion species using 1H NMR-and GC-MS-based metabolite 563 profiling. Analyst 2011, 136, (20), 4222-4231. 564 24. Xia, J.; Wishart, D. S., MSEA: a web-based tool to identify biologically 565 meaningful patterns in quantitative metabolomic data. Nucleic Acids Res. 2010, 38, 566 (suppl 2), W71-W77. 567

25. Zhao, L.; Peralta-Videa, J. R.; Ren, M.; Varela-Ramirez, A.; Li, C.; Hernandez-568 Viezcas, J. A.; Aguilera, R. J.; Gardea-Torresdey, J. L., Transport of Zn in a sandy loam 569

soil treated with ZnO NPs and uptake by corn plants: Electron microprobe and confocal 570 microscopy studies. Chemical Engineering Journal 2012, 184, 1-8. 571

26. Navabpour, S.; Morris, K.; Allen, R.; Harrison, E.; A‐H‐Mackerness, S.; 572

Buchanan‐Wollaston, V., Expression of senescence‐enhanced genes in response to 573

oxidative stress. Journal of Experimental Botany 2003, 54, (391), 2285-2292. 574

27. Holmes, E.; Antti, H., Chemometric contributions to the evolution of 575 metabonomics: mathematical solutions to characterising and interpreting complex 576 biological NMR spectra. Analyst 2002, 127, (12), 1549-1557. 577 28. Zhao, L.; Huang, Y.; Keller, A. A., Comparative Metabolic Response between 578 Cucumber (Cucumis sativus) and Corn (Zea mays) to a Cu(OH)2 Nanopesticide. Journal 579

of Agricultural and Food Chemistry 2017. 580 29. Shulaev, V.; Cortes, D.; Miller, G.; Mittler, R., Metabolomics for plant stress 581 response. 2008; Vol. 132, p 199-208. 582

Page 24 of 35



25

30. Xie, Q.; Michaeli, S.; Peled-Zehavi, H.; Galili, G., Chloroplast degradation: one 583

organelle, multiple degradation pathways. Trends in Plant Science 20, (5), 264-265. 584 31. Spicher, L.; Almeida, J.; Gutbrod, K.; Pipitone, R.; Dörmann, P.; Glauser, G.; 585 Rossi, M.; Kessler, F., Essential role for phytol kinase and tocopherol in tolerance to 586 combined light and temperature stress in tomato. 2017; Vol. 68. 587

32. Martín‐Romero, F. J.; García‐Martín, E.; Gutiérrez‐Merino, C., Inhibition of 588

oxidative stress produced by plasma membrane NADH oxidase delays low‐potassium589

‐induced apoptosis of cerebellar granule cells. Journal of Neurochemistry 2002, 82, (3), 590

705-715. 591 33. Hou, R. C.-W.; Chen, Y.-S.; Chen, C.-H.; Chen, Y.-H.; Jeng, K.-C. G., Protective 592 effect of 1,2,4-benzenetriol on LPS-induced NO production by BV2 microglial cells. 593 Journal of Biomedical Science 2006, 13, (1), 89-99. 594 34. Avase, S. A.; Srivastava, S.; Vishal, K.; Ashok, H. V.; Varghese, G., Effect of 595

Pyrogallol as an Antioxidant on the Performance and Emission Characteristics of 596 Biodiesel Derived from Waste Cooking Oil. Procedia Earth and Planetary Science 2015, 597 11, 437-444. 598 35. Taguri, T.; Tanaka, T.; Kouno, I., Antibacterial Spectrum of Plant Polyphenols 599 and Extracts Depending upon Hydroxyphenyl Structure. Biological and Pharmaceutical 600 Bulletin 2006, 29, (11), 2226-2235. 601 36. Wurzinger, B.; Nukarinen, E.; Nägele, T.; Weckwerth, W.; Teige, M., The 602 SnRK1 Kinase as Central Mediator of Energy Signaling between Different Organelles. 603 Plant Physiology 2018, 176, (2), 1085-1094. 604

37. Wingler, A.; Lea, P. J.; Quick, W. P.; Leegood, R. C., Photorespiration: metabolic 605 pathways and their role in stress protection. Philosophical Transactions of the Royal 606 Society B: Biological Sciences 2000, 355, (1402), 1517-1529. 607

38. Li, L.; Zhao, J.; Zhao, Y.; Lu, X.; Zhou, Z.; Zhao, C.; Xu, G., Comprehensive 608 investigation of tobacco leaves during natural early senescence via multi-platform 609 metabolomics analyses. Scientific Reports 2016, 6, 37976. 610 39. Florencio-Ortiz, V.; Sellés-Marchart, S.; Zubcoff-Vallejo, J.; Jander, G.; Casas, J. 611 L., Changes in the free amino acid composition of Capsicum annuum (pepper) leaves in 612 response to Myzus persicae (green peach aphid) infestation. A comparison with water 613 stress. PLOS ONE 2018, 13, (6), e0198093. 614 40. Diaz, C.; Purdy, S.; Christ, A.; Morot-Gaudry, J.-F.; Wingler, A.; Masclaux-615 Daubresse, C., Characterization of Markers to Determine the Extent and Variability of 616 Leaf Senescence in Arabidopsis. A Metabolic Profiling Approach. Plant Physiology 617

2005, 138, (2), 898-908. 618 41. Upchurch, R. G., Fatty acid unsaturation, mobilization, and regulation in the 619

response of plants to stress. Biotechnology Letters 2008, 30, (6), 967-977. 620 42. Chen, Z.; Silva, H.; Klessig, D., Active oxygen species in the induction of plant 621 systemic acquired resistance by salicylic acid. Science 1993, 262, (5141), 1883-1886. 622 43. Ford, K. A.; Casida, J. E.; Chandran, D.; Gulevich, A. G.; Okrent, R. A.; Durkin, 623 K. A.; Sarpong, R.; Bunnelle, E. M.; Wildermuth, M. C., Neonicotinoid insecticides 624

induce salicylate-associated plant defense responses. Proceedings of the National 625 Academy of Sciences 2010, 107, (41), 17527-17532. 626 44. Miyazawa, J.; Kawabata, T.; Ogasawara, N., Induction of an acidic isozyme of 627

peroxidase and acquired resistance to wilt disease in response to treatment of tomato 628

Page 25 of 35



26

roots with 2-furoic acid, 4-hydroxybenzoic hydrazide or salicylic hydrazide. 629

Physiological and Molecular Plant Pathology 1998, 52, (2), 115-126. 630 45. Tachibana, Y.; Kikuzaki, H.; Lajis, N. H.; Nakatani, N., Antioxidative Activity of 631 Carbazoles from Murraya koenigii Leaves. Journal of Agricultural and Food Chemistry 632 2001, 49, (11), 5589-5594. 633 46. Kaplan, F.; Guy, C. L., <em>β</em>-Amylase Induction and the Protective Role 634 of Maltose during Temperature Shock. Plant Physiology 2004, 135, (3), 1674-1684. 635 47. Nishizawa, A.; Yabuta, Y.; Shigeoka, S., Galactinol and Raffinose Constitute a 636 Novel Function to Protect Plants from Oxidative Damage. Plant Physiology 2008, 147, 637 (3), 1251-1263. 638 48. Molinari, H. B. C.; Marur, C. J.; Filho, J. C. B.; Kobayashi, A. K.; Pileggi, M.; 639 Júnior, R. P. L.; Pereira, L. F. P.; Vieira, L. G. E., Osmotic adjustment in transgenic 640 citrus rootstock Carrizo citrange (Citrus sinensis Osb. x Poncirus trifoliata L. Raf.) 641

overproducing proline. Plant Science 2004, 167, (6), 1375-1381. 642

643

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Figure legends 659

Figure 1. Biochemical detection of lipid peroxidation in cucumber leaves exposed to 660

different doses of AgNPs and Ag+ for 7 days. All data show the means of 5 replicates. 661

Error bars represent standard deviation. Means with same letters are not significantly 662

different (Tukey’s HSD multiple comparison at p < 0.05). 663

Figure 2. Principle Component Analysis (PCA) (A) and Partial least squares-664

discriminate analysis (PLS-DA) (B) score plots of metabolic profiles in cucumber leaves 665

treated with different doses of AgNPs (4 and 40 mg per plant) and Ag+ (0.04 and 0.4 mg 666

per plant). A, B, C, D, E represent control, 0.04 mg and 0.4 mg Ag ions, 4 and 40 mg 667

AgNPs, respectively. 668

Figure 3. Schematic diagram of the proposed metabolic pathways in cucumber leaves 669

exposed to AgNPs. Red and green represent the up- and downregulated metabolites, 670

respectively. 671

Figure 4. Box plot of relative abundance of antioxidant metabolites in 4-week-old 672

cucumber leaves exposed to different doses of AgNPs (4 and 40 mg per plant) and Ag+ 673

(0.04 and 0.4 mg per plant) (n=5). A, B, C, D, E represent control, 0.04 mg and 0.4 mg 674

Ag ions, 4 and 40 mg AgNPs, respectively. 675

Figure 5. Box plot of relative abundance of carbohydrates in 4-week-old cucumber 676

leaves exposed to different doses of AgNPs (4 and 40 mg per plant) and Ag+ (0.04 and 677

0.4 mg per plant) (n=5). A, B, C, D, E represent control, 0.04 mg and 0.4 mg Ag ions, 4 678

and 40 mg AgNPs, respectively. 679

Figure 6. Box plot of relative abundance of amino acids in 4-week-old cucumber leaves 680

exposed to different doses of AgNPs (4 and 40 mg per plant) and Ag+ (0.04 and 0.4 mg 681

Page 27 of 35



28

per plant) (n=5). A, B, C, D, E represent control, 0.04 mg and 0.4 mg Ag ions, 4 and 40 682

mg AgNPs, respectively. 683

Figure 7. Box plot of relative abundance of fatty acids in 4-week-old cucumber leaves 684

exposed to different doses of AgNPs (4 and 40 mg per plant) and Ag+ (0.04 and 0.4 mg 685

per plant) (n=5). A, B, C, D, E represent control, 0.04 mg and 0.4 mg Ag ions, 4 and 40 686

mg AgNPs, respectively. 687

688

689

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704

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705

Control

0.1 ppm Ag ions

1 ppm Ag ions

10 ppm AgNPs

100 ppm AgNPs

MD

A c

onte

nt

in c

ucu

mber

leav

es (

μm

ol

g-1

)

0.000

0.005

0.010

0.015

0.020

0.025

0.030

a abbc

c

ab

706

Figure 1. 707

708

709

710

711

712

713

714

715

716

717

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718

719

720

Figure 2. 721

722

723

724

725

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731

732

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733

Figure 3. 734

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735

736 Figure 4. 737

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738

739

Figure 5. 740

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743 Figure 6. 744

745

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Figure 7. 750

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