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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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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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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
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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
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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
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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 down- regulated 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
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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
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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
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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 down- regulated 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
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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
690
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695
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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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728
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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
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Figure 7. 750
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