Metabolomics Reveals How Cucumber (Cucumis sativus

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Ecotoxicology and Human Environmental Health

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

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Environmental Science & Technology

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Metabolomics Reveals How Cucumber (Cucumis

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sativus) Reprograms Metabolites to Cope with

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Silver Ions and Silver Nanoparticle-Induced

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Oxidative Stress

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

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

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§

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

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Nanjing University, Nanjing 210023, China ζ

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Avenue, El Paso, Texas 79968, United States ¶

Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station

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

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

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

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

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

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ACS Paragon Plus Environment


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TOC

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ABSTRACT

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

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

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

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

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

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

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metabolomics enabled the identification and quantification of 268 metabolites in

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cucumber leaves. Multivariate analysis revealed that all the treatments significantly

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

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

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

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

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

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

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

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

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

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

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

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

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agriculture.

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INTRODUCTION

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

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

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

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Nanotechnology is emerging as a promising strategy to sustainably increase food

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

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

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

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environmental impacts while saving valuable water and energy inputs.

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

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

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

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

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

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

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

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

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significant uncertainty remains concerning the application of AgNPs in agriculture.

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

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

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

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

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

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

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

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sought to characterize the underlying toxicity and detoxification mechanisms of plants

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

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

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

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reports have demonstrated that AgNPs trigger the overproduction of reactive oxygen

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

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

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

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

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

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

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

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

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

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providing an essentially unbiased, comprehensive qualitative and quantitative overview

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

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

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molecular level.18-20

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

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

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

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

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

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mechanisms will not only advance understanding of environmental implications of these

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

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AgNPs in agriculture.

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EXPERIMENTAL SECTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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respectively.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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MS analysis, and multivariate analysis are shown below.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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°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

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

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

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

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

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

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analytical run.

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

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

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

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

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

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

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

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

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

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

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

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

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

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value threshold calculated for pathway identification was set at 0.1.24

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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study.

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

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

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

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

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also causing dehydration (Figure S2).

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

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

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

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

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

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

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increase indicates potentially significant membrane damage as a function of AgNPs

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exposure.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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discussion of this data follows below.

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

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

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the importance of ion release to plant response.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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of plant defense against stress.

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

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

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

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

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

Metabolomics Reveals How Cucumber (Cucumis sativus

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