Impact of polystyrene nanoplastics (PSNPs) on seed germination and seedling growth of wheat (Triticum aestivum L.)

Journal Pre-proof Impact of polystyrene nanoplastics (PSNPs) on seed germination and seedling growth of wheat (Triticum aestivum L.) Jiapan Lian, Jiani Wu, Hongxia Xiong, Aurang Zeb, Tianzhi Yang, Xiangmiao Su, Lijuan Su, Weitao Liu

PII:

S0304-3894(19)31574-2

DOI:

https://doi.org/10.1016/j.jhazmat.2019.121620

Reference:

HAZMAT 121620

To appear in:

Journal of Hazardous Materials

Received Date:

19 August 2019

Revised Date:

29 October 2019

Accepted Date:

5 November 2019

Please cite this article as: Lian J, Wu J, Xiong H, Zeb A, Yang T, Su X, Su L, Liu W, Impact of polystyrene nanoplastics (PSNPs) on seed germination and seedling growth of wheat (Triticum aestivum L.), Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121620

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Impact of polystyrene nanoplastics (PSNPs) on seed germination and seedling growth of wheat (Triticum aestivum L.)

Jiapan Lian a, Jiani Wu a, Hongxia Xiong b, Aurang Zeb a, Tianzhi Yang a, Xiangmiao

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Su a, Lijuan Su a, Weitao Liua,*

MOE Key Laboratory of Pollution Processes and Environmental Criteria, Tianjin

Key Laboratory of Environmental Technology for Complex Trans-Media Pollution,

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College of Environmental Science and Engineering, Nankai University, Tianjin 300350, P R China

Tianjin Research Institute for Water Transport Engineering, Laboratory of

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b

*Corresponding

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Environmental Protection in Water Transport Engineering, Tianjin 300456, P R China author: Weitao Liu

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 Full postal Address: No. 38 Tongyan Road, College of Environmental Science and Engineering, Nankai University, Tianjin 300350, P R China  Phone: +86 22 23501117

+86 22 23501117

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 Fax:

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 E-mail: [email protected]

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Graphical Abstract

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Highlights:  PSNPs exhibited no effect on seed germination rate but significantly enhanced

wheat seedling growth.  Growth parameters and chlorophyll content were greatly increased.  PSNPs reduced the shoot to root biomass ratio and micronutrients contents.

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 The metabolic profiles in wheat leaves were profoundly altered by PSNPs.

Abstract: Microplastics and nanoplastics are emerging pollutants of global concern. However, the understanding of their ecological effects on terrestrial plants is still

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limited. We conducted the systematic research to reveal the impact of polystyrene nanoplastics (PSNPs) (0.01-10 mg/L) on seed germination and seedling growth of

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wheat (Triticum aestivum L.). The results showed that PSNPs had no discernible

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effect on seed germination rate whereas significantly (p<0.01) increased root elongation by 88.6%122.6% when compared with the control. Similarly, remarkable

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increases in carbon, nitrogen contents, and plant biomass were also observed after exposure to PSNPs. Moreover, PSNPs could reduce the shoot to root biomass ratio

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(S:R ratio) of wheat seedlings. Furthermore, the imagings of a 3D laser confocal

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scanning microscopy (LCSM) and scanning electron microscopy (SEM) indicated that PSNPs were taken up and subsequently down-top transported to shoot. The absorption and accumulation of four micronutrients (Fe, Mn, Cu and Zn) in wheat were generally reduced in varying degrees. Notably, metabolomics analysis revealed that all PSNPs treatments altered the leaf metabolic profiles mainly by regulating energy metabolisms and amino acid metabolisms. These findings are expected to 3

provide new insights into the effects of PSNPs on crop plants.

Keywords: Polystyrene nanoplastics (PSNPs); wheat (Triticum aestivum L.); plant growth; uptake; nutrient element; metabolomics

1. Introduction

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Microplastics pollution in terrestrial ecosystems has become an emerging concern in recent years [1, 2]. Although there is a broad and general consensus on the definition of microplastic (MP) whose particle size is below 5 mm, an international

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controversy over the definition of nanoplastic (NP) has been brewing. Several authors defined nanoplastics as particles that exhibit a colloidal behavior with the size ranging

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from 1 nm to 1 m [3, 4]. NPs also can be derived from abrasion or environmental

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degradation (where UV light could render the material brittle) of MPs or larger-sized pieces [5-7]. Sewage treatment plants are an important source of soil microplastic

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pollution because over 90% of the coming microplastics are retained in the sewage sludge [8]. However, the current sludge treatment processes (anaerobic digestion,

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thermal drying, or lime stabilization) could not effectively remove them [9]. For

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example, lime stabilization of sludge would shear MPs during treatment process, resulting in significantly higher abundances of smaller size MPs. Owing to the fact that most plastics have been produced, used, and often discarded on land, MPs pollution on land might be 4-23-fold larger than that in the ocean [10]. MPs have been found in various terrestrial environmental matrixes, including agricultural farmlands [11, 12], municipal and industrialized soil [13], and also rather 4

remote regions [14]. In agricultural systems, sewage sludge is considered as a significant source for terrestrial ecosystems because the biosolids, containing a large number of fibers and micro- and nanoplastic beads from clothing or personal care products, are often applied to soil as fertilizer [9, 15]. Recent studies estimated that a total of 63,000-43,0000 and 44,000-300,000 tons MPs were released directly into European and North American farmlands per year [16], respectively, and between

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2,800 and 19,000 tons per year in Australia agroecosystems [17]. In southwestern China, Zhang and Liu [11] reported that the concentration of MPs in arable soil ranges

from 7,100 to 42,960 particles/kg (mean 18,760 particles/kg). There is no report of

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nanoplastics detection in the environmental matrixes due to the limitations of

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analytical techniques, but it has been proved that the nanoplastics can be easily

laboratory conditions [18].

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generated from the degradation of polystyrene disposable coffee cup lids under

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Terrestrial ecosystems have received rather far less scientific attention to MP/NPs than their aquatic counterparts [1, 2]. Growth reduction or oxidative stress has been

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much reported in aquatic biota induced by MP/NPs, including algae [19-22], sediment-rooted macrophytes (Myriophyllum spicatum and Elodea sp.) [23], and

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duckweed (Lemna minor) [24]. However, the impact of micro- and/or nanoplastics on terrestrial plants is still not well explored, as only few reports are available up to now. In the terrestrial environment, microplastic mulch film residues could negatively affect shoot and root growth of wheat (Triticum aestivum) [25]. Exposure to both micro- and nanoplastics significantly reduced the germination rate and root growth of 5

Lepidium sativum at an early stage, but no obvious effects were found after 48 or 72 h of exposure [26]. Nevertheless, even though MPs pollution in terrestrial environment has received increasing attention, the researches on the interactions between MPs/NPs and plants are quite scarce [2, 27]. Wheat (Triticum aestivum L.) is the predominant cereal crop and a staple food source for more than half of the world’s population [28]. On the other hand, it is one

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of the most common plant indicators for determining the potential ecological effects

of nanomaterials [29]. Although the effects of two types of microplastic mulch film on wheat growth performance have been studied [25], the knowledge regarding the

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toxicity of polystyrene nanoplastics (PSNPs) to wheat is still unknown. Therefore, we

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selected wheat as a tested plant to reveal (1) the effect of PSNPs on seed germination and root elongation; (2) the biological responses of wheat seedling to PSNPs at the

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endpoints: growth, photosynthesis, nutrients accumulation and metabolic profiles. To

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our knowledge, this paper is the first report highlighting the effect of PSNPs on seed germination and seeding growth of wheat, and will contribute to a new understanding

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of the potential health risks and ecological effects of nanoplastics on crop plants.

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2. Materials and methods 2.1. PSNPs test solution characterization and sodium dodecyl sulfate (SDS) Fluorescent (Nile red) and non-fluorescent nanospheres (100 nm), supplied as 5% (w/v) mono-dispersed in deionized water, were purchased from Shanghai Huge Biotech Co., China. Morphology and particle size of PSNPs were characterized by 6

scanning electron microscopy (SEM) (JSM-7800F, JEOL, Japan) operated at 10 kV (Fig. S2A). The composition and surface functional groups they carried were analyzed by Laser Confocal Raman Spectrometer (LCRS) (InVia Reflex, Renishaw, UK) (Fig. S2B). Depending on the product information provided by the manufacturer, the PSNPs were synthesized from styrene monomers with ammonium persulfate as initiator. The

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synthesized emulsion product (PSNPs) was thoroughly washed with deionized water and then dispersed in deionized water with sodium dodecyl sulfate (SDS) for a long storage time. To eliminate the interference of SDS to the development of wheat

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seedlings, the toxicity of SDS to wheat was therefore considered in separate pilot tests.

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The concentrations of SDS in PSNPs test dispersions were below “1.2 mg/L” determined as described by Hayashi [30]. Detailed protocols are provided in Text S1.

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Additionally, the colloidal behavior of PSNPs in two different culture media

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(deionized water & 25% Hoagland solution) was also studied over experimental time. After 10 min of ultrasonication (100 W) (SB25-12, SCIENTZ, China), hydrodynamic

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size distribution and zeta potential of SPNPs in culture solutions were measured using a Dynamic Light Scattering (DLS) instrument (Zetasizer, Nano-ZS 90, Malvern).

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Subsequently, the agglomeration processes of PSNPs were evaluated at 1 mg/L over time 1, 7, 14 and 21 d in two suspensions. 2.2. Seed germination assay Wheat seeds (Triticum aestivum L. cv. Xiaoyan 22) in this study were obtained from Northwest Agriculture & Forestry University, China and stored in a sealed kraft 7

paper bag at 4-8 C until use. The cultivar is a new wheat variety with the largest planting area in China’s Shanxi province. The germinating experiment was carried out according to the protocol of Lin and Xing [31]. Wheat seeds were surface-sterilized by 2% (v/v) hydrogen peroxide solution for 30 min prior to being rinsed three times with deionized water. They were subsequently soaked in non-fluorescent PSNPs test dispersions (0.01, 0.1, 1.0, 10

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mg/L) or corresponding SDS solution (0.0012, 0.012, 0.12, 1.2 mg/L) for about 2 h at

room temperature. The exposure solutions were prepared by stock solution and then agitated by ultrasonic vibration (100 W, 25 kHz) for 10 min, in which the

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concentrations of PSNPs are chosen from the previous studies [24, 32, 33].

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Subsequently, 25 seeds were transferred onto a Petri dish (90 mm in diameter) containing one piece of qualitative filter paper, and 1 cm or larger distance was kept

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away from each other. Finally, 5 ml of a test medium was added and Petri dishes were sealed with parafilm. After 5 days’ incubation in a growth chamber under dark

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condition at 25 C, the germination of wheat seeds was recorded and the control seed

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had more than 80% germination. The sprout and root length of wheat seedlings were measured using a millimeter-scale ruler.

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Relative seed germination (RSG), relative root elongation (E) and seedling vigour

index (SVI) were calculated as described by Barrena, et al. [34]: 𝑠𝑒𝑒𝑑𝑠 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑤𝑖𝑡ℎ 𝑃𝑆𝑁𝑃𝑠 × 100 𝑠𝑒𝑒𝑑𝑠 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑤𝑖𝑡ℎ 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 mean root length with PSNPs Relative root elongation (E) = × 100 𝑚𝑒𝑎𝑛 𝑟𝑜𝑜𝑡 𝑙𝑒𝑛𝑔𝑡ℎ 𝑤𝑖𝑡ℎ 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 Relative seed germination (RSG) =

Seed vigour index (SVI) = Germination% × seedling length (Root + Shoot) 8

Water uptake by wheat seeds during imbibition process was determined following the previous methods [35]. 25 seeds were placed on Petri dish with a 5 ml test medium and then incubated at 25 C in a growth chamber under dark. At different imbibition times, all germinating seeds were carefully removed, blotted dry and weighed quickly (Sartorius, BSA124S-CW). The water uptake by seeds was calculated based on the weight changes as a result of imbibition. [(Fresh weight of seeds − Dry weight of seeds)] × 100 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑒𝑒𝑑𝑠

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Water uptake (WU%) =

2.3. Hydroponic experiment

Wheat seeds were sterilized using 2% hydrogen peroxide solution for 30 min and

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were then thoroughly rinsed with deionized water several times. The sterilized wheat

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seeds were placed on absorbent cotton gauze drenched in sterile water on an enameled dish and later germinated for 5 days at 25 C in the dark growth chamber. Seedlings

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of uniform growth were transferred to 8 L of 25% strength Hoagland solution for 7

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days, which was replaced every day. Afterward, uniform seedlings were transferred to 500 ml glass beakers with different concentrations of PSNPs test solution (0, 0.01, 0.1,

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1.0, 10 mg/L) dispersed by 25% strength Hoagland solution. Each treatment had four replicates in this experiment. The beakers were carefully covered with tin foil paper to

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avoid algae growth and water evaporation. The seedlings were cultured at room temperature with a light intensity of 6000 lux and 14 h photoperiod for 21 days. Water loss due to evaporation or plant transpiration was compensated by adding nutrient solution every three days. At the harvest time, the seedlings were washed with tap water and then rinsed three times with deionized water. Shoot and root tissues were 9

separated, and their weights were measured respectively. The isolated roots were scanned and the root morphological parameters were analyzed with WinRHIZO Pro 2012b (Regent Instruments Inc., Canada). Dry biomass was obtained after freeze-dried for 48 h and the shoot to root ratio (S:R ratio) was calculated on a dry weight basis. 2.4. Photosynthetic gas exchange parameters and chlorophyll content

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The photosynthetic gas exchange parameters were determined on May 9, 2019

using a Li-6800 portable photosynthesis system (Li-COR Biosciences, Lincoln, NE,

USA). The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular

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carbon dioxide concentration (Ci) and transpiration rate (Tr) were measured from 9:00

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to 11:30 am under an ambient light source (190 mol/m2s) on the clamp-on leaf chamber (1.5 cm2). The flow rate was at 500 mol/s and the CO2 concentration in the

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leaf chamber was maintained at 400 mol/mol by a CO2 cylinder. The pressure value

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in the pipeline was 0.1 kPa and the leaf temperature was set at 25 C. The instantaneous water use efficiency (WUE) is expressed as WUE=Pn/Tr, whereas the

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limiting value of stomata (Ls)=1-Ci/Ca, where Ca is the CO2 concentration of air outside leaf.

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The relative chlorophyll content was measured with a SPAD-502 chlorophyll

meter (SPAD, Minolta Camera, Japan). Briefly, SPAD values of six fully expanded leaves from the top of the plant were measured. The average of all SPAD values from 24 leaves was regarded as the chlorophyll content of different treatments. 2.5. Microscopic observation 10

As the culture temperature in these experiments is far below the glass transition temperature of PSNPs [36], the leakage of immobilized fluorescent dye from the polymer matrix is basically negligible. Nevertheless, no direct research focusing on the toxicity of Nile red dye on wheat seedling was retrieved. Therefore, we intended to avoid the potential or side effects of fluorescent dye on wheat seedlings in a main hydroponic experiment and only the highest concentration (10 mg/L) of PSNPs was

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chosen for uptake characterization in a separate test. Hence, any observed impacts on the growth of wheat seedling could be considered as caused by PSNPs. Seedlings

exposed to fluorescent PSNPs were cultivated in the same procedures described above.

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After 21 days’ exposure, the location of PSNPs in root tips was examined using a 3D

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laser confocal scanning microscopy (LSM880, Zeiss, Germany). The observation was made on an inverted glass slide with the excitation and emission wavelengths of 535

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and 610 nm. Images were acquired with the operating software Zeiss Zen 2010 (Zeiss,

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Germany).

The root and leaf sections were prepared as described by Zhao, et al. [37]. Briefly,

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root tips and leaves prefixed with phosphate (pH 7.2) buffered 2.5% glutaraldehyde and 2% paraformaldehydes and then fixed in 1% osmium tetroxide for 1 h. Then, the

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dehydrated specimens were cut in transverse sections, coated with gold for 60 s (ca. 1nm gold layer) by using a Sputter Coater (HITACHI E-1010, Japan). The cross-section was observed at an acceleration voltage of 5 kV. With regard to the morphology of starch grain in germinating seed, the protocol for SEM observation was listed in Text S2 and examined following a modified method of Xu, et al. [38]. 11

2.6. Foliar carbon and nitrogen (%) contents The freeze-dried leaves were firstly cut into small segments and then ground into fine powder in a ball mill (Jxfstprp-24, Jinxin, China). The C, N contents were analyzed using an EA3000 automatic elemental analyzer (Leeman, Italy). Briefly, 13 mg of leaf powder was weighed into tin containers and the tin cups were then dropped in a tube where flash combustion occurred in the presence of external oxygen (at

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1800 °C). Two tests from one sample were done with the relative error below 1%. 2.7. Metal elements contents

Approximately 0.10.2 g dry samples were weighed and then digested at 200 °C

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with a mixed acid containing concentrated HNO3 and HClO4 (4:3 v/v) on DigiBlock

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digestion system (EH35A, LabTech, UK) [39]. After cooling to room temperature, remained samples were diluted to 25 ml with 2% (v/v) nitric acid and filtered through

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a 0.22 m filters. The contents of micro- and macroelements (Mn, Cu, Zn, Fe and Mg)

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in the digest solution from all treatments were determined using inductively coupled plasma mass spectrometry (ICP-MS) (Elan drc-e, Perkin Elmer, USA) or inductively

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coupled plasmas atomic emissive spectrometry (ICP-AES) (IRIS Intrepid II XSP, Thermo Elemental, USA). A certified reference material, orange leaves (GBW10020,

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Beijing, China), was used to monitor the recovery of metals from the plant samples. The recovery rates of metal elements of the ICP-MS method were between 90 and 99%. 2.8. Metabolic profiling of wheat Freeze-dried leaf samples of the control and PSNPs-treated plants were used for 12

metabolic analysis through a gas chromatography−mass spectrometry (GC−MS). The samples were prepared according to our previously described method [40]. In the simplest terms, the metabolites were extracted from 10 mg of powdered samples using a single-phase solvent mixture (2 ml) of methanol, water and chloroform at a ratio of 5:2:2 (v/v/v) under ultrasonication (300 W, 30 min) in an ice bath, followed by centrifugation (12000 rpm, 4 C) to collect the supernatant. The centrifugal

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sediment was again extracted and mixed with the first supernatant. Subsequently, the

resulting supernatant was filtered through a 5 cm silica gel column and then

concentrated to dry via nitrogen blow-off and lyophilization. For derivatization, 50 L

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of methoxyamine hydrochloride in pyridine (20 mg/ml) was added, and the mixture

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was vigorously vortexed for 1 min and then incubated at 30 C for 90 min. The mixture was then silylated at 37 C for 30 min by adding 80 L of

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N-methyl-N-(trimethylsilyl) tri-fluoroacetamide (MSTFA).

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The samples were analyzed using a GC (6890 N, Agilent, USA) equipped with a quadrupole MS (model 5973, Agilent). The GC parameters were as follows:

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capillary-column chromatograph, HP-5MS; temperature, 6 min at 320 C with a speed of 15 C /min after 80 C for 2 min. Mass spectroscopy parameters were as follows:

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ion source temperature, 250 C; full scan range, from 50 to 500 m/z. The metabolites were

identified

using

the

NIST

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library.

Then,

Metaboanalyst

3.0

(http://www.metaboanalyst.ca/MetaboAnalyst/) was used for multivariate analysis and identification of perturbed metabolic pathways. 2.9. Data analysis 13

All the experiments were conducted in at least quadruplicates. Data were expressed as mean ± SD (standard deviation) and analyzed using SPSS 17.0 software (IBM, USA). Statistical differences between treatments were analyzed with a one-way ANOVA followed by Duncan's post hoc test. Statistical significance was accepted at p<0.05. Comparisons between PSNPs and SDS treatments were performed by an

3. Results 3.1 Primary and secondary characterization of PSNPs

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independent samples t-Test at the p<0.05 level.

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The morphology and primary size of PSNPs are shown in Fig.S2A. The shape of

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PSNPs is spherical and their surface is quite smooth. After counting more than 200 individual particles by ImageJ software, the average size of PSNPs is 87.88.6 nm

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(Fig. S2C). Fig.S2B confirms the Raman spectra of PSNPs with a range of 100 to

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3500 cm-1. The LCRS spectrum of polystyrene displays three main characteristic bands at 3045 cm-1, arising from aromatic C-H moieties, 1587 cm-1, from asymmetric

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C-C stretching vibration in benzene ring, and 987 cm-1 from ring breathing vibration (C-C symmetric motion), whereas the other weak peaks are mainly due to C-C/C-H

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stretching or the bending vibration of CH2, and no peaks assigned to other surface functional groups were found. The size distribution of PSNPs in two culture media was studied (Table S1). In deionized water, no differences were observed in the hydrodynamic diameter of PSNPs agglomerates among four concentrations at time 0. The mean and main peak 14

of four size distributions is around 120 nm in deionized water. However, the mean hydrodynamic diameter of PSNPs agglomerates significantly varies with the concentration gradients. The mean peak of the size distribution for 10 mg/L PSNPs reached the lowest value of 254.416.54 nm in 25% Hoagland solution with a PDI value greater than 0.6. For another, almost no agglomeration process was found in deionized water over experiment time. The curve of size distribution was similar with

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a mean and main peak of 120 nm before 14 d while slightly shifted to right (140 nm)

at 21 d (Fig.S3A). At the same time, the zeta potential of colloidal dispersion was relatively stable (about -35 mV) during the experimental time course. By contrast, the

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absolute value of zeta potential was continuously decreased from 1st day in 25%

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Hoagland solution. Obvious right shifts in the main peak of size distribution were also observed in Hoagland solution during the experiment period (Fig.S3C), increasing the

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agglomeration process of PSNPs.

3.2 Seed germination and root elongation induced by PSNPs

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The RSG of wheat was not significantly changed when exposed to all concentrations of PSNPs or SDS (Fig. 1A). Compared to the control, both PSNPs and

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SDS could slightly but not significantly (p>0.05) improve seed germination rate under

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high-dose treatment. Due to the high germinating seed lot (over 80%), seed vigour was expected to provide more information as to the potential seed lot performance. PSNPs markedly (p<0.05) improved the seed vigour index at the concentration of 0.01 and 10 mg/L whereas the SVI values were unaffected by SDS compared with the control (Fig. 1C). In addition, no statistically significant differences in the RSG and SVI index of wheat were observed between all PSNPs and corresponding SDS 15

treatments. Figure 1B presents the results obtained for the root length in the seed germination test. Clearly, PSNPs produced a significant (p<0.01) positive effect on the root elongation and dramatically increased root elongation 1.9-2.2 times. SDS, in contrast, produced no observable effect on root length. In all cases, PSNPs and their SDS counterparts presented significant differences (p<0.001) in root length. Thus, these

of PSNPs. 3.3 Changes in seed water uptake during germination

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results indicated that the remarkable positive effect observed was due to the presence

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Seed water uptake was evaluated in the early germination stage (the first three

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days). It was found that imbibition process in our study was a rapid initial uptake phase when seeds soaked in PSNPs absorbed water significantly faster (p<0.05 at

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0.01 mg/L; p<0.01 at 0.1,1, and 10 mg/L) than controls, especially within 3 h of

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imbibition (Fig. 1D). As the imbibition time increased, seed water uptake of only 10 mg/L PSNPs treatment was noticeably higher (p<0.05) after 14 h when compared

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with the control. Afterward, no differences in seed water uptake among all PSNPs treatments were found with prolonged exposure time.

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3.4 Influence of PSNPs on growth parameters and chlorophyll content PSNPs exposure resulted in a sharp growth of wheat seedlings but did not reveal

any signs of overt stress (Fig. 2A). The chlorophyll content in wheat seeding leaf significantly increased at 0.1 mg/L (p<0.01) and 1.0 mg/L (p<0.05) after exposure to PSNPs (Fig. 2B). PSNPs significantly (p<0.01) increased shoot biomass by 70.5% 16

and 87.1% when exposure to 0.01 and 0.1 mg/L, respectively. Similarly, root biomass increased markedly by 98.9% at 0.01 mg/L (p<0.01), 116.5% at 0.1 mg/L (p<0.01) and 52.5% at 1 mg/L (p<0.05) respectively (Fig. 2C). Additionally, compared to the control, PSNPs exposure reduced the shoot to root ratio (S:R ratio) at all concentrations, while the S:R ratio decreased significantly (up to 27.3%, p<0.05) under the treatment of 0.01 mg/L (Fig. 2D).

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Root morphology (length, SurfArea, AvgDiam, RootVolume and Tips) was

profoundly changed when exposed to PSNPs. Root length, average diameter, surface

area and root volume were increased significantly (p<0.05) by PSNPs exposure as

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compared to the unexposed treatment, except for 10 mg/L PSNPs. However, root tips

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were not profoundly affected by PSNPs exposure (Table S2). 3.5 Effect of PSNPs on photosynthetic parameters

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PSNPs exposure promoted the photosynthesis of wheat seedlings (Table 1). The

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net photosynthetic rate of wheat exposed to 0.1 mg/L PSNPs was 1.85 times higher than controls, whereas the intercellular CO2 concentration did not change after

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exposure to PSNPs. Similarly, exposure to 0.1 mg/L PSNPs significantly increased the stomatal conductance (p<0.05) and transpiration rate (p<0.01) of plants to 2.13

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and 3.77-folds as compared with the control. The WUE of wheat seedlings was greatly decreased when exposure to 0.1 mg/L PSNPs, which was 38.96% of the control. Although the Gs and Tr of plant exposure to PSNPs were increased, the Ls value of wheat seedlings was not significantly changed. 3.6 Influence of PSNPs on carbon and nitrogen contents 17

After 21 days of PSNPs exposure, the nitrogen and carbon contents in leaf were generally increased with increasing PSNPs concentrations (Fig. 5). Except for 0.01 mg/L PSNPs, the leaf nitrogen contents were raised significantly (p<0.01). Similarly, both 0.1 mg/L and 1 mg/L PSNPs treatments markedly (p<0.01) increased carbon contents in the leaves. 3.7 Effect of PSNPs on nutrients accumulation

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The contents of macro- and micronutrients were measured in wheat shoots and

roots across all the treatments (Fig. 6). For macronutrient, magnesium (Mg) concentrations in the shoots are much higher than those in roots and no significant

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differences in Mg concentration were observed with respect to the control (Fig. 6A).

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However, compared with the unexposed group, the accumulation of Mg in root tissues was markedly (p<0.05) elevated by 10.9% at 0.01 mg/L and 11.7% at 0.1 mg/L,

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respectively.

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The concentration of iron (Fe) in shoot and root tissues was altered in the same manner and an only significant decrease at 1 mg/L was found (Fig. 6B). After 21 days

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of exposure, 1 mg/L PSNPs treatment markedly reduced Fe concentration from 386.2 mg/kg to 344.7 mg/kg (10.8%) in shoots (p<0.05) and from 2472.1 mg/kg to 1791.6

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mg/kg (27.5%) in roots (p<0.01), respectively, as compared to the unexposed treatment. Shoot Mn contents were markedly (p<0.01) decreased by 28.3%53.2% after exposure to PSNPs when compared with the controls. On the contrary, Mn concentrations in roots dramatically increased, especially at 0.1 and 10 mg/L (Fig. 6C). Similarly, PSNPs significantly (p<0.01) depressed the accumulation of Cu in roots by 18

26.0% 65.1% with respect to the control, while the shoot Cu concentration was just affected by PSNPs at 10 mg/L. In addition, PSNPs had no impact on the shoot Zn content whereas the root Zn content was strikingly (p<0.05) reduced by 80.9% after exposure to PSNPs as compared with the control. 3.8 Metabolic profiling of wheat Using GC-MS, a total of 203 peaks were detected and 59 metabolites were

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identified in wheat leaves based on retention time and the results of mass spectra

(Table S3). To visualize general grouping information with the concentration of PSNPs treatment, the hierarchical cluster analysis (HCA) was performed based on the

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similarities in metabolic profiles. As shown in Fig. 7A, the control group and PSNPs

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treatments were distinctly separated via a Pearson correlation analysis. Under PSNPs treatments, the group exposed to 1 mg/L was clustered close to the group of 10 mg/L

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PSNPs, whereas the group treated with 0.1 mg/L PSNPs clearly differentiated itself

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from the control group and 0.01 mg/L group. In conclusion, PSNPs significantly (p<0.05) altered the metabolic profiles in wheat, especially under the treatment of 0.1

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mg/L.

The identified metabolites included a large number of primary metabolites, such

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as amino acids, sugars, organic acids, alcohols and other compounds (Table S3). After exposure to PSNPs, a large portion of metabolites (around 65%) in leaves were downregulated, whereas about 18% of metabolites were upregulated which primarily included threonic acid, boric acid, butanedioic acid, glycolic acid, aconitic acid, malic acid, mannose, and tagatose. By multivariate component analysis, most of the 19

significantly affected metabolites were lactic acid, tagatose, talose, fructose, alanine and Myo-inositol. In the group exposed to 1 mg/L PSNPs, the concentration of these metabolites varied to 85.4%, -122.5%, 61.1%, 73.3%, 69.9%, and 94.6% of that in the control group, respectively. Additionally, the metabolic pathways involved in wheat responded to PSNPs are shown in Fig. 7B and C. Galactose metabolism, the tricarboxylic acid cycle (TCA cycle), starch and sucrose metabolism, glyoxylate and

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dicarboxylate metabolism, as well as alanine, aspartate and glutamate metabolism were significantly (p<0.01) affected.

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4. Discussion

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Once entering agroecosystems, MPs/NPs will persist, accumulate, and eventually reach to the levels affecting the biodiversity and functioning. However, potential

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biological effects of MPs/NPs and the mechanisms behind nanoplastics actions on

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plant development are largely unknown. Seed germination is a critical stage of plant life cycle and has therefore been widely used as an index to evaluate the phytotoxicity

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of toxic chemicals [41], although some studies indicated that seed germination may be a poor indicator of toxicity [42]. The effect of chemicals on seed germination depends

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on multiple factors like chemical species, chemical particle size, and plant species. In this study, we observed that PSNPs had no obvious effects on seed germination rate whereas significantly promoted the root elongation of wheat seedlings. Similarly, a nano-priming effect on seed germination and seedling growth of rice was caused by carbon nanotubes (CNTs), since CNTs were able to penetrate the seed coat by creating 20

small pores, inducing in the up-regulation of the water-channel gene [43]. Our study also revealed that exposure to PNSPs significantly increased seed water uptake in the rapid imbibition phase, in which it quickly launches the resumption of basic metabolism resulted from the gradual increase in hydration. However, an opposite result showed that the plastic particles (500 and 4800 nm) could partially accumulate in the pores of the testa of L. sativum, slowing down water uptake and thus delaying

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seed germination [26]. The relative root elongation is more sensitive than the germination index and it can be a sign of the presence of stress effects or other

non-acute toxicological effects in the plant evolution [34]. Hence, the resultant

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increase in root elongation indicated that PSNPs could not affect the wheat root

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development at environmentally relevant concentrations. However, the mechanisms underlying the promotion of root elongation by PSNPs are still unclear. During seed

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germination, -amylase generally pitted the starch granule surface first and then

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penetrated into the interior and hydrolyzed the granule from the inside out [44]. In the present study, it can be observed that the starch granules became rougher and visibly

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eroded in germinating seeds when exposure to PSNPs (Fig S5), which implied that -amylase activity was induced higher in PSNPs-treated seeds, resulting in higher

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decomposition of starch. Thus, we reasonably hypothesize that PSNPs could positively induce the activity of α-amylase to accelerate the hydrolysis of starch granules like a "nanocatalyst", producing more availability of soluble sugars or more energy for seedling growth. The better understanding of uptake and transport of PSNPs in plant is vital for 21

assessment their phytotoxicity and potential risks to human health. Previous studies showed that a large number of plastics nanoparticles could enter root tips through rhizodermis or epidermis via an intercell-wall route, which is a lignified epidermis path [45, 46]. However, the transport mechanisms of PSNPs crossing over cortex or Casparian band to reach xylem are still unclear. In this study, both a 3D LCSM and SEM were used to fully confirm the uptake and translocation of PSNPs in wheat. The

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3D LCSM imaging, as direct evidence, clearly showed that PSNPs were indeed taken up by root tips whereas the fluorescent signal of PSNPs in leaves was totally covered

by the autofluorescence of plant tissues (Fig. 3; Fig. S6). In addition, the absorbed

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PSNPs in root and shoot xylem were also observed by SEM. PSNPs (100 nm) could

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be down-top transported from roots to leaves via xylem pathway (Fig. 4), which may be potentially transported into grain or livestock via feeding, thus causing nanoplastic

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pollution of agricultural products and generating potential ecological and health risks.

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Although wheat biomass increased under PSNPs treatments, S:R ratio notably decreased especially at low concentration. According to the theory of Poorter, et al.

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[47], plants with a low nutrient supply show increased allocation to roots, resulting in lower S:R ratio in plants in response to their environment so as to optimize their

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resource use. Similarly, Weert, et al. [23] also reported a significant decreasing trend in S:R ratio in Elodea sp. with the increasing PSNPs concentration. The lowest S:R biomass ratio was also found in wheat when exposed to 1% microplastics for four months [25]. One of the most common mechanisms for the effect of PSNPs on plants might be 22

the blockage for essential nutrients uptake and transport. The uptake and translocation of metal elements in plant tissues is a complex biological process, which involves several steps, such as: (a) transport of metals across the plasma membrane of root cells; (b) xylem loading and translocation; and (c) distribution at the whole plant and at the cellular levels [48]. In our study, the concentrations of four micronutrients (Fe, Mn, Cu and Zn) in wheat root were all decreased while no similar "blockage effect"

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was found on Mg concentration (Fig. 6A), which suggested that the uptake and

translocation of metal elements were selectively inhibited as PSNPs could discriminatively regulate the expression of genes involved in metal ion transport.

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There is little direct evidence for depicting the interaction between PSNPs and

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photosynthesis up to now. We hence tried to discuss the possible mechanisms based on our results and previous reports. As shown in Table 1, the photosynthetic

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parameters were stimulated by PSNPs at a low dose while decreased at a high dose,

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which seems to be a dose-response phenomenon of hormesis [49]. In a separate research, the higher concentrations (>5 mg/L) of polypropylene (PP) and polyvinyl

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chloride (PVC) microplastics would inhibit algae photosynthetic activity by lowering the quantum yield or potential photosynthetic activity of PS II reaction centers

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respectively [50]. In our study, we observed that the nitrogen content in leaves was markedly increased with increasing PSNPs concentration, indicating that PSNPs could significantly promote wheat to absorb nitrate and accelerate the conversion of inorganic nitrogen (NO3-N and NH4+-N) into organic nitrogen (protein and chlorophyll). In leaves of C3 plant, there are strong linear relationships between 23

nitrogen and Ribulose 1,5-bisphosphate (RuBP) carboxylase [51], which may catalyze the rate of photosynthetic carbon reaction to improve photosynthetic capacity. Consistently, the net photosynthetic rate and carbon content of wheat increased by 1.85 times and 1.6%, respectively, at 0.1 mg/L PSNPs compared with the control. In addition, Mg plays a crucial role in chlorophyll biosynthesis. Thus, the increased Mg uptake in wheat would facilitate the biosynthesis of chlorophyll content in leaves.

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Besides, it was worth noting that the values of Ls in wheat leaves were unaffected after exposure to PSNPs whereas the values of WUE were decreased. These results

suggested that photosynthetic capacity induced by PSNPs was positively associated

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with stomatal transpiration, not the intercellular CO2 concentration. However, no

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obvious or detrimental effect on photosynthesis in algae or duckweed was mainly attributed to physical damage or oxidative stress [20, 22, 24], of which the mechanism

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seems largely different in wheat.

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The TCA cycle plays an important role in energy production and glyoxylate and dicarboxylate metabolism is a pathway that synthesized carbohydrates from fatty

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acids [52]. In this study, the upregulation of galactose metabolism was actually observed (Fig. 7B). PSNPs might have a stimulating effect for plants to synthesize

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more ATP for energy-consuming cellular activity. However, the starch and sucrose metabolism was significantly downregulated, which simultaneously resulted in a decrease of intermediates (sucrose, fructose, maltose and glucose). Generally, the biomass of plants will reduce when the energy consumption from decomposition of sucrose or maltose surpasses the substrate accumulation. Interestingly, the increased 24

biomass was observed in our study which may be due to the enhancement of photosynthesis (Table 1), indicating that PSNPs not only certainly accelerated the energy production process but also improved photosynthesis process to a greater extent. Furthermore, the upregulated amino acid metabolism (alanine, aspartate and glutamate metabolism) plays a central role in nitrogen metabolism because their biosynthesis, degradation, and transport are tightly regulated to meet demand in

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response to nitrogen and carbon availability [53]. Additionally, previous studies reported that PSNPs had the potential capability to induce the generation of reactive oxygen species (ROS) in plants [19, 46], which are chemically reactive chemical

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species containing oxygen such like superoxide, hydrogen peroxide, and hydroxyl

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radical. The lower contents of non-enzymatic antioxidants (proline, glycine, glutamine) in leaves were determined due to PSNPs exposure, which may be the

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defense strategy of wheat to protect the cell from ROS.

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In summary, the present study revealed that PSNPs had no negative effects on seed germination rate whereas significantly increased root elongation. PSNPs could

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significantly enhance seedling growth by improving photosynthetic parameters, whereas the absorption and translocation of nutrient metal elements (Fe, Mn, Cu and

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Zn) in wheat were partially hindered. The results of SEM and LCSM suggested that PSNPs can be taken up by root tips and then subsequently down-top transported to shoot via xylem pathway. Furthermore, the metabolic analysis revealed that PSNPs could significantly manage wheat growth or development by modulating plant carbon and nitrogen status and regulating several metabolic pathways, including galactose 25

metabolism, TCA cycle, starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, as well as alanine, aspartate and glutamate metabolism. It should be emphasized that the observed no direct toxicity effects are mainly based on our short-term PSNPs exposure test. Therefore, numbers of long-term PSNPs exposure experiments focused on other crop plants are still urgently needed.

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Conflict of interest

We declare that we have no financial and personal relationships with other people or

organizations that can inappropriately influence our work submitted, there is no

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professional or other personal interest of any nature or kind in any product, service

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Acknowledgements

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the review of, the manuscript entitled.

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and/or company that could be construed as influencing the position presented in, or

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This work was supported by the National Natural Science Foundation of China (41471411), the National Natural Science Foundation of China as a key project

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(U1806216) jointed with the Shandong Provincial Government, and the Fundamental Research Funds for the Central Public Welfare Research Institutes (TKS 160226).

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Figures Captions

Figure 1. Effect of polystyrene nanoplastics (PSNPs) and sodium dodecyl sulfate (SDS) on relative seed germination (A), relative root elongation (B), seed vigour index (C) and seed water uptake (D) of wheat. Values were represented as mean ± SD (n=4).  indicates the statistical significant at p<0.05 while  shows the statistical significant at p<0.01 between the control and PSNPs treatments.

Figure 2. Growth photographs (A), SPAD value (B), plant biomass (C) and shoot to root ratio

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(S:R) (D) of wheat seedlings. Values were represented as mean ± SD (n=4).  represents the statistical significant at p<0.05 while  shows the statistical significant at p<0.01 between the control and PSNPs treatments.

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Figure 3. The 3D laser confocal scanning micrographs (LCSM) of wheat root tips treated with the control (A, B) and PSNPs (C, D).Ortho images (B、D) revealed that PSNPs have been taken up by root.

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Figure 4. Scanning electron microscopy (SEM) images of transversal cut of wheat roots (G-L) and leaves (A-F). The images of A-C and G-I came from the unexposed treatment while images of D-F and J-L were from 10 mg/L PSNPs treatment. PSNPs were observed and dispersed in the root and leaf vein of the wheat plant. (C), (F), (I) and (L) showed an enlargement of the pane in (A), (D), (G) and (J), as marked by the red square. Figure 5. Foliar carbon (A) and nitrogen (B) contents (%) when exposed to PSNPs. Data were

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represented as mean ± SD (n=4).  shows the statistical significant at p<0.01 between the control and PSNPs treatments. Figure 6. Concentrations of Mg (A), Fe (B), Mn (C), Cu (D) and Zn (E) in the shoot and root of wheat plants cultivated for 21 days in hydroponic solution amended with PSNPs. Values were

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represented as mean ± SD (n=4).  indicates statistical significant at p<0.05 while statistical significant at p<0.01 between the control and PSNPs treatments.



shows the

Figure 7. Metabolic changes (A) and upregulated (B) or downregulated (c) metabolic pathways in wheat leaves when exposure to PSNPs.

30

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-p

re

lP

na

ur

Jo Figure 1

31

ro of

-p

re

lP

na

ur

Jo Figure 2

32

33

ro of

-p

re

lP

na

ur

Jo Figure 3

ro of

-p

re

lP

na

ur

Jo Figure 4

34

ro of

-p

re

lP

na

ur

Jo

Figure 5

35

ro of

-p

re

lP

na

ur

Jo

Figure 6

36

ro of

-p

re

lP

na

ur

Jo Figure 7

37

Table 1 Influence of PSNPs on photosynthetic parameters of wheat leaves. Treatments

Pn

Gs

Ci

Tr

WUE

Ls

(mg/L)

µmol m-2 s-1

mol m-2 s-1

µmol mol-1

mmol m-2 s-1

mmol mol-1

1-Ci/Ca

0

5.500.39b

0.150.04b

313.8211.57a

2.400.35b

2.900.42a

0.220.03a

0.01

8.041.37ab

0.210.03b

323.095.31a

3.220.35b

2.610.22a

0.190.01a

0.1

10.201.71a

0.320.07a

329.302.71a

9.051.68a

1.130.04b

0.180.01a

1.0

5.902.90b

0.190.11b

336.5518.76a

2.681.15b

2.530.86a

0.170.07a

10

6.250.80b

0.140.03b

305.9414.02a

2.210.50b

3.180.50a

0.220.02a

Jo

ur

na

lP

re

-p

ro of

Values were represented as mean ± SD. Different lowercase letters in the same column represent significant differences among the treatment means using using Duncan's test (p<0.05). leaves.

38