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Agricultural and Environmental Chemistry
Molybdenum sulfide induce growth enhancement effect of rice (Oryza sativa L.) through regulating the synthesis of chlorophyll and the ex-pression of aquaporin gene yadong li, Qian Jin, Desong Yang, and Jianghu Cui J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018
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Journal of Agricultural and Food Chemistry
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Molybdenum sulfide induce growth enhancement effect of rice (Oryza
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sativa L.) through regulating the synthesis of chlorophyll and the
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expression of aquaporin gene
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Yadong Li‡§, Qian Jin‡§, Desong Yang‡†*, Jianghu Cui§*
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§
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and Management, Guangdong Institute of Eco-environmental Science & Technology,
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Guangzhou 510650, China
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‡
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†
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Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control
College of Agriculture, Shihezi University, Shihezi 832000, Xinjiang, P.R. China Engineering Research Center of Materials-Oriented Chemical Engineering of
Xinjiang Bintuan, Shihezi University, Shihezi 832000, Xinjiang, P.R. China
11 12 13
*
Corresponding Authors:
[email protected] [email protected] Tel: (+86) 0993-2058769 Tel: (+86) 020-87025240
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Abstract
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Molybdenum sulfide (MoS2) has been applied widely in industrial and environmental
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application, leading to increasing release into environment. So far, no studies have been
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investigated with regard to the potential effect of MoS2 on plant. Herein, we studied the impact of
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MoS2 on the growth, chlorophyll content, lipid peroxidation, antioxidase system, and aquaporins
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of rice for the first time. Results showed that MoS2 did not significantly affect the germination of
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rice seeds, malonaldehyde (MDA) content and the antioxidant enzyme activity. While the length
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and biomass of rice root and shoot, chlorophyll content index (CCI) and expression of aquaporin
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genes were significantly increased. Based on these results, we concluded that MoS2 promoted rice
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growth through: (i) the promotion of nitrogen source assimilation, (ii) the enhancement of
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photosynthesis, enzymatic-related biochemical reactions and metabolic processes, subsequently,
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(iii) the acceleration of cell division and expansion, furthermore (iv) no abiotic stress and favorable
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condition of antioxidant enzyme system. These results provided an important insight into the
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further application of MoS2 on agriculture and environment.
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Key words: molybdenum sulfide; rice germination and growth; chlorophyll; antioxidant enzyme;
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aquaporin
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Introduction
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Molybdenum disulfide (MoS2) as a typical layered 2D nanomaterial has received increasing
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attentions due to its excellent properties in electrical,1 mechanical,2 and environment.3 The
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foreseeable increasing application suggests the unavoidable exposure of organism to MoS2 in
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environment. The potential effect of MoS2 on human and environmental organisms has been
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assessed previously. The exfoliated MoS2 showed excellent antibacterial activity against bacteria
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and fungi by inducing reactive oxygen species (ROS) and affecting their metabolic profile.4, 5
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Different from microorganisms, MoS2 nanosheets showed low cytotoxicity to numerous
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mammalian cell lines without necrosis and/or apoptosis.6-8 Plants are a vital component of
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ecological systems and play an important role in ecological balance. Besides, plants provide
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abundant food for human survival and a potential route for the exposure of nanomaterials to
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human. So far, no studies have been investigated with regard to the potential effect of MoS2 on
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plant.
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Upon exposure to plants, engineering nanomaterials (ENMs) can penetrate cells via bases of
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transporter proteins (small size), endocytosis and the natural orifices (large size) of root (cell wall
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pore) and leaf (stomata).9 Subsequently, the ENMs are translocated through the vascular system
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and react with plant cell and organelles. Therefore, the treatment of MoS2 could be internalized in
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the rice seedlings. It was reported that the elements of molybdenum (Mo) and sulfur (S) are
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important nutrient elements for plant growth and development.10 Mo is an important component of
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nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase in plant.10 It is
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acknowledged that nitrate is the major form of soil nitrogen absorbed by plant. As a functional
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component of nitrate reductase, Mo is of great importance for the reduction of nitrate (NO3 ) to
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nitrite (NO2 ) in the assimilation of nitrogen by plants. In plant, the oxidation of sulfite (SO32-) to
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sulfate (SO42-) is mediated by the molybdoenzyme and sulfite oxidase, which play a role in sulfur
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metabolism in plants. Xanthine dehydrogenase is involved in the synthesis of ureide and the
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catabolism of purine to uric acid in plants. The uric acid is a substrate for the synthesis of ureides,
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allantoin, and allantoic acid, which are involved in the translocation of N2 fixed symbiotically by
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plant from root to shoot. Aldehyde oxidase catalyzes the final step in the biosynthesis of the
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phytohormones indoleacetic acid (IAA) and abscisic acid (ABA) in the cytoplasm. These
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hormones have the function of regulating diverse biological processes and responses such as
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stomatal aperture, seed germination, seedling growth, apical dominance, and the regulation of
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phototropic and gravitropic behavior. Therefore, as a functional component of these enzymes
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mentioned above, Mo can promote these physiological and biochemical processes involved in
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these enzymes. In addition, the application of Mo could increase the activities of the antioxidative
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enzymes in winter wheat and the resistance to cold.11 Sulfur is a macroelement and plays critical
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role in the growth and physiological functioning of plants. The vegetative parts of crops contains
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sulfur content varying between 0.1 to 2% of the dry weight. Sulfur-containing amino acids are
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components of almost all protein.12 These sulfur compounds are of great significance in plant
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functioning and food quality and yield. Therefore, we hypothesize that the exposure of MoS2 may
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be beneficial to plant growth by supplying the sufficient Mo and S element.
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In light of the irreplaceable role of plant in providing food and maintaining the ecological
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balance of the natural environment, the present study aimed to assess the possible risk of MoS2 on
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plant using rice. This research was designed for two aims: (i) study the effect of MoS2 on the seed
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germination and the growth of rice seedlings by hydroponics culture; (ii) investigate the potential
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mechanism underlying these apparent impact, including the chlorophyll content index (CCI), the
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aquaporins gene expression, lipid peroxidation and antioxidant enzyme activities. To the best of
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our knowledge, it is the first study about the potential impact of MoS2 on plant, which should be
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helpful for the future application of MoS2 on agriculture and environment.
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Experimental section
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Raw MoS2 powder with average particle size of 1.5 µm was purchased from Nanjing
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XFNANO Materials Tech Co., Ltd (Nanjing, China). Prior to use, the raw MoS2 powder was
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sonicated in an ultrasonic bath (Bransonic, Danbury, USA) for 2 h to form a homogeneous
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suspension. The rice variety of Huanghuazhan was obtained from College of Life Science, South
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China Agricultural University (Guangzhou, China). The kits for the content of Malondialdehyde
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(MDA) and activities of antioxidase was purchased from Beijing Solarbio Science & Technology
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Co., Ltd (Beijing, China). Other chemical reagents were analytical grade without further
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purification.
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Germination Assays
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The effect of MoS2 on seed germination was determined as described in the previous study
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with some modifications.13 Petri dishes (90 mm in diameter) were sterilized in an autoclave at
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121 °C for 30 min and dried in an oven at 65 °C. Rice seeds were screened by NaCl aqueous
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solution (1.15 g/mL), surface sterilized with 1500 times metalaxyl·hymexazol (30 %) for 3 h,
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rinsed thoroughly with deionized water, and immersed in MoS2 suspension at different
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concentrations for 8 h. Then 20 of rice seeds were evenly placed into Petri dish containing 0.5
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wt % agar and different concentration of MoS2. These Petri dishes were placed green house (65%
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relative humidity) and incubated 27 ± 2 °C for in dark, randomly designed with 3 replications and
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20 seeds for each replicate. The emergence of a radicle (1-2 mm) was considered to be the
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criterion for seeds germination. After 24 h, 36 h, 48h, the following germination parameters were
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calculated:
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(1) Germination percentages (GP) = (n/N) × 100 %
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Where n is the germination number accumulated until the last evaluation, and N is the total
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number rice seed. (2) Mean germination times (MGT) = ∑(∆n×t)/ (∑∆n)
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Where ∆n is the number of rice seed newly germinated at time t, and t is the number of hours from sowing. (3) Germination rates (GR) = ∑ (∆n/t) Plant materials and growth conditions
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The hydroponics of rice was conducted according to Wang et al previous report with some
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modifications.14 Surface sterile rice seeds were scattered on moist filter paper in a plastic container
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with moderate water. Then the plastic container was placed in dark at 30 °C. After 3 days, the
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seedlings were transferred to light at 30 °C. The 6-day-old homogeneous seedlings were
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transferred to specialized plastic containers containing Kimura B solution. These rice seedlings
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were placed in a growth chamber with a day/night temperature regime of 27 °C (14 h): 23 °C (10 h)
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and a light intensity of 400 pmol·m-2·s-1. The Kimura B solution contains the following
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macronutrients: 0.37 mM (NH4)2SO4, 0.55 mM MgSO4·7H20, 0.18 mM KNO3, 0.18 mM KH2PO4,
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0.37 mM Ca(NO3)2·4H20, 0.09 mM K2SO4 and the micronutrients 50 µM Fe(Ⅱ)-EDTA, 1 µm
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ZnSO4·7H20, 1 µM CuSO4·5H20, 5 µm MnSO4·H20, 10 µM H3BO3, 0.5 µM Na2Mo04·2H20, 100
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µM NaCl, 0.2 µM CoSO4·7H2O. The Kimura B solution was prepared with purified water (Heal
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Force, Shanghai, China) and adjusted pH to 5.5 with dilute HCl and/or KOH. The nutrient solution
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was renewed once a week.
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Foliar applications and growth of rice plants
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After moving the rice seedlings to growth chamber, a series of concentrations of MoS2
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suspension (32-500 µg/mL) was used once a day. Rice seedlings sprayed with same volume of
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deionized water served as control. After 20-days treatment, these seedlings were used for the
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following measurement. 10 of rice seedlings from each treatment were harvested freshly, removed
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the excess water by absorbent paper, and measured for the length and fresh weight of root and
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shoot. Dry biomass of root and shoot was determined after drying them at 65 °C.
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The uptake and distribution of MoS2 in the leaf of rice
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The distribution of MoS2 in the treated rice seedlings was investigated using transmission
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electron microscopy (TEM). The samples were cut into 1×2 mm pieces and fixed in a mixture of
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2% paraformaldehyde and 2.5% glutaraldehyde for 24 h at 4°C. After thoroughly washed with 0.1
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M phosphate buffer (pH=7.2) at 4°C, samples were then fixed in 1% OsO4 at room temperature for
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4 h. Then the samples were thoroughly washed with phosphate buffer and dehydrated with an
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ascending ethanol series (30%, 50%, 70%, 80%, 90% and 100%). After infiltration and embedding
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in araldite, the samples were cut into ultrathin sections (70-80 nm thick).These sections were
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stained with 4% uranyl acetate and 0.2 % lead citrate, and loaded on copper grids for the
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observation with TEM (TECNAI SPIRIT, USA).
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Determination of antioxidant enzyme activities
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The determination of antioxidant enzyme activity was conducted according to the reported
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literatures.15, 16 Fresh leaf samples (0.1 g) were collected from treated seedlings and cut into pieces,
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then leaf samples were pestled with liquid nitrogen in a mortar and homogenized in 50 mM
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potassium phosphate buffer (pH 7.4). The homogenate was centrifuged at 8000×g for 10 min at
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4 °C. Then the supernatant was collected for the measurement of antioxidase activity. All enzyme
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specific activities were calculated according to the fresh weight of the samples. The principle for
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each enzyme activity is described as follows:15, 16
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Superoxide dismutase (SOD): The reaction of xanthine oxidase with xanthine produces
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superoxide anion (O2-). O2- can shift nitroblue tetrazolium (NBT) to blue formazan which has a
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characteristic absorption peak at 560 nm. SOD can eliminated O2- to inhibit the production of blue
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formazan. Therefore, SOD activity can be indirectly measured through measuring the absorbance
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at 560 nm wavelength.
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Catalase (CAT): CAT decomposes hydrogen peroxide (H2O2, extinction coefficient of
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4.36×104 L/mol/cm), leading to decrease in absorbance at 240 nm with time. So that the specific
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activity of CAT can be calculated from the change rate of absorbance at 240 nm.
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Peroxidase (POD): POD catalyzes the oxidation reaction between H2O2 and phenols/amines
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to eliminate the H2O2 and phenols/amines. Guaiacol can be oxidized by H2O2 to red-brown
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4-o-methoxyphenol under the catalysis of POD. The POD enzyme activity was determined as the
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change rate of the absorbance value of 4-o-methoxyphenol at 470 nm.
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Determination of lipid peroxidation
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Lipid peroxidation was determined using Malondialdehyde (MDA) analysis as described
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previously.17 Briefly, fresh rice leaf samples were prepared as the ground using liquid nitrogen and
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homogenized in 1 mL of 10 % trichloroacetic (TCA) solution. The supernatants were separated by
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centrifugation at 8000 × g for 10 min at 4 °C. Then the supernatants were mixed 0.5 %
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thiobarbituric acid (TBA) and followed by boiling water bath for 1 h. Then the mixtures were
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quickly cooled by ice bath. The absorbance of the samples was measured at 450, 532 and 600 nm.
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The content of MDA was expressed as nmol/g fresh weight.
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Determination of chlorophyll content index (CCI)
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After the rice plants were treated with MoS2 for 20 days, the CCI in new and mature leaves
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was measured using a portable analyzer (Chlorophyll Content Meter CCM-200, Opti-Science,
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USA) as reported.18 The CCI is the proportion of the light transmission at 653 nm and 931 nm.
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Thirty of new and mature leaves were measured for each treatment.
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Gene expression of aquaporin
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Determination of gene expression of aquaporin was carried out as described previously.19
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Three uniform root samples from each treatment were collected and cryopreserved at -80 °C. Then,
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these root samples (0.1 g) were ground in liquid nitrogen and total RNAs were isolated using
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TRIzol (Invitrogen, USA) according to a standard protocol. Then the extracted RNAs were
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converted to cDNA by PrimScript RT reagent Kit (Takara, Kyoto, Japan). Expression levels of
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aquaporin genes (OsPIP1;1, OsPIP1;2, OsPIP1;3, OsPIP2;5) were studied using iQTM 5 real-time
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PCR system (Bio.Pad,Hercules,CA,USA) by 1 µL of cDNA sample. The designed primers for
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target genes are listed in Table S1. In this process, SYBR Premix Ex TaqTM (Takara, Kyoto,
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Japan) was used as a fluorescent dye and actin as an internal reference. The data was analyzed
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using AAcycle threshold method.
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Statistical analysis
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Values presented in this manuscript were expressed as means ± standard deviation (SD).
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Statistical significance of all data was determined using one-way analysis of variance (ANOVA)
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and compared using Duncan’s test at p < 0.05 levels in SPSS 19.
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Results
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Characterization of MoS2
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Detailed characterization information for raw MoS2 nanosheets was carried out as described
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in SI-1. From the images of SEM and TEM, the bulk of MoS2 was composed of numerous
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monolayers MoS2 sheets (Figure S1 A and B). From the AFM image shown in Figure S1 C, the
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thickness of the raw MoS2 flakes was determined as approximately 3.64 nm in topographic height,
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the theoretical value of monolayer MoS2 was 0.65 nm.20 Therefore, the MoS2 flakes consist of 5 or
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6 of single MoS2 layers binding to each other by weak van der Waals forces. The crystal structures
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of MoS2 powder were revealed by XRD patterns shown in Figure S1 D. It was identified as 2H
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MoS2 with a dominant peak appearing at 2θ=14.4, reflecting the (002) plane.21 All the detectable
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diffraction peaks matched well with the crystal planes of the standard XRD data for the 2H MoS2
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(PDF#37–1492). It proved that the raw MoS2 powder possesses good hierarchical structure and
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was well crystallised. Raman spectrum of the raw MoS2 powder is shown in Figure S1 E. The
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spectrum clearly shows two distinct first-order Raman peaks at 378 cm−1 and 405 cm−1 for the E2g
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1
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to in-plane optical vibration of Mo-S bond (E2g1 ) and out-plane optical vibration of S atoms (A1g)
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in 2H MoS2.22
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Effects of MoS2 on germination in rice seed
and A1g vibrational modes, respectively.21 Individually, the two Raman active modes correspond
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In the whole growth period of the plant, germination of seeds is the weakest stage and firstly
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contact with ENMs released into environment. Therefore, the effect of MoS2 at different
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concentrations (32, 62.5,125 and 500 µg/mL) on the germination of rice seeds was investigated. As
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shown in Figure 1, the percentage, mean time and the rate of germination of the rice seeds were
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not significantly increased by the treatments of MoS2, although some numerical increase of was
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observed. The best treatment of MoS2 at 62.5 µg/mL improved the germination percentages for
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16.67 %, 13.34 % and 11.66 % after 24 h, 36 h and 48h respectively (Figure 1A). The average
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time for a seed to germination was shortened 6.87% (Figure 1B), and the germination rate was
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increased 26.06 % in the treatment of MoS2 at 62.5 µg/mL (Figure 1C). The results indicated that
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MoS2 at 62.5 µg/mL had the potential to accelerate the germination of rice seed.
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Effects of MoS2 on seedling development in rice
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The growth of rice seedlings was further used to evaluate the potential effect of MoS2. MoS2
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suspension was foliar applied on rice seedlings. After 20-day exposure, the lengths and biomass of
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root and shoot were recorded. The average root and shoot lengths of control seedlings reached
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(13.35 ± 1.78) cm and (35.26 ± 1.44) cm, respectively (Figure 2). After treated with MoS2
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suspension, the lengths of root and shoot were significantly increased with increasing treated
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concentration, peaked at 125 µg/mL, but decreased at 500 µg/mL (Figure 2 A and B). The lengths
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of root and shoot were increased by 19.48 % and 7.2 % in the treatment of MoS2 at 125 µg/mL in
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relation to the control, respectively. As concentration continually increased (500 µg/mL), the
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promotion effect was inhibited, but no negative effect was observed. In comparison with shoot,
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MoS2 have more potential to improve the elongation of rice root, which is conducive to the
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nutrients absorption for rice plant growth. The effect of MoS2 on the biomass of rice seedlings was
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simultaneously determined. Both root and shoot biomasses were significantly increased by the
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treatments of MoS2 as showed in Figure 3. In general, the highest values of fresh and dry weight of
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root, as well as dry weight of shoot were realized by the treatment of 62.5 µg/mL, which were
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increased by 50.34 %, 48.62 % and 35.50 %. Exceptionally, the treatment of 125 µg/mL
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maximized the fresh weight of shoot, which was increased by 28.17 %. Therefore, the exposure of
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MoS2 has great potential to promote the growth of rice.
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The uptake and distribution of MoS2 in the leaf of rice
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In order to investigate the uptake and distribution of MoS2 in the leaf of treated rice seedlings,
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TEM technique was used. It was indicated that the structure and morphology of rice cells had no
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change without the treatment of MoS2 (Figure 4A-C). For the treated rice seedlings (Figure 4D-F),
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the small size of MoS2 entered into the cell and located in the cell wall, mitochondria, cytosol and
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vacuole. However, the structure and morphology of cell was not affected with the exposure of
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MoS2.
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Effects of MoS2 on lipid peroxidation and antioxidant enzymes in rice seedling
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Previous study has suggested that engineering nanomaterials usually induced oxidative stress
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by generating reactive oxygen species (ROS) as a primary impact in plant.23 The oxidative stress
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easily causes membrane lipid peroxidation, which usually is used to evaluate the oxidative stress
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in plants.24 Therefore, lipid peroxidation in rice seedlings was measured by analysed the MDA
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content in this study. As shown in Figure 5A, 20-day treatments of MoS2 at all concentrations did
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not significantly affect the MDA content in rice seedlings in comparison to the control. This result
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suggested that MoS2 exposure did not cause oxidative stress in rice seedlings. Antioxidant
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enzymes such as SOD, POD, and CAT play crucial roles in scavenging the ROS generated by
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environmental stress.25 In the present work, the activities of SOD, POD, and CAT in treated rice
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leaves were measured to examine the effect of MoS2 on the antioxidant system. The activity of
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SOD in rice seedlings was not significantly affected by MoS2 at all concentrations compared to the
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control (Figure 5 B). The activity of POD was first suppressed significantly (32 µg/mL) and then
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recovered (62.5-500 µg/mL) (Figure 5 C). CAT activity showed a reverse trend in compared with
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that of POD, which was first remarkably increased (32 µg/mL) and then recovered (62.5-500
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µg/mL) (Figure 5 D).
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Effects of MoS2 on chlorophyll content index (CCI) in rice seedling
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Plant growth largely depends on photosynthesis, which is closely related to chlorophyll
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content. In addition, chlorophyll content is commonly used as a significant biomarker that reflects
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the status of plant physiology. Thus, CCI was measured in the present study which was shown in
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Figure 6. The chlorophyll contents both in new and mature leaves were increased with treated
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concentration increased. Specifically, the highest concentration of MoS2 (500 µg/mL) obviously
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increased the CCI by 21.7% and 22.4% for new and mature leaves, respectively. Therefore, the
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treatment of MoS2 improved the chlorophyll synthesis and following the photosynthesis in rice
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seedlings.
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Effects of MoS2 on aquaporin gene expression in rice seedling
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Plant growth and development depend largely on the tight regulation of water movement and
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homeostasis.26 Aquaporin plays a key role in maintaining the whole plant water and nutrient status.
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27
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the translocation of water across the membrane.28 In the present study, we analyzed the expression
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levels of five OsPIP genes in rice seedlings. As shown in Figure 7, MoS2 increased the expression
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levels of all OsPIPs in a concentration-dependent way. When treated with MoS2 at 500 µg/mL (the
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highest concentration), the transcript levels of the aquaporins OsPIP 1;1, OsPIP 1;2, OsPIP 1;3,
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OsPIP 2;5 increased approximately 3.97, 3.62, 3.51, 3.81 folds over the control levels, respectively.
Plasma membrane intrinsic proteins (PIPs) are a subfamily of aquaporin that are responsible for
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This result indicated that the water absorption ability of the rice root can be significantly facilitated
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by MoS2 application.
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Discussion
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According to the previous reports, MoS2 has been widely applied in industry and has great
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potential application in environment due to their extraordinary properties.3 Thus, a large number of
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MoS2 related materials would be released into environment and thereby cause unforeseen health
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and environment hazards. In the present study, we examined the potential impact of MoS2 on crop
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plant using rice for the first time.
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Seed germination, accompanied by water uptake, is a critical phase for plant survival. Our
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findings demonstrated that exposure of MoS2 insignificant improved the germination of rice seed
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without significantly difference (Figure 1). This could be due to the short exposure period. After a
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20-day exposure to MoS2, the lengths and biomasses of root and shoot were obviously increased
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without phytotoxicity (Figure 2 and 3). Therefore, the non-phytotoxicity and improvement for rice
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seedling growth observed for MoS2 makes it a very promising material for applications involving
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plant exposure. These results were similar with those of reported previously. It was reported that
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carbon nanotubes (CNTs) dramatically accelerated seed germination and increased the fresh
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weight of total biomass (leaves, stems, and roots).29 Anjali et al found that exposure of
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multi-walled carbon nanotubes (MWCNTs) to wheat could improve the grain yield by 63 %.30 For
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metal oxide nanoparticle, the treatment of titanium dioxide (TiO2) could increase the rate of
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germination and the vigor indexes of aged spinach seeds.31
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From the TEM images (Figure 4), it was observed that MoS2 sheets were internalized by rice
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cells and distributed in organelle, cytosol and vacuole. We speculated that the observed activation
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of MoS2 to rice seedling growth may be resulted from the assimilation of Mo and S elements,
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which are essential nutrient elements for plants.10 It has been reported that Mo is an ingredient of
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numerous enzymes involved in plant growth, such as nitrate reductase, which catalyzes the NO3-
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reduction in nitrogen assimilation of plant.10 When appropriately applied extra Mo source, the
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period of high activity of nitrate reductase was extended and the nitrogen assimilation was
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promoted in beans.32 In addition, sulfur-containing amino acids are components of almost all
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protein.12 Especially, the thioredoxin, iron-sulfur protein and nitrogenase play important role in
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plant photosynthesis and nitrogen fixation.33, 34 Therefore, foliar application of MoS2 possibly
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provide sufficient Mo and S source for the synthesis of intracellular compounds in plant.35 In
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present study, the CCI in rice seedlings was enhanced with the increasing concentration of MoS2
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(Figure 6). Chlorophyll is the most essential photosynthetic pigments, which converts light energy
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to stored chemical energy. The higher content of chlorophyll could result in more absorption of
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solar radiation by leaves, followed by higher photosynthetic potential and more photosynthate.36
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Thus, the growth of treated rice seedlings was accelerated (Figure 2 and 3). In addition,
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chlorophyll contains much of leaf nitrogen,37, 38 indicating the enhancement of nitrogen source
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assimilation by MoS2 treatment.
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Khodakovskaya et al found that the carbon nanotubes were able to support water uptake
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inside tomato seeds.29 According to previous reports, aquaporin plays crucial role in root water
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uptake, followed by affecting seed germination, cell division and expansion, reproduction, and
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photosynthesis.39 However, studies on the effect of nanomaterials on the expression of aquaporin
315
genes are few. Our data suggested that the expression levels of aquaporins (OsPIP1;1, OsPIP1;2,
316
OsPIP1;3, OsPIP2;5) in treated rice seedlings were significantly increased in a dose-dependent
317
way (Figure 7). OsPIP2;5 has significant osmotic water channel activity and plays a crucial role in
318
maintaining water homeostasis of rice as water channels.19 The water homeostasis can facilitate
319
the biochemical reactions, metabolic processes, nutrient uptake.26, 40 Besides, with the enhanced
320
chlorophyll content, strong water homeostasis promoted the photosynthesis and produced more
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photosynthate (Figure 3). Moreover, the incessant uptake of water accelerated the cell division and
322
expansion, leading to elongation of root and shoot (Figure 2).26 Furthermore, it has been revealed
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that OsPIP1;1 and OsPIP1;3 tightly regulate seed germination.41, 42 Upon imbibition of water, the
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quiescent dry seed rapidly resumes metabolic activity and leads to germination. Khodakovskaya et
325
al has reported that CNTs were able to penetrate into tomato seed and enhance water uptake,
326
leading to the acceleration of seed germination and growth of tomato seedlings.29 However, the
327
treatments of MoS2 did not remarkably promote the germination of rice seed in this study (Figure
328
1). It may be resulted from the thicker seed coat of rice, leading to the difficult entrance of MoS2
329
into the seed. Besides seed germination, over-expression of OsPIP1;1, OsPIP1;2 and OsPIP1;3
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genes could enhance the tolerance of plant to environmental stress, such as salt, drought and
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chilling stress.19 High resistance to abiotic stress can prevent plant from adverse condition.
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Oxidative stress has been suggested as one primary mechanism of nanomaterial affecting plant
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growth. 9 Oxidative stress can further affect cell organelles and structures, proteins, carbohydrates,
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lipids, secondary metabolites and DNA in plants. Lipid peroxidation is often used as a parameter
335
to evaluate the level of oxidative stress in plant.43 For example, graphene was reported to induce
336
the production of oxidative stress and increase the content of MDA in wheat.44 In present study,
337
the treatment of MoS2 did not affect the content of MDA (Figure 5 A), indicating that no oxidative
338
stress was caused by MoS2.45 The contrary results should result from the different chemical
339
reactions in the plant, which is still yet to be studied in our future study. Antioxidant enzymes are
340
the principal defense system in plant to scavenge excess ROS induced by environmental stress.25
341
SOD catalyzes the disproportionation of superoxide radicals to produce oxygen and H2O2 in plants.
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H2O2 can be degraded by both POD and CAT to non-toxic substance. This process plays crucial
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role in maintaining the homeostasis of intracellular ROS. With the exposure of nanomaterials, the
344
activities of antioxidant enzymes in plant largely depended on the level of their toxicity.46 In our
345
study, the activities of SOD, POD and CAT in treated seedlings were not affected (Figure 5 B, C
346
and D), which were inconsistent with the enhanced tolerance of rice to environmental stress
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regulated by OsPIP1 genes. The decrease of POD activity and the increase of CAT activity in
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present study caused by MoS2 at 32 µg/mL is likely own to their coordination effect to metabolize
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intracellular H2O2. When suffered from drought and low temperature stress, the application of Mo
350
increased the activities of SOD, POD and CAT and the decreased the MDA content in wheat.16 The
351
different results in our study may due to the absence of abiotic stress.
352
Based on our results and previous reports, we concluded that MoS2 accelerated rice seedling
353
growth by the supplement of Mo and S, which is in good agreement with our hypothesis. In
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summary, MoS2 promoted the rice growth through a complex physiological processes. Firstly,
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supplement of Mo and S sources accelerated the synthesis of some growth related enzymes,
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followed by promoting the nitrogen assimilation and enzymatic reactions. Moreover, up-regulated
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expression of aquaporins genes promoted the water uptake and homeostasis in plant. Alone with
358
the high chlorophyll content, the metabolic, nutrient uptake and photosynthesis were accelerated.
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Furthermore, the normal MDA content and antioxidant enzyme activity further indicated the
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favorable growth condition of rice plant. Therefore, the growth of rice plant was promoted by
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these synergistic effect. According to these findings, MoS2 has great potential in the agriculture
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application as a nanofertilizer of Mo and S, which can reduce application rates through enhanced
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reactivity due to its high surface area and appropriate sorption properties.47
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This study demonstrated that the exposure of MoS2 could improve the growth of rice without
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phytotoxicity. This information has important significance in the application of agriculture and
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environment. However, the interaction between plants and nanomaterials is highly complex and
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depends on the shape, size, concentration, surface features of nanomaterials, the species, genotype
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and age of plant, and the environmental conditions.48 Previous studies indicated that the treatment
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of MoS2 showed low toxicity with mammalian cell.6, 7 For example, Liu et al found that the
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functionalized MoS2 was used for as a multifunctional drug delivery system for the therapy of
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cancer.49 But the potential toxicity and possible metabolism of MoS2 in the longer-term exposure
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for human is not investigated. Thus, it is necessary to further study the potential toxicity of human
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and plant.
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Abbreviations used
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MoS2-Molybdenum disulfide; FETs-field-effect transistors; ROS-reactive oxygen species;
376
PEG-polyethylene
glycol;
CCI-chlorophyll
content
index;
MDA-Malondialdehyde;
377
SEM-Scanning electron microscopy; TEM-transmission electron microscopy; AFM-atomic force
378
microscopy; XRD-X-ray diffractometer; GP-Germination percentages; MGT-Mean germination
379
times; GR-Germination rates; SOD-Superoxide dismutase; CAT-Catalase; POD-eroxidase;
380
TCA-trichloroacetic; TBA-thiobarbituric acid; SD-standard deviation.
381
Acknowledgements
382
This work was supported by National Natural Science Foundation of China (Grant No.
383
41401564), Opening Fund of Key Laboratory of Integrated Pest Management on Crops in
384
Northwestern Oasis of Ministry of Agriculture (KFJJ20170105) and the fund of Engineering
385
Research
386
(2016BTRC002).
387
Supporting Information Available
388 389
Center
of
Materials-Oriented
Chemical
Engineering
of
Xinjiang
Bintuan
This characterization of MoS2 and the designed primers for target genes in real-time PCR is available free of charge via the Internet at http://pubs.acs.org.
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Plant Biol. 2008, 51, 167-173. 26. Maurel, C.; Santoni, V.; Luu, D.-T.; Wudick, M. M.; Verdoucq, L. The cellular dynamics of plant aquaporin expression and functions. Curr. Opin. Plant Biol. 2009, 12, 690-698. 27. Preston, G. M.; Agre, P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Sci 1992, 256, 385. 28. Kaldenhoff, R.; Fischer, M. Functional aquaporin diversity in plants. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 1134-1141. 29. Khodakovskaya, M.; Dervishi, E.; Mahmood, M.; Xu, Y.; Li, Z.; Watanabe, F.; Biris, A. S. Carbon nanotubes are able to penetrate plant seed coat and dramatically affect seed germination and plant growth. ACS Nano 2009, 3, 3221-3227. 30. Joshi, A.; Kaur, S.; Dharamvir, K.; Nayyar, H.; Verma, G. Multi‐walled carbon nanotubes applied through seed‐priming influence early germination, root hair, growth and yield of bread wheat (Triticum aestivum L.). J. Sci. Food Agric. 2017, (In press), DOI: 10.1002/jsfa.8818. 31. Zheng, L.; Hong, F.; Lu, S.; Liu, C. Effect of nano-TiO2 on strength of naturally aged seeds and growth of spinach. Biol. Trace Elem. Res. 2005, 104, 83-91. 32. Vieira, R. F.; Cardoso, E. J. B. N.; Vieira, C.; Cassini, S. T. A. Foliar application of molybdenum in common beans. I. Nitrogenase and reductase activities in a soil of high fertility. J. Plant Nutr. 1998, 21, 169-180. 33. Balk, J.; Lobréaux, S. Biogenesis of iron–sulfur proteins in plants. Trends Plant Sci. 2005, 10, 324-331. 34. Jacquot, J.-P.; Lancelin, J.-M.; Meyer, Y. Thioredoxins: structure and function in plant cells. New Phytol. 1997, 136, 543-570. 35. Zhang, M.; Hu, C.; Sun, X.; Zhao, X.; Tan, Q.; Zhang, Y.; Li, N. Molybdenum affects photosynthesis and ionic homeostasis of chinese cabbage under salinity stress. Commun. Soil Sci. Plant Anal. 2014, 45, 2660-2672. 36. Curran, P. J.; Dungan, J. L.; Gholz, H. L. Exploring the relationship between reflectance red edge and chlorophyll content in slash pine. Tree Physiol. 1990, 7, 33-48. 37. Yuan, Z.; Ata-Ul-Karim, S. T.; Cao, Q.; Lu, Z.; Cao, W.; Zhu, Y.; Liu, X. Indicators for diagnosing nitrogen status of rice based on chlorophyll meter readings. Field Crops Res 2016, 185, 12-20. 38. Ali, A. M.; Thind, H. S.; Sharma, S.; Singh, Y. Site-specific nitrogen management in dry direct-seeded rice using chlorophyll meter and leaf colour chart. Pedosphere 2015, 25, 72-81. 39. Kaldenhoff, R.; Fischer, M. Aquaporins in plants. Acta Physiol. 2006, 187, 169-176. 40. Turner, N. C.; Begg, J. E. Plant-water relations and adaptation to stress. Plant Soil 1981, 58, 97-131. 41. Liu, H.-Y.; Yu, X.; Cui, D.-Y.; Sun, M.-H.; Sun, W.-N.; Tang, Z.-C.; Kwak, S.-S.; Su, W.-A. The role of water channel proteins and nitric oxide signaling in rice seed germination. Cell Res. 2007, 17, 638-649. 42. Liu, C.; Fukumoto, T.; Matsumoto, T.; Gena, P.; Frascaria, D.; Kaneko, T.; Katsuhara, M.; Zhong, S.; Sun, X.; Zhu, Y.; Iwasaki, I.; Ding, X.; Calamita, G.; Kitagawa, Y. Aquaporin OsPIP1;1 promotes rice salt resistance and seed germination. Plant Physiol. Biochem. 2013, 63, 151-158. 43. Stone, V.; Johnston, H.; Schins, R. P. F. Development of in vitro systems for nanotoxicology: methodological considerations. Crit. Rev. Toxicol. 2009, 39, 613-626. 44. Zhang, P.; Zhang, R.; Fang, X.; Song, T.; Cai, X.; Liu, H.; Du, S. Toxic effects of graphene on the growth and nutritional levels of wheat (Triticum aestivum L.): short- and long-term exposure studies. J. Hazard. Mater. 2016, 317, 543-551. 45. Gitelson, A. A.; Gritz †, Y.; Merzlyak, M. N. Relationships between leaf chlorophyll content and spectral reflectance and algorithms for non-destructive chlorophyll assessment in higher plant leaves. J. Plant Physiol. 2003, 160, 271-282. 46. Rizwan, M.; Ali, S.; Qayyum, M. F.; Ok, Y. S.; Adrees, M.; Ibrahim, M.; Zia-ur-Rehman, M.; Farid, M.; Abbas, F. Effect of metal and metal oxide nanoparticles on growth and physiology of globally important food crops: A critical review. J. Hazard. Mater. 2017, 322, 2-16. 47. Gogos, A.; Knauer, K.; Bucheli, T. D. Nanomaterials in plant protection and fertilization: current state, foreseen applications, and research priorities. J. Agric. Food Chem. 2012, 60, 9781-9792. 48. Tripathi, D. K.; Shweta; Singh, S.; Singh, S.; Pandey, R.; Singh, V. P.; Sharma, N. C.; Prasad, S. M.; Dubey, N. K.; Chauhan, D. K. An overview on manufactured nanoparticles in plants: Uptake, translocation, accumulation and phytotoxicity. Plant Physiol. Biochem. 2017, 110, 2-12. 49. Liu, T.; Wang, C.; Gu, X.; Gong, H.; Cheng, L.; Shi, X.; Feng, L.; Sun, B.; Liu, Z. Drug delivery with PEGylated MoS2 nano‐sheets for combined photothermal and chemotherapy of cancer. Adv. Mater. 2014, 26, 3433-3440.
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Figure captions
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Figure 1. Germination percentage (A), mean germination time (B), and germination rate (C) of rice
507
seeds treated with MoS2. Means values ± SD (n=20). Means in the column followed by the same
508
letter do not differ significantly (p ≤ 0.05).
509 510
Figure 2. Length of (A) roots and (B) shoots treated with MoS2. Means values ± SD (n=10).
511
Means in the column followed by the same letter do not differ significantly (p ≤ 0.05). (C)
512
Seedling growth images of rice seedlings in the treatments of control and MoS2.
513 514
Figure 3. Fresh weight of (A) roots and (C) shoots, dry weight of: (B) roots, (D) shoots for rice
515
seedlings treated with MoS2 suspensions. Means values ± SD (n=10). Means in the column
516
followed by the same letter do not differ significantly (p ≤ 0.05).
517 518
Figure 4. TME image of the leaf of rice treated under various conditions: (A-C) Control, (D-F)
519
MoS2 treatment. v, vacuole (blue arrowheads); w, cell wall (red arrowheads); m, mitochondria
520
(white arrowheads); c, cytosol (green arrowheads). The concentration of MoS2 was 500 µg/L.
521
Arrowheads indicate the localization of MoS2.
522 523
Figure 5. MDA content (A) and SOD (B), POD (C), CAT (D) activities of rice seedlings treated
524
with different concentrations of MoS2. Means values ± SD (n=3). Means in the column followed
525
by the same letter do not differ significantly (p ≤ 0.05).
526 527
Figure 6. CCI content in leaves of rice seedlings treated with MoS2 suspensions. Means values ±
528
SD (n=10). Means in the column followed by the same letter do not differ significantly (p ≤ 0.05).
529
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Figure 7. Relative expression of OsPIPs in rice seedlings treated with MoS2 suspension for 20
531
days. Mean values ± SD (n = 3). Means in the column followed by the different letter differ
532
significantly at p ≤ 0.05.
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