Environmental Science and Pollution Research Effects of Cr2O3 Nanoparticles on the Chlorophyll Fluorescence and Chloroplast Ultrastructure of Soybean (Glycine max) --Manuscript Draft-Manuscript Number:
ESPR-D-17-06324R1
Full Title:
Effects of Cr2O3 Nanoparticles on the Chlorophyll Fluorescence and Chloroplast Ultrastructure of Soybean (Glycine max)
Article Type:
Research Article
Corresponding Author:
Tingqiang Li, PhD Zhejiang University CHINA
Corresponding Author Secondary Information: Corresponding Author's Institution:
Zhejiang University
Corresponding Author's Secondary Institution: First Author:
Jinxing LI, Ph.D candidate
First Author Secondary Information: Order of Authors:
Jinxing LI, Ph.D candidate Yuchao Song, Master candidate Keren Wu, Master candidate Qi Tao Yongchao Liang Tingqiang Li, PhD
Order of Authors Secondary Information: Funding Information:
Abstract:
National Natural Science Foundation of China (#41271333) National Natural Science Foundation of China (#21477104) National Natural Science Foundation of China (#41671315)
Dr Tingqiang Li
Dr Tingqiang Li
Dr Tingqiang Li
Abstract Chromic oxide nanoparticles (Cr2O3 NPs) are widely used in commercial factories and can cause serious environmental problems. However, the mechanism behind Cr2O3 NPs induced phytotoxicity remains unknown. In this study, the effects of Cr2O3 NPs on the growth, chlorophyll fluorescence, SEM-EDS analysis and chloroplast ultrastructure of Soybean (Glycine max) were investigated to evaluate its phytotoxicity. The growth of soybean treated with various Cr2O3 NPs suspension (0.01, 0.05, 0.1, 0.5 g/L) was significantly inhibited. Specially shoot and root biomass decreased by 9.9% and 46.3% respectively. Besides, the maximum quantum yield of PS II (Fv/Fm) as well as the photochemical quenching (qP) decreased by 8-22% and 30-37% respectively, indicating that the photosynthetic system was damaged when treated with Cr2O3 NPs. Moreover, the inhibition was confirmed by the reduction of Rubisco and MDH enzymes activity (by 54.5-86.4% and 26.7-96.5%, respectively). Overall, results indicated that the damage was caused by destruction of chloroplast thylakoid structure, which subsequently reduced the photosynthetic rate. Our research suggests that Cr2O3 NPs can be transported and cause irreversible damage to soybean plants by inhibiting the activity of electron acceptors (NADP+) and destroying ultrastructure of chloroplasts,
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
providing insights into estimate plant toxicity issues. Response to Reviewers:
see attachment
Additional Information: Question
Response
§Are you submitting to a Special Issue?
No
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Authors
Click here to download Authors' Response to Reviewers' Comments Response MS ESPR-D-17-06324R1.docx
Response to the reviewers’ comments on MS ESPR-D-17-06324R1
We greatly appreciate the reviewers for their affirmation on our work and for their constructive comments. All these comments are carefully considered with changes incorporated into the revised version. The following are the responses to the individual comments of the reviewers. Response to the comments of Reviewer #1 A recent study is beging with the following sentence: "Chromium oxide nanoparticles are used for industrial applications such as catalysts and pigments. Thus, it is very important to find a simple and cost-effective method for the synthesis of nanoparticles...." However only a few studies for evaluating environmental risk of Cr2O3 NPs on plants, especially the effect on the photosynthesis system. Thus, the topic of the MS is interesting and up to date. The presentation is excellent, understandable and logical. Re: We greatly appreciate the reviewer for his/her affirmation on our work. 1. I recommend a very recent review about this topic to the authors: Rastogi et al, 2017 Oktober, Impact of Metal and Metal Oxide Nanoparticles on Plant: A Critical Review, in addition a recent study about ultrastructural changes: Puerari et al., 2016, Synthesis, characterization and toxicological evaluation of Cr2O3 nanoparticles using Daphnia magna and Aliivibrio fischeri. Re: Thanks for recommendation, the review article is very useful for my research field. We have cited them in the revision. 2. Just one question, but not critism: Did Cr2O3 Nanoparticles decrease the rate of root branching?
Re: The branching of soybean plants is not affected by Cr2O3 Nanoparticles, but the plant growth weakened with the addition of Cr2O3 Nanoparticles.
Response to the comments of Reviewer #2 General comments The manuscript "Effects of Cr2O3 Nanoparticles on the Chlorophyll Fluorescence and Chloroplast Ultrastructure of Soybean (Glicine max)", addresses the study of the phytotoxicity of Cr2O3 NPs on soybean. The subject of the manuscript is very interesting and of current relevance in the field of environmental sciences. The experiments are well planned and the conclusions are clear. However, I have some comments that the authors should answer and clarify before the paper can be published. Re: We greatly appreciate the reviewer for his/her positive comments on our work. We also wish to thank the reviewers for their thorough work and useful comments which have been considered carefully and incorporated into the revised version. 1. Page 7. Lines 107-110. The nanoparticles used in this work were purchased and they were not prepared as part of the work. I am struck by the relatively large size chosen (500 nm), when sizes from 1-100 nm are usually preferred. Do the authors have any comment on this point? Re: Thank you for your kind work on our research. The size of Cr2O3 NPs (0.5 μm) mentioned in the article was a type error, we checked and corrected it in the revision. Cr2O3 NPs were purchased from Jianglai Biology Ltd, Shanghai, China. The size of which were originally between 20-50 nm. However, we re-characterized the NPs by transmission electron microscopy in the aggregate and dispersed state; the results can be seen in Fig. S1. According to previous studies, the size of aggregates can be hundreds of times or even thousands of times the original size. We chose one aggregate (Fig. S1
A.) to determine the level of aggregation. On the other hand, we characterized Cr2O3 NPs dispersion after ultrasonic agitation for 15 minutes (Fig. S1 B.), results showed the original size of the Cr2O3 NPs is about 50nm and below, which meets the experimental requirements. 2. Page 11. Section Chlorophyll fluorescence measurements needs to be described in more detail. For example, Fq 'and Fm' measurements, that are reported later in results, are not sufficiently explained. Re: Thank you for your comment, I have re-written the measurements. According to CF Imager calculation measurement method (Hernandez-Viezcas, 2013), after dark recovery, dark-adapted minimum fluorescence (Fo), dark-adapted maximum fluorescence (Fm) were measure, maximum quantum yield of PSII (Fv/Fm) can be calculated. Fo’ and Fm’ were measured after 30 min of light adaption. nonphotochemical quenching Fm/Fm'-1 (NPQ) and the capture rate of excitation energy of PSII reaction center Fv'/Fm (XE'), photochemical quenching coefficients Fq'/Fv' (qP) and Fq'/Fm' (φPSII) can directly be calculated and read. 3. Page 12. Results. The section "NPs characterization" should be re-written to improve its clarity and understanding Re: Thank you for your suggestion. I have re-written the ‘NPs characterization’ section. 4. Page 36. Fig. 4. Numerical values should be included next to the chromatic scale to clearly explain if red indicates greater values or viceversa. The authors should also state in the Figure caption what exactly A and B graphs are. Explain in detail which parameter is mapped as leaf damage. Regarding the table at the bottom part of the figure: are there significative differences among the parameters as concentration is increased? The figure caption is confusing as it is actually written. Please re-write it for clarity.
Re: Figure A is leaf damage evaluation mapping based on potential energy capture efficiency of the reaction center after dark recovery for 30 min. Figure B is leaf damage evaluation mapping based on non-photochemical quenching Fm/Fm’-1 (NPQ) under illumination. Figure C is leaf damage evaluation mapping based on photosynthesis quenching coefficiency Fq'/Fv' (qP). According to the data, the difference of parameters are significative as concentration increases, so we added letters next to the data, and rewrote it in the revision. Minor comments: Line 19. Add a hyphen between NPs and induced: Cr2O3 NPs-induced phytotoxicity. Re: We added a hyphen between NPs and induced. Line 31-32. …providing insights into estimate plant toxicity issues. (Delete estimate) Re: We deleted estimate in the revision. Line 72. "AgNPs were reported" and not "was reported". Re: Thank you. We have corrected the mistake in the revision. Line 83. To date, may studies only investigated the chlorophyll content and ROS damage, while only a small number of which have studied the changes in plant morphology and even less were focused on the ultrastructural changes. Change to: To date, many studies only investigated the chlorophyll content and ROS damage, while only a small number of them have studied the changes in plant morphology and even less were focused on the ultrastructural changes. Re: Thanks. We re-wrote the phrase in the revision. Lines 85-88. The phrase "Although…" should be re-written for clarity. Re: Thank you for your advice. We have re-written the phrase.
Line 117. Does CK stand for the concentration of suspension? The letter K is a bit confusing in this notation. I wonder why the authors have chosen CK for this case. Re: Group CK is blank control group. Word ‘CK’ is short for ‘control check’ respectively. Line 132. …light intensity 15000 lx Re: We corrected the mistake. Lines 166-168. Re-write the phrase beginning with: Photochemical quenching…It is not clear. Re: Thanks. We re-wrote the phrase. Line 177. Replace "Taking" by "A mass of" Re: Thank. We changed the word “Taking” to “A mass of”. Line 188-190. The sentence "Zeta potential…" should be grammatically revised and should be re-written to explain better the results. Re: Thanks. We re-wrote the phrase. Line 213. Figure 3A appears in the text before Figure 2. Unless the authors have a very good justification for maintaining that order, the numbers in figures 2 and 3 should be interchanged Re: Thank you for your suggestion. We changed the figure order. Line 225. Fq´/Fm´appears in this section but it was not introduced in the experimental section. Re: We added experimental methods in the section. Line 228 … was 0.874. Notice that in the table (Figure 4) appears 0.854 instead. Re: Thanks. We checked the number and corrected it. Line 271. Revise: "Also, soybean phloem catheter less nanoparticles…" Re: We checked the sentence and corrected it.
Line 274. Change "To determine the uptake and accumulation of NPs by plants which to show consistency with previous studies" By "To determine the uptake and accumulation of NPs by plants that show consistency with previous studies" Re: Thanks. We corrected the sentence. Line 278. Replace " In consistent" by "Consistently" Re: We re-wrote the sentence in the revision. Line 281 Replace "exposure" by " treatment" to avoid the repetition "exposed… for 14 days exposure". Re: Thanks. We re-wrote the sentence in the revision. Line 291. Delete point before "shrunken" Re: We deleted the point before “shrunken”. Line 305. Replace "In consistent" by "Consistently" and re-write correctly the whole sentence. Re: We checked the sentence and corrected it. Line 524. Add a space before (C). Re: We added a space before (C).
Supplementary material Line 11. Add "on" between "NPs" and "the germination" Re: We added “on” between “NPs” and “the germination”. Page 4. Fig. S2. In the figure legend it is stated: Bars with different letters are… But no letters appear next to the bars in the figure.
Re: According to the result, there is no significant difference among the treatment groups, so there should be no letters on the bars. We re-wrote the phrase in the revision.
Reference Anshu, R., Marek, Z., Oksana, S., Kalaji, H. M., He, X., & Sonia, M., et al. (2017). Impact of metal and metal oxide nanoparticles on plant: a critical review. Frontiers in Chemistry, 5, 78. Hernandez-Viezcas, J.A., Castillo-Michel, H., Andrews, J.C., Cotte, M., Rico, C., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2013. In situ synchrotron X-ray fluorescence mapping and speciation of CeO2 and ZnO nanoparticles in soil cultivated soybean (Glycine max), ACS Nano. 7(2), 1415-1423. Ondřej Jankovský, David Sedmidubský, Zdeněk Sofer, Jan Luxa, & Vilém Bartůněk. (2014). Simple synthesis of Cr2O3, nanoparticles with a tunable particle size. Ceramics International, 41(3), 4644-4650. Puerari, R. C., Costa, C. H. D., Vicentini, D. S., Fuzinatto, C. F., Melegari, S. P., & Éder C. Schmidt, et al. (2016). Synthesis, characterization and toxicological evaluation of cr 2 o 3, nanoparticles using daphnia magna, and aliivibrio fischeri. Ecotoxicology & Environmental Safety, 128(June 2016), 36-43.
Manuscript
Click here to download Manuscript REVISED manuscript MS ESPR-D-17-06324R1.docx
Click here to view linked References
11 2 3 4 52 6 7 8 93 10 11 12 4 13 14 15 165 17 18 19 206 21 22 23 247 25 26 278 28 29 30 319 32 33 34 10 35 36 37 38 11 39 40 41 42 12 43 44 45 46 13 47 48 49 14 50 51 52 53 15 54 55 56 57 16 58 59 60 61 62 63 64 65
Title: Effects of Cr2O3 Nanoparticles on the Chlorophyll Fluorescence and Chloroplast Ultrastructure of
Soybean (Glycine max)
Authors: Jinxing Li, Yuchao Song, Keren Wu, Qi Tao, Yongchao Liang, Tingqiang Li*
The affiliation(s) and address (es) of the author(s):
Ministry of Education Key Laboratory of Environmental Remediation and Ecological Health, College
of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
Corresponding author
Dr. Tingqiang Li
Ministry of Education Key Laboratory of Environmental Remediation and Ecological Health, College
of Environmental and Resource Sciences
Zhejiang University, Hangzhou, 310058, China
Tel: +86-571-88982907
Fax: +86-571-88982907
E-mail:
[email protected]
1
1 17 2 3 4 18 5 6 7 8 19 9 10 11 12 20 13 14 15 16 21 17 18 19 22 20 21 22 23 23 24 25 26 27 24 28 29 30 31 25 32 33 34 26 35 36 37 38 27 39 40 41 42 28 43 44 45 46 29 47 48 49 30 50 51 52 53 31 54 55 56 57 32 58 59 60 61 62 63 64 65
Abstract Chromic oxide nanoparticles (Cr2O3 NPs) are widely used in commercial factories and can cause
serious environmental problems. However, the mechanism behind Cr2O3 NPs-induced phytotoxicity
remains unknown. In this study, the effects of Cr2O3 NPs on the growth, chlorophyll fluorescence,
SEM-EDS analysis and chloroplast ultrastructure of Soybean (Glycine max) were investigated to
evaluate its phytotoxicity. The growth of soybean treated with various Cr2O3 NPs suspension (0.01,
0.05, 0.1, 0.5 g/L) was significantly inhibited. Specially shoot and root biomass decreased by 9.9%
and 46.3% respectively. Besides, the maximum quantum yield of PS II (Fv/Fm) as well as the
photochemical quenching (qP) decreased by 8-22% and 30-37% respectively, indicating that the
photosynthetic system was damaged when treated with Cr2O3 NPs. Moreover, the inhibition was
confirmed by the reduction of Rubisco and MDH enzymes activity (by 54.5-86.4% and 26.7-96.5%,
respectively). Overall, results indicated that the damage was caused by destruction of chloroplast
thylakoid structure, which subsequently reduced the photosynthetic rate. Our research suggests that
Cr2O3 NPs can be transported and cause irreversible damage to soybean plants by inhibiting the activity
of electron acceptors (NADP+) and destroying ultrastructure of chloroplasts, providing insights into
plant toxicity issues.
2
1 33 2 3 4 34 5 6 7 8 35 9 10 11 12 36 13 14 15 16 37 17 18 19 38 20 21 22 23 39 24 25 26 27 40 28 29 30 31 41 32 33 34 42 35 36 37 38 43 39 40 41 42 44 43 44 45 46 45 47 48 49 46 50 51 52 53 47 54 55 56 57 48 58 59 60 61 62 63 64 65
Keywords: chlorophyll fluorescence; photosynthesis; photosynthetic enzymes; Cr2O3 NPs
Introduction
Metal oxide nanoparticles (NPs) have smaller size (particles with at least one dimension of less
than 100 nm) and larger surface area, which lead to the unique physico-chemical patterns and high
reactivity. NPs have recently been the focus of intense research because of its wide application in
industrial, agricultural and household use (Meshram et al., 2012; Zhu et al., 2004). Previous studies
have proved that NPs can enter the environment via waste water from industrial sites or through
domestic sewage (Hernandez-Viezcas et al., 2013; Rico et al., 2011). Besides, NPs can be transported
to soil via sewage sludge, then ultimately be absorbed by plants (Petersen et al., 2014), raising concerns
of the environment safety and the threaten of NPs to plants (Hawthorne et al., 2014), living organisms
and even humans as a function of direct or indirect exposure (Rickerby and Morrison, 2007; Ma et al.,
2010).
The potential toxicity and bioaccumulation of NPs motivates the investigation of their fate and
transportation in the uptake system. Plants are first produced in the ecosystem and therefore they may
easily act as intermediaries for the transfer of NPs to the organism (Hawthorne et al., 2014). It is
reported that NPs can be species specific absorbed by various plants (Zhang et al., 2011; Wang et al.,
3
1 49 2 3 4 50 5 6 7 8 51 9 10 11 12 52 13 14 15 16 53 17 18 19 54 20 21 22 23 55 24 25 26 27 56 28 29 30 31 57 32 33 34 58 35 36 37 38 59 39 40 41 42 60 43 44 45 46 61 47 48 49 62 50 51 52 53 63 54 55 56 57 64 58 59 60 61 62 63 64 65
2013; Ebbs et al., 2016; Cui et al., 2014) and allocated to the roots, stems, and leaves (Harris et al.,
2008; Parsons et al., 2010; Navarro et al., 2008). Evidence showed that CeO2 cannot be absorbed into
maize through root resorption (Birbaum et al., 2010) but ZnO and CeO2 can be accumulated into both
shoot and root of soybean and corn. Besides, the accumulation of ZnO was higher than CeO2 (Zhu et
al., 2004; Priester et al., 2012; Zhao et al., 2012). Also, the cumulative amount of Ag NPs in soybean
plant exposed to Ag NPs was significantly lower than those in the plants exposed to CeO2 NPs and
ZnO NPs (De La Torre-Roche et al., 2013). Therefore, it is necessary to verify the uptake of different
NPs in the plants.
Some studies argue that the varying degrees of toxicity responding to nanoparticles is related to
the migration process inside the plants. CuO NPs can be transported to the shoots through xylem sap
and translocated back to roots trough phloem-based transport in maize (Wang et al., 2012), which is
similar to Au NPs in woody poplar (Zhai et al., 2014), expressing a root-shoot-root distribution loop
and providing evidence for the bioaccumulation and detoxification of NPs in plants. The migration
process of NPs in plants may also be accompanied by ion release, change of redox state, combined
with increased reactive oxygen species, thus results in irreversible damage to gene expression, causing
root swelling and inhibition of root elongation (Wang et al., 2016; Ebbs et al., 2016). In addition to
4
1 65 2 3 4 66 5 6 7 8 67 9 10 11 12 68 13 14 15 16 69 17 18 19 70 20 21 22 23 71 24 25 26 27 72 28 29 30 31 73 32 33 34 74 35 36 37 38 75 39 40 41 42 76 43 44 45 46 77 47 48 49 78 50 51 52 53 79 54 55 56 57 80 58 59 60 61 62 63 64 65
morphology, changes in the valence of nanoparticles can be found in plants. The redox state of a small
number of Cu were found changed from Cu (Ⅱ) to Cu (Ⅰ) in maize (Wang et al., 2012), and the redox
state of small part of Ce NPs were found reduced from Ce(Ⅳ) to Ce(Ⅲ) in soybean (Hernandez-
Viezcas et al., 2013); metal oxidation process is often accompanied with changes in plant organic
metabolism, pH and reactive oxygen species. CuO NPs were reported to raise reactive oxygen species
(ROS) in rice (Shaw et al., 2013). In Arabidopsis thaliana, CuO NPs can cause the generation and
accumulation of ROS in the chloroplasts, affecting the electron transfer, causing gene damage (Wang
et al., 2016); Ag NPs were reported to induce the up-regulated expression of ROS associated genes
(Kohan-Baghkheirati et al., 2013).
However, most of the studies focused on the toxicity and crop yield reduction induced by NPs, only
few of them focused on the effects of nanoparticles on plant photosynthetic system. A life circle
experiment on cucumber pointed that NPs showed no effect on chlorophyll and gas exchange (Zhao
et al., 2013), while CuO NPs were reported to cause damage to Arabidopsis thaliana chloroplasts,
inhibiting the electron transfer during photosynthetic process (Wang et al., 2016). Besides, the
chlorophyll content was reported significantly reduced in wheat once exposed to ZnO NPs and CuO
NPs (Dimkpa et al., 2012), but not significantly changed in soybean plants exposed to CeO2 and ZnO
5
1 81 2 3 4 82 5 6 7 8 83 9 10 11 12 84 13 14 15 16 85 17 18 19 86 20 21 22 23 87 24 25 26 27 88 28 29 30 31 89 32 33 34 90 35 36 37 38 91 39 40 41 42 92 43 44 45 46 93 47 48 49 94 50 51 52 53 95 54 55 56 57 96 58 59 60 61 62 63 64 65
(Zhao et al., 2013). Ag NPs only inhibited chlorophyll b content in rice (Mirzajani et al., 2013). In
addition, TiO2, ZnO, and Au NPs were reported to enhance the chlorophyll content in some edible
plants (Ma et al., 2015). To date, many studies only investigated the chlorophyll content and ROS
damage, while only a small number of them have studied the changes in plant morphology and even
less were focused on the ultrastructural changes. Photosynthesis-related organelles can be found by
observing the subcellular structure of plants. Chlorophyll fluorescence imaging can visually show the
damage of leaf photosynthesis system, including electron transportation, opening degree of light
system, as well as dark reaction rate. These two methods could provide insight into the way that
nanoparticles affect the photosynthesis of different plants.
Cr2O3 is versatile as it is an excellent green dye material and stainless steel raw material (Farinati
et al., 2011), and are used for industrial applications such as catalysts and pigments (Ondřej et al.,
2014). In recent years, due to increasing technological requirements, the use of Cr2O3 NPs inputs was
rising, especially in magnetic materials, refractories and environmental catalysis (Park et al., 2016),
but little attention has been paid to the environmental risks that Cr2O3 NPs could pose to plants and
even humans. The transportation form and the toxicity of Cr2O3 NPs could pose to the growth and
photosynthesis of plants still remain uncertain. Soybean plants, as one of the largest farm species
6
1 97 2 3 4 98 5 6 7 8 99 9 10 11 12 100 13 14 15 16 101 17 18 19 102 20 21 22 23 103 24 25 26 27 104 28 29 30 31 105 32 33 34 106 35 36 37 38 107 39 40 41 42 43 108 44 45 46 47 109 48 49 50 51 110 52 53 54 55 111 56 57 58 59 112 60 61 62 63 64 65
around the world, were chosen because of its popularity as food and also economical crops, which
have directly connections with human life. To get a comprehensive understanding of the direct effects
of nanoparticles on plants, SEM/TEM and CF Imager were conducted in the present study.
Experiments were performed and differences in Cr2O3 NPs intensity were confirmed by various
analysis. This study was aimed to 1) confirm that Cr2O3 NPs can be absorbed and translocated by
soybean plants; 2) measure the activity of photosynthetic enzymes and observe the substructure
damage of chloroplasts with the addition of Cr2O3 NPs; 3) collect and analyze the photosynthetic data
along with the chlorophyll fluorescence image to reveal the electron transport and the damage of dark
reaction. The findings in this work will help to gain insight into the negative effects induced by Cr2O3
NPs and provide evidence to assess the risk.
Materials and methods
Cr2O3 Nanoparticle suspension characterization
Cr2O3 NPs were purchased from Jianglai Biology Ltd., Shanghai, China. The morphology of the
Cr2O3 NPs was examined by transmission electron microscopy, operated at 200 kV, and the characteristics of Cr2O3 NPs (0.05 μm) are shown in Figure S1. Cr2O3 NPs of different concentrations
were added to nutrient solution and agitated by ultrasonic vibration (100W, 40 kHz) for 30 minutes to 7
1 113 2 3 4 114 5 6 7 8 115 9 10 11 12 116 13 14 15 16 117 17 18 19 118 20 21 22 23 119 24 25 26 27 120 28 29 30 31 121 32 33 34 122 35 36 37 38 123 39 40 41 42 124 43 44 45 46 125 47 48 49 126 50 51 52 53 127 54 55 56 57 128 58 59 60 61 62 63 64 65
increase dispersion. The stability of NPs in the nutrient solution dispersion system was determined by
Zeta potential.
Germination experiment
Soybean seeds were sterilized by 0.7% NaClO solution for 10 min, and then rinsed with deionized
water for three times to ensure the surface was clean. Then the seeds were immersed in Cr2O3 NPs-
water with different concentrations of suspension (CK, 0.01, 0.05, 0.1, 0.5, g/L), and subsequently placed in the dark at 25 ℃, making sure that the seeds maintained humidity. After germination (3d),
the germination rate was calculated.
Plant culture
Soybean seeds were rinsed with deionized water for three times after sterilizing by 0.7% NaClO
solution for 10 min. Then the seeds were immersed in deionized water for germination, and subsequently placed in the dark at 25 ℃, making sure the seeds maintained humidity. After 3 days,
buds whose lengths surpassed half of the seed length were chosen and then placed to the nutrient
solution with the addition of Cr2O3 NPs and cultured under natural conditions.
Uniform seedlings were selected and moved to plastic containers amended with strength of the following nutrient solution (mmol L-1): Ca(NO3)2, 2.0; KH2PO4, 0.1; MgSO4, 0.5; KCl, 0.1; K2SO4,
8
1 129 2 3 4 130 5 6 7 8 131 9 10 11 12 132 13 14 15 16 133 17 18 19 134 20 21 22 23 135 24 25 26 27 136 28 29 30 31 137 32 33 34 138 35 36 37 38 139 39 40 41 42 140 43 44 45 46 141 47 48 49 142 50 51 52 53 143 54 55 56 57 58 59 60 61 62 63 64 65
0.7; H3BO3,10×10-3; MnSO4, 5×10-4; ZnSO4, 5×10-4; CuSO4, 2×10-4; (NH4)6MoO24,1×10-4; Fe-EDTA, 20×10-3. Cr2O3 NPs were added to the nutrient solution, and five experimental treatment groups were
initiated (g/L): 0.01, 0.05, 0.1, 0.5 and control (CK). The pH of the nutrient solution was adjusted to
6.0. Each experiment had four replicates. The seedlings grew for 14 days under natural conditions: at 25-30 / 20-25 ℃ (day/night) with relative humidity 60-70%, and a light intensity 15000 lx. The
suspensions with NPs were renewed every 3 days. The exposure time was 14 days; shoot and root
tissue were washed by ultrasonic vibration (output frequency 53 kHz, power 500 W, SK20GT, Ishine,
China) to remove the NPs on the surface, subsequently separated and dried by Bal-Tec CPD 030 a critical point dryer (Leica, Wetzlar, Germany) at 65 ℃ for 72 h. Fresh biomass of shoots and roots of
tissues from all treatments were measured, and Cr content in plant tissues were analyzed by ICP-MS
(Agilent 7500a, USA) after digestion with HNO3-HClO4.
SEM-EDS, TEM observation
SEM-EDS was performed to observe the transport of nanoparticles in plants and their effects on
plant morphology. TEM was used to analyze the structure change of chloroplast, thus revealing the
reasons for inhibition of photosynthetic system.
9
1 144 2 3 4 145 5 6 7 8 146 9 10 11 12 147 13 14 15 16 148 17 18 19 149 20 21 22 23 150 24 25 26 27 151 28 29 30 31 152 32 33 34 153 35 36 37 38 154 39 40 41 42 155 43 44 45 46 156 47 48 49 157 50 51 52 53 158 54 55 56 57 159 58 59 60 61 62 63 64 65
Fresh soybean root and shoot tissues were separated and the shoots were placed in 4℃ refrigerator
to avoid light overnight for dark recovery. The samples were first fixed with 2.5% glutaraldehyde in
phosphate buffer (0.1 M, pH 7.0) for more than 4h, washed three times in the phosphate buffer (0.1 M,
pH7.0) for 15 min at each step, then post fixed with 1% OsO4 in phosphate buffer for 1-2h and washed
three times in the phosphate buffer (0.1 M, pH 7.0) for 15 min at each step. The samples were prepared
for Dehydration: the samples were first dehydrated by graded series of ethanol (30%, 50%, 70%, 80%,
90%,95% and 100%) for about 15 to 20 min at each step.
For SEM scanning, the samples were transferred to the mixture of alcohol and iso-amyl acetate
(v:v=1:1) for about 30 min, and then transferred to pure iso-amyl acetate for about 1h. In the end, the
samples were dehydrated in Hitachi Model HCP-2 critical point dryer with liquid CO2. The dehydrated
samples were coated with gold-palladium in Hitachi Model E-1010 ion sputter for 4-5 min and
observed in Hitachi Model TEM-1000 SEM and INCA100 EDS (Oxfordshire, U.K.).
For TEM scanning, the specimen was placed in 1:1 mixture of absolute acetone and the final
Spurr resin mixture for 1h at room temperature, then transferred to 1:3 mixture of absolute acetone
and the final resin mixture for 3 h and lastly to the final Spurr resin mixture for overnight. The specimen
was placed in Eppendorf contained Spurr resin and heated at 70℃ for more than 9 h. The specimen
10
1 160 2 3 4 161 5 6 7 8 162 9 10 11 12 163 13 14 15 16 164 17 18 19 165 20 21 22 23 166 24 25 26 27 167 28 29 30 31 168 32 33 34 169 35 36 37 38 170 39 40 41 42 171 43 44 45 46 172 47 48 49 173 50 51 52 53 174 54 55 56 57 175 58 59 60 61 62 63 64 65
was sectioned in LEICA EM UC7 ultratome and sections were stained by uranyl acetate and alkaline
lead citrate for 5 to 10 min respectively and observed in Hitachi Model H-7650 TEM.
Chlorophyll fluorescence measurement
After being cultured in complete nutrient solution with Cr2O3 NPs, the samples were picked and
ready to be used to evaluate the effect of Cr2O3 NPs on PSII electron transport. Soybean leaves were
placed in the dark for 30 min for dark adaptation to evaluate the dark-adapted minimum fluorescence
(Fo), dark-adapted maximum fluorescence (Fm), variable fluorescence Fv (Fv=Fm-Fo). Fo’ and Fm’ were measured after 30 min of light intensity1000 μmol / (m2·s) for the potential capture efficiencies
of Fv/Fm (XE), non-photochemical quenching Fm/Fm'-1 (NPQ) and the capture rate of excitation
energy of PSII reaction center Fv'/Fm (XE'), photochemical quenching coefficients Fq'/Fv' (qP) and Fq'/Fm' (φPSII). State was ready for monitoring in leaves in vivo with CF Imager (CF0056, TNC,
America). All the leaves were measured under same condition.
Photosynthesis rate, intercellular CO2 concentration and stomatal conductance were measured by using
the LI-6400/LI-6400XT Portable Photosynthesis System (LI-6400XT; Li-Cor Environmental, Lincoln,
NE, USA).
Activity of the photosynthetic enzyme (MDH, Rubisco) and determination of chlorophyll content
11
1 176 2 3 4 177 5 6 7 8 178 9 10 11 12 179 13 14 15 16 180 17 18 19 181 20 21 22 23 182 24 25 26 27 183 28 29 30 31 184 32 33 34 185 35 36 37 38 186 39 40 41 42 187 43 44 45 46 188 47 48 49 189 50 51 52 53 190 54 55 56 57 191 58 59 60 61 62 63 64 65
This experiment adopted the tissue kit (Suzhou Comin Biotechnology Co. Ltd) and followed the
protocol provided to measure the activity of Rubisco (Rubisco, EC 4.1.1.39) in the soybean leaves.
Malate dehydrogenase (MDH) tissue kit was from Nanjing Jiancheng Bioengineering Institute,
the activity of MDH was examined according to the protocol provided (Lv et al., 2013).
Fresh soybean leaves were collected and washed for sampling. Amass of 0.1g crushed leaves
were put into 25ml colorimetric tube and 15ml of extract (acetone: ethanol = 1: 1) were added. After
24-hour dark soaking, the leaves turned to be transparent. The extract was used as blank control.
Chlorophyll was examined by Spectrophotometer (721-100) at the wavelength of 649 nm and 665 nm.
Statistical analysis
Data were analyzed statistically using the SPSS package (version 11.0; SPSS Inc., Chicago, IL,
USA). Analysis of variance (ANOVA) was performed on the datasets. Means of significant difference
were separated by t test or Duncan’s multiple range test at the p
