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Identifying plant stress responses to roxarsone in soybean root exudates: New insights from two-dimensional correlation spectroscopy Qing-Long Fu, Lee Blaney, and Dong-Mei Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04706 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017
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Identifying plant stress responses to roxarsone in soybean root
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exudates:
3
spectroscopy
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Qing-Long Fu†,‡,§, Lee Blaneyǁ, Dong-Mei Zhou†,*
5 6 7
†
New
insights
from
two-dimensional
correlation
Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil
Science, Chinese Academy of Sciences, Nanjing 210008, China ‡
University of Chinese Academy of Sciences, Beijing 100049, China
8
ǁ
9
Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
Department of Chemical, Biochemical and Environmental Engineering, University of
10 11
§
12
Institute of Technology, Ookayama, Meguroku, Tokyo 152-8552, Japan
13
*
14
[email protected] Present address: Department of Civil and Environmental Engineering, Tokyo
Corresponding author. Tel.: +86-25-86881180; Fax: +86-25-86881000; E-mail:
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ABSTRACT
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Roxarsone (ROX) is an organoarsenic feed additive of increasing interest used in
18
the poultry industry. Soybean responses to ROX stress were investigated in root
19
exudates (REs) using two-dimensional correlation spectroscopy (2D-COS) with
20
fluorescence and Fourier transform infrared spectra. Environmentally-relevant ROX
21
concentrations caused negligible toxicity to crop growth and photosynthesis activity,
22
but blackened soybean roots at high concentrations. 2D-COS analysis revealed that
23
the protein-like fluorophore and C=C and C=O, aliphatic-OH, and polysaccharide
24
C-O-H moieties in soybean REs were most sensitive to ROX stress. Hetero-spectral
25
2D-COS results suggested that aromatic, amide I, quinone, ketone, and aliphatic
26
functional groups were the foundational components of protein-like and short
27
wavelength excited humic-like fluorophores in soybean REs. Carboxyl and phenolic
28
moieties were related to the long wavelength excited humic-like fluorophore. Overall,
29
2D-COS combined with molecular-based spectral analysis of REs provided an
30
innovative approach to characterize the physiological responses of crops to
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contaminants at sub-lethal levels.
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KEYWORDS: roxarsone, root exudates, plant responses, two-dimensional
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correlation spectroscopy
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INTRODUCTION
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Two-dimensional correlation spectroscopy (2D-COS) is a powerful mathematical
37
tool that can be used to analyze spectral intensities as a function of external
38
perturbations (e.g., time and pH)1. Compared to conventional spectroscopic
39
techniques, such as infrared and fluorescence, 2D-COS can not only solve the issue of
40
overlapping spectral peaks in heterogeneous mixtures (e.g., dissolved organic matter,
41
DOM) by extending spectra along a second dimension, but also identify the sequential
42
order of perturbation-induced spectral intensity changes1-5. As an emerging technique,
43
2D-COS has been used to investigate the binding characteristics of DOM and
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microbial extracellular polymeric substances to heavy metals3, 5-7, organic pollutants4,
45
8
46
microbial degradation of fluorescent components in DOM derived from cyanobacteria
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blooms10, elucidate the biomineralization pathway of nano-scale minerals11, explore
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the interaction of microbial cells with iron mineral surfaces12, and reveal the influence
49
of pH on fluorescent components in rainwater13. However, the application of 2D-COS
50
to ecotoxicology and plant science has not yet been reported.
, and nanoparticles2, 9. Recently, 2D-COS has also been employed to analyze
51
Roxarsone (ROX) is a water-soluble organoarsenic feed additive used in poultry
52
operations to control intestinal parasites, improve feed efficiency, and promote animal
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growth4,
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ultimately introduced to the environment through land application of poultry litter,
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causing elevated ROX levels in agricultural soil. The degradation of ROX varies with
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soil properties. For instance, the half-life of ROX in red and yellow-brown soil was
14-19
. ROX undergoes minimal metabolism within poultry birds and is
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reported as 130-394 d and 4-94 d, respectively20. Given the toxicity associated with
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arsenic-containing compounds16, it is reasonable to expect that crops grown in
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poultry-litter amended soils may be affected by ROX. A previous study demonstrated
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that ROX accumulated in wheat seedlings was subsequently metabolized into arsenite
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(As(III)) and arsenate (As(V))16. However, no study has been performed to
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characterize plant physiological responses to ROX stress.
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The secretion of root exudates (REs), which are composed of low molecular
64
weight compounds (e.g., amino acids and organic acids) and high molecular weight
65
compounds (e.g., polysaccharides and proteins), is a critical physiological response of
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plants to stress caused by toxic contaminants and biotic factors 21-24. For example, the
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bioavailability of hexachlorobenzene in biochar-amended soils was enhanced by
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ryegrass REs due to their facilitated role in desorbing hexachlorobenzene from the
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soil
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releasing phytate, which in turn promoted As and Fe uptake and formation of
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insoluble FeAsO424. By forming stable complexes with pollutants, REs play an
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important role in immobilizing and reducing the bioavailability of contaminants in
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plants25. Furthermore, the fluorescent fractions of REs exhibit high binding affinities
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to organic and inorganic contaminants26-27. However, changes in the fluorescent
75
components of REs have not been studied in crops stressed by organoarsenic feed
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additives.
23
. In another study, it was found that Pteris vittata responded to As stress by
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In this work, the transformation of ROX in soybean, a globally cultivated crop,
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was assessed using high performance liquid chromatography with inductively coupled
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plasma-mass spectrometry (HPLC-ICP-MS). Moreover, ROX toxicity to the
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photosystem II (PSII) activity of soybean leaves was probed using chlorophyll
81
fluorescence. Finally, the sequential order of changes to fluorescent components and
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functional groups present in REs from soybean plants stressed by incremental, but
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environmentally-relevant levels of ROX was investigated using Fourier transform
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infrared (FTIR) and synchronous fluorescence spectroscopies integrated with
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2D-COS. The corresponding results provide critical insights into crop RE responses to
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lethal and sub-lethal levels of ROX and demonstrate the utility of 2D-COS in
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agricultural and ecotoxicology research.
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MATERIALS AND METHODS
89 90
Plant Cultivation. The soybean (Glycine max L. wandou 15) plants used in this study were hydroponically cultivated according to the method detailed in Content S1.
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ROX Treatment. Following pre-cultivation, soybean plants of uniform height
92
were treated with environmentally-relevant ROX levels (0-25 mg/L, always expressed
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as the mass of As)
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diluting ROX (Acros Organics, 98% purity) directly in 0.25 strength Hoagland
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nutrient solution (0.25-HS). The pH of ROX-containing solutions was adjusted to
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5.5±0.1 using concentrated HNO3 and 1 M NaOH. Each treatment was performed in
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triplicate with every replicate containing three acclimated soybean plants, which were
98
transferred to 800-mL opaque plastic pots. Plants were then cultivated under the same
99
conditions described above. To minimize volume loss, ROX test solutions were
100
28-29
. Experimental solutions were prepared by dissolving and/or
replenished every 12 h and renewed with deionized (DI) water every 24 h.
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Chlorophyll a Fluorescence Transients and Pigment Content Measurements.
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After a 7-d exposure to ROX, the center section of the second set of leaves was
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exposed to 2 s pulses of blue light (455 nm, 3000 µM photon/(m2·s); 25±1 °C) to
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record chlorophyll a fluorescence OJIP transients (JIP-test) using a handheld
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fluorometer (PAR-FluorPen FP 100-MAX-LM-D, PSI, CZ). All tested leaves were
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dark-adapted for 60 min before measurement. Three individual leaves from each plant
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were tested for a total of 27 independent leaves per treatment scenario. JIP-test
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parameters quantifying PSII photosynthetic performance were calculated using the
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FluorPen 1.0.5.1 software (PSI, CZ) with previously detailed formulae30. Chlorophyll
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a and b contents of leaves collected at the end of JIP-tests were extracted using 80%
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acetone and measured using a UV-visible spectrometer (Shimadzu UV-2700, Japan)31.
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RE Collection. REs from soybean plants treated with 0-25 mg/L ROX for 10 d 25
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were collected following slightly modified procedures from Zhao et al. (2001)
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Liu et al. (2016) 24. Roots were thoroughly rinsed 3-5 times with DI water and then
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placed in a 10 mM NaH2PO4 solution with 0.125% NaClO (v/v) for 1 h to desorb
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ROX from root surfaces and inactivate microbial growth. Subsequently, soybean roots
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were washed 3-5 times with DI water and transferred to 50-mL centrifuge tubes,
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containing 40 mL of freshly prepared 0.1 mM CaCl2 solution, to collect RE over a 10
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h period at the aforementioned incubation conditions. The solution volume was
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replenished every 2.5 h with 0.1 mM CaCl2 to offset volume loss due to plant
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transpiration. All tubes were covered with black tape to minimize photodegradation of
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RE components. The collected REs were immediately filtered (0.45 µm), lyophilized,
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and stored at -60 °C.
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Spectroscopic Measurement. FTIR spectra of lyophilized REs were recorded for
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samples mixed with 100 mg KBr using the Nicolet iS10 FTIR spectrometer (Thermo,
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USA) with 64 scans and a 4 cm-1 resolution. Reference spectra for ROX and KNO3
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standards were acquired in a similar manner. Baseline corrections of raw FTIR spectra
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were performed by the Nicolet OMNIC software.
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To prevent inner-filter effects during fluorescence measurement, the lyophilized
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REs were dissolved in DI water and diluted until the UV absorbance at 254 nm was
131
below
132
excitation-emission matrices (EEMs) of diluted REs were measured at room
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temperature using a fluorescence spectrophotometer (F-7000; Hitachi, Japan). The
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instrumental parameters for both techniques have been previously reported
135
not repeated here. A constant offset of ∆λ (λemission - λexcitation = 60 nm) was adopted for
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synchronous fluorescence spectra measurement with excitation wavelengths ranging
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from 250 to 550 nm4, 32. The fluorescence spectrum of DI water was subtracted from
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the spectra of experimental samples.
0.3.
The
synchronous
fluorescence
spectra
and
three-dimensional
4
and are
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Arsenic Extraction and Analysis. The total As content in test solutions and
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soybean tissues stressed with ROX for 10 d were analyzed by atomic fluorescence
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spectrophotometry (AFS-230; Haiguang, China) after digestion with 5 mL
142
concentrated HNO3 at 210 °C. Full details of the As extraction methods are included
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in Content S2. As speciation in the samples was simultaneously analyzed by HPLC
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(Agilent 1260, USA), with an anion exchange column (Hamilton PRP-X100,
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250 × 4.1 mm, 10 µm), coupled to ICP-MS (Agilent 7700×, USA). The operational
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conditions for the HPLC-ICP-MS method were described elsewhere33.
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2D-COS Analysis. In this work, 2D-COS was performed using synchronous
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fluorescence and baseline-corrected FTIR spectra with ROX concentration as the
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external perturbation. This analysis allowed observation of changes in fluorescent
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fractions and functional groups in the soybean REs due to stress responses caused by
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incremental ROX exposure. The 2D-COS maps, including synchronous and
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asynchronous spectra, were processed using MATLAB 7.8.0 (Mathworks, USA) with
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the MIDAS 2010 toolbox (University of Saskatchewan, Canada). The hetero-spectral
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2D-COS maps were calculated in 2D-Shige (Kwansei-Gakuin University, Japan) and
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plotted in MATLAB. The sequential order of spectral changes banded at λ1 and λ2
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were compared with the signs of the synchronous Φ(λ1, λ2) and asynchronous Ψ(λ1, λ2)
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peaks based on Noda’s rule1. Briefly, a positive product of Φ(λ1, λ2) and Ψ(λ1, λ2)
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indicates that the change in spectral intensity at band λ1 occurs prior to that at band λ2,
159
while the order is reversed for a negative product of Φ(λ1, λ2) and Ψ(λ1, λ2). If the
160
product of Φ(λ1, λ2) and Ψ(λ1, λ2) Ψ(λ1, λ2) equals zero, the intensity changes at λ1 and
161
λ2 concurrently occur.
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Data Analysis. The concentration-dependent uptake kinetics of ROX in soybean
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roots were fit to the two-parameter Michaelis-Menten model with Origin 9.0
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(OriginLab, USA)16.
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All experiments were conducted in triplicate, and data are reported/plotted as
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mean ± standard deviation. The significance of differences between experimental data
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sets was determined with the two-tailed Student's t-test (Origin 9.0) at p < 0.05.
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RESULTS AND DISCUSSION
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Uptake and Distribution of ROX in Soybean Plants. Figure 1 indicates that
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ROX uptake by soybean roots was well described by the Michaelis-Menten equation
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(R2 = 0.997) with calculated maximum uptake rate, Vmax, and Michaelis-Menten rate
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constant, Km, of 1251 ± 37 mg/(kgDW·7d) and 6.49 ± 0.56 mg/L, respectively. ROX
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uptake in soybeans differed from the previously reported linear model for wheat
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seedlings16. These contrasting ROX accumulation patterns can be attributed to
175
different uptake mechanisms and exposure periods for soybeans and wheat. For
176
example, the uptake period in this study was five times that used in the wheat seedling
177
experiment16; therefore, the linear portion of the Michaelis-Menten expression may
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only dominate at shorter times in the wheat seedlings. Additionally, the total As
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content in soybean leaves was linearly correlated with the ROX concentration of the
180
test solutions, suggesting rapid distribution of ROX within the soybean plant.
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The transfer factor (TF) was calculated as the slope of the ROX content in the
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leaves plotted against that in the roots16. ROX translocation in soybean plants was
183
dependent on the ROX concentration in the test solution (not shown). TF values
184
between soybean leaf and root at low (i.e., 0-5 mg/L) and high (i.e., 10-25 mg/L)
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external ROX concentrations were found to be (8.57 ± 2.48)×10-3 and (8.94 ±
186
2.69)×10-2, respectively. These TF values indicate that ROX was more easily
187
transferred to above-ground tissue, such as leaves, in the high ROX scenarios. The
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translocation of ROX from soybean roots to leaves for high external ROX
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concentrations was comparable to that found in wheat seedlings16. Furthermore, ROX
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translocation was similar to previous findings for uptake and distribution of inorganic
191
As in soybean plants34.
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Figure 1
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Arsenic Speciation in Soybean Roots and Leaves. Figure 2 and Table S2
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confirm As speciation changes in soybean roots and leaves following cultivation in
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the 2.5 and 10 mg/L ROX-laden test solutions. The predominant As species in
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soybean roots and leaves was identified as ROX, which accounted for 62%-109% and
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102%-111% of total As content, respectively. The next most abundant As species were
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As(III) and As(V). Methylated arsenicals were not detected in this study; note that the
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detection limits for monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)
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were 0.35 and 0.24 µg/L, respectively. These findings are consistent with previous
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reports indicating that As in Tropaeolum majus and wheat seedlings is mainly present
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as ROX with low levels of As(III) and As(V)16, 35. Compared with the extraction
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method using methanol + water (v/v, 1:1)16, 0.1 mM NaH2PO4 + methanol (v/v, 1:1)
204
demonstrated better extraction for arsenicals due to the additional As desorption from
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plant tissues caused by phosphate, yielding As extraction efficiencies of 79-121%.
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An unknown As peak was observed at 2.91 min in the HPLC-ICP-MS
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chromatograms for soybean leaf extracts from the 2.5 and 10 mg/L ROX conditions
208
(Figure 2A), but was absent for root extracts. For reference, arsenocholine (AsC) and
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arsenobetaine (AsB) standards (not shown) eluted at 1.91 and 2.35 min, respectively.
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Because the retention time of the unknown As compound on the anion exchange
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column was closer to that of As(III) compared to AsB and AsC, the unknown As
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species may result from complexation of inorganic As with low molecular weight
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organic substances in soybean leaf extracts; note that the leaf extracts exhibited high
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levels of green substances that were most likely associated with plant pigments31.
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Consistent with the insignificant (p > 0.05) variation in ROX concentration shown in
216
Figure S1, changes in As speciation (Figure S2) were negligible during the 48-h
217
incubation/renewal period. Therefore, the As(III), As(V), and unknown As species
218
detected in soybean root and leaf extracts indicate metabolic transformation of ROX
219
within the soybean plant. Future study of the metabolic pathway of ROX within
220
soybean plants is warranted to further understand these important mechanisms that
221
may influence toxicological responses.
222
Figure 2.
223
ROX Toxicity to Soybeans at Environmentally-Relevant Concentrations.
224
Physiological activities of plants are affected by ROX accumulation into root systems.
225
The toxicity of environmentally-relevant ROX concentrations (i.e., 1-25 mg/L)28-29 to
226
soybean plants was examined by measuring photosynthetic properties in the leaves.
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As depicted in Figure 3A, the chlorophyll a and b levels in soybean leaves did not
228
significantly change during 7-d incubation periods in test solutions with 1 mg/L ROX
229
(p = 0.815 and 0.646, respectively) or 25 mg/L ROX (p = 0.240 and 0.347,
230
respectively). While, an insignificant decreasing trend was observed for chlorophyll
231
content variation with increasing ROX levels. The maximum quantum yield for PSII
232
primary photochemistry (Fv/Fm) and the performance index on an absorption basis
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(PIabs) were used to quantify the maximum theoretical and actual photosynthetic
234
performance, respectively36. Figure 3B shows that no significant changes (p > 0.05)
235
were observed in the Fv/Fm and PIabs values for soybeans during a 7-d exposure to
236
ROX compared with the control. These findings signify the low toxicity of ROX to
237
soybean plants. Our previous study reported that the median effective concentration of
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ROX for inhibition of wheat root elongation was approximately 20 mg/L16. Figure
239
S3A further demonstrates that the growth of soybean plants, including the
240
above-ground tissues, was not affected by environmental levels of ROX as all plants
241
had similar stem structure and leaf size. In contrast, the soybean roots (shown in
242
Figure S3B) were darkened with increasing ROX concentration, implying potential
243
influences of ROX on the physiology of soybean roots. These findings are consistent
244
with a previous report, which found that roots are more sensitive than above-ground
245
plant tissues to ROX stress because of the greater ROX concentrations at plant roots
246
(Figure S4)16.
247
Figure 3.
248
Fluorescent Properties of Soybean REs Affected by ROX. The EEMs for
249
soybean REs are presented in Figure S5, and the peak excitation/emission
250
wavelengths and assignments are tabulated in Table 1. According to the peak
251
assignments37-38, seven distinct fluorescence domains were observed in the EEMs
252
measured for REs collected from soybean plants incubated with ROX. The peaks can
253
be classified into three main categories, namely fulvic acid-like, humic acid-like, and
254
protein-like fluorescence. These results differ from previous reports indicating that
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only protein-like fluorophores were detected in REs from other plants (e.g., maize,
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sunflower, and wetland halophyte)26-27,
257
inherent chemistry of other plant systems or variation in the incubation conditions
258
and/or the RE extraction protocols between studies. The A, B, C, D, E, F, and G peaks
259
identified in the EEMs (Figure S5) correspond to the RE fluorophores A, B, C, D, E, F,
260
and G, respectively, reported in Table 1. As suggested by Fu et al. (2016) 4, the fulvic
261
acid-like (i.e., A and E) and humic acid-like (i.e., B, C, D, and F) fluorophores are
262
broadly identified as humic-like fluorophores in this study. Fluorophore G was further
263
characterized as having tryptophan protein-like fluorescence37. Figure S5 illustrates
264
that the fluorescence intensities of the humic-like fluorophores were greater than those
265
for the protein-like fluorophore, suggesting that humic-like substances were the
266
dominant fluorescent components in soybean REs.
39
. These differences may stem from the
267
EEMs for soybean REs exposed to different ROX concentrations were divided
268
into four groups based on the overall fluorescence signature and associated peak
269
assignments. Group I is represented in Figure S5A by the control RE (no ROX), and
270
contains one fulvic acid-like (A) and two humic-acid like fluorophores (B, C). Group
271
II includes soybean REs from the low-level (1.0-5.0 mg/L) ROX exposures.
272
Compared to Group I, the Group II EEMs contained an additional fulvic acid-like
273
fluorophore D, as shown in Figure S5B-D. For soybean plants treated with 2.5 and 5.0
274
mg/L ROX, the excitation and emission wavelengths of fluorophore B showed blue
275
and red shifts, respectively, compared to the control EEM, indicating changes in
276
conformation or rigidity4. Groups III (Figure S5E-F) and IV (Figure S5G-H) are
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comprised of EEMs generated for soybean REs exposed to 10-15 mg/L and 20-25
278
mg/L ROX, respectively. Comparison of the EEMs from Groups III and IV with
279
Group I shows the disappearance of fluorophores A and B and the appearance of
280
fluorophores D, E, F, and G at intensities that vary with the ROX concentration in the
281
test solution. These findings verify that the fluorescence signature of soybean REs is
282
significantly altered by ROX concentrations greater than 10 mg/L. Protein-like
283
substances are reported to exhibit a high binding affinity for ROX4. Therefore, the
284
excretion of protein-like fluorophores from soybean roots may represent a coping
285
mechanism for the plant to deal with stress caused by ROX.
286
Four peaks were observed in the synchronous fluorescence spectra of REs from
287
soybean plants incubated with various ROX concentrations (Figure S6). Peak I
288
(λExcitation = 279-289 nm) was associated with protein-like fluorescence and
289
fluorophore A. Peaks II (λExcitation = 329-336 nm), III (λExcitation = 372-401 nm), and IV
290
(λExcitation = 469-485 nm) corresponded to humic-like substances3-4, 32, 40, and these
291
peaks were further characterized as short-, long-, and far-wavelength excited
292
humic-like (i.e., S-humic-like, L-humic-like, and F-humic-like, respectively)
293
fluorophores. Peaks II and III were associated with the humic acid-like fluorophores B
294
and C, respectively. Spectral changes in synchronous fluorescence with increasing
295
ROX concentration are in accordance with those identified above using the EEMs
296
from soybean REs.
297 298
Table 1 Functional Groups of Soybean REs Affected by ROX. Soybean REs are
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composed of the following: nitrate; low molecular weight organic acids (LMWOA),
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such as citric acid, succinic acid, acetic acid, and ascorbic acid; and, other unknown
301
substances. Moreover, soybean RE fluorophores contain carboxylic, carbonyl,
302
hydroxyl, and other functional groups4. Given this compositional information, the
303
effects of ROX stress on the functional groups present in soybean REs were
304
elucidated using FTIR. The FTIR spectra of soybean REs, along with ROX and KNO3
305
standards, are provided in Figure S7, and the corresponding band assignments are
306
summarized in Table 2 according to previous studies2, 41-43. The position of the sharp
307
band at 1384 cm-1 in soybean REs was comparable to that of the KNO3 standard
308
(1384 cm-1) and nitrate (1381 cm-1)42, suggesting that this band was derived from the
309
high nitrate levels in the REs. This assignment is reinforced by the HPLC analysis in
310
Figure S8, which showed high nitrate content in the soybean REs. Importantly, the
311
FTIR spectra of soybean REs differed from the ROX standard (Figure S7B). For
312
example, the asymmetric stretching of -NO2 at 1508 cm-1 in the ROX standard was
313
not observed in any of the soybean REs, implying negligible ROX excretion from
314
soybean roots during RE collection.
315
Table 2
316
Figure S7 shows that the FTIR spectra of soybean REs were greatly affected by
317
10-25 mg/L of ROX. Compared with the control solution (ROX = 0 mg/L), the bands
318
for symmetric stretching of N-H and O-H shifted from 3048 cm-1 to 3446 cm-1, and
319
the bands for asymmetric stretching of C-H (2497-2952 cm-1) and stretching of C=O
320
in carboxyl and aliphatic groups (1719-1763 cm-1) disappeared for the 25 mg/L ROX
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treatment. Furthermore, a new peak was identified at 1257 cm-1 in the FTIR spectrum
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for soybean plants treated with 25 mg/L ROX due to stretching of C-C, C-O-C, and
323
C-N in carboxyl and amide III groups. These structural changes further confirm the
324
presence of the protein-like fluorophore G shown in Figure S5; furthermore, these
325
data reinforce the hypothesis that more amide III-containing protein-like substances
326
were induced by the soybean plant to alleviate rhizotoxicity to ROX.
327
2D-COS Analysis of Soybean REs.
328
Two-Dimensional
Fluorescence
Correlation
Spectroscopy
(2D-F-COS)
329
Analysis. Figure 4 shows the synchronous and asynchronous 2D-F-COS maps for
330
soybean REs at 250-550 nm. Three striking auto-peaks were identified at 282, 330,
331
and 384 nm along the 1:1 line of the synchronous map (Figure 4A) and associated
332
with peaks I, II, and III, respectively, from the synchronous fluorescence spectra. The
333
auto-peak intensity followed the order of 330 nm > 384 nm > 282 nm, suggesting that
334
changes in fluorescence intensity decreased from the S-humic-like to L-humic-like to
335
protein-like fluorophores4, 44. The spectral changes of peaks I, II, and III proceeded in
336
the same order with increasing ROX concentrations because all cross-peaks were
337
positive in Figure 4A.
338
Figure 4.
339
The asynchronous maps provide additional information to qualitatively compare
340
the sequential order of different spectral origins affected by external perturbations.
341
Two positive cross-peak areas, Ψ(325-360 nm, 290-326 nm) and Ψ(385-430 nm,
342
365-390 nm), and two negative cross-peak areas, Ψ(296-336 nm, 272-285 nm) and
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343
Ψ(375-440 nm, 260-350 nm), were observed in the asynchronous 2D-F-COS map in
344
Figure 4B. The presence of the two positive cross-peak areas signifies peak overlap in
345
the S-humic-like and L-humic-like fluorophores from Figure S6, suggesting that these
346
fractions of soybean REs can be further divided into two components, namely
347
S-humic-like/L-humic-like fractions excited at relatively shorter and longer
348
wavelengths3-4. According to Noda’s rule1, the two subdivided components for the
349
S-humic-like and L-humic-like fractions are responsible for ROX stress in the
350
sequence:
351
wavelengths > S-humic-like/L-humic-like fraction excited at relatively longer
352
wavelengths.
S-humic-like/L-humic-like
fraction
excited
at
relatively
shorter
353
The negative cross-peak area, Ψ(296-336 nm, 272-285 nm), implies that peak
354
intensities change in the order of 272-285 nm > 296-336 nm. This result indicates that
355
the protein-like fraction in the RE is more sensitive to ROX stress than the
356
S-humic-like component. The broad, negative cross-peak area, Ψ(375-440 nm,
357
260-350 nm), suggests that the 260-350 nm peak intensity changes before that of
358
375-440 nm, which means that the protein-like and S-humic-like fractions change
359
faster at lower ROX concentrations than the L-humic-like component. The
360
F-humic-like component is affected by ROX concentrations more significantly than
361
the L-humic-like fraction because of Ψ(480 nm, 400 nm) < 0. Overall, the results from
362
the 2D-F-COS maps suggest the following order of RE fluorescence changes due to
363
soybean stress responses to ROX: protein-like fraction > S-humic-like fraction >
364
L-humic-like fraction > F-humic-like fraction.
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365
Recall that the F-humic-like fraction was associated with fluorophore C in the
366
EEMs. Therefore, the relatively slow changes in the F-humic-like fraction agree with
367
the minor positional changes observed for peak C at increasing ROX concentrations.
368
Consistent with the EEM results, the protein-like fraction was found to be the most
369
sensitive fluorescent component in soybean REs to ROX stress, presumably due to the
370
higher binding capacity of protein-like RE fractions to ROX than that of humic-like
371
components 4. The complexation activation energy between ROX and a model protein,
372
glutathione, was calculated as 36.9 kJ/mol45. Similarly, long-chain ionic liquids
373
interacted with dissolved organic matter (DOM) fractions in the following sequence:
374
protein-like > humic-like 8. The protein-like fluorophore in soybean REs also shows a
375
high binding affinity to heavy metals and organic contaminants, forming stable
376
protein-contaminant complexes and, subsequently, inhibiting uptake of these
377
contaminants by plant roots26-27,
378
fluorophores in soil-derived DOM through a static quenching mechanism to yield
379
stable DOM-ROX complexes4. For these reasons, the protein-like fluorophore is
380
expected to be the first fluorescent fraction in soybean REs to alleviate ROX stress.
39
. Similarly, ROX complexes with protein-like
381
Two-Dimensional FTIR Correlation Spectroscopy (2D-FTIR-COS) Analysis.
382
The FTIR spectra for REs overlap due to the numerous functional groups present in
383
these complex mixtures, leading to ambiguous band assignments and incorrect
384
conformational analysis. Figure 5 illustrates the synchronous and asynchronous
385
2D-FTIR-COS maps for the 1800-400 cm-1 region in soybean REs following ROX
386
perturbation. The predominant auto-peak at 1384 cm-1 in the synchronous map
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387
(Figure 5A) is ascribed to the symmetric stretching of nitrate and carboxylic C-O
388
bonds and the in-plane bending vibration of phenolic O-H groups. Another weak
389
auto-peak occurred at 880 cm-1 due to deformation stretching of C-H. Four
390
cross-peaks, Φ(1384 cm-1, 1648 cm-1), Φ(1050 cm-1, 1384 cm-1), Φ(880 cm-1, 1384
391
cm-1), and Φ(480 cm-1, 1384 cm-1), exhibited positive magnitudes in the synchronous
392
map, revealing that the vibrational intensities of the corresponding FTIR peaks
393
assigned in Table 2 change in the same direction with increasing ROX levels1.
394
Figure 5.
395
Two positive cross-peaks, Ψ(1050 cm-1, 1384 cm-1) and Ψ(1377 cm-1, 1384 cm-1),
396
and two negative cross-peaks, Ψ(1384 cm-1, 1648 cm-1) and Ψ(1384 cm-1, 1393 cm-1),
397
were observed in the asynchronous 2D-FTIR-COS map in Figure 5B. According to
398
the positive synchronous peaks and Noda’s rule1, the positive Ψ(1050 cm-1, 1384 cm-1)
399
and negative Ψ(1384 cm-1, 1648 cm-1) suggest that the FTIR peak intensities at 1050
400
cm-1 and 1648 cm-1 change before that at 1384 cm-1 under ROX-induced stress. These
401
findings indicate that C=C and C=O stretching of aromatic, amide I, quinone, and
402
ketone functional groups and C-O-H stretching of aliphatic OH and polysaccharide
403
molecules occur before N-O stretching of nitrate, carboxylic C-O stretching, and
404
phenolic O-H in-plane bending vibrations. The neutral spectral signs of Ψ(880 cm-1,
405
1384 cm-1) and Ψ(1050 cm-1, 1648 cm-1) indicate that the bands at 880 cm-1 and 1050
406
cm-1 change in a similar manner as those at 1384 cm-1 and 1648 cm-1, respectively, for
407
the same ROX level.
408
The appearance of the cross-peaks, Ψ(1377 cm-1, 1384 cm-1) and Ψ(1384 cm-1,
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409
1393 cm-1), manifests in the sharp band at 1384 cm-1 in Figure S7 and originates from
410
three different band vibrations of soybean RE functional groups. The spectral signs of
411
cross-peaks Ψ(1377 cm-1, 1384 cm-1) and Ψ(880 cm-1, 1384 cm-1) are positive and
412
negative, respectively, indicating that the band variations at 1377 and 1393 cm-1 are
413
higher than that at 1384 cm-1 under ROX stress. These findings highlight
414
heterogeneous distribution of functional groups in the 1384 cm-1 range. As observed
415
above, the sharp peak at 1384 cm-1 was attributed to nitrate N-O stretching, carboxylic
416
C-O stretching, and in-plane bending vibration of phenolic O-H (Table 2).
417
Additionally, Figure S7B showed that the KNO3 standard generated a predominant
418
peak for N-O stretching at 1384 cm-1. For these reasons, the striking band at 1384
419
cm-1 stems from N-O stretching of nitrate in the soybean REs. The overlapping bands
420
at 1377 and 1393 cm-1 were assigned to C-O stretching and O-H in-plane bending
421
vibration of carboxylic and phenolic substances present in the soybean REs. These
422
results suggest that carboxylic and phenolic moieties in the soybean RE are more
423
susceptible to ROX stress than nitrate.
424
In conclusion, the structural variation sequence of soybean REs due to ROX
425
stress follows the order: C=C and C=O (aromatic rings, amide I, quinones, and
426
ketones) ≈ aliphatic OH, polysaccharide C-O-H > carboxylic C-O, phenolic O-H >
427
nitrate N-O ≈ aliphatic C-H. DOM can complex ROX through hydroxyl, amide II,
428
carboxyl, and aliphatic CH groups, thereby inhibiting ROX uptake by plants4, 16. Here,
429
the 2D-FTIR-COS results reveal additional information that C=C and C=O (e.g.,
430
aromatic rings, amide I, quinones, and ketones), aliphatic OH, and polysaccharide
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431
C-O-H in soybean REs are the most sensitive functional groups to ROX-related stress.
432
It is important to note that non-fluorescent polysaccharides in soybean REs may also
433
contribute to the physiological responses of soybean plants to ROX stress. The
434
sequential order identified here is in accordance with that reported for changes in
435
humic-acid functional groups as a function of solution pH2. Therefore, the RE
436
functional group variations may be partially attributed to changes in test solution pH
437
affected by ROX-induced production of LMWOA.
438
Hetero-Spectral 2D-COS Analysis. Hetero-spectral 2D-COS based on the FTIR
439
and synchronous fluorescence spectra was employed to analyze the co-variations or
440
correlations between soybean RE functional groups and fluorescent fractions. The
441
synchronous and asynchronous hetero-spectral 2D-COS maps are illustrated in Figure
442
S9, with FTIR wavenumber and fluorescence wavelength along the abscissa and
443
ordinate, respectively. As shown in Figure S9A, FTIR bands at 1635-1653, 1377-1393,
444
and 1050-1150 cm-1 were positively correlated with fluorescence shifts at 310-335,
445
260-370, and 280-360 nm, respectively. These observations indicate that aromatic,
446
amide I, quinone, ketone, and aliphatic functional groups are the foundational units
447
for the protein-like and S-humic-like fluorophores. The negative cross-peak identified
448
in the FTIR band at 1377-1397 cm-1 and the fluorescence at 400-425 nm in the
449
synchronous map signify that the L-humic-like fraction is comprised of carboxyl and
450
phenolic groups.
451
The asynchronous map from hetero-spectral 2D-COS analysis reveals slight
452
differences between the FTIR and fluorescence spectra of soybean REs as a function
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453
of ROX stress. Figure S9B exhibits two positive, Ψ(1377-1393 cm-1, 275 nm) and
454
Ψ(1377-1393 cm-1, 325-360 nm), and one negative, Ψ(1377-1393 cm-1, 375-440 nm),
455
cross-peak areas. Following Noda’s rule 1, these results suggest that carboxylic and
456
phenolic groups are related to the fluorescence response of both protein-like and
457
S-humic-like components. However, the fluorescence response of the L-humic-like
458
fraction to ROX stress occurred prior to the spectral response of carboxylic and
459
phenolic groups. The hetero-spectral and homo-spectral 2D-COS results discussed
460
above signify that aromatic, amide I, quinone, ketone, and aliphatic groups for
461
protein-like and S-humic-like fluorophores are the most susceptible functional groups
462
in soybean REs and may, therefore, be used as molecular-based spectral properties to
463
characterize the physiological responses of plants to ROX stress.
464
Environmental Significance
465
ROX, the blockbuster organoarsenic feed additive, is extensively used in many
466
developing countries and is highly persistent in agricultural soils. However, few
467
studies
468
environmentally-relevant concentrations16,
469
exhibit negligible toxicity to the above-ground tissues of soybean seedlings, but the
470
blackened soybean roots suggested changes in rhizosphere chemistry. The responses
471
of soybean REs to a gradient of ROX concentrations were elucidated using 2D-COS,
472
providing new insights to changes in soybean RE fluorescent components and
473
structural groups during exposure to ROX.
474
have
investigated
crop
responses
to
ROX-induced
stress
at
46-47
. In this work, ROX was shown to
The spectral overlap of the S-humic-like fluorophore and the dominant FTIR
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475
peak at 1384 cm-1 was resolved by 2D-COS, demonstrating the unique abilities and
476
high resolution of 2D-COS (compared to conventional one-dimensional techniques) to
477
probe the sequential order of fluorescence and FTIR spectral variations for changes in
478
RE chemistry caused by ROX stress. To date, the physiological responses of plants to
479
toxic contaminants have been focused on leaf photosynthesis parameters, cell
480
antioxidant systems, metabolic product identification, and gene expression36,
481
This work has established the application of fluorescence and FTIR measurements in
482
soybean REs to characterize physiological responses to contaminants, especially
483
under circumstances that present limited toxicological effects on plant growth.
484
Moreover, the elucidation of the RE spectral responses to ROX stress may facilitate
485
extension of 2D-COS to other fields, including agricultural/plant science and
486
ecotoxicology. The LMWOA in REs play pivotal roles in alleviating and detoxifying
487
the stress responses of plants to contaminants24. The 2D-COS results described in this
488
work highlight the equally important role of high molecular weight organics,
489
especially protein-like substances, in the phytoremediation and detoxification of ROX
490
in soybean plants.
491
Supporting Information
48-50
.
492
Plant cultivation (Content S1); arsenic extraction and analysis (Content S2);
493
composition of 0.25 strength Hoagland nutrient solution (Table S1); arsenic content in
494
soybean roots and leaves (Table S2); ROX concentration during a 48-h incubation
495
period (Figure S1); arsenic speciation before and after incubation (Figure S2);
496
photographs of soybean plants and roots following exposure to ROX (Figure S3);
23
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497
total As content in soybean tissues (Figure S4); EEMs for soybean REs (Figure S5);
498
synchronous fluorescence spectra of REs (Figure S6); FTIR spectra for soybean REs
499
(Figure S7); HPLC chromatograms for soybean REs (Figure S8); and, hetero-spectral
500
2D-COS plots showing synchronous and asynchronous maps generated from the
501
synchronous fluorescence and FTIR spectra of soybean REs (Figure S9).
502
AUTHOR INFORMATION *
Corresponding author. Tel.: +86-25-86881180; Fax: +86-25-86881000; E-mail:
503 504
[email protected] §
505
Present address: Department of Civil and Environmental Engineering, Tokyo
506
Institute of Technology, Ookayama, Meguroku, Tokyo 152-8552, Japan
507
ACKNOWLEDGEMENTS
508
This work was financially supported by National Natural Science Foundation of
509
China (41430752). The authors are grateful to the editor and anonymous referees for
510
their valuable suggestions that have improved the manuscript.
511
Notes
512
The authors declare no competing financial interest.
513
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molecular weight (HMW) fluorescent substances in root exudates from a wetland
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spectroscopy. Bioresour. Technol. 2014, 159, 412-420.
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Table and Figure Captions
681
Table 1. Positions of fluorescence peaks in the EEMs of REs from soybean plants
682
exposed to ROX.
683
Table 2. Assignment of FTIR bands from the REs of soybean plants stressed by ROX.
684 685
Figure 1. The total As content in soybean root and leaf extracts as a function of ROX
686
concentrations in the test solutions.
687
Figure 2. Arsenic speciation in the (A) leaves and (B) roots of soybean plants
688
cultivated in the 2.5 and 10 mg/L ROX test solutions.
689
Figure 3. (A) Chlorophyll a, b, and a + b content and (B) Fv/Fm and PIabs values for
690
soybean leaves plotted as a function of ROX concentration. The upper-case letters
691
represent statistical differences (p < 0.05) for the control and 1-25 mg/L ROX
692
treatments.
693
Figure 4. (A) Synchronous and (B) asynchronous 2D-F-COS maps generated from
694
the synchronous fluorescence spectra of REs from soybean plants stressed by ROX.
695
Figure 5. (A) Synchronous and (B) asynchronous 2D-FTIR-COS maps generated
696
from the FTIR spectra of REs from soybean plants stressed by a gradient of ROX
697
concentrations.
698
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Table 1. Positions of fluorescence peaks in the EEMs of REs from soybean plants exposed to ROX. Peak position
ROX level (mg/L)
a
Excitation wavelength (nm) / Emission wavelength (nm) Peak A
Peak B
Peak C
Peak D
Peak E
Peak F
Peak G
a
a
a
0
245.0/393.0
290.0/393.0
405.0/447.0
a
1.0
245.0/393.0
290.0/390.0
405.0/447.0
315.0-320.0/390.0-399.0
a
a
a
a
a
2.5
245.0/388.0
285.0-290.0/391.0-397.0
405.0/449.0
325.0-330.0/403.0-405.0
a
5.0
245.0/392.0
285.0-290.0/400.0-401.0
405.0-446.0
310.0-315.0/412.0-417.0
a
a
a
10.0
a
a
405.0/444.0
310.0/433.0
235.0/441.0
a
a
15.0
a
a
400.0-405.0/443.0-444.0
305.0/431.0
235.0/432.0
a
a
20.0
a
a
405.0-410.0/453.0-458.0
a
a
275.0/444.0
275.0/356.0
25.0
a
a
400.0-405.0/444.0-447.0
a
a
275.0/443.0
285.0-290.0/384.0-388.0
not detected
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Table 2. Assignment of FTIR bands from the REs of soybean plants stressed by ROX. Wavenumber (cm-1)
Vibration type
Functional group
3397-3447
Symmetric stretching of N-H and O-H
Phenol, carboxyl, amide
2942-2952
Asymmetric stretching of C-H
1763-1719
Stretching of C=O
1635-1653
Stretching of C=C and C=O
1534
1384 1257-1260 1074-1097 943-986 825-840
Deformation stretching of N-H; stretching of C-N Symmetric stretching of NO3-; stretching of C-O; in-plane bending vibration of O-H Stretching of C-C, C-O-C, and C-N Stretching of C-O-H Stretching of C-O Deformation stretching of C-H
aliphatic and carboxylic CH3 or CH2 Carboxylic, aliphatic Aromatic rings, amide I, quinones, ketones Amide II
NO3-, carboxylic, phenolic Carboxylic, amide III Aliphatic OH, polysaccharide Polysaccharide Aliphatic C-H
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As content in the root (mg/(kgDW·7d))
As content in the root As content in the leaf Mechaelis-Menten Linear
800
30
600 20 400 10 200
0
As content in the leaf (mg/(kgDW·7d))
40 1000
0 0
5
10
15
20
25
ROX concentration (mg/L)
Figure 1. The total As content in soybean root and leaf extracts as a function of ROX concentrations in the test solutions.
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10000
10000
(A)
2.5 mg/L ROX 10 mg/L ROX 8000
ROX
8000
Signal intensity (cps)
Signal intensity (cps)
(B)
2.5 mg/L ROX 10 mg/L ROX
6000
ROX 4000
2000
As(III)
As(III) 6000
4000
2000
Unknow As As(V)
As(V)
0
0 0
5
10
15
20
25
0
5
10
15
20
Time (min)
Time (min)
Figure 2. Arsenic speciation in the (A) leaves and (B) roots of soybean plants cultivated in the 2.5 and 10 mg/L ROX test solutions.
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Figure 3. (A) Chlorophyll a, b, and a + b content and (B) Fv/Fm and PIabs values for soybean leaves plotted as a function of ROX concentration. The upper-case letters represent statistical differences (p < 0.05) for the control and 1-25 mg/L ROX treatments.
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Figure 4. (A) Synchronous and (B) asynchronous 2D-F-COS maps generated from the synchronous fluorescence spectra of REs from soybean plants stressed by ROX.
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Figure 5. (A) Synchronous and (B) asynchronous 2D-FTIR-COS maps generated from the FTIR spectra of REs from soybean plants stressed by a gradient of ROX concentrations.
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