Identifying Plant Stress Responses to Roxarsone in Soybean Root

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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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Journal of Agricultural and Food Chemistry

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Identifying plant stress responses to roxarsone in soybean root

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exudates:

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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

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ǁ

9

Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA

Department of Chemical, Biochemical and Environmental Engineering, University of

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§

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Institute of Technology, Ookayama, Meguroku, Tokyo 152-8552, Japan

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*

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[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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1

ACS Paragon Plus Environment


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ABSTRACT

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Roxarsone (ROX) is an organoarsenic feed additive of increasing interest used in

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the poultry industry. Soybean responses to ROX stress were investigated in root

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exudates (REs) using two-dimensional correlation spectroscopy (2D-COS) with

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fluorescence and Fourier transform infrared spectra. Environmentally-relevant ROX

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concentrations caused negligible toxicity to crop growth and photosynthesis activity,

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but blackened soybean roots at high concentrations. 2D-COS analysis revealed that

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the protein-like fluorophore and C=C and C=O, aliphatic-OH, and polysaccharide

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C-O-H moieties in soybean REs were most sensitive to ROX stress. Hetero-spectral

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2D-COS results suggested that aromatic, amide I, quinone, ketone, and aliphatic

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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,

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2D-COS combined with molecular-based spectral analysis of REs provided an

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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

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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,

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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,

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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

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of pH on fluorescent components in rainwater13. However, the application of 2D-COS

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to ecotoxicology and plant science has not yet been reported.

, and nanoparticles2, 9. Recently, 2D-COS has also been employed to analyze

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Roxarsone (ROX) is a water-soluble organoarsenic feed additive used in poultry

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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

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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

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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

77

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

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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

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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

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were treated with environmentally-relevant ROX levels (0-25 mg/L, always expressed

93

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%

111

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

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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

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excitation-emission matrices (EEMs) of diluted REs were measured at room

133

temperature using a fluorescence spectrophotometer (F-7000; Hitachi, Japan). The

134

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

138

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

141

spectrophotometry (AFS-230; Haiguang, China) after digestion with 5 mL

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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

144

(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

146

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,

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while the order is reversed for a negative product of Φ(λ1, λ2) and Ψ(λ1, λ2). If the

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product of Φ(λ1, λ2) and Ψ(λ1, λ2) Ψ(λ1, λ2) equals zero, the intensity changes at λ1 and

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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

164

(OriginLab, USA)16.

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All experiments were conducted in triplicate, and data are reported/plotted as

166

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

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different uptake mechanisms and exposure periods for soybeans and wheat. For

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example, the uptake period in this study was five times that used in the wheat seedling

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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

179

content in soybean leaves was linearly correlated with the ROX concentration of the

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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

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dependent on the ROX concentration in the test solution (not shown). TF values

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between soybean leaf and root at low (i.e., 0-5 mg/L) and high (i.e., 10-25 mg/L)

185

external ROX concentrations were found to be (8.57 ± 2.48)×10-3 and (8.94 ±

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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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189

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

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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)

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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

207

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

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Figure S1, changes in As speciation (Figure S2) were negligible during the 48-h

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incubation/renewal period. Therefore, the As(III), As(V), and unknown As species

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detected in soybean root and leaf extracts indicate metabolic transformation of ROX

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within the soybean plant. Future study of the metabolic pathway of ROX within

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soybean plants is warranted to further understand these important mechanisms that

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may influence toxicological responses.

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Figure 2.

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ROX Toxicity to Soybeans at Environmentally-Relevant Concentrations.

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Physiological activities of plants are affected by ROX accumulation into root systems.

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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

11



233

(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

238

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

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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.

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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,

256

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

13



277

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),

300

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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321

treatment. Furthermore, a new peak was identified at 1257 cm-1 in the FTIR spectrum

322

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

16


Page 17 of 41


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.

17



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

18


Page 18 of 41

Page 19 of 41


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,

19



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

20


Page 20 of 41

Page 21 of 41


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

21



Page 22 of 41

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

22


Page 23 of 41


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



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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sewage sludge using EEM-PARAFAC and two-dimensional FTIR correlation

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680

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

33



Page 34 of 41

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

34


Page 35 of 41


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

35



Page 36 of 41

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.

36


Page 37 of 41


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.

37


25


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.

38


Page 38 of 41

Page 39 of 41


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.

39



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.

40


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TOC

41


Identifying Plant Stress Responses to Roxarsone in Soybean Root

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