1
Supporting Information
2
Transformation
3
Arbuscular Mycorrhizal Fungi as Revealed by SEM-EDS,
4
TEM-EDS and XAFS
5
Songlin Wu†,#,¶, Xin Zhang†,¶, Yuqing Sun†, #, Zhaoxiang Wu†, #, Tao Li†, Yajun Hu†,⊥,
6
Dan Su†,#, JitaoLv‡, Gang Li‡, Zhensong Zhang‡, Lirong Zheng§, Jing Zhang§,
7
Baodong Chen†,*
8
9
10
11
12
13
†
and
Immobilization
of
Chromium
by
State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental
Sciences, Chinese Academy of Sciences, Beijing, 100085, People’s Republic of China
#
University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China
⊥
Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical
Agriculture, Chinese Academy of Sciences, Changsha, 410125, People’s Republic of China
‡
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for
14
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, People’s Republic
15
of China
16
17
18
§
Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of
Sciences, Beijing 100049, People’s Republic of China
¶
These authors contributed equally to this work.
19
*Corresponding author, Baodong Chen, Phone: 0086-10-62849068; Fax: 0086-10-62923549;
20
E-mail: [email protected].
21
This Supporting Information (SI) contains 18 pages, 6 figures, and 5 tables.
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23
1
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SR µ-XRF Methods
25
The synchrotron radiation micro-focused X-ray fluorescence (SR μ-XRF) microspectroscopy
26
experiment was carried out at beamline 4W1B, Beijing synchrotron Radiation Facility (BSRF),
27
which runs 2.5 GeV electron with current from 150 mA to 250 mA. The incident X-ray energy
28
was monochromatized by W/B4C Double-Multilayer-Monochromator (DMM) at 15 keV and was
29
focused on an area with 50 μm in diameter by the polycapillary lens. The two-dimensional
30
mapping data were acquired by a stepsize of 50 μm. The Si (Li) solid state detector is used to
31
detect X-ray fluorescence emission lines with live time of 20 s. SR μ-XRF spectra were processed
32
using the PyMCA software package.1
33
34
XAFS Analysis Methods
35
The XAFS spectra of mycelium and root samples were acquired at beamline 1W1B of the
36
Beijing Synchrotron Radiation Facility (BSRF). In order to unravel different Cr oxidation states
37
and chemical compositions, 9 Cr standard compounds, including K2CrO4, K2Cr2O7, Cr2O3,
38
Cr(III)-phosphate
39
[Cr2(SO4)3·6H2O], Cr(III)-cysteine, Cr(III)-histidine, and Cr(III)-acetate were employed in this
40
study. K2CrO4, K2Cr2O7, Cr2O3 and Cr(III)-nitrate were purchased from Sinopharm Chemical
41
Reagent Beijing Co., Ltd (Beijing, China). CrPO4·4H2O was purchased from Alfa Aesar (A
42
Johnson Matthey Company, Stanbul, Turkey). Cr(III)-sulfate was purchased from Shanghai Laize
43
Chemical Co., Ltd. (Shanghai, China) K2CrO4, K2Cr2O7, Cr2O3, Cr(III)-phosphate, and
44
Cr(III)-nitrate were diluted to 1% Cr (w/w) with NaCl,2 and ground to a fine powder.
45
Cr(III)-Cysteine was synthesized according to De Meester et al. (1977),3: Cr(NO3)3·9H2O
(CrPO4·4H2O),
Cr(III)-nitrate
2
[Cr(NO3)3·9H2O],
Cr(III)-sulfate
46
(Sinopharm Chemical Reagent Beijing Co., Ltd, Beijing, China) (5×10-3 mol) in 25 mL of water
47
was mixed with 25×10-3 mol of L-cysteine (CAS number 52-90-4) in 25 mL of water. The
48
resulting solution was boiled for a few minutes and solid NaOH was added until the solution’s pH
49
reached 6.0. Cr(III)-histidine was prepared according to Levina et al.(2007)4: a solution of
50
Cr(NO3)3·9H2O (5×10-3 mol in 15 mL of water) was added dropwise with heating (352 K) and
51
stirring to a solution of L-histidine (CAS number 71-00-1) (25×10-3mol in 35 mL of water). The
52
reaction was continued for 1 h at 352 K, then the pH was adjusted to 6.0 using 1 M NaOH.
53
Cr(III)-acetate was synthesized as follows: Cr(NO3)3·9H2O (5×10-3mol) in 25 mL of water was
54
mixed with 25×10-3 mol of Sodium Acetate (Sinopharm Chemical Reagent Beijing Co., Ltd.,
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Beijing, China) in 25 mL of water, the resulting solution pH was adjusted to 6.0 using 1 M NaOH
56
solution.2
57
An energy range of -200 to 700 eV relative to the Cr K edge absorption was used to acquire
58
spectra of each mycelium and reference compounds in fluorescence mode under ambient
59
conditions. Data were acquired using the following energy ranges: -200 — -20 eV (4 eV steps
60
with 1s per interval); -20 — 50 eV (0.5 eV steps with 1s per interval); 50 — 150 eV (1 eV steps
61
with 2s per interval); 150 — 300 eV (2 eV steps with 2s per interval); 300 — 600 eV (3 eV steps
62
with 2s per interval); 600 — 700 eV (4 eV steps with 3s per interval). For root samples, the Cr
63
K-edge spectra were collected using a 19 element Ge array solid state detector at an energy range
64
of -160 to 400 eV relative to the Cr K edge absorption. And data were acquired using the
65
following energy ranges: -160 — -20 eV (5 eV steps with 5s per interval); -20 — 50 eV (0.5 eV
66
steps with 5s per interval); 50 — 100 eV (1 eV steps with 5s per interval); 100 — 400 eV (5 eV
67
steps with 5s per interval).
3
68
Two scans of each sample were collected to improve the signal to noise ratios. The energy scale
69
was calibrated using a stainless steel foil as an internal standard (calibration energy, 5989.0 eV,
70
corresponded). The foil was placed between the I1 and I2 ion chambers and collected
71
simultaneously with each sample spectra. The XAFS data (average of two scans) were normalized
72
(baseline and background corrections), and the X-ray adsorption near-edge spectroscopy (XANES)
73
spectra (extracted from -20 eV — 70 eV) were analyzed by principal component analysis (PCA)
74
and linear combination fitting (LCF) employing the ATHENA and SixPACK from IFEFFIT
75
software package (CARS, University of Chicago).5 The minimal R-factor, Chi-square and
76
Reduced Chi-square were used in the quality control of the fitting. The correctness of the LCF is
77
controlled by many factors6 and therefore the results we report are not free of uncertainties and
78
limitations. The extended X-ray absorption fine structure (EXAFS) analysis was performed by
79
converting spectra of standard compounds and samples into k space (weighted to 3), and
80
subsequently Fourier transformed by using a modified Hanning window. The transformed EXAFS
81
were then fitted using ARTEMIS from IFEFFIT software package.5 The FEFF input files were
82
created by Stand-alone Atoms softwares from crystallographic data of the model compounds. In
83
the fit, amplitude reduction factor (S02) was set to 0.87, the coordination numbers (N), the
84
interatomic distances (R) and the Debye–Waller factor (σ2) etc were calculated.
4
85
Figure Legends:
86
Figure S1 Diagram of the two-compartment in vitro cultivation system. Root compartment was
87
filled with solid M medium gelled with 0.4% (w/v) phytagel, allowing development of
88
mycorrhizal roots, then extraradical mycelium (ERM) ramified into hyphal compartment filled
89
with liquid M medium without sucrose and phytagel, and the roots that crossed the central wall
90
were trimmed to prevent their growth in hyphal compartment.
91
92
Figure S2. Synchrotron radiation micro-focused X-ray fluorescence (SR µ-XRF) imaging of Cr
93
and X-ray fluorescence spectrum of typical area in control extraradical mycelium (treatment
94
“+M-Cr”) (A) and extraradical mycelium treated with 0.05 mmol L-1 Cr (VI) (treatment “+M+Cr”)
95
(B). Notes: treatment “+M-Cr” represents inoculation in root compartment, and no Cr(VI)
96
addition in hyphal compartment, treatments “+M+Cr” represents inoculation in root compartment,
97
and 0.05 mmol L-1 Cr(VI) addition in hyphal compartment.
98
99
Figure S3. Cr K-edge XANES spectra of standard Cr compounds and Cr(VI) treated hyphae;
100
Notes: a, K2CrO4; b, K2Cr2O7; c, Cr2O3; d, Cr(III)-phosphate (CrPO4·4H2O); e, Cr(III)-nitrate
101
[Cr(NO3)3·9H2O]; f, Cr(III)-sulfate [Cr2(SO4)3·6H2O]; g, Cr(III)-cysteine; h, Cr(III)-histidine; i,
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Cr(III)-acetate; j, hyphae in hyphal compartment treated with 0.05 mmol L-1 Cr(VI) (treatment
103
“+M+Cr”); k, hyphae in hyphal compartment treated with 0.05 mmol L-1 Cr(VI) after 24-h 2%
104
(v/v) formaldehyde treatment (treatment “+M+CrF”); l, hyphae in hyphal compartment treated
105
with 0.5 mmol L-1 DNP and 0.05 mmol L-1 Cr(VI) together (treatment “+M+CrD”).
106
5
107
Figure S4. Cr K-edge extended X-ray absorption fine structure (EXAFS) spectra of Cr(III)
108
standard compounds and hyphae of different treatments. The solid lines represent experimental
109
data, dashed lines represent simulations.
110
111
Figure S5. Linear combination analysis of Cr K edge XANES spectra of hyphae treated with 0.1
112
mmol L-1 Cr(VI). Fitting parameters are R-factor=0.00015, Chi-square=0.018, Reduced
113
Chi-square= 0.00013.
114
115
Figure S6. Cr K-edge EXAFS spectra of hyphae treated with 0.1 mmol L-1 Cr(VI) and Cr(III)
116
standard compounds. The solid lines represent experimental data, dashed lines represent
117
simulations.
118
119
120
6
121
Figure S1
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7
123
Figure S2
124
125
8
126
Figure S3
127
128
9
129
Figure S4
130
131
132
10
133
Figure S5
134
135
136
11
137
138
Figure S6
139
12
140
141
142
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Table S1. Intensity of the mycorrhizal colonization on the root system (M%) and dry weight of
carrot roots, number of spores in root compartment and pH, hyphal dry weight and spore number
in hyphal compartment. Values are mean ± SD (n=8). Means followed with the same letter are not
significantly different (Duncan test, p < 0.05).
Treatment M (%)
Root dry
weight in
Number of
pH in hyphal
root
spores in root
compartment
compartment compartment
(mg)
ERM dry
weight in
hyphal
compartment
(mg)
Number of
spores
in hyphal
compartment
-M-Cr
-M+Cr
+M-Cr
+M+Cr
+M+CrF
+M+CrD
23.5±12.7b
21.0±11.4b
45.8±9.81a
43.6±4.36a
42.2±3.24a
44.8±8.08a
0
0
2.10±1.27a
1.90±0.63a
2.70±1.46a
2.05±1.26a
0
0
2257±964a
1168±1025b
1250±1033b
1022±597b
0
0
52.6±14.5a
52.7±10.2a
44.3±9.56a
47.0±8.52a
0
0
2370±733a
2456±718a
2507±724a
2083±377a
144
145
146
13
5.68±0.25c
5.72±0.28c
7.66±0.19a
7.26±0.24ab
5.39±0.36cd
5.29±0.74cd
147
148
149
Table S2. Phosphorus (P) concentration and content of carrot roots in root compartment of
different treatments. Values are mean ± SD (n=8). Means followed with the same letter are not
significantly different (Duncan test, p < 0.05).
Treatment
Root P concentration in
root compartment (mg g-1)
Root P content in root
compartment (μg)
-M-Cr
-M+Cr
+M-Cr
+M+Cr
+M+CrF
+M+CrD
0.81±0.10a
0.82±0.08a
0.84±0.13a
0.84±0.06a
0.82±0.06a
0.86±0.04a
19.6±2.77b
13.5±5.56b
35.7±7.93a
37.7±5.79a
34.7±2.06a
38.7±7.85a
150
151
14
152
153
154
Table S3. Extraradical mycelium (ERM) Cr content, root Cr content, and percentage of ERM Cr
content in total Cr content of roots and ERM of different treatments. Values are mean ± SD (n=8).
Means followed with the same letter are not significantly different (Duncan test, P < 0.05).
Treatment
Cr content of
ERM in hyphal
compartment
(μg)
Cr content of
mycorrhizal roots in
root compartment
(μg)
Percentage of ERM Cr
content in total Cr content
of roots and ERM (%)
+M+Cr
+M+CrF
+M+CrD
2.17±1.37b
6.96±3.14a
3.39±1.24b
0.62±0.23a
0.25±0.08b
0.27±0.12b
73.6±17.1c
96.5±0.98a
93.3±2.11ab
155
156
157
15
158
159
160
161
Table S4. First and/or second shell structural parameters derived from the Cr K EXAFS spectra of
Cr model compounds and hyphae of different treatments. “N” represents the coordination number,
“R” represents the interatomic distance, “σ2” represents the Debye–Waller factor, “R factor” value
indicates goodness of fit parameters.
Sample
Bond
N
R(Å)
σ2(Å)
Cr(III)-phosphate
Cr-O
Cr-P
6.2±0.4
4.0±1.0
1.98±0.01
3.12±0.02
0.0040±0.0008 1.3%
0.0066±0.0013
Cr(III)-histidine
Cr-O/N
5.9±0.3
2.02±0.01
0.0027±0.0007 0.3%
Hyphae,
0.05mM Cr(VI)
Cr-O/N
Cr-P
7.1±1.6
3.8±1.0
1.97±0.02
2.95±0.07
0.0008±0.0025 3.9%
0.0015±0.0033
Hyphae,
0.05mM Cr(VI),
2%(v/v) HCHO
Cr-O/N
Cr-P
7.1±1.3
3.5±1.0
1.98±0.02
3.08±0.05
0.0037±0.0026 3.9%
0.0083±0.0068
Hyphae,
0.05mM Cr(VI),
0.5mM DNP
Cr-O/N
Cr-P
7.1±1.8
2.8±0.5
1.95±0.02
3.01±0.03
0.0025±0.0032 5.5%
0.0004±0.0041
162
163
164
16
R factor
165
166
167
168
Table S5. First and/or second shell structural parameters derived from the Cr K EXAFS spectra of
hyphae treated with 0.1 mmol L-1 Cr(VI). “N” represents the coordination number, “R” represents
the interatomic distance, “σ2” represents the Debye–Waller factor, “R factor” value indicates
goodness of fit parameters.
Sample
Hyphae,
0.1 mM Cr(VI)
Bond
Cr-O/N
Cr-P
N
6.6±1.1
1.5±0.3
R(Å)
1.96±0.01
2.96±0.08
169
170
17
σ2(Å)
R factor
0.0021±0.0019 2.1%
0.0003±0.0041
171
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