Subscriber access provided by Stockholm University Library
Article
Transformation and Immobilization of Chromium by Arbuscular Mycorrhizal Fungi as Revealed by SEM-EDS, TEM-EDS and XAFS Songlin Wu, Xin Zhang, Yuqing Sun, Zhaoxiang Wu, Tao Li, Yajun Hu, Dan Su, Jitao Lv, Gang Li, Zhensong Zhang, Lirong Zheng, Jing Zhang, and Baodong Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03659 • Publication Date (Web): 09 Nov 2015 Downloaded from http://pubs.acs.org on November 19, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 44
Environmental Science & Technology
1 2 3 4
Transformation and Immobilization of Chromium by Arbuscular Mycorrhizal Fungi as Revealed by SEM-EDS, TEM-EDS and XAFS
5 6
Songlin Wu†,#,¶, Xin Zhang†,¶, Yuqing Sun†, #, Zhaoxiang Wu†, #, Tao Li†,
7
Yajun Hu†,⊥, Dan Su†,#, Jitao Lv‡, Gang Li‡, Zhensong Zhang‡, Lirong
8
Zheng§, Jing Zhang§, Baodong Chen†,*
9 10
†
State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental
11
Sciences, Chinese Academy of Sciences, Beijing, 100085, People’s Republic of China
12
#
13 14 15
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
16
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, People’s Republic
17
of China
18 19 20
§
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.
21
*Corresponding author, Baodong Chen, Phone: 0086-10-62849068; Fax: 0086-10-62923549;
22
E-mail:
[email protected]. 1
ACS Paragon Plus Environment
Environmental Science & Technology
23
TOC Art
24
2
ACS Paragon Plus Environment
Page 2 of 44
Page 3 of 44
Environmental Science & Technology
25
ABSTRACT:
26
Arbuscular mycorrhizal fungi (AMF), as ubiquitous soil fungi that form symbiotic relationships
27
with the majority of terrestrial plants, are known to play an important role in plant tolerance to
28
chromium (Cr) contamination. However, the underlying mechanisms, especially the direct
29
influences of AMF on translocation and transformation of Cr in the soil-plant continuum, are still
30
unresolved. In a two-compartment root-organ cultivation system, the extraradical mycelium (ERM)
31
of mycorrhizal roots was treated with 0.05 mmol L-1 Cr(VI) for 12 days to investigate the uptake,
32
translocation, and transformation of Cr(VI) by AMF using inductively coupled plasma mass
33
spectrometry (ICP-MS), scanning electron microscope equipped with energy dispersive
34
spectroscopy (SEM-EDS), transmission electron microscope equipped with energy dispersive
35
spectroscopy (TEM-EDS) and X-ray absorption fine structure (XAFS) technologies. The results
36
indicated that AMF can immobilize quantities of Cr via reduction of Cr(VI) to Cr(III), forming
37
Cr(III)-phosphate analogues, likely on the fungal surface. Besides, we also confirmed that the
38
extraradical mycelium (ERM) can actively take up Cr [either in the form of Cr(VI) or Cr(III)], and
39
transport Cr [potentially in the form of Cr(III)-histidine analogues] to mycorrhizal roots, but
40
immobilize most of the Cr(III) in the fungal structures. Based on a XANES analysis of
41
Cr(VI)-treated roots, we proposed that the intraradical fungal structures can also immobilize Cr
42
within mycorrhizal roots. Our findings confirmed the immobilization of Cr by AMF, which plays
43
an essential role in the Cr(VI) tolerance of AM symbioses.
44
3
ACS Paragon Plus Environment
Environmental Science & Technology
45
INTRODUCTION
46
Chromium (Cr) is a valuable metal commonly used in leather production, electroplating and steel
47
manufacturing. Cr naturally exists in two stable forms, hexavalent chromium [Cr(VI)] and
48
trivalent chromium [Cr(III)].1 Cr(VI) is highly mobile and more toxic to organisms than Cr(III).1, 2
49
In recent decades, large amounts of Cr(VI) have been released into the environment by a wide
50
range of industrial and agricultural activities, resulting in Cr contamination of soil and water.2 A
51
recent survey of soil contamination in China showed that Cr is one of the eight key inorganic
52
pollutants (Cd, Hg, As, Cu, Pb, Cr, Zn and Ni) in Chinese soils.3 Although Cr is an essential
53
element for human beings and animals, excessive Cr can be toxic to all living organisms.4 As a
54
non-essential element for plants, Cr usually disturbs plant physiological processes, including
55
photosynthesis and respiration. Soil Cr contamination can produce negative effects on crop yield
56
and threaten the food safety of human beings.5,6
57
Plant rhizospheres usually serve as a favorable habitat for soil microorganisms, and some
58
microorganisms can establish intimate relationships with plants. Arbuscular mycorrhizal fungi
59
(AMF) are ubiquitous soil fungi that form symbiotic relationships with the majority of terrestrial
60
plants.7 The fungi survive on carbohydrates from the host plants, and they in return provide
61
mineral nutrients [especially phosphorus (P)] and water to their plant partner.7-9 Additionally, AMF
62
can stabilize soil structure,10 relieve drought stress on plants,11 protect host plants from
63
pathogens,12 and even take an active part in maintaining plant biodiversity and ecosystem
64
stability.13 Many studies have also demonstrated that AM symbiosis plays an important role in
65
plant resistance to contamination by heavy metals14, 15 such as As,16 Cd,17 Cu,18 Zn,19 Pb,20 Cr,21
66
etc. Our recent work has shown that AM symbiosis can greatly enhance plant Cr tolerance, 4
ACS Paragon Plus Environment
Page 4 of 44
Page 5 of 44
Environmental Science & Technology
67
especially at high levels of Cr(VI) contamination.22
68
However, to date, the underlying mechanisms of the enhanced plant Cr(VI) tolerance by AM
69
symbioses are largely unknown. One potential mechanism is that AM symbiosis can improve plant
70
growth via improving plant mineral nutrition (e.g., phosphorus, nitrogen, etc.), thereby indirectly
71
enhancing plant Cr(VI) resistance. For example, in our recent study, AM symbiosis dramatically
72
increased the dry weight of dandelion plants in Cr(VI)-contaminated soils by increasing plant P
73
uptake.22 The larger plant biomass may dilute Cr in the plants, thus minimizing the Cr
74
phytotoxicity, resulting in the so-called “growth dilution effect”.23 Another possible mechanism is
75
that the extraradical mycelium (ERM) of mycorrhizal roots may directly reduce Cr(VI) to Cr(III),
76
immobilize Cr(III) and restrict Cr(III) transfer to plants, similar to Cd24 and U.25,26 However, solid
77
evidence is needed to test this hypothesis. Because Cr(VI) is much more toxic than Cr(III),
78
organisms usually reduce Cr(VI) to Cr(III) to relieve Cr toxicity,27, 28, 29 and this reduction process
79
may also occur in ERM. Besides, the ERM is known to have a high cation exchange capacity
80
(CEC) and can retain large quantities of metals (especially those metal cations),30,31 thus
81
obstructing the entrance of toxic metals into the root cytoplasm. Furthermore, even if the heavy
82
metals taken up by the ERM are transported to the roots, the metals may not actually enter root
83
cells across the symbiotic surface.24,32 Therefore, we hypothesized that the ERM can take up
84
Cr(VI), and reduce it to Cr(III), then transport Cr(III) to mycorrhizal roots but retains most of the
85
Cr(III) in its own structures. The next question is how AMF immobilize Cr(III). As Cr(III) can be
86
complexed with phosphate, carboxylate, amine acids, thiols, hydroxide, etc., within organisms,33-35
87
therefore, we further hypothesized that AMF diminish Cr(III) toxicity by combining Cr(III) with
88
phosphate, thiols, histidine, or carboxylate. 5
ACS Paragon Plus Environment
Environmental Science & Technology
89
The objectives of this study were therefore to assess Cr(VI) uptake, translocation and
90
transformation by AMF and to uncover the underlying mechanisms of enhanced plant resistance to
91
Cr(VI) by AM symbiosis. A two-compartment root-organ cultivation system36 was adopted,
92
through which we investigated not only the direct Cr(VI) absorption and translocation by ERM
93
without interference from undesirable microorganisms but also obtained ERM material for further
94
mechanism studies. Inductively coupled plasma mass spectrometry (ICP-MS) was used to
95
determine Cr concentrations in the roots and the ERM, and synchrotron radiation micro-focused
96
X-ray fluorescence (SR µ-XRF) was used to detect Cr in the ERM in situ. To localize Cr in the
97
ERM and the mycorrhizal roots, a scanning electron microscope equipped with energy dispersive
98
spectroscopy (SEM-EDS) and a transmission electron microscope equipped with energy
99
dispersive spectroscopy (TEM-EDS) were used. Additionally, chemical speciation of Cr in the
100
ERM was analyzed by using X-ray absorption fine structure (XAFS) spectroscopy with
101
synchrotron radiation, which has developed in recent years into a powerful tool for the study of
102
metal speciation in biological and environmental samples.29,33,37
103 104
MATERIALS AND METHODS
105
Root-Organ Cultures. Agrobacterium rhizogenes (Ri T-DNA)-transformed carrot (Daucus
106
carotaL.) roots were cultivated in a minimal (M) medium38 solidified by 0.4% (w/v) phytagel
107
(Sigma-Aldrich).36,39 The AM symbiosis in the solidified M medium was then established
108
according to the methods described by St.-Arnaud et al.36 Briefly, the spores of the AM fungus
109
Rhizophagus irregularis DAOM 197198 were first surface-sterilized with Tween 80 and
110
chloramine T solutions, then rinsed in a solution of 1% (w/v) streptomycin sulfate and 0.5% (w/v) 6
ACS Paragon Plus Environment
Page 6 of 44
Page 7 of 44
Environmental Science & Technology
111
gentamycin sulfate, and finally spread on a 1.5% (w/v) water agar plate. The Ri
112
T-DNA-transformed carrot roots were initiated on the modified M medium. Colonization was
113
achieved by placing 10-15 germinated AMF spores near the apex of a 2-cm-long carrot root piece.
114
The monoxenic culture was then incubated in the dark at 25℃. After 3 months, approximately
115
4,000 spores associated with approximately 80 mg (dry weight) of roots had developed in each
116
Petri plate. The transformed carrot roots without AMF inoculation were also maintained as
117
experimental controls.
118
Before the formal experiment, the Cr concentrations in solidified and liquid M media and
119
nonmycorrhizal and mycorrhizal roots were determined using an ICP-MS (7500a Agilent
120
Technologies, California, USA) after digestion by HNO3. The Cr concentrations were as follows:
121
solidified M medium, 1.92×10-4 mmol L-1; liquid M medium, 1.92×10-4 mmol L-1; nonmycorrhizal
122
roots, 0.57 mg kg-1 (dry weight); mycorrhizal roots, 0.39 mg kg-1 (dry weight).
123
Experimental Design. Two-compartment Petri plates (9 cm in diameter, Figure S1)36 were used
124
for the formal experiment. Cultures were initiated in a root compartment composed of 30 mL M
125
medium gelled with 0.4% (w/v) phytagel. The counterpart hyphal compartment allowed only
126
growth of extraradical hyphae, allowing us to investigate the direct interactions of hyphae with
127
Cr(VI).
128
We tested two root compartment treatments: mycorrhizal or nonmycorrhizal. For the mycorrhizal
129
treatment (“+M”), a 2-cm2 piece of solid M medium containing carrot roots colonized by
130
Rhizophagus irregularis was introduced, whereas for the nonmycorrhizal treatment (“-M”), a
131
2-cm2 piece of solid M medium containing uninoculated carrot roots was introduced.39 The initial
132
dry weight of both mycorrhizal and nonmycorrhizal roots was approximately 0.90 mg. The Petri 7
ACS Paragon Plus Environment
Environmental Science & Technology
133
plates were incubated horizontally in an inverted position at 25℃ in the dark for 6 weeks. The
134
Petri plates were then set upright for 3 additional weeks, and the hyphal compartments were filled
135
with 10 mL liquid M medium without sucrose and phytagel. The extraradical mycelium (ERM) of
136
the “+M” treatment started to cross the central wall between the root compartment and the hyphal
137
compartment and proliferated in the liquid medium. The cultures were examined weekly, and the
138
roots that crossed the central wall were trimmed to prevent their growth into the hyphal
139
compartment. After three additional weeks, the cultures were ready for the formal experiment.
140
Cr(VI) Treatment. For each“-M” and “+M” treatment, we applied two treatments in the hyphal
141
compartment: additions with or without Cr(VI) (i.e., the “+Cr” or “-Cr” treatments). In the “+Cr”
142
treatment, 10 mL of liquid M medium without sucrose, vitamins, potassium iodide, phosphate and
143
EDTA-Fe (in the form of NaFeEDTA) [to avoid potential Cr(VI) reduction by vitamins or
144
potassium iodide, and Cr(III) precipitation by phosphate, or complexation with EDTA in the
145
medium] but with Cr(VI) (in the form of K2CrO4 at a concentration of 0.05 mmol L-1; the pH was
146
adjusted to 5.5) was added to the hyphal compartment after removal of the old medium via pipette.
147
Similarly, 10 mL of the same liquid M medium without Cr(VI) but with the same amount of K (in
148
the form of KCl) as the K2CrO4 in the “+Cr” treatment was applied in the “-Cr” treatment. To
149
investigate whether Cr(VI) uptake and translocation by hyphae was an active process and whether
150
Cr(VI) transformation by AMF was a metabolic process, two control treatments were arranged.
151
For one control, metabolic activity of the hyphae was inhibited by formaldehyde (2% v/v) for 24 h
152
according to Rufyikiri et al.25 After 24 h, we removed the formaldehyde solution and washed the
153
hyphae carefully with Milli-Q water before Cr(VI) was applied to avoid direct interaction between
154
formaldehyde and Cr(VI). In the second control, the hyphal activity was inhibited by 0.5 mmol L-1 8
ACS Paragon Plus Environment
Page 8 of 44
Page 9 of 44
Environmental Science & Technology
155
2,4-dinitrophenol (DNP), a respiration inhibitor and an uncoupler of oxidative phosphorylation,
156
which can cause dissipation of the proton motive force across membranes, thereby inhibiting
157
active metal uptake by fungi.40 By comparing the Cr concentrations in the DNP- and
158
Cr(VI)-treated hyphae with the Cr concentrations in the solely Cr(VI)-treated hyphae, we aimed to
159
identify whether Cr became adsorbed onto the fungal surface. Therefore, we performed a total of 6
160
treatments, as shown in Table 1. Treatments “-M-Cr” [noninoculation in the root compartment and
161
no Cr(VI) addition in the hyphal compartment] and “-M+Cr” [noninoculation in the root
162
compartment and 0.05 mmol L-1 Cr(VI) addition in the hyphal compartment] were replicated 8
163
times, whereas treatments “+M-Cr” [inoculation in the root compartment and no Cr(VI) addition
164
in the hyphal compartment], “+M+Cr” [inoculation in the root compartment and 0.05 mmol L-1
165
Cr(VI) addition in the hyphal compartment], “+M+CrF” [inoculation in the root compartment and
166
0.05 mmol L-1 Cr(VI) addition in the hyphal compartment after addition of ERM inhibition by 2%
167
(v/v) formaldehyde] and “+M+CrD” [inoculation in the root compartment and 0.05 mmol L-1
168
Cr(VI) plus 0.5 mmol L-1 DNP addition in the hyphal compartment] were replicated twelve times
169
(with four treatments primarily for microscopy observations and spectroscopy studies). The Petri
170
plates with the various treatments were arranged in a completely randomized order and incubated
171
in the dark at 25℃ for 12 days.
172
Assessment of Variables. By the end of the experiment, the total number of spores in the hyphal
173
compartment and the root compartment were assessed using 1-cm grids marked on the bottom of
174
each Petri plate.25 The roots in the root compartment were separated from the medium by
175
solubilizing the solidified M media in 10 volumes of citrate buffer (pH 6.0), and the roots were
176
then washed carefully with Milli-Q water. The ERM and liquid M medium in the hyphal 9
ACS Paragon Plus Environment
Environmental Science & Technology
177
compartment were collected simultaneously. The ERM samples were washed thoroughly, first
178
with cold 0.5 mmol L-1 Ca(NO3)2 (4℃) for 10 min to remove apoplastic Cr and then with Milli-Q
179
water. A small portion of the fresh roots and hyphae were used for microscopy observations and
180
determination of mycorrhizal colonization, and the remaining portions were frozen in liquid
181
nitrogen and stored at -80℃ for subsequent ICP-MS and spectroscopy analysis.
182
The collected liquid M medium from the hyphal compartment was filtered through a 0.45-µm
183
millipore filter. The pH of the medium was determined by a pH meter (FE20-FiveEasy Plus,
184
Mettler Toledo, Zurich, Switzerland). Cr(VI) was analyzed by the Cr(VI)-specific colorimetric
185
reagent 1,5-diphenylcarbazide (DPC) method.41 The total Cr concentrations were determined with
186
an inductively coupled plasma atomic emission spectrometer (ICP-AES, Prodigy, Leemans, New
187
hampshire, USA) after acidification by HNO3. The Cr(III) concentration was calculated as the
188
difference between the total Cr concentration and the Cr(VI) concentration.
189
The root and ERM samples that were stored at -80℃ were lyophilized with a freeze dryer at -50℃
190
for 48 h. The dried samples were motor-homogenized in liquid nitrogen after weighing. To
191
analyze the Cr concentrations, dried root and hyphal samples were digested in HNO3 using a
192
microwave-accelerated reaction system (Mars 5, CEM Microwave Technology Ltd., Matthews,
193
North Carolina, USA) in a three-step digestion program. The temperature was raised to 120℃ over
194
8 min, held for 3 min, then raised to 160℃ over 11 min, held for 7 min, and finally raised to 180℃
195
over 8 min and held for 15 min. The digested solutions were then held at 140℃ for 4 h to remove
196
the nitric acid. The dissolved samples were then diluted to 10 mL with Milli-Q water. The Cr
197
concentrations were determined using an ICP-MS (model 7500a, Agilent Technologies, California,
198
USA), and the P concentrations were determined using an ICP-AES (Prodigy, Leemans, New 10
ACS Paragon Plus Environment
Page 10 of 44
Page 11 of 44
Environmental Science & Technology
199
hampshire, USA). Blanks and internal standards of bush leaves (GBW07603, China Standard
200
Research Center) and tea leaves (GBW10016, China Standard Research Center) were used to
201
ensure the accuracy of the chemical analyses.
202
To determine AMF colonization, samples of fresh roots were cleared in 10% KOH and stained
203
with Trypan blue following a modified procedure of Phillips and Hayman42 and omitting phenol
204
from the solutions. The intensity of the mycorrhizal colonization on the root system (M%) was
205
determined by the method of Trouvelot et al.(1986) 43 using “mycocalc”software.
206
Scanning Electron Microscopy (SEM) Analysis. Morphological changes in the hyphae exposed
207
to Cr(VI) were examined using SEM. Fresh hyphae in the hyphal compartment of the treatments
208
“+M+Cr”
209
piperazine-1,4-bisethanesulfonic acid (PIPES) (Amresco 0169, Ohio, USA) buffer solution (pH
210
7.2) overnight and then thoroughly washed 3 times with the same buffer solution. The hyphae
211
were then treated with 1% osmium tetroxide for 2 h and dehydrated in a graded acetone series
212
(30%, 50%, 70%, 80%, 90%, 100%). Then, the mycelia were dried, sputter-coated and analyzed
213
using a field emission scanning electron microscope equipped with an energy dispersive X-ray
214
spectrometer (FE-SEM-EDS, SU-8020, Hitachi, Tokyo, Japan).
215
Synchrotron Radiation Micro-focused X-Ray Fluorescence (SR µ-XRF) Analysis. To detect
216
Cr in the hyphae in situ without digestion by HNO3, SR µ-XRF analyses were performed. Fresh
217
hyphae in the hyphal compartment of the treatments “+M+Cr” and “+M-Cr” were attached to 3M
218
tape (cat. 810, Minnesota Mining and Manufacturing Company, Minn., USA) and freeze dried at
219
-25℃ for 72 h. The SR µ-XRF analyses were performed at beamline 4W1B, Beijing Synchrotron
220
Radiation Facility (BSRF). The detailed procedure is described in the Supporting Information (SI).
and
“+M-Cr”
were
fixed
with
2.5%
11
ACS Paragon Plus Environment
glutaraldehyde
in
a
Environmental Science & Technology
221
Transmission Electron Microscope (TEM) Analysis. The TEM analyses were performed to
222
preliminarily identify the locations of Cr at the subcellular level in the mycorrhizal roots in
223
treatment “+M+Cr”, in which only the living ERM in the hyphal compartment was treated with
224
Cr(VI). Fresh roots in the root compartment of treatment “+M+Cr” were fixed overnight with 2.5%
225
glutaraldehyde in a boric acid buffer solution (pH 7.4) and then thoroughly washed 3 times with
226
the same buffer solution, followed by fixation with 1% osmium tetroxide for 2 h. Then, the roots
227
were washed 3 times with the boric acid buffer, dehydrated in a graded acetone series (30%, 50%,
228
70%, 80%, 90%, 100%), and finally embedded in Spurr's resin. Ultrathin sections (90 nm) were
229
obtained using a Leica Ultracut UCT (Leica, Solms, Germany) with a glass knife. The sections
230
were positioned on copper grids, stained with uranyl acetate and lead citrate, and observed under a
231
transmission electron microscope (TEM, H-7500, Hitachi, Tokyo, Japan) operating at 80 keV. The
232
TEM-EDS spectra were collected on a TEM (JEM-2011, JEOL, Tokyo, Japan) equipped with an
233
energy dispersive X-ray spectrophotometer. More than four sections cut from different roots were
234
examined.
235
X-Ray Absorption Fine Structure (XAFS) Spectroscopy Analysis. The lyophilized and
236
homogenized samples were used for XAFS analysis to detect Cr speciation. Roots from the root
237
compartment of treatment “+M+Cr” were pressed into thin slices with a diameter of 10 mm and a
238
thickness of 2 mm and then attached to 3M tape. The ERM samples from the treatments “+M+Cr”,
239
“+M+CrF” and “+M+CrD” were attached directly and uniformly to 3M tape. The XAFS spectra
240
of the ERM and root samples were collected on beamline 1W1B at the Beijing Synchrotron
241
Radiation Facility (BSRF). Detailed methods are described in the SI.
242
Cr Speciation in Mycorrhizal Roots Exposed to Cr(VI): The XANES Study. The XANES 12
ACS Paragon Plus Environment
Page 12 of 44
Page 13 of 44
Environmental Science & Technology
243
experiment was a supplement to the above XAFS study of the ERM exposed to Cr(VI) and aimed
244
to investigate Cr speciation changes in the roots after AMF colonization. Petri plates 9 cm in
245
diameter were filled with 40 mL solidified M medium with an addition of 0.02 mmol L-1 Cr(VI).
246
Mycorrhizal and nonmycorrhizal roots were then introduced separately, resulting in 2 treatments
247
with 8 replicates each. All cultures were incubated in the dark at 25°C for 3 months. The roots
248
were then collected and lyophilized. The Cr concentrations were determined via ICP-MS, and the
249
Cr speciation was analyzed via XANES.
250
Statistical Analysis. Data on the AM development parameters and Cr concentrations in biological
251
samples were subjected to one-way analysis of variance (ANOVA) tests, followed by Duncan’s
252
test (p < 0.05), to determine the significance of the differences between the treatments.
253 254
RESULTS AND DISCUSSION
255
AM Development. The intensity of the mycorrhizal colonization on the root systems (M%) was
256
generally greater than 40%, and no differences were observed among the inoculated treatments
257
(Table S1). The root dry weights ranged from 20 to 45 mg in the root compartment, and AM
258
symbiosis correlated with higher root biomass (p < 0.05) but not with higher plant phosphorus (P)
259
concentration (Table S2). The greater root growth may due to the beneficial effects of AM
260
symbiosis because AM symbiosis can enhance plant uptake of nitrogen and certain micronutrients,
261
such as Cu, Zn, etc.7 The spore numbers in the root compartment of inoculated treatments were
262
between 2000 and 2500, and no differences were observed among the treatments. Numerous
263
hyphae crossed the barrier between the two compartments and developed in the hyphal
264
compartment. The mycelium dry weight in the hyphal compartment was greater than 1.90 mg. The 13
ACS Paragon Plus Environment
Environmental Science & Technology
265
spore numbers in the hyphal compartment were greater than 1000 per plate with no significant
266
differences among the inoculated treatments with the exception of the decreased spore numbers
267
due to Cr(VI) stress (Table S1, p < 0.05). In the noninoculated control treatments, no mycorrhizal
268
colonization was observed; thus, no ERM or spores were present in the hyphal compartment
269
(Table S1).
270
Starting at an initial pH of 5.5, the pH in the hyphal compartment increased significantly in the
271
treatments “+M-Cr” and “+M+Cr” (Table S1, p < 0.05). However, the pH in the hyphal
272
compartment did not change when the ERM was treated with formaldehyde or DNP. The dramatic
273
alkalization of the medium associated with ERM development may result from the active uptake
274
of NO3--N, including the NO3-/H+ symport or NO3–/OH– antiport mechanisms exerted by the
275
fungus. These processes likely overwhelm any other ERM-promoted acidification, leading to a net
276
alkalinization.44,45 The formaldehyde and DNP inhibited this process by inhibiting hyphal activity;
277
thus, the pH of the medium did not change. These results also confirm the effectiveness of the two
278
inhibitors on AMF activity.
279
Cr Concentrations in the Hyphal Compartment. In the treatments “-M-Cr” and “+M-Cr”, Cr
280
was not detected via ICP-AES in the hyphal compartment, demonstrating that no Cr
281
contamination was present in the cultivation system (Figure 1). Compared with the control
282
treatment “-M+Cr”, the total Cr concentrations in the hyphal compartments of the treatments
283
“+M+Cr”, “+M+CrF” and “+M+CrD” were significantly lower (Figure 1, p< 0.05), indicating
284
possible uptake/sorption of Cr by the ERM. Moreover, in the hyphal compartment of the
285
inoculated treatments, some Cr(VI) was reduced to Cr(III) (Figure 1), especially for treatment
286
“+M+CrF”, in which nearly half of the residual Cr(VI) was reduced to Cr(III). This reduction 14
ACS Paragon Plus Environment
Page 14 of 44
Page 15 of 44
Environmental Science & Technology
287
likely resulted from the high reduction abilities of dissolved organic carbons (e.g. polysaccharides,
288
peptides and glycoproteins etc) derived from the fungal biomass (especially the dead or
289
inactivated fungal biomass).46 Alternatively, the Cr(VI) may have been reduced by extracellular
290
soluble reductase excreted by the fungi, similar to that of certain Gram-negative bacteria.47
291
Another possibility is that Cr(VI) was firstly reduced in the fungi or on the fungal surface by
292
chromate reductase or intracellular compounds (e.g., cysteine, glutathione and sulfite), then some
293
Cr(III) in the fungal biomass was released into the growth medium due to hyphal break or
294
complexation of Cr(III) with complexing ligands.48
295
The ERM Adsorbs, Accumulates, and Actively Transports Cr to Mycorrhizal Roots. Figure
296
2A shows that the ERM treated with Cr(VI) can accumulate more than 1000 mg kg-1 Cr. The SR
297
µ-XRF analysis also showed high Cr signals in Cr(VI)-treated in situ ERM (Figure S2),
298
corroborating the results from the ICP-MS. These results are similar to the results from a previous
299
study that documented large accumulations of Cu and Zn in AMF biomass.31 Moreover, the total
300
Cr concentrations in the hyphal compartment solution of the treatments “+M+CrF” and “+M+CrD”
301
were significantly lower than the total Cr concentrations in “+M+Cr” (Figure 1, p < 0.05).
302
Accordingly, the inactivated ERM in the hyphal compartment contained higher Cr concentrations
303
than the living hyphae treated with the same quantity of Cr(VI) (Figure 2A, p < 0.05). Just as
304
mentioned above, Cr(VI) was probably reduced to Cr(III) in the medium, in the fungi or on the
305
fungal surface by chromate reductase or compounds that can serve as electron donors (e.g.,
306
dissolved organic carbons, cysteine, glutathione or sulfite).48 The Cr concentrations in the
307
inactivated hyphae were likely associated with Cr adsorption by the fungal biomass, as the
308
inactivated hyphae exhibited a higher adsorption capacity than living hyphae.30 Similar to 15
ACS Paragon Plus Environment
Environmental Science & Technology
309
previous studies on Serratia spp.49 and Rhizopus arrhizus,50 extracellular polymeric substances
310
(EPSs), including carboxyl or phosphate groups, may play an important role in Cr(III) adsorption.
311
On the other hand, it is essential to point out that Cr(VI) may first be bound to the positively
312
charged complexing groups on fungal surface, and then reduced to Cr(III) by adjacent
313
electron-donor compounds (such as glutathione, cysteine, amino acid, etc)51. This occurs
314
especially under acid conditions, as low pH can make the biomass surface more positive.
315
Therefore the Cr(VI) adsorption may preferably occur on the surface of inactivated AM fungi, as
316
they maintained an acid condition (the pH was 5.3-5.4) (Table S1). The SEM observation showed
317
that the mycelium without Cr(VI) stress was rod shaped and had a smooth surface (Figure 3A).
318
However, when treated with Cr(VI), the hyphae became rougher with small irregular structures on
319
the surface (Figure 3B), which may result from EPS production. The EDS spectrum also
320
supported the presence of Cr on the edge (surface) of mycelia treated with Cr(VI) (Figure 3c),
321
whereas no Cr signal was detected in the mycelia free of Cr(VI) treatment (Figure 3a and Figure
322
3b).
323
The root Cr concentrations were higher in treatment “+M+Cr” than in other treatments, including
324
“+M+CrF” and “+M+CrD” (Figure 2B, p < 0.05), which confirms that living ERM likely can take
325
up and transfer Cr to mycorrhizal roots via a process at least partially metabolic.
326
To reveal the pattern of Cr partitioning between the ERM and the roots, we calculated the
327
percentage of Cr content in the ERM in the hyphal compartment relative to the total Cr content in
328
the ERM and mycorrhizal roots (Table S3). The majority of the Cr was immobilized in the fungal
329
biomass: more than 70% of the Cr taken up or adsorbed by the ERM was retained in the ERM,
330
and less than 30% was translocated to the mycorrhizal roots. 16
ACS Paragon Plus Environment
Page 16 of 44
Page 17 of 44
Environmental Science & Technology
331
TEM-EDS Study of Cr in Mycorrhizal Roots. Using ICP-MS analyses of the Cr concentrations
332
in roots from different treatments (Figure 2B), we found that the ERM can actively transport Cr
333
from the hyphal compartment to the mycorrhizal roots in the root compartment. However, it was
334
unclear whether Cr was actually transferred to the root cells. Like Cd, which was found to be
335
retained in the fungal structures of mycorrhizal Lotus japonicus,24 we hypothesized that the Cr
336
taken up by the ERM was retained primarily in the fungal structures of the mycorrhizal roots. The
337
TEM image showed that certain root cells in the mycorrhizal roots in the root compartment
338
contained arbuscules or intracellular mycelium (Figures 4D and 4E). The EDS data indicated a
339
high Cu signal in the root samples, which may be due to the copper grid used to hold the samples.
340
Although the Cr concentration was low in the mycorrhizal roots, Cr Kα peaks (5.41 keV) were
341
found in both the fungal structures and the nearby root cell cytoplasm (Figures 4a and 4b) but not
342
in the background area. Moreover, the fungal structures tended to have a higher Cr peak (5.41 keV)
343
(with 162 net counts) than the cytoplasm of plant cells (with 110 net counts), potentially indicating
344
Cr immobilization by the fungal structures within the mycorrhizal roots. However, EDS has its
345
own drawbacks with respect to metal mapping, i.e., its low resolution and high detection limit.
346
Therefore, the subcellular localization of Cr via more advanced techniques with higher resolutions
347
and lower detection limits is urgently needed. Regardless, the present TEM-EDS analysis
348
confirms the presence of Cr in the mycorrhizal roots, thus corroborating the ICP-MS results
349
showing that the ERM can actively transport Cr from a distance to the mycorrhizal roots.
350
Reduction of Cr(VI) to Cr(III)-Phosphate Analogues by the Hyphae. Normalized Cr-K-edge
351
X-ray adsorption near-edge spectroscopy (XANES) spectra of standard chemical compounds and
352
the hyphal samples from different treatments are shown in Figure S3. The Cr(VI) standard 17
ACS Paragon Plus Environment
Environmental Science & Technology
353
compounds (K2CrO4 and K2Cr2O7) exhibited a peak at 5993.3 eV, which is significantly different
354
from the Cr(III) standard compounds that produced no peak at 5993.3 eV. The pre-edge feature of
355
Cr(VI) was attributed to the transition of 1s electrons to an empty p–d hybridized orbit of Cr(VI)
356
in a tetrahedral coordination.52 Based on the measurement of this peak, the Cr(III)/Cr(VI) ratio can
357
be assessed in the mixture.53 No hyphal samples exhibited a peak at 5993.3 eV, indicating that Cr
358
in the ERM or on fungal surface was in the form of Cr(III). Same to our previous assumption,
359
Cr(VI) may be reduced by certain enzymes54 or specific biochemical groups (electron donors, e.g.
360
glutathione, carboxyl and amino groups).51 The high Cr(VI) reduction capability of inactivated
361
hyphae demonstrates that Cr(VI) reduction can be a nonmetabolic process,55 possibly resulting
362
from dissolved organic carbons derived from the AMF.46 Because DNP inhibited active Cr(VI)
363
uptake by inhibiting the respiration process,40 the Cr(III) adsorbed on the fungal surface indicated
364
that Cr(VI) reduction likely occur on the fungal surface, or in the hyphosphere and adsorbed on
365
fungal surface.51, 56, 57 Besides, considering the fact that a small proportion of Cr (about 26.4%)
366
was transported to mycorrhizal roots via living ERM (Figure S3) and the root Cr concentration
367
decreased when the ERM in hyphal compartment was inactivated by formaldehyde or DNP
368
(Figure 2), we predict that the living hyphae may also absorb a small proportion of Cr either in the
369
form of Cr(VI) [through sulfate transporter,58 once Cr(VI) is taken up, it will be reduced to Cr(III)
370
immediately in the fungi by enzymes or biochemical groups] or Cr(III) (through iron uptake
371
system59), and Cr(III) is then transported to mycorrhizal roots.
372
To obtain quantitative information regarding Cr speciation in the fungal hyphae, a principal
373
component analysis (PCA) and a linear combination fitting (LCF) analysis were performed on the
374
normalized XANES spectra. The PCA provided a statistical basis for selecting the proper Cr 18
ACS Paragon Plus Environment
Page 18 of 44
Page 19 of 44
Environmental Science & Technology
375
species for inclusion in the LCF model.60 The results suggested that Cr(III)-phosphate,
376
Cr(III)-nitrate, Cr(III)-sulfate, Cr(III)-cysteine, Cr(III)-histidine and Cr(III)-acetate could be
377
selected for LCF analysis because these compounds showed low Chi Sq(