Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Thioarsenate toxicity and tolerance in the model system Arabidopsis thaliana Britta Planer-Friedrich, Tanja Kühnlenz, Dipti Halder, Regina Lohmayer, Nathaniel Wilson, Colleen Rafferty, and Stephan Clemens Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 21, 2017
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 24
Environmental Science & Technology
1
Thioarsenate toxicity and tolerance in the model system Arabidopsis thaliana
2 3 4 5
Britta Planer-Friedrich1*, Tanja Kühnlenz2, Dipti Halder1, Regina Lohmayer1, Nathaniel Wilson3, Colleen Rafferty2, Stephan Clemens2 1
Environmental Geochemistry, Bayreuth Center for Ecology and Environmental Research (BayCEER),
6
University Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany
7
2
8
Germany
9
3
Department of Plant Physiology, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth,
formerly at Environmental Geochemistry, Bayreuth Center for Ecology and Environmental Research
10
(BayCEER), University Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany; now at Babbage
11
Consultants Limited, 1010 Auckland, New Zealand
12 13 14
* corresponding author: address: Environmental Geochemistry, Bayreuth Center for Ecology and
15
Environmental Research (BayCEER), University Bayreuth, Universitätsstraße 30, 95447 Bayreuth,
16
Germany; phone: +49 921 553999, fax: +49 921 552334; email:
[email protected] 17
1 Environment ACS Paragon Plus
Environmental Science & Technology
Col-0 As
As
=
cad1-3 As
PC > As
PC
11, so it is generally not environmentally relevant 48.
96
Investigating thioarsenates is difficult because the species are pH- and oxygen-sensitive and require
97
specific stabilization (flash-freezing 33 or, in the presence of iron, anoxic storage with ethanol addition
98
36
99
Acidification induces AsS-mineral precipitations
or SPE-based separation 50) and analytical techniques (chromatographic separation at pH 12-13 33). 51
and the transformation of thioarsenates to
48
100
arsenite or arsenate . Therefore, most studies to date only analyze for arsenite and arsenate even
101
in the presence of free sulfide, e.g. Jia and Bao
102
incubations amended with 100 mM sulfate at pH 7.2 but did not look for thioarsenates. Interestingly,
103
in his study on microbially catalyzed As transformation in the rice rhizosphere, Somenahally
104
reported an unidentified As species eluting after arsenate which he speculated was
105
monothioarsenate. In preliminary studies, we have, for the first time, been able to detect mono- and
106
dithioarsenate in paddy soils, both under natural conditions in the field (maximum 10% of total As),
107
as well as in laboratory incubation experiments mimicking sulfate fertilization (maximum 63% of total
108
As).
43
who detected 1.4 mM sulfide in paddy soil
5 Environment ACS Paragon Plus
52
Environmental Science & Technology
Page 6 of 24
109
In summary, both geochemical considerations and direct analytical evidence suggest that
110
thioarsenates form in rice paddy soils, and previous studies confirmed that they can even occur in
111
the presence of iron. Moreover, thioarsenates are expected to show less sorption than arsenite and
112
arsenate to iron plaque in the rhizosphere and so they may be more bioavailable for uptake into the
113
rice plant.
114
There is, to date, no information about uptake, accumulation, toxicity or tolerance of any
115
thioarsenate species in plants. We therefore aimed to obtain the first mechanistic insights into the
116
potential toxicity of monothioarsenate (in comparison to its structural oxyanion analogue arsenate),
117
as the most stable and – based on our own preliminary studies – the dominant thioarsenate species
118
in soil incubations under sulfate-reducing conditions. We chose the model plant A. thaliana for this
119
pilot study because of ease of cultivation and, more importantly, the availability of very useful
120
biological material such as defined mutants for relevant pathways. Many components of As transport
121
and metabolism in plants have been identified and characterized in A. thaliana. For instance, besides
122
the wild type (Col-0), a GSH-deficient (cad2) mutant, a PC-deficient mutant (cad1-3), and a double
123
knock-out mutant for the PC vacuolar transporters (abcc12) are available 53. Direct comparison of
124
Col-0 and these mutants enables direct investigations on the potential role of PCs in detoxification of
125
thioarsenic species. For rice, such mutants are not readily available, yet. The toxicity was tested by
126
growing A. thaliana on agar plates for tolerance assays and in hydroponic cultures containing
127
arsenate and monothioarsenate, measuring growth parameters such as root length and seedling
128
fresh weight as well as accumulation of As and formation of PCs in roots and shoots.
129 130
MATERIALS AND METHODS
131
Plant growth and As treatment. The A. thaliana wild-type Col-0 and the AtPCS1 mutant cad1-3
132
were used for testing toxicity of monothioarsenate on plant growth. Before use in As tolerance
133
assays on agar plates, or cultivation in hydroponic culture, A. thaliana seeds were surface-sterilized
134
by exposure to chlorine gas for 35 min. The medium used for plant growth was based on the
135
modified Hoagland's solution No 2 as described previously 55 (0.28 mM Ca(NO3)2, 0.6 mM KNO3, 0.1
136
mM NH4H2PO4, 0.2 mM MgSO4, 4.63 µM H3BO3, 32 nM CuSO4, 915 nM MnCl2, 77 nM ZnSO4, 11 nM
137
MoO3). According to Chaney 56, Fe was supplied as N,N′-di-(2-hydroxybenzoyl)-ethylenediamine-N,N′-
138
diacetic acid (Fe-HBED) to a final concentration of 5 µM.
139
For tolerance assays on vertical agar plates, medium containing 1% (w/v) sucrose and 0.05% (w/v) 2-
140
(N-morpholino)ethanesulfonic acid (MES) at pH 5.7 that was devoid of microelements, except for Fe,
141
was used. Arsenate [sodium arsenate dibasic heptahydrate (Na2HAsO4 × 7H2O, Fluka)] and
142
monothioarsenate [(Na3AsVO3S × 7 H2O, synthesized in our laboratory according to a previously
6 Environment ACS Paragon Plus
54
Page 7 of 24
Environmental Science & Technology
57
143
described method
; purity 97%, the remainder being arsenate] were added at indicated
144
concentrations between 1 and 200 µM. For comparison, 5 µM arsenite (AsCl3, Sigma-Aldrich, St Louis,
145
MO, USA) were also tested. The medium was solidified with 1% (w/v) Agar Type A (Sigma-Aldrich, St
146
Louis, MO, USA). Plates were sealed with Leucopore tape (Duchefa Biochemie, Haarlem, NLD)
147
followed by stratification for 2 d at 4°C. For plant growth, plates were then incubated for 14 d under
148
long-day conditions (16 h light/ 8 h darkness) at 23°C and 75 µE light intensity. After 14 days, primary
149
root length and seedling fresh weight was determined. To investigate the PC-based detoxification
150
pathway in greater detail, tolerance assays were not only conducted with Col-0 and cad1-3 but also
151
with the mutants cad2 (a GSH biosynthesis mutant)
152
mutant) 25.
153
For analysis of PC and As contents, plants were grown hydroponically for 6 weeks in medium without
154
sucrose, containing 0.05% (w/v) MES and microelements at pH 5.7 as described before
155
stratification, cultivation started in agar-filled PCR tubes in pipette tip boxes for 3 weeks, followed by
156
a transfer into 50 mL tubes (Greiner Bio-One, Kremsmuenster, AUT) for another 3 weeks under short-
157
day conditions (8 h light at 22°C/ 16 h darkness at 17°C) at a light intensity of 150 µE. Medium was
158
changed weekly in order to guarantee a sufficient mineral and oxygen supply. Addition of As species
159
started after 5.5 weeks at approximately same-effect concentrations of 200 µM to Col-0 and 20 μM
160
to cad1-3. Arsenic-exposure lasted for 4 days. The stability of arsenate and monothioarsenate in the
161
externally applied medium was confirmed by analyzing test solutions (without plants) at day 0 and
162
day 4 (Table S1) by ion chromatography (Dionex ICS-3000; AG/AS16 IonPac column, 20-100 mM
163
NaOH at a flow rate of 1.2 mL/min) coupled to the ICP-MS as described earlier 33. Due to a lack of a
164
commercial standard, monothioarsenate was quantified via the arsenate (Na2HAsO4×7H2O, Fluka)
165
calibration curve. The validity of this approach has been shown in a previous publication 33.
166
To investigate the extent of efflux, Col-0 plants that had been grown for 14 days in hydroponic
167
culture at 6.7 µM arsenate and monothioarsenate, respectively, and showed no signs of reduced
168
plant growth, were transferred to As-free medium. After 0, 1, 2, 4, 7, 9, and 11 days 1 mL of aqueous
169
medium was removed and analyzed for total As by inductively coupled plasma-mass spectrometry
170
(ICP-MS, XSeries2, Thermo-Fisher) using oxygen as reaction cell gas (AsO+, m/z 91). The percentage of
171
As released was calculated by multiplying the measured As concentrations in solution with the
172
respective medium volume at each sampling time and referring this absolute concentration to the
173
original concentration of total As stored in the plants after the initial 14 days of As-exposure.
174
Analysis of total As and PCs in roots and shoots. Roots were first carefully washed with distilled
175
water, followed by 2 washing steps with 0.1 M sodium phosphate buffer solution (pH 5.7) and a final
176
washing step with water. Each washing step was performed with 25 mL per root sample at 4°C for 10
58
and abcc12 (a PC vacuolar transport double
7 Environment ACS Paragon Plus
55
. After
Environmental Science & Technology
177
minutes to remove any surface-bound As. Frozen root and shoot samples were ground in liquid
178
nitrogen and the powder stored in the freezer until analysis.
179
For the determination of total As, approximately 100 mg of sample were digested in 3 mL
180
concentrated HNO3 and 2 ml 30% H2O2 in a CEM Mars 5 microwave digestion system (CEM
181
Corporation, Matthews, NC, USA). Digested samples were analyzed for total As by ICP-MS as
182
described above. The microwave blank (HNO3/H2O2) was subtracted from all concentrations and
183
results are reported as mean concentrations with standard deviation of three biological replicates.
184
PCs were determined from pooled root samples and pooled leaves samples including shoots by
185
UPLC-ESI-QTOF-MS as described earlier 55. Briefly, plant material was frozen in liquid nitrogen and
186
ground to homogenous powder followed by extraction with 0.1 % (v/v) trifluoroacetic acid (TFA)
187
containing 6.3 mM diethylene triamine pentaacetic acid (DTPA) and 40.04 µM N-acetylcysteine (NAC)
188
as internal standard. Before derivatization with monobromobimane (mBrb) at 45°C for 30 min, thiol
189
groups were reduced with Tris-(2-carboxyethyl)-phosphine (TCEP). The separation of the mBrb-
190
labelled thiols was performed by a Waters Aquity UPLC systems equipped with a HSS T3 column (1.8
191
μm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA) at an injection volume of 5 µL and the
192
linear binary gradient of water (A) and acetonitrile (B), both acidified with 0.1% (v/v) formic acid, at a
193
flow of 0.5 mL min–1: 99.5 % A, 0.5 % B for 1 min, a linear gradient to 60.5 % B at 10 min, gradient to
194
99.5 % B at 12 min, flushing with 99.5 % B for 1 min, a gradient back to initial conditions in 1 min and
195
an additional re-equilibration for 1 min. The column temperature was set to 40°C. Thiols were
196
detected with a Q-TOF Premier mass spectrometer equipped with an ESI-source (Waters
197
Corporation) operated in the V+ mode. Data were acquired from m/z 300–2000 with a scan time of
198
0.3 s and an inter-scan delay of 0.05 s. For quantification the QuanLynx module of the MarkerLynx
199
software was used. All samples were measured in three technical replicates.
200
Statistical analysis. Root lengths and seedling fresh weights data were statistically analyzed by pair-
201
wise analysis using post-hoc analysis (Tukey Honest Significant Differences (TukeyHSD)) following
202
two-way ANOVA (significance defined as p 1 µM upon exposure to monothioarsenate and
293
> 5 µM upon exposure to arsenate (Figure 1b, d and Figure S2b, d). At concentrations above 100 µM,
294
where Col-0 still had less than 50% growth reduction, cad1-3 was not able to germinate anymore.
295
The IC50 value calculated from the dose response curve of relative root length versus individual As
296
species concentration (Figure S4) was 14.9 ± 0.8 µM and 16.7 ± 1.2 µM for cad1-3 for exposure to
297
monothioarsenate and to arsenate, respectively. The result of an almost 10 times lower tolerance of
298
cad1-3 compared to Col-0 observed in our study is, at least for arsenate, comparable to previous
299
results by Ha et al. 53 and Liu et al. 23 who assessed the role of PC formation in arsenite and arsenate
300
tolerance. For monothioarsenate, our results are the first direct evidence that PC formation plays an
301
important role in thioarsenate detoxification as well.
302
Along with significantly lower growth rates, the arsenic concentration in the roots was 15 times
303
lower in cad 1-3 than in Col-0 for exposure to arsenate, and below detection limit (factor > 50) in cad
304
1-3 exposed to monothioarsenate. The higher accumulation of As in the roots of Col-0 gives further
12, 13
11 Environment ACS Paragon Plus
, enzymatic monothioarsenate
49
and also in the present
Environmental Science & Technology
305
indirect evidence for efficient As-PC complexation and vacuolar sequestration in the roots as it has
306
been described before 23.
307
Direct analytical evidence for PC formation was found in the roots (Figure 4a) and, to a lesser extent,
308
also in the shoots (Figure 4b) of Col-0 both upon exposure to arsenate and monothioarsenate.
309
Analysis using MANOVA indicates that there is a significant relationship between arsenic species and
310
plant genotype with both PC2 and PC3 formation. Absolute PC concentrations were significantly
311
lower after exposure to monothioarsenate compared to exposure to arsenate both in the roots and
312
in the shoots (p0.05), while
314
concentration of PC3 was significantly higher (350 mg/kg fw) than PC2 (172 mg/kg fw) for exposure
315
to monothioarsenate (p
