Subscriber access provided by Kaohsiung Medical University
Environmental Processes
Phosphorus stress-induced changes in plant root exudation could potentially facilitate uranium mobilization from stable mineral forms. Nimisha Edayilam, Dawn A Montgomery, Brennan O Ferguson, Amith Sadananda Maroli, Nicole Martinez, Brian A. Powell, and Nishanth Tharayil Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05836 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018
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 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 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.
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 39
Environmental Science & Technology
1
Phosphorus stress-induced changes in plant root exudation could
2
potentially facilitate uranium mobilization from stable mineral forms.
3
Nimisha Edayilam†, Dawn Montgomery‡, Brennan Ferguson‡, Amith S. Maroli†, Nicole
4
Martinez‡, Brian A. Powell‡, Nishanth Tharayil*†
5
†Department of Plant & Environmental Sciences, Clemson University, Clemson, SC 29634, US;
6
‡Department of Environmental Engineering and Earth Sciences, Clemson University, 342
7
Computer Court, Anderson, SC 29625, USA.
8
ABSTRACT
9
Apparent deficiency of soil mineral nutrients often triggers specific physio-morphological
10
changes in plants, and some of these changes could also inadvertently increase the ability of
11
plants to mobilize radionuclides from stable mineral forms. This work, through a series of sand-
12
culture, hydroponics and batch-equilibration experiments, investigated the differential ability of
13
root exudates of Andropogon virginicus grown under conditions with variable phosphorus (P)
14
availability (KH2PO4, FePO4, Ca3(PO4)2, and no P) to solubilize uranium (U) from the uranyl
15
phosphate mineral Chernikovite. The mineral form of P, and hence the bioavailability of P,
16
affected the overall composition of the root exudates. The lower bioavailable forms of P (FePO4
17
and Ca3(PO4)2), but not the complete absence of P, resulted in a higher abundance of root
18
metabolites with chelating capacity. With the lower P-bioavailability the physiological amino
19
acid concentration inside of the roots increased, whereas the concentration of organic acids in the
20
roots decreased due to the active exudation. In batch dissolution experiments, the organic acids,
21
but not amino acids, increase the dissolution U from Chernikovite. The root exudate matrix of
22
plants exposed to low available forms of P induced a >60% increase in U dissolution from
23
Chernikovite due to 5-16 times greater abundance of organic acids in these treatments. However,
ACS Paragon Plus Environment
Environmental Science & Technology
24
this was ca. 70% of the theoretical dissolution achievable by this exudate matrix. These results
25
highlight the potential of using active management of soil P as an effective tool to alter the plant-
26
mediated mobilization of U in contaminated soil.
27
INTRODUCTION
28
Long-lived radionuclides released into the biosphere from the nuclear fuel cycle or processing of
29
defense-related legacy wastes have the potential to lead to long-term subsurface soil
30
contamination.1-2 The occurrence and fate of these long-lived radionuclides such as U in the
31
environment is of growing concern because of the associated potential health and ecological
32
effects.
33
concentration of the radionuclide source, the type and strength of the radiation being emitted, and
34
the mobility of radionuclides in soil.6-9 Due to adsorption and co-precipitation reactions,
35
radionuclides in the soil are often present in chemical forms that are insoluble.10-11 Although
36
these immobile forms are of limited environmental concern, various physical, chemical and
37
biological process in soil could enhance the solubility and thus the mobility of these insoluble
38
contaminants. The various process and the extent to which they influence the mobility of
39
radionuclides are of key interest for environmental monitoring since the mobilized radionuclides
40
pose a potential hazard.11 A finer level understanding of the processes that facilitate the mobility
41
of radionuclides in the soil will not only assist in creating a predictive framework for
42
radionuclide transport in the environment, but also will assist in formulating robust management
43
practices that preclude the reactions that mobilize the radionuclide at the source.
3-5
The degree of hazard associated with radioactive contamination is determined by the
44
The biochemical behavior of radionuclides in soils is correlated to its oxidation state.12,13
45
Radionuclides such as Uranium (U), technetium (Tc), and plutonium (Pu) exhibit multiple
46
oxidation states, and unlike the reduced forms that are insoluble, their oxidized forms are highly
ACS Paragon Plus Environment
Page 2 of 39
Page 3 of 39
Environmental Science & Technology
47
soluble.9.14 For instance, tetra and hexavalent states of U are dominant under environmental
48
conditions with hexavalent, U(VI), U is the most stable state under oxidizing conditions and
49
persists in solution as the dioxycation UO22+, whereas tetravalent U(IV) state hydrolyzes readily
50
in water, precipitates as U(IV)-(hydr)oxide phases, and sorbs strongly to mineral surfaces. Thus,
51
U(IV) is generally much less mobile than U(VI) in the environment.15 In addition to the
52
oxidation state, the mobility of radionuclides in soil is also regulated by their sorption onto
53
organic matter and mineral surfaces.16,17 Even though the chemical, physical, and geochemical
54
processes that enhance the mobility of radionuclides in soils have been well studied across
55
different model systems,12,18,19 relatively little is known about the plant-mediated processes that
56
influence the mobility of radionuclides in soils.
57
Potential mechanisms of plant-mediated transport of radionuclides in the soil can be
58
broadly classified into the initial dissolution of radionuclides from the unavailable mineral forms
59
(mobilization), followed by the uptake and accumulation of the mobilized radionuclides within
60
the plant tissues,20 both of which are significantly influenced by the physiology of the plant. The
61
plant uptake and transport of radionuclides in solution has been demonstrated in several lab-scale
62
hydroponic studies.13,14 A few studies also have recorded significant upward movement of
63
radionuclides in plants growing in contaminated soils.21-23 However, plant-mediated processes
64
that regulate the mobilization of radionuclides at the source, and the influence of growing
65
environments in modulating this plant-mediated radionuclide mobilization remains less explored.
66
Soil nutrient deficiency is one of the key environmental stressors that limits growth and
67
productivity of plants.24,25 Plants respond to the availability and distribution of nutrients in soils
68
by modulating their root architecture in response to the resource availability.26-28 Along with the
69
morphological changes, under nutrient deficiency plants also employ efficient physiological
ACS Paragon Plus Environment
Environmental Science & Technology
Page 4 of 39
70
strategies to acquire nutrients from sources that are less soluble.29,30 At the physiological level,
71
much research has focused on the components of root exudates that play important roles in
72
increasing the bioavailability of nutrients.31 A common strategy across many plant species in the
73
face of nutrient deficiency is rhizosphere acidification through proton extrusion.32 In addition,
74
many plants exude organic acid anions and TCA cycle intermediates through root exudates that
75
lower the rhizosphere pH. This lower soil pH potentially mobilizes the metal-bound P and
76
enhances P acquisition capabilities of plants.32 Chelating compounds present in root exudates
77
increase the mobilization and subsequent availability of nutrients because of their ability to form
78
soluble complexes.33 Similarly, enhanced secretion of amino acids and organic acids have been
79
reported under iron (Fe) deficiency.34,35 Recent experimental studies also have demonstrated the
80
physiological capacity of plant roots to effectively remobilize mineral associated soil carbon.
81
Root exudates released into soil respond to elevated CO2 concentration promotes removal of
82
crosslinking metal cation from metal organic complex. 36 Root exudates have also been shown to
83
alter the sorption/desorption dynamics of various contaminants. For instance, organic acids
84
promoted desorption of phenanthrene and naphthalene has been reported recently.
85
the above reactions including chelation and rhizosphere acidification could also influence the
86
dissolution of radionuclides, especially from mineral forms where the radionuclides are
87
complexed with nutrient elements that are essential for plant growth. Thus, nutrient deficiency
88
could potentially influence the ability of plants to mobilize radionuclides. However, little is
89
known regarding the role of nutrient stress in facilitating plant-mediated radionuclide
90
mobilization in the soil. Many of the soils subjected to radionuclide contamination are marginal
91
lands that challenge plant growth, and these sites are often dominated by ruderal plant species
92
that are known for their ability to actively forage for nutrients39, a process that inadvertently
ACS Paragon Plus Environment
37,38
Many of
Page 5 of 39
Environmental Science & Technology
93
could enhance the mobility of radionuclides from mineral forms that are otherwise unavailable.
94
However, the ability of plants to mobilize radionuclides from stable mineral forms, and the
95
environmental factors that regulate the magnitude of this mobilization remains less known.
96
The current study focuses on the plant-induced mobilization of radionuclides under
97
varying availability of P. Phosphorus is a key mineral required for plant growth, and unlike other
98
macronutrients, most P in soil is inaccessible to plants due to the pH-dependent complexation
99
with Ca, Fe and Al.29 To address this apparent deficiency, plants have evolved efficient
100
morphological and physiological strategies for obtaining this important macro-nutrient. These P
101
foraging strategies include changes in root architecture, rhizosphere acidification and exudation
102
of organic compounds with chelating moieties.29 As U is a ubiquitous (∼2−4 mg/kg of soil or
103
sediment) naturally occurring radionuclide.40 Uranium has a strong affinity to associate with P,
104
and is a major constituent in rock phosphate used for making P fertilizer.41 Also, injecting
105
soluble P to soils for the sequestration of U as uranyl phosphate is an in situ remediation strategy
106
that is widely practiced.42 Thus, under P limiting conditions the plant physiological adaptations
107
aimed at mobilizing P could potentially result in a collateral mobilization of U from uranyl
108
phosphates; but the magnitude and regulators of such mobilization remain unknown.
109
In this work, a native bunch grass, Andropogon virginicus, exposed to different mineral
110
forms of P was examined with respect to their potential for U mobilization. Andropogon
111
virginicus is a ruderal, perennial grass species with an extensive deep root system (>2 m) that is
112
tolerant to nutrient poor soil 43 and is one of the dominant ground covers in several radionuclide
113
contaminated areas along Southeastern United States.44 The effect of P stress on U mobilization
114
was determined using a series of sand, hydroponic culture, and batch dissolution studies. We
115
hypothesized that, i) mineral forms of P will alter the root exudate profile of A. virginicus, and
ACS Paragon Plus Environment
Environmental Science & Technology
116
this change in exudate profile would be a function of the bioavailability of P; ii) root exudates of
117
P deficient plants will be proportionally abundant in compound with a greater chelating capacity,
118
and iii) root exudates of A. virginicus produced under P deficiency and will enhance the
119
dissolution U from uranyl phosphate.
120
MATERIALS AND METHODS
121
1. Plant response under resource limitations
122
1.1 Sand culture study
123
The influence of bioavailability and spatial localization of P on root distribution patterns
124
and chemical composition of root exudates of A. virginicus was studied using sand culture study.
125
The experiment was conducted in 25 cm long, 5 cm diameter plastic tubes (PETG, Polyethylene
126
Terephthalate Glycol) filled with autoclaved sand (600 g per tube, 0.5 to 1mm grain size).
127
Phosphorus treatments included three mineral forms with varying bioavailability of P: KH2PO4
128
that represented a completely water-soluble form of P (Ksp = 28), and FePO4 and Ca3(PO4)2 that
129
are sparingly water soluble (Ksp = 1.3×10–22 and 2.0×10–29, respectively). The amount of the P
130
minerals across the three treatments were normalized to supply 36 mg P per 600 g of sand. Both
131
FePO4 and Ca3(PO4)2 were provided in two distinct spatial distribution patterns, minerals were
132
either uniformly distributed throughout the sand or concentrated in a 5 cm patch (Figure 1). In
133
uniform distribution treatments, the tubes were first filled with pure autoclaved sand to a height
134
of 5 cm, and then topped with sand premixed with FePO4 or Ca3(PO4)2 to a height of 17 cm. For
135
patch treatments, the same amount of FePO4 or Ca3(PO4)2 as that in the uniform treatment was
136
distributed in a 5 cm wide band 7 cm below the surface, resulting in a concentrated P distribution
137
3.6 times that of the P in the uniform treatment (Figure 1). The control treatment contained pure
138
autoclaved sand throughout the root zone, and KH2PO4 was provided along with Hoagland’s
ACS Paragon Plus Environment
Page 6 of 39
Page 7 of 39
Environmental Science & Technology
139
nutrient solution to form uniform distribution of P. Due to the high solubility of KH2PO4, patch
140
application was not practical for this treatment. The background P concentration of the
141
autoclaved sand was below the detection limit (0.1 mg P per kg sand).
142
To investigate the role of the rhizosphere microbial community in aiding P mobilization,
143
half the tubes in each of the two treatments (mineral form and spatial distribution of P) were re-
144
inoculated with microbial inoculum native to the rhizosphere of A. virginicus. Details on
145
rhizosphere soil collection and inoculum preparation is given in the supplementary information
146
(SI Section S1). Four replicates were maintained for each of the 12 treatments (three forms of P
147
minerals, two spatial distribution of P, and both autoclaved and re-inoculated soils). Seeds from
148
A. virginicus, collected from Clemson, SC, were planted in a 16 x 8 tray containing a sterile
149
germination mixture. Two-week-old seedlings were transplanted into these tubes and irrigated as
150
needed with distilled water. Nutrient solutions specific to each treatment were supplied at weekly
151
intervals: the control (KH2PO4) treatment received a complete nutrient solution providing 200
152
mg L-1 Ca as CaNO3, 234 mg L-1 K as KNO3 and KH2PO4, 30 mg L-1 P as KH2PO4, 48 mg L-1
153
Mg as MgSO4 ,140 mg L-1 N as NH4NO3, KNO3 and CaNO3 and micronutrients, 0.5 mg L-1 B as
154
H3BO4, 4 mg L-1 Fe as FeSO4, 0.5 mg L-1 Zn as ZnSO4, 0.5 mg L-1 Mn as MnCl2, 0.02 mg L-1 Cu
155
as CuSO4 , 0.01 mg L-1 Mo as (NH4)6MO7O. Chelating compounds including EDTA was
156
avoided from the micronutrient preparations.
157
The calcium phosphate and iron phosphate treatments received a nutrient solution without P
158
providing 200mg L-1 Ca as CaNO3, 195 mg L-1 as KNO3, 48mg L-1 Mg as MgSO4 and 140 mg
159
L-1 N as NH4NO3 and KNO3. The plants were grown in a greenhouse maintained at 30/20 °C
160
day/night temperatures with a 14 h photoperiod for 16 weeks. The carbon assimilation capacity
161
of each plant was measured using Li-COR to compare the carbon assimilation rate across the
ACS Paragon Plus Environment
Environmental Science & Technology
162
treatments. At the termination of experiment, the plants were harvested, sectioned into leaves and
163
roots, and fresh biomass was recorded. The bulk sand was discarded and the sand adhering to the
164
roots were gently removed and collected. Subsamples of root were stored in 70% ethanol for
165
determining the percent mycorrhizal colonization across different P treatments. Remaining root,
166
shoot, and rhizosphere soils were rapidly frozen on dry ice and stored in a freezer at -80°C for
167
further analyses.
168
Extraction and analysis of metabolites
169
Metabolites from the roots exposed to the P treatments were extracted using the method reported
170
in previous studies45,46 with minor modifications. Briefly, frozen root samples were finely ground
171
with dry ice using a mortar and pestle and stored at -800 C. Approximately, 1.0 g of the ground
172
samples were placed in a 15-mL centrifuge tube, and 4mL of ice-cold methanol: propanol
173
(1:1, v/v) was added to each. The tubes were vortexed for 20 s and then homogenized by
174
sonication at 50% amplitude for 3 min and vortexed again for 20 s. This mixture was
175
centrifuged for 5 min at 2500 rpm and the supernatant was collected. A 200 µL aliquot of the
176
supernatant was transferred to a glass insert containing 20 µL of a mix of retention time lock d27-
177
myristic acid (2 mg mL–1) and internal standard ribitol (500 µg mL–1) and dried completely under
178
nitrogen. Twenty µl of freshly prepared methoxylamine (20 mg mL–1) solution in pyridine was
179
added, and the vials were incubated at 60°C for 90 min followed by trimethyl silylation with 90
180
µL of N-methyl-N (trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane
181
(TMCS) for 30 min at 40 °C. These derivatized samples were then analyzed using a GC-MS
182
(Agilent 7980, Agilent Technologies, Santa Clara, CA). Details on GC-MS parameters and
183
spectral identification is provided in the Supporting Information (SI, section S2).
ACS Paragon Plus Environment
Page 8 of 39
Page 9 of 39
Environmental Science & Technology
184
1.2 Hydroponic study
185
To assess the U dissolution efficiency of root exudates of A. virginicus released under P
186
deficiency a hydroponic study was conducted using various mineral forms that differed in their P
187
availability. The exudates from the hydroponic study were further characterized to identify the
188
major compounds that could be responsible for the dissolution of uranyl- phosphate. Following
189
germination, each seedling was transplanted to 2 L glass jars containing half-strength Hoagland’s
190
solution that contained all macro and micro nutrients, but without any EDTA. The jars were
191
covered with aluminum foil to prevent light from interfering with root growth. The nutrient
192
solution was aerated continuously using an air pump and the nutrient solution was replaced with
193
fresh solution every three days to reduce the microbial buildup. The plants were grown in this
194
media for 40 days to facilitate the development of an extensive, healthy root system (SI Image
195
S1). Before the application of the P treatments, roots of the plants were gently but thoroughly
196
rinsed with deionized water multiple times, and all plants were kept in a P free, continuously
197
aerated, nutrient solution for 8 hours in dark to remove any traces of P that adhered to the roots.
198
Plants of uniform visual characteristics (number of tillers, root length, and stem height) were
199
selected and randomly assigned to four treatment groups, P supplied as KH2PO4, Ca3(PO4)2,
200
FePO4, and no P, with five replicates per treatment. The plants were grown in their appropriate
201
modified Hoagland’s solution (SI section S3) for 72 hours with constant aeration; then 50 mL of
202
the solution in each flask was sampled using a syringe. Immediately after collection, the solution
203
was filter sterilized using 0.22-micron nylon membrane filters, and stored at -80℃ until analysis
204
of root exudates. Because of the co-occurring high salt content of the nutrient media, instead of
205
the MSTFA derivatization, the samples were subjected to ethyl chloroformate (ECF)
206
derivatization prior to GC-MS analysis to quantify the metabolites in the hydroponic solution.
ACS Paragon Plus Environment
Environmental Science & Technology
207
The quantity of specific amino acids and organic acids was measured using external calibration
208
curves. Details on ECF derivatization method is provided in the Supporting Information (SI
209
section 4).
210
2. Effect of compounds identified in root exudates on the dissolution of uranyl-phosphate
211
Batch dissolution experiments were conducted to investigate the ability of various
212
compounds identified in the root exudates of A. virginicus to mobilize U from Chernikovite
213
(UO2HPO4•4H2O, lg Ksp; -24). A preliminary batch dissolution experiment was conducted using
214
citric acid,47 thereby optimizing (i) the amount of substrate (UO2HPO4•4H2O, (ii) the
215
concentration of the ligand and (iii) the substrate/solution ratio that would result in 50-60%
216
dissolution of the U from the UO2HPO4•4H2O. Based on the preliminary results (SI Figure S4)
217
0.5 mM of ligand, 25 mg uranyl-phosphate, and a 1:400 (0.0025g mL-1) solids/solution ratio was
218
used for the batch dissolution studies. Following optimization, batch dissolution experiments
219
were conducted on 13 compounds (ligands) identified as abundant in the root exudates of A.
220
virginicus. A background electrolyte solution of 0.01 M NaCl was used in the preparation of the
221
individual ligand stock solutions. Uranyl phosphate (0.025 g) was added to each of the 15 ml
222
centrifuge tubes, followed by 10 mL of the appropriate ligand solution. The control samples were
223
prepared in the same manner but with distilled water. The pH of the solution was then adjusted to
224
6 by adding small volumes of 0.1 M HCl or 0.1M NaOH. All samples were placed on an end-
225
over-end mixer for 24 hours at room temperature. After the mixing period, a 1.3 mL aliquot from
226
each tube was centrifuged at 8000 rpm for 20 min. The supernatant was filtered through a 0.22
227
µm syringe filter, and a 0.1 mL aliquot was diluted with 9 mL of 2% HNO3 for analysis via
228
inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Scientific XSeries 2). The
ACS Paragon Plus Environment
Page 10 of 39
Page 11 of 39
Environmental Science & Technology
229
release of U from the uranyl-phosphate was expressed as the percentage of U released in relation
230
to the total U content in the uranyl phosphate.
231
3. Root exudates under P deficiency after uranium dissolution
232
The ability of the overall root exudate matrix to mobilize U from uranyl-phosphate was
233
explored through a batch dissolution study similar to the one described previously. However,
234
instead of an individual ligand solution, we used hydroponic solutions exposed to various
235
nutrient treatments collected from Study 1.2. A batch-dissolution study was conducted using
236
uranyl-phosphate and collected root exudates in a solid/solution ratio 1:400; the pH of the
237
solution was adjusted to 6 by adding small volumes of 0.1 M HCl or 0.1 M NaOH. The samples
238
were incubated on a rotator for 24 hours. The control samples were prepared with fresh
239
Hoagland solution using the same process. The release of U from uranyl-phosphate was analyzed
240
as described above.
241
Statistical analysis
242
A two-way analysis of variance (ANOVA) was used to compare the main and interactive effects
243
of inoculum (with and without inoculum) and nutrient treatments (KH2PO4, FePO4, Ca3(PO4)2,
244
and no P) on the biomass (root and shoot) and carbon assimilation capacity (photosynthesis)
245
followed by Tukey’s HSD post hoc test. All differences were reported to be significant at P
amino acids. Although the
377
possibility of amino acids being involved in the chelation of metal ions has been discussed in
378
previous studies,61,71 observations from the current study indicate that, compared to organic
379
acids, amino acids have a lower effect on the release of U from uranyl-phosphate. The ligands
380
selected for this study contain COOH and NH2 groups as electron pair donor sites that attract
381
electron-deficient metals. The net charges of individual ligands vary depending on the degree of
382
dissociation of these functional groups, which is a function of the pH of the media.72 Most
383
chelating agents are less reactive at low pH,73 and pH range 5-6 generally leads to the
384
dissociation of the carboxyl groups, and as the pH increases further, the amino group are
385
deprotonated at pH 9-10.72 The organic acids used in this study carry 2-3 negatively charged
386
carboxyl groups, which form strong chelates with metals.72 Thus, at the pH used in this study (pH
387
6) the dissolution potential of organic acids was higher than the other ligands. In addition, types
388
and position of functional groups in the organic acids are most important in regulating metal
389
dissolution and subsequent leaching.73 For instance, comparing three dicarboxylic acids such as
ACS Paragon Plus Environment
Environmental Science & Technology
390
malic acid, fumaric acid and succinic acid; malic acid was the most effective in mobilizing the U
391
from uranyl-phosphate due to the presence of alpha-hydroxyl group. These results emphasize the
392
importance of type of organic acids produced under P stress in dissolving/mobilizing U from
393
uranyl-phosphate in the soil.
394
3. Differential influence of root exudates produced under varying P-bioavailability on U
395
dissolution
396
Root exudates present a complex, multi-compound matrix, where individual compounds could
397
facilitate or antagonize the chelating capacity of the companion compounds. This, in turn, will
398
alter the chelation capacity of the overall exudate matrix. Batch dissolution studies were
399
conducted with the root exudates of A. virginicus exposed to different P treatments to determine
400
the differential ability of the overall exudate matrix to influence the dissolution of Chernikovite.
401
Root exudates from plant grown under no-phosphate and KH2PO4 had a lower effect on U
402
dissolution (20% dissolution). However, the root exudates collected from A. virginicus exposed
403
to less available forms of P treatments (FePO4) resulted in 70% dissolution of U from
404
Chernikovite (Figure 6b). The amount of U dissolution could be partly explained by the root
405
exudate composition- plants exposed to no-P and KH2PO4 treatments had a lower concentration
406
of organic acids whereas, the organic acids in the root exudates of plants exposed to FePO4
407
treatments were 5-16 times higher. The hydroponic study was not conducted in an axenic
408
condition since the presence of microbial cells and metabolites are required triggers to elicit
409
natural root exudation. Thus, the possibility that part of the observed mobilization could have
410
been from microbial exudates in the medium cannot be ruled out. The batch dissolution study
411
using the root exudates from hydroponic experiment, though instrumental in elucidating the
412
potential of multi-compound root exudates in mobilizing U from mineral forms, would
ACS Paragon Plus Environment
Page 18 of 39
Page 19 of 39
Environmental Science & Technology
413
overestimate the root exudate mobilization of U from soil matrices. This is because compounds
414
in soils the root exudates will be actively degraded by microbes, sorbed on to soil minerals, or
415
reabsorbed by plants, which in turn reduce the amount of chelating compounds in the
416
rhizosphere.
417
The percent dissolution of U from Chernikovite by the root exudates was 25 % lower than the
418
sum total of the dissolution potential of individual organic compounds (SI Figure S5). While the
419
concentration of individual organic compounds used for the batch dissolution study was
420
~0.5mM, total organic acid exuded under low P availability (FePO4) was 10 times greater (SI
421
Figure S3). Under P stress, chelation appears to be the major mechanism for P solubilization
422
form its metal-bound complexes (FePO4, Ca3(PO4)2), and thus would reduce the chelating
423
compounds that are available to complex with U. For instance, experimental results show that
424
presence of citric acid increases dissolution of U due to the formation of U(VI)-citrate
425
complexes. However, the formation of U(VI)-citric acid complexes could be regulated by the
426
dissolution of iron from FePO4 in the hydroponic solution as a consequence of the formation of
427
iron-citric acid complexes. Citrate can also form complexes with other micronutrients in the
428
hydroponic solution such as manganese, zinc, and copper.74 Thus, the presence of other metal
429
elements in the system can be one of the factors limiting the mobilization and potential leaching
430
of U in the soil. In addition, the natural exudates from plant roots are a complex mixture of
431
different class of biomolecules 37, and will vary substantially from the individual organic ligands
432
in function and character. Only small proportions compounds in the exudates, mostly low
433
molecular substances such as organic acids, sugars, amino acids, can be identified with a greater
434
molecular-level accuracy, whereas polymeric compounds such as proteins and carbohydrates are
435
often difficult to characterize. This has been shown previously for real and artificial root
ACS Paragon Plus Environment
Environmental Science & Technology
Page 20 of 39
37
436
exudates (ARE), where ARE contained substantially higher DOC than plant root exudates.
437
Thus, there could be many more compounds in the root exudates of A. virginicus, some of which
438
could synergize or antagonize the overall dissolution of U form uranyl-phosphate, which would
439
have resulted in the observed lower mobilization of U from uranyl-phosphate by root exudates.
440
Although heavy metals dissolution as a function of root exudation has been studied before,74 our
441
study links the plant stress response to U mobilization via changes in the composition of root
442
exudates. Overall, the results indicate that when exposed to moderate nutrient stress, contributed
443
by low available forms of P, plants increase the amounts of chelating compounds in root
444
exudates to mobilize the sparingly available form of P in the soil. This, in turn, could potentially
445
facilitate the mobilization of U from uranyl-phosphate. Our results suggest that nutrient stress
446
could be one of the key factors that regulate plant-mediated mobilization of radionuclides in
447
soils. However, there are multiple fates for root exudates including microbial degradation, and
448
only a smaller fraction of the overall exuded compounds take part in nutrient foraging. Hence the
449
process of root exudate mediated mobilization of U would be slower under field conditions, but
450
could contribute significantly to the long-term soil transport of U at decadal time scale,
451
especially since the half-life of U could far exceed this time frame. Uranium is a key
452
contaminant of concern at several Department of Energy sites including the Savannah River Site
453
(SRS) in South Carolina. Additionally, soil injection of P is a remediation strategy that is
454
actively pursued across contaminated sites to decrease the mobility of U by forming a uranyl-
455
phosphate precipitate. Findings from this study suggest a possibility of enhanced plant-mediated
456
remobilization of U from the sequestered uranyl-phosphate, especially in soils with low P
457
availability. More importantly, though species-specific, the results from our study system
458
highlight the potential to use active soil P management as a viable strategy to regulate the plant-
ACS Paragon Plus Environment
Page 21 of 39
Environmental Science & Technology
459
mediated U mobilization from contaminated soils. Although plant nutrient foraging processes
460
could inadvertently enhance the mobility of U from uranyl-phosphate, there are multiple fates
461
associated with mobilized U under natural environmental conditions. For example, once
462
mobilized the uptake of U from solution has been shown to occur across many plant species.75,76
463
However, if the mobilization of radionuclides exceeds their uptake then the radionuclides could
464
migrate down and potentially contaminate groundwater. Thus, future studies are still needed to
465
gain better insight into the relationship between soil P status in terms of plant bioavailability and
466
the associated U uptake and transport in plants and soil.
467
AUTHOR INFORMATION
468
Corresponding Author
469
*Phone: (864)656-4453 E-mail:
[email protected] 470
Notes
471
The authors declare no competing financial interest.
472
ACKNOWLEDGEMENTS
473
We thank Dr. Vidya Suseela for assisting with the photosynthesis measurements and analysis of
474
root metabolomics data. This material is based upon work supported by the U.S. Department of
475
Energy Office of Science, Office of Basic Energy Sciences and Office of Biological and
476
Environmental Research under Award Number DE-SC-0001253, and DE-SC-0010832. This is
477
technical contribution No. ----- of the Clemson University Experiment Station.
478
Supporting Information.
ACS Paragon Plus Environment
Environmental Science & Technology
479
Section S1: Preparation of soil inoculum, tissue phosphorus analysis. Section S2: GC parameters.
480
Section S3: Modified hoagland solution. Section S4: ECF derivatization. Image S1: Root image
481
from hydroponic culture study. Image S2: Mycorrhizal staining. Figure S1: Effect of phosphorus
482
treatment on root to shoot weight ratio. Figure S2: Abundance of organic acids in the rhizosphere
483
sand. Figure S3: Concentration of identified organic acids and amino acids in the root exudates
484
in hydroponic media. Figure S4: Dissolution of U form uranyl-phosphate using different
485
concentration of citric acid and uranyl-phosphate. Figure S5: Comparison between observed U
486
dissolution with root exudate matrix and expected dissolution with all individual ligands together
487
Reference
488
(1) Vodyanitskii, Y. N. Chemical Aspects of U Behavior in Soils: A Review. Eurasian Soil Sci.
489
2011, 44 (8), 862–873.
490
(2) Gupta, D. K., & Walther, C. Radionuclide contamination and remediation through plants.
491
Springer International Publishing. 2014
492
(3) Keith, S.; Doyle, JR. et al. Agency for Toxic Substances and Disease Registry (ATSDR).
493
2012. Toxicological profile for Radon. Atlanta, GA: U.S. Department of Health and Human
494
Services, Public Health Service.
495
(4) Keepax, R. E.; Moyes, L. N.; Livens, F. R. Speciation of Heavy Metals and Radioisotopes. In
496
Environmental and Ecological Chemistry Volume II, Aleksandar, S., Ed. Encyclopedia of Life
497
Support Systems (EOLSS) 2009, 165-199.
498
(5) Laplace, G.J.; Gilek, M.; Sundell-Bergman, S.;Larsson, C. M. Assessing ecological effects of
499
radionuclides: data gaps and extrapolation issues. J. Radiol.Prot, 2004. 24 (4A), A139.
ACS Paragon Plus Environment
Page 22 of 39
Page 23 of 39
Environmental Science & Technology
500
(6) Gombert, D., Carter, J., Cozzi, A., Jones, R., Matthern, G., Nutt, M., ... & Sorenson, K.
501
Global nuclear energy partnership integrated waste management strategy 2008, US DOE
502
(7) von Gunten, H. R.; Beneš, P. Speciation of Radionuclides in the Environment. Radiochim.
503
Acta, 1995, 69 (1), 1-30.
504
(8) Violante, A.; Cozzolino, V.; Perelomov, L.; Caporale, A. G.; Pigna, M. Mobility
505
Bioavailability of Heavy Metals and Metalloids in Soil Environments. J. Soil Sci. Plant Nutr.
506
2010, 10 (3), 268–292.
507
(9)Cumberland, S. A.; Douglas, G.; Grice, K.; Moreau, J. W. U Mobility in Organic Matter-Rich
508
Sediments: A Review of Geological and Geochemical Processes. Earth-Sci. Rev. 2016, 159,
509
160–185.
510
(10) Knox, A. S.; Seaman, J. C.; Mench, M. J.; Vangronsveld, J. Remediation of Metal- and
511
Radionuclides-Contaminated Soils by In Situ Stabilization Techniques. In Environmental
512
Restoration of Metals-Contaminated Soils. Iskandar, I. K. Ed. CRC Press 2000. pp. 21.
513
(11) Landa, E. Isolation of U Mill Tailings and Their Component Radionuclides from the
514
Biosphere; Some Earth Science Perspectives; Dept. of the Interior, Geological Survey, 1980.
515
(12) Mehta, V. S.; Maillot, F.; Wang, Z.; Catalano, J. G.; Giammar, D. E. Effect of Reaction
516
Pathway on the Extent and Mechanism of U(VI) Immobilization with Calcium and Phosphate.
517
Environ. Sci. Technol. 2016, 50 (6), 3128–3136.
518
(13) Mitchell, N.; Pérez-Sánchez, D.; Thorne, M. C. A Review of the Behaviour of U-238 Series
519
Radionuclides in Soils and Plants. J. Radiol. Prot. 2013, 33(2), R17–R48.
520
(14) Molz, F.; Demirkanli, I.; Thompson, S.; Kaplan, D.; Powell, B. Plutonium Transport in Soil
521
and Plants. In Fluid Dynamics in Complex Fractured-Porous Systems. Faybishenko, B.; Benson,
522
S. M. and Gale, J. E. Eds. John Wiley & Sons, Inc. Hoboken, NJ 2015; pp. 181.
ACS Paragon Plus Environment
and
Environmental Science & Technology
523
(15) Zavodska, L.; Kosorinova, E.; Scerbakova, L.; Lesny, J. Environmental Chemistry of U. HV
524
ISSN 2008, 1418–7108.
525
(16) Santschi, P. H.; Xu, C.; Zhang, S.; Schwehr, K. A.; Grandbois, R.; Kaplan, D. I.; Yeager,
526
C. M. Iodine and Plutonium Association with Natural Organic Matter: A Review of Recent
527
Advances. Appl. Geochem, 2016.
528
(17) Luo, W.; Gu, B. Dissolution of U-bearing minerals and mobilization of U by organic
529
ligands in a biologically reduced sediment. Environ. Sci. Technol . 2011, 45 (7), 2994-2999
530
(18) Goncharova, NV. Availability of Radiocasium in Plant from Soil: Facts, Mechanisms and
531
Modelling. Global Nest J. 2009, 11 (3), 260-266.
532
(19) Pulhani, V. A.; Dafauti, S.; Hegde, A. G.; Sharma, R. M.; Mishra, U. C. Uptake and
533
Distribution of Natural Radioactivity in Wheat Plants from Soil. J. Environ. Radioact. 2005, 79
534
(3), 331–346.
535
(20) McGrath, S. P.; Zhao, J.; Lombi, E. Phytoremediation of Metals, Metalloids, and
536
Radionuclides. Adv Agron, 2002;75, pp. 1–56.
537
(21) Soudek, P.; Petrová, S.; Benešová, D.; Dvořáková, M.; Vaněk, T. U Uptake by
538
Hydroponically Cultivated Crop Plants. J. Environ. Radioact. 2011, 102 (6), 598–604.
539
(22) Velasco, H.; Dos Anjos, R.M.; Ayub, J.J. Radionuclide Uptake by Plants: Soil-to-Plant
540
Transfer Factors, Kinetics of Absorption, and Internal Radionuclide Distribution of 137Cs and
541
40K in South American Species. In. Radionuclide Contamination and Remediation Through
542
Plants, Gupta, D. K. and Walther, C. Eds.; Springer International Publishing; Switzerland 2014;
543
pp 125 .
ACS Paragon Plus Environment
Page 24 of 39
Page 25 of 39
Environmental Science & Technology
544
(23)Mehta, V. S.; Maillot, F.; Wang, Z.; Catalano, J. G.; Giammar, D. E. Effect of Reaction
545
Pathway on the Extent and Mechanism of U(VI) Immobilization with Calcium and Phosphate.
546
Environ. Sci. Technol. 2016, 50 (6), 3128–3136.
547
(24) Zhu, Y.; Fan, X.; Hou, X.; Wu, J.; & Wang, T. Effect of different levels of nitrogen
548
deficiency on switch grass seedling growth. The Crop Journal, 2014; 2 (4), 223-234.
549
(25) He, M.; Dijkstra, F. A.; Zhang, K.; Li, X.; Tan, H.; Gao, Y.; Li, G. Leaf Nitrogen and
550
Phosphorus of Temperate Desert Plants in Response to Climate and Soil Nutrient Availability.
551
Sci. Rep. 2014, 4, 6932.
552
(26) Ahmed, E.; Holmström, S. J. M. Siderophores in Environmental Research: Roles and
553
Applications. Microb. Biotechnol. 2014, 7 (3), 196–208.
554
(27) Theodorou, M. E.; Plaxton, W. C. Metabolic Adaptations of Plant Respiration to Nutritional
555
Phosphate Deprivation. Plant Physiol. 1993, 101(2), 339–344.
556
(28)Lyons, E. M.; Snyder, R. H.; Lynch, J. P. Regulation of Root Distribution and Depth by
557
Phosphorus Localization in Agrostis stolonifera. HortScience. 2008, 43 (7),2203-2209.
558
(29)Müller, J.; Gödde, V.; Niehaus, K.; Zörb, C. Metabolic Adaptations of White Lupin Roots
559
and Shoots under Phosphorus Deficiency. Front. Plant Sci. 2015, 6, 1014.
560
(30)Guy, C.; Kopka, J.; Moritz, T. Plant Metabolomics Coming of Age. Physiol. Plant. 2008,
561
132 (2), 113–116.
562
(31)Giehl, R. F. H.; von Wirén, N. Root Nutrient Foraging. Plant Physiol. 2014, 166 (2), 509–
563
517.
564
(32)Ryan, P. R.; Delhaize, E.; Jones, D. L. Function and Mechanism of Organic Anion
565
Exudation From Plant Roots. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52 (1), 527–560.
ACS Paragon Plus Environment
Environmental Science & Technology
566
(33)Watt, M.; Evans, J. R. Proteoid Roots. Physiology and Development. Plant Physiol. 1999,
567
121 (2), 317–324.
568
(34) Carvalhais, L. C.; Dennis, P. G.; Fedoseyenko, D.; Hajirezaei, M.-R.; Borriss, R.; von
569
Wirén, N. Root Exudation of Sugars, Amino Acids, and Organic Acids by Maize as Affected by
570
Nitrogen, Phosphorus, Potassium, and Iron Deficiency. Z. Pflanzenernähr. Bodenk. 2011, 174
571
(1), 3–11.
572
(35) Li, G.; Kronzucker, H. J.; Shi, W. The Response of the Root Apex in Plant Adaptation to
573
Iron Heterogeneity in Soil. Front. Plant Sci. 2016, 7, 344.
574
(36) Keiluweit, M.; Bougoure, J. J; Nico, P. S.; Pett-Ridge, J.; Weber, P. K., & Kleber, M.
575
Mineral protection of soil carbon counteracted by root exudates. Nat. Clim. Change. 2015. 5(6),
576
588.
577
(37) Sun, B.; Gao, Y.; Liu, J.; Sun, Y. The impact of different root exudate components on
578
phenanthrene availability in soil. Sci Soc Am J, 2012 76(6), 2041-2050.
579
(38) LeFevre, G. H.; Hozalski, R. M.; Novak, P. J. Root exudate enhanced contaminant
580
desorption: an abiotic contribution to the rhizosphere effect. Environ. Sci. Technol, 2013 47(20),
581
11545-11553.
582
(39) Im, J.; Jensen, J. R.; Coleman, M., & Nelson, E. Hyperspectral remote sensing analysis of
583
short rotation woody crops grown with controlled nutrient and irrigation treatments. Geocarto
584
International. 2009, 24 (4), 293-312.
585
(40) Nolan, J.; & Weber, K. A. Natural U contamination in major US aquifers linked to
586
nitrate. Environmental Science & Technology Letters. 2015, 2 (8), 215-220.
587
(41) Martinez, R. J.; Beazley, M. J.; Sobecky, P. A. Phosphate-Mediated Remediation of Metals
588
and Radionuclides. Advances in Ecology. 2014.
ACS Paragon Plus Environment
Page 26 of 39
Page 27 of 39
Environmental Science & Technology
589
(42) Vermeul, V. R.; Bjornstad, B. N.; Fritz, B. G.; Fruchter, J. S.; Mackley, R. D.Mendoza, D.
590
P.; Newcomer, D. R.; Rockhold, M. L.; Wellman, D. M.; Williams, M. D. 300 Area U
591
Stabilization through Polyphosphate Injection: Final Report. PNNL-18529. Prepared for the US
592
Department of Energy under Contract DE-AC05-76RL01830 2009.
593
(43) de Oliveira Xavier, R.; D’Antonio, C. M. Multiple Ecological Strategies Explain the
594
Distribution of Exotic and Native C4 Grasses in Heterogeneous Early Successional Sites in
595
Hawai’i. J. Plant Ecol 2016, 10 (3), 426-439.
596
(44) Anderson, G. E.; Gentry, J. B.; Smith, M. H. Relationships between Levels of Radiocesium
597
in Dominant Plants and Arthropods in a Contaminated Streambed Community. Oikos. 1973, 24,
598
165–170.
599
(45) Maroli, A.S.; Nandula, V.K.; Dayan, F.E.; Duke, S.O.; Gerard, P.; Tharayil, N. Metabolic
600
profiling and enzyme analyses indicate a potential role of antioxidant systems in complementing
601
glyphosate resistance in an Amaranthus palmeri biotype. J. Agric. Food Chem. 2015, 63 (41),
602
9199-9209.
603
(46) Maroli, A.; Nandula, V.; Duke, S.; Tharayil, N. Stable Isotope Resolved Metabolomics
604
Reveals the Role of Anabolic and Catabolic Processes in Glyphosate-Induced Amino Acid
605
Accumulation in Amaranthus palmeri Biotypes. J. Agric. Food Chem. 2016, 64 (37), 7040–
606
7048.
607
(47) Neaman, A., Chorover, J., & Brantley, S. L.; Effects of organic ligands on granite
608
dissolution in batch experiments at pH 6. American Journal of Science. 2004, 306 (6), 451-473
609
(48) Jacob, J.; Lawlor, DW. Dependence of photosynthesis of sunflower and maize leaves on
610
phosphate supply, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and ribulose-1,5-
611
bisphosphate pool size. Plant Physiol. 1992, 98 (10), 801–807
ACS Paragon Plus Environment
Environmental Science & Technology
612
(49) Pieters, AJ.; Paul, MJ.; Lawlor,DW. Low sink demand limits photosynthesis under Pi
613
deficiency. J Exp Bot. 2001, 52 (52): 1083–1091.
614
(50) Lambers, H.; Shane, M. W.; Cramer, M. D.; Pearse, S. J.; Veneklaas, E. J. Root Structure
615
and Functioning for Efficient Acquisition of Phosphorus: Matching Morphological and
616
Physiological Traits. Ann. Bot. 2006, 98 (4), 693–713.
617
(51) Rao, I. M.; Miles, J. W.; Beebe, S. E.; Horst, W. J. Root Adaptations to Soils with Low
618
Fertility and Aluminium Toxicity. Ann. Bot. 2016, 118 (4), 593-605 .
619
(52) Vance, C. P.; Uhde-Stone, C.; Allan, D. L. Phosphorus Acquisition and Use: Critical
620
Adaptations by Plants for Securing a Nonrenewable Resource. New Phytol. 2003, 157 (3), 423–
621
447.
622
(53) Mc murtrey, J. E. Symptoms on Field-Grown Tobacco Characteristic of the Deficient
623
Supply of Each of Several Essential Chemical Elements, US Department of Agriculture. (No. 04;
624
USDA, FOLLETO 586.). 1938.
625
(54) Hernández, G.; Ramírez, M.; Valdés-López, O.; Tesfaye, M.; Graham, M. A.; Czechowski,
626
T.; Schlereth, A.; Wandrey, M.; Erban, A.; Cheung, F.; et al. Phosphorus Stress in Common
627
Bean: Root Transcript and Metabolic Responses. Plant Physiol. 2007, 144 (2), 752–767.
628
(55) Morcuende, R.; Bari, R.; Gibon, Y.; Zheng, W.; Pant, B. D.; Bläsing, O.; Usadel, B.;
629
Czechowski, T.; Udvardi, M. K.; Stitt, M.; et al. Genome-Wide Reprogramming of Metabolism
630
and Regulatory Networks of Arabidopsis in Response to Phosphorus. Plant Cell Environ. 2007,
631
30 (1), 85–112.
ACS Paragon Plus Environment
Page 28 of 39
Page 29 of 39
Environmental Science & Technology
632
(56) Ning,J.; Cumming, J.R. Arbuscular Mycorrhizal Fungi Alter Phosphorus Relations of
633
Broomsedge (Andropogon virginicus L.) Plants. Journal of experimental botany. 2001, 52 (362),
634
1883-1891.
635
(57) Anderson, R. C.; Hetrick, B.A.D.; Wilson, G.W.T. Mycorrhizal Dependence of Andropogon
636
gerardii and Schizachyrium scoparium in Two Prairie Soils. American Midland Naturalist.1994,
637
2 (132) 366-376.
638
(58) Johnson, N. C.; Graham, J. H.; & Smith, F. A. Functioning of mycorrhizal associations
639
along the mutualism–parasitism continuum. The New Phytologist, 1997,135 (4), 575-585.
640
(59) Reid, M.H.; Bieleski R.L. Response of Spirodela oligorrhiza to Phosphorus Deficiency.
641
Plant Physiol.1970, 46 (4), 609-613
642
(60) Feng, T.-Y.; Yang, Z.-K.; Zheng, J.-W.; Xie, Y.; Li, D.-W.; Murugan, S. B.; Yang, W.-D.;
643
Liu, J.-S.; Li, H.-Y. Examination of Metabolic Responses to Phosphorus Limitation via
644
Proteomic Analyses in the Marine Diatom Phaeodactylum tricornutum. Sci. Rep. 2015, 5, 10373.
645
(61) Sajadi, S. A. A. Metal ion-binding properties of L-glutamic acid and L-aspartic acid, a
646
comparative investigation. Natural Science. 2010, 2 (02), 85.
647
(62) Pennanen, T.; Caul, S.; Daniell, T. J.; Griffiths, B. S.; Ritz, K.; Wheatley, R. E. Community-
648
Level Responses of Metabolically-Active Soil Microorganisms to the Quantity and Quality of
649
Substrate Inputs. Soil Biol. Biochem. 2004, 36 (5), 841–848.
650
(63) Cumming, J. R.; Ning, J. Arbuscular Mycorrhizal Fungi Enhance Aluminium Resistance of
651
Broomsedge (Andropogon Virginicus L.). J. Exp. Bot. 2003, 54 (386), 1447–1459.
ACS Paragon Plus Environment
Environmental Science & Technology
652
(64) Jones, D. L. Organic acids in the rhizosphere–a critical review. 1998, Plant and soil, 205
653
(1), 25-44
654
(65) Arai, Y.; Sparks, D. L. Phosphate reaction dynamics in soils and soil components: A
655
multiscale approach. Adv. Agron. 2007, 94, 135-179.
656
(66) Johnson, S. E.; Loeppert, R. H. Role of organic acids in phosphate mobilization from iron
657
oxide. Soil Sci. Soc. Am. J. 2006, 70(1), 222-234.
658
(67) Jones, D. L. Organic acids in the rhizosphere–a critical review. Plant and soil. 1998, 205(1),
659
25-44. Griffiths, B. S.; Ritz, K.; Ebblewhite, N.; Dobson, G. Soil Microbial Community
660
Structure: Effects of Substrate Loading Rates. Soil Biol. Biochem. 1998, 31 (1), 145–153.
661
(68) Bowsher, A. W.; Ali, R.; Harding, S. A.; Tsai, C.-J.; Donovan, L. A. Evolutionary
662
Divergences in Root Exudate Composition among Ecologically-Contrasting Helianthus Species.
663
PLoS One 2016, 11 (1), e0148280.
664
(69) Zhao, K.; Wu, Y. Rhizosphere calcareous soil P-extraction at the expense of organic carbon
665
from root-exuded organic acids induced by phosphorus deficiency in several plant species. Soil
666
Sci. Plant Nutr, 2014, 60(5), 640-650.
667
(70) Pearse, S. J.; Veneklaas, E. J.; Cawthray, G.; Bolland, M. D.; Lambers, H. Carboxylate
668
composition of root exudates does not relate consistently to a crop species’ ability to use
669
phosphorus from aluminium, iron or calcium phosphate sources. New Phytol, 2007, 173(1), 181-
670
190.
ACS Paragon Plus Environment
Page 30 of 39
Page 31 of 39
Environmental Science & Technology
671
(71) Sharma, S. S.; Dietz, K.-J. The Significance of Amino Acids and Amino Acid-Derived
672
Molecules in Plant Responses and Adaptation to Heavy Metal Stress. J. Exp. Bot. 2006, 57 (4),
673
711–726.
674
(72) Evans, L. J. Chemistry of metal retention by soils. Environ. Sci. Technol. 1989, 23 (9),
675
1046-1056.
676
(73) Gregor, J. E.; Powell, H. K. J.; Town, R. M. Metal-fulvic acid complexing: evidence
677
supporting an aliphatic carboxylate mode of coordination. Science of the total environment,
678
1989, 81, 597-606.
679
(74) Bais, H.P.; Weir, T.L.; Perry, L.G.; Gilroy, S.; Vivanco, J. The Role of Root Exudates in
680
Rhizosphere Interactions with Plants and Other Organisms. Annual Review of Plant Biology.
681
2006, 57, 233-66
682
(75) Soudek, P.; Petrová, Š.; Benešová, D.; Dvořáková, M.; Vaněk, T. U uptake by
683
hydroponically cultivated crop plants. J. Environ. Radioact. 2011, 102(6), 598-604.
684 685
(76) Thompson, S. W.; Molz, F. J.; Fjeld, R. A.; Kaplan, D. I. Uptake, distribution, and velocity
686
of organically complexed plutonium in corn (Zea mays). J. Environ. Radioact, 2012, 112, 133-140
ACS Paragon Plus Environment
Environmental Science & Technology
687
FIGURES
688
689
(a)
(b)
690
691
692
693
694
695
Figure 1. Schematic of nutrient distribution used for the spatial localization of phosphorus. a)
696
Patch treatment b) Uniform treatment. Both treatments received the same amount of P.
ACS Paragon Plus Environment
Page 32 of 39
Page 33 of 39
697
Environmental Science & Technology
(a)
698 699 700 701 702
703
704
705
(b) 706 707 708 709 710 711
(c)
712 713 714 715 716
717
Fig 2. Effect of different phosphorus (KH2PO4, FePO4, Ca3PO4) and reinoculation treatments on
718
(a) root biomass (b) shoot biomass and (c) photosynthesis of Andropogon virginicus grown in
ACS Paragon Plus Environment
Environmental Science & Technology
719
sand culture. Different letters indicate significant difference between treatments (Tukey’s HSD
720
multiple comparison at P ≤ 0.05). Bars represent means ± SE (n = 4)
721
(a)
722 723 724 725 726 727
(b) 728 729 730 731 732 733 734
Fig.3 Concentration of phosphorus in the leaf (a) and root (b) of Andropogon virginicus grown
735
in sand culture exposed to P treatments (KH2PO4, FePO4 and Ca3(PO4)2). Different letters
736
indicate significant difference between treatments (Tukey’s HSD multiple comparison at P ≤
737
0.05). Bars represent means ± SE (n = 4)
ACS Paragon Plus Environment
Page 34 of 39
Page 35 of 39
Environmental Science & Technology
(a)
738
739
740
741
ACS Paragon Plus Environment
Environmental Science & Technology
742
(b)
743 744
Fig.4 Principle component analysis (PCA) and heat map showing changes in 73 metabolites in
745
Andropogon virginicus root tissue grown in sand culture exposed to P treatments (KH2PO4,
746
FePO4 and Ca3(PO4)2). a. PCA score plots of 73 metabolites. Data point represents biological
747
replicates, and the ellipses represent 95% confidence interval. b. Heat map and two-way
748
hierarchical cluster analysis of root metabolites highlighting the metabolite differences in
749
Andropogon virginicus in response to P treatments (KH2PO4, FePO4 and Ca3(PO4)2). The color
ACS Paragon Plus Environment
Page 36 of 39
Page 37 of 39
Environmental Science & Technology
750
of each cell depicts the abundance of individual metabolites, blue indicates a significant decrease
751
and red represent a significant increase in metabolic content. 752 753 754 755 756 757 758 759 760 761 762 763 764 765
766
Fig.5 Average concentration of identified organic acids and amino acids in the root exudates of
767
Andropogon virginicus exposed to P treatments (KH2PO4, FePO4, Ca3(PO4)2 and no P) in
768
hydroponic media for 72 h. Bars represent means ± SE (n = 4).
769 770 771
ACS Paragon Plus Environment
Environmental Science & Technology
772
Page 38 of 39
(a)
773 774 775 776 777 778 779 780 781 782 783 784
(b)
785 786 787 788 789 790 791 792
KH2PO4
No P
FePO4 Ca3(PO4)2
Blank
793
Fig 6. a. Uranium dissolution from uranyl-phosphate at pH 5 following treatment with different
794
solution matrix for 24 hours. a. 0.5 mM organic ligands; b. hydroponic solution from 72 hours of
ACS Paragon Plus Environment
Page 39 of 39
Environmental Science & Technology
795
P treatments (KH2PO4, FePO4, Ca3(PO4)2 and No P). Different letters indicate significant
796
difference between treatments (Tukey’s HSD multiple comparison at P ≤ 0.05). Bars represent
797
means ± SE (n = 4)
798 799 800 801 802 803
Abstract figure
804
ACS Paragon Plus Environment