Phosphorus Stress-Induced Changes in Plant Root Exudation Could

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


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


Page 2 of 39

Page 3 of 39


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



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


37,38

Many of

Page 5 of 39


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



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


Page 6 of 39

Page 7 of 39


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



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).


Page 8 of 39

Page 9 of 39


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.



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


Page 10 of 39

Page 11 of 39


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



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


Page 18 of 39

Page 19 of 39


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



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-


Page 21 of 39


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.



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.


Page 22 of 39

Page 23 of 39


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.


and


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 .


Page 24 of 39

Page 25 of 39


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.



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.


Page 26 of 39

Page 27 of 39


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



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.


Page 28 of 39

Page 29 of 39


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.



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.


Page 30 of 39

Page 31 of 39


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



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.


Page 32 of 39

Page 33 of 39

697


(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



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)


Page 34 of 39

Page 35 of 39


(a)

738

739

740

741



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


Page 36 of 39

Page 37 of 39


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



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


Page 39 of 39


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


Phosphorus Stress-Induced Changes in Plant Root Exudation Could

Recommend Documents