Phosphorus Stress-Induced Changes in Plant Root Exudation Could

Article Cite This: Environ. Sci. Technol. 2018, 52, 7652−7662

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Phosphorus Stress-Induced Changes in Plant Root Exudation Could Potentially Facilitate Uranium Mobilization from Stable Mineral Forms Nimisha Edayilam,† Dawn Montgomery,‡ Brennan Ferguson,‡ Amith S. Maroli,† Nicole Martinez,‡ Brian A. Powell,‡ and Nishanth Tharayil*,† †

Department of Plant & Environmental Sciences, Clemson University, Clemson, South Carolina 29634, United States Department of Environmental Engineering and Earth Sciences, Clemson University, 342 Computer Court, Anderson, South Carolina 29625, United States

Environ. Sci. Technol. 2018.52:7652-7662. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/27/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: Apparent deficiency of soil mineral nutrients often triggers specific physio-morphological changes in plants, and some of these changes could also inadvertently increase the ability of plants to mobilize radionuclides from stable mineral forms. This work, through a series of sand-culture, hydroponics, and batch-equilibration experiments, investigated the differential ability of root exudates of Andropogon virginicus grown under conditions with variable phosphorus (P) availability (KH2PO4, FePO4, Ca3(PO4)2, and no P) to solubilize uranium (U) from the uranyl phosphate mineral Chernikovite. The mineral form of P, and hence the bioavailability of P, affected the overall composition of the root exudates. The lower bioavailable forms of P (FePO4 and Ca3(PO4)2), but not the complete absence of P, resulted in a higher abundance of root metabolites with chelating capacity at 72 hrs after treatment application. In treatments with lower P-bioavailability, the physiological amino acid concentration inside of the roots increased, whereas the concentration of organic acids in the roots decreased due to the active exudation. In batch dissolution experiments, the organic acids, but not amino acids, increase the dissolution U from Chernikovite. The root exudate matrix of plants exposed to low available forms of P induced a >60% increase in U dissolution from Chernikovite due to 5−16 times greater abundance of organic acids in these treatments. However, this was ca. 70% of the theoretical dissolution achievable by this exudate matrix. These results highlight the potential of using active management of soil P as an effective tool to alter the plant-mediated mobilization of U in contaminated soil.

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INTRODUCTION Long-lived radionuclides released into the biosphere from the nuclear fuel cycle or processing of defense-related legacy wastes have the potential to lead to long-term subsurface soil contamination.1,2 The occurrence and fate of these long-lived radionuclides such as U in the environment is of growing concern because of the associated potential health and ecological effects.3−5 The degree of hazard associated with radioactive contamination is determined by the concentration of the radionuclide source, the type and strength of the radiation being emitted, and the mobility of radionuclides in soil.6−9 Due to adsorption and coprecipitation reactions, radionuclides in the soil are often present in chemical forms that are insoluble.10,11 Although these immobile forms are of limited environmental concern, various physical, chemical, and biological processes in soil could enhance the solubility and thus the mobility of these insoluble contaminants. The various processes and the extent to which they influence the mobility of radionuclides are of key interest for environmental monitoring because the mobilized radionuclides pose a potential hazard.11 A finer level understanding of the processes that facilitate the © 2018 American Chemical Society

mobility of radionuclides in the soil will assist in not only creating a predictive framework for radionuclide transport in the environment but also formulating robust management practices that preclude the reactions that mobilize the radionuclide at the source. The biochemical behavior of radionuclides in soils is correlated to its oxidation state.12,13 Radionuclides such as uranium (U), technetium (Tc), and plutonium (Pu) exhibit multiple oxidation states, and unlike the reduced forms that are insoluble, their oxidized forms are highly soluble.9,14 For instance, tetra- and hexavalent states of U are dominant under environmental conditions with hexavalent, U(VI), U is the most stable state under oxidizing conditions and persists in solution as the dioxycation UO22+, whereas tetravalent U(IV) state hydrolyzes readily in water, precipitates as U(IV)(hydr)oxide phases, and sorbs strongly to mineral surfaces. Received: Revised: Accepted: Published: 7652

November 14, 2017 April 27, 2018 May 3, 2018 May 3, 2018 DOI: 10.1021/acs.est.7b05836 Environ. Sci. Technol. 2018, 52, 7652−7662

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Environmental Science & Technology

plant growth, and these sites are often dominated by ruderal plant species that are known for their ability to actively forage for nutrients,39 a process that inadvertently could enhance the mobility of radionuclides from mineral forms that are otherwise unavailable. However, the ability of plants to mobilize radionuclides from stable mineral forms and the environmental factors that regulate the magnitude of this mobilization remains less known. The current study focuses on the plant-induced mobilization of radionuclides under varying availability of P. Phosphorus is a key mineral required for plant growth, and unlike other macronutrients, most P in soil is inaccessible to plants due to the pH-dependent complexation with Ca, Fe, and Al.29 To address this apparent deficiency, plants have evolved efficient morphological and physiological strategies for obtaining this important macronutrient. These P foraging strategies include changes in root architecture, rhizosphere acidification, and exudation of organic compounds with chelating moieties.29 U is a ubiquitous (∼2−4 mg/kg of soil or sediment) naturally occurring radionuclide.40 Uranium has a strong affinity to associate with P and is a major constituent in rock phosphate used for making P fertilizer.41 Also, injecting soluble P to soils for the sequestration of U as uranyl phosphate is an in situ remediation strategy that is widely practiced.42 Thus, under P limiting conditions, the plant physiological adaptations aimed at mobilizing P could potentially result in a collateral mobilization of U from uranyl phosphates, but the magnitude and regulators of such mobilization remain unknown. In this work, a native bunch grass, Andropogon virginicus, exposed to different mineral forms of P, was examined with respect to its potential for U mobilization. A. virginicus is a ruderal, perennial grass species with an extensive deep root system (>2 m) that is tolerant to nutrient poor soil43 and is one of the dominant ground covers in several radionuclide contaminated areas along the southeastern United States.44 The effect of P stress on U mobilization was determined using a series of sand, hydroponic culture, and batch dissolution studies. We hypothesized that (i) mineral forms of P will alter the root exudate profile of A. virginicus, and this change in exudate profile would be a function of the bioavailability of P; (ii) root exudates of P deficient plants will be proportionally abundant in compounds with a greater chelating capacity, and (iii) root exudates of A. virginicus produced under P deficiency and will enhance the dissolution U from uranyl phosphate.

Thus, U(IV) is generally much less mobile than U(VI) in the environment.15 In addition to the oxidation state, the mobility of radionuclides in soil is also regulated by their sorption onto organic matter and mineral surfaces.16,17 Even though the chemical, physical, and geochemical processes that enhance the mobility of radionuclides in soils have been well studied across different model systems,12,18,19 relatively little is known about the plant-mediated processes that influence the mobility of radionuclides in soils. Potential mechanisms of plant-mediated transport of radionuclides in the soil can be broadly classified into the initial dissolution of radionuclides from the unavailable mineral forms (mobilization), followed by the uptake and accumulation of the mobilized radionuclides within the plant tissues,20 both of which are significantly influenced by the physiology of the plant. The plant uptake and transport of radionuclides in solution has been demonstrated in several lab-scale hydroponic studies.13,14 A few studies also have recorded significant upward movement of radionuclides in plants growing in contaminated soils.21−23 However, plant-mediated processes that regulate the mobilization of radionuclides at the source, and the influence of growing environments in modulating this plant-mediated radionuclide mobilization remain less explored. Soil nutrient deficiency is one of the key environmental stressors that limits growth and productivity of plants.24,25 Plants respond to the availability and distribution of nutrients in soils by modulating their root architecture so as to enhance the upake of the limiting nutrient.26−28 Along with the morphological changes, under nutrient deficiency, plants also employ efficient physiological strategies to acquire nutrients from sources that are less soluble.29,30 At the physiological level, much research has focused on the components of root exudates that play important roles in increasing the bioavailability of nutrients.31 A common strategy across many plant species in the face of nutrient deficiency is rhizosphere acidification through proton extrusion.32 In addition, many plants exude organic acid anions and TCA cycle intermediates through root exudates that lower the rhizosphere pH. This lower soil pH potentially mobilizes the metal-bound P and enhances P acquisition capabilities of plants.32 Chelating compounds present in root exudates increase the mobilization and subsequent availability of nutrients because of their ability to form soluble complexes.33 Similarly, enhanced secretion of amino acids and organic acids has been reported under iron (Fe) deficiency.34,35 Recent experimental studies also have demonstrated the physiological capacity of plant roots to effectively remobilize mineral associated soil carbon. Root exudates released into soil respond to elevated CO 2 concentration and promote removal of cross-linking metal cations from metal organic complexes.36 Root exudates have also been shown to alter the sorption/desorption dynamics of various contaminants. For instance, organic acid promoted desorption of phenanthrene and naphthalene has been reported recently.37,38 Many of the above reactions, including chelation and rhizosphere acidification, could also influence the dissolution of radionuclides, especially from mineral forms where the radionuclides are complexed with nutrient elements that are essential for plant growth. Thus, nutrient deficiency could potentially influence the ability of plants to mobilize radionuclides. However, little is known regarding the role of nutrient stress in facilitating plant-mediated radionuclide mobilization in the soil. Many of the soils subjected to radionuclide contamination are marginal lands that challenge

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MATERIALS AND METHODS 1. Plant Response under Resource Limitations. 1.1. Sand Culture Study. The influence of bioavailability and spatial localization of P on root distribution patterns and chemical composition of root exudates of A. virginicus was studied using sand culture study. The experiment was conducted in 25 cm long, 5 cm diameter plastic tubes (PETG, Polyethylene Terephthalate Glycol) filled with autoclaved sand (600 g per tube, 0.5−1 mm grain size). Phosphorus treatments included three mineral forms with varying bioavailability of P: KH2PO4 that represented a completely water-soluble form of P (Ksp = 28) and FePO4 and Ca3(PO4)2 that are sparingly water-soluble (Ksp = 1.3 × 10−22 and 2.0 × 10−29, respectively). The amount of the P minerals across the 3 treatments were normalized to supply 36 mg P per 600 g of sand. Both FePO4 and Ca3(PO4)2 were provided in two distinct spatial distribution patterns, and minerals were either uniformly distributed throughout the sand 7653

DOI: 10.1021/acs.est.7b05836 Environ. Sci. Technol. 2018, 52, 7652−7662

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grown in a greenhouse maintained at 30/20 °C day/night temperatures with a 14 h photoperiod for 16 weeks. The carbon assimilation capacity of each plant was measured using Li-COR to compare the carbon assimilation rate across the treatments. At the termination of the experiment, the plants were harvested and sectioned into leaves and roots, and fresh biomass was recorded. The bulk sand was discarded, and the sand adhering to the roots was gently removed and collected. Subsamples of root were stored in 70% ethanol for determining the percent mycorrhizal colonization across different P treatments. Remaining root, shoot, and rhizosphere soils were rapidly frozen on dry ice and stored in a freezer at −80 °C for further analyses. Extraction and Analysis of Metabolites. Metabolites from the roots exposed to the P treatments were extracted using the method reported in previous studies 45,46 with minor modifications. Briefly, frozen root samples were finely ground with dry ice using a mortar and pestle and stored at −80 °C. Approximately 1.0 g of the ground samples was placed in a 15 mL centrifuge tube, and 4 mL of ice-cold methanol:propanol (1:1, v/v) was added to each. The tubes were vortexed for 20 s and then homogenized by sonication at 50% amplitude for 3 min and vortexed again for 20 s. This mixture was centrifuged for 5 min at 2500 rpm, and the supernatant was collected. A 200 μL aliquot of the supernatant was transferred to a glass insert containing 20 μL of a mix of retention time lock d27myristic acid (2 mg mL−1) and internal standard ribitol (500 μg mL−1) and dried completely under nitrogen. Twenty microliters of freshly prepared methoxylamine (20 mg mL−1) solution in pyridine was added, and the vials were incubated at 40 °C for 90 min followed by trimethylsilylation with 90 μL of N-methyl-N (trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) for 30 min at 40 °C. These derivatized samples were then analyzed using a GC-MS (Agilent 7980, Agilent Technologies, Santa Clara, CA). Details on GC-MS parameters and spectral identification is provided in the Supporting Information (Section S2). 1.2. Hydroponic Study. To assess the U dissolution efficiency of root exudates of A. virginicus released under P deficiency, a hydroponic study was conducted using various mineral forms that differed in their P availability. The exudates from the hydroponic study were further characterized to identify the major compounds that could facilitate the dissolution of uranyl-phosphate. Following germination, each seedling was transplanted to 2 L glass jars containing halfstrength Hoagland’s solution that contained all macro- and micronutrients but without any EDTA. The jars were covered with aluminum foil to prevent light from interfering with root growth. The nutrient solution was aerated continuously using an air pump, and the nutrient solution was replaced with fresh solution every three days to reduce the microbial buildup. The plants were grown in this media for 40 days to facilitate the development of an extensive, healthy root system (Supporting Information Image S1). Before the application of the P treatments, roots of the plants were gently but thoroughly rinsed with deionized water multiple times, and all plants were kept in a P-free, continuously aerated nutrient solution for 8 h in the dark to remove any traces of P that adhered to the roots. Plants of uniform visual characteristics (number of tillers, root length, and stem height) were selected and randomly assigned to four treatment groups: P supplied as KH2PO4, Ca3(PO4)2, FePO4, and no P, with five replicates per treatment. The plants were grown in their appropriate modified Hoagland’s solution

or concentrated in a 5 cm patch (Figure 1). In uniform distribution treatments, the tubes were first filled with pure

Figure 1. Schematic of nutrient distribution used for the spatial localization of phosphorus. (a) Patch treatment. (b) Uniform treatment. Both treatments received the same amount of P.

autoclaved sand to a height of 5 cm and then topped with sand premixed with FePO4 or Ca3(PO4)2 to a height of 17 cm. For patch treatments, the same amount of FePO4 or Ca3(PO4)2 as that in the uniform treatment was distributed in a 5 cm wide band 7 cm below the surface, resulting in a concentrated P distribution 3.6 times that of the P in the uniform treatment (Figure 1). The control treatment contained pure autoclaved sand throughout the root zone, and KH2PO4 was provided along with Hoagland’s nutrient solution to form uniform distribution of P. Due to the high solubility of KH2PO4, patch application was not practical for this treatment. The background P concentration of the autoclaved sand was below the detection limit (0.1 mg P per kg sand). To investigate the role of the rhizosphere microbial community in aiding P mobilization, half the tubes in each of the two treatment groups (mineral form and spatial distribution of P) were reinoculated with microbial inoculum native to the rhizosphere of A. virginicus. Details on rhizosphere soil collection and inoculum preparation are given in the Supporting Information (Section S1). Four replicates were maintained for each of the 12 treatments (three forms of P minerals, two spatial distribution of P, and both autoclaved and reinoculated soils). Seeds from A. virginicus, collected from Clemson, SC, were planted in a 16 × 8 tray containing a sterile germination mixture. Two-week-old seedlings were transplanted into these tubes and irrigated as needed with distilled water. Nutrient solutions specific to each treatment were supplied at weekly intervals: the control (KH2PO4) treatment received a complete nutrient solution providing 200 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 Mg as MgSO4; 140 mg L−1 N as NH4NO3, KNO3, and CaNO3 and micronutrients; 0.5 mg L−1 B as 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 as CuSO4; and 0.01 mg L−1 Mo as (NH4)6MO7O. Chelating compounds including EDTA were avoided from the micronutrient preparations. The calcium phosphate and iron phosphate treatments received a nutrient solution without P, providing 200 mg L−1 Ca as CaNO3, 195 mg L−1 as KNO3, 48 mg L−1 Mg as MgSO4, and 140 mg L−1 N as NH4NO3 and KNO3. The plants were 7654


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Figure 2. Effect of different phosphorus (KH2PO4, FePO4, and Ca3PO4) and reinoculation treatments on (a) root biomass, (b) shoot biomass, and (c) photosynthesis of A. virginicus grown in sand culture. Different letters indicate significant difference between treatments (Tukey’s HSD multiple comparison at P ≤ 0.05). Bars represent means ± SE (n = 4).

(Supporting Information Section S3) for 72 h with constant aeration; then, 50 mL of the solution in each flask was sampled using a syringe. Immediately after collection, the solution was filter sterilized using 0.22-μm nylon membrane filters and stored at −80 °C until analysis of root exudates. Because of the co-occurring high salt content of the nutrient media, instead of the MSTFA derivatization, the samples were subjected to ethyl chloroformate (ECF) derivatization prior to GC-MS analysis to

quantify the metabolites in the hydroponic solution. The quantity of specific amino acids and organic acids was measured using external calibration curves. Details on ECF derivatization method are provided in the Supporting Information (Section 4). 2. Effect of Compounds Identified in Root Exudates on the Dissolution of Uranyl-Phosphate. Batch dissolution experiments were conducted to investigate the ability of various 7655


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culture, both the shoot and root biomass of A. virginicus supplied with FePO4 (Ksp = 1.3 × 10−22) were similar to that of KH2PO4 (Ksp = 28) treatments (Figures 2a and b). Compared to KH2PO4 treatment, the plants supplied with Ca3(PO4)2 (Ksp = 2.0 × 10−29) had 41% lower root biomass (P = 0.008, Figure 2a) and 15% lower shoot biomass (Figure 2b). The similar biomass between KH2PO4 and FePO4 was accompanied by similar tissue P concentration, which indicates active P acquisition from FePO4 (Figure 3). Although P deficiency

compounds identified in the root exudates of A. virginicus to mobilize U from Chernikovite (UO2HPO4·4H2O, lg Ksp; −24). A preliminary batch dissolution experiment was conducted using citric acid,47 thereby optimizing (i) the amount of substrate (UO2HPO4·4H2O, (ii) the concentration of the ligand, and (iii) the substrate/solution ratio that would result in 50−60% dissolution of the U from the UO2HPO4·4H2O. Based on the preliminary results (Supporting Information Figure S4), 0.5 mM of ligand, 25 mg uranyl-phosphate, and a 1:400 (0.0025 g mL−1) solids/solution ratio was used for the batch dissolution studies. Following optimization, batch dissolution experiments were conducted on 13 compounds (ligands) identified as abundant in the root exudates of A. virginicus. A background electrolyte solution of 0.01 M NaCl was used in the preparation of the individual ligand stock solutions. Uranyl phosphate (0.025 g) was added to each of the 15 mL centrifuge tubes, followed by 10 mL of the appropriate ligand solution. The control samples were prepared in the same manner but with distilled water. The pH of the solution was then adjusted to 6 by adding small volumes of 0.1 M HCl or 0.1 M NaOH. All samples were placed on an end-overend mixer for 24 h at room temperature. After the mixing period, a 1.3 mL aliquot from each tube was centrifuged at 8000 rpm for 20 min. The supernatant was filtered through a 0.22 μm syringe filter, and a 0.1 mL aliquot was diluted with 9 mL of 2% HNO3 for analysis via inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Scientific XSeries 2). The release of U from the uranylphosphate was expressed as the percentage of U released in relation to the total U content in the uranyl phosphate. 3. Root Exudates under P Deficiency after Uranium Dissolution. The ability of the overall root exudate matrix to mobilize U from uranyl-phosphate was explored through a batch dissolution study similar to the one described previously. However, instead of an individual ligand solution, we used hydroponic solutions exposed to various nutrient treatments collected from Study 1.2. A batch-dissolution study was conducted using uranyl-phosphate at a solid:solution ratio of 1:400; the pH of the solution was adjusted to 6 by adding small volumes of 0.1 M HCl or 0.1 M NaOH. The samples were incubated on a rotator for 24 h. The control samples were prepared with fresh Hoagland solution using the same process. The release of U from uranyl-phosphate was analyzed as described above. Statistical Analysis. A two-way analysis of variance (ANOVA) was used to compare the main and interactive effects of inoculum (with and without inoculum) and nutrient treatments (KH2PO4, FePO4, Ca3(PO4)2, and no P) on the biomass (root and shoot) and carbon assimilation capacity (photosynthesis) followed by Tukey’s HSD post hoc test. All differences were reported to be significant at P < 0.05. Principal component analysis (PCA) was used to analyze the composition of root exudates (MetaboAnalyst 3.0).45,46 Hierarchical cluster analysis of the metabolite responses with respect to the treatments were visualized using heat maps. Oneway analysis of variance (ANOVA) was conducted to analyze the effect of individual organic acids and hydroponic solution under P treatments on the percentage dissolution of U from uranyl-phosphate. All differences were reported to be significant at P < 0.05.

Figure 3. Concentration of phosphorus in the leaf (a) and root (b) of A. virginicus grown in sand culture exposed to P treatments (KH2PO4, FePO4, and Ca3(PO4)2). Different letters indicate significant difference between treatments (Tukey’s HSD multiple comparison at P ≤ 0.05). Bars represent means ± SE (n = 4).

reduced shoot biomass in Ca3(PO4)2 (Figure 2b), the root/ shoot ratio was similar (∼2.5) across all P treatments (Supporting Information Figure S1). Carbon assimilation capacity of A. virginicus was also similar across P treatments (Figure 2c). Inhibition of plant CO2 assimilation capacity by P limitation has often been explained by a lower activity of the Calvin cycle, low amount of Rubisco, and low regeneration of RuBP.48,49 Because all the treatments contained P forms with varying bioavailability, the above similarity in root/shoot ratio and CO2 assimilation capacity could indicate the presence of an active P foraging strategy of A. virginicus. In natural environments, soil nutrient availability varies in time and space, and plants respond to low availability of nutrients in the soil by altering physiological or morphological attributes of their roots.50,51 In P-deficient soils, plants allocate more carbon to the root system, resulting in increased root/shoot ratio and greater exploration of the surface soil, thereby enhancing the acquisition of the less-available P.51,52 Plants also enhance P acquisition by modifying physiological traits and rely on increased root exudation to mobilize soil P.53,54 The similarity of root/shoot ratio irrespective of the bioavailability of P indicates that, in our study system, A. virginicus could rely more on physiological modification (root exudates) for P foraging. Similar results have been reported in other plants under P deficiency. For example, Lupinus albus and Cicer arietinum were

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RESULTS AND DISCUSSION Plant Response under Resource Limitations. Plant Growth Parameters and Phosphorus Content. In sand7656


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Figure 4. Principle component analysis (PCA) and heat map showing changes in 73 metabolites in A. virginicus root tissue grown in sand culture exposed to P treatments (KH2PO4, FePO4, and Ca3(PO4)2). (a) PCA score plots of 73 metabolites. Data point represents biological replicates, and the ellipses represent 95% confidence interval. (b) Heat map and two-way hierarchical cluster analysis of root metabolites highlighting the metabolite differences in A. virginicus in response to P treatments (KH2PO4, FePO4, and Ca3(PO4)2). The color of each cell depicts the abundance of individual metabolites; blue indicates a significant decrease, and red represent a significant increase in metabolic content.

Composition of Root Metabolites and Exudates under Various P Sources. Metabolite profiling elucidated the changes in composition in roots of A. virginicus grown in sand culture in response to the bioavailability and distribution of P-forms. Analysis based on both fragmentation pattern and retentionindex matches positively identified 73 metabolites across all samples. The PCA plot (Figure 4a) revealed a distinct clustering of treatments along the principle component axes that explained 56.2% of the variation in the data, indicating that each P treatment influenced the root metabolites in A. virginicus in a unique manner. Many of the metabolites identified in the roots of A. virginicus exposed to various P treatments were primary metabolites belonging to organic acids, amino acids, sugars, polyols, and polyhydroxy acids. Based on hierarchical cluster analysis, major organic acids (citric acid, malic acid, ketoglutarate, fumaric acid, linolenic acid, maleic acid, succinate, isocitrate, and gluconic acid) and amino acids (glutamic acid, aspartic acid, pyro glutamic acid, asparagine, tryptophan, isoleucine, and phenylalanine) were observed to be the primary drivers of this nutrient response (Figure 4b). Compared to KH2PO4 treatments, the plants exposed to the low available forms of P source (FePO4, Ca3(PO4)2) in sand culture had a higher abundance of 12 amino acids (Figure 4b). This observation is also supported by the hydroponic culture studies where a significant increase of some amino acids was observed; notably, glutamine, glutamic acid, valine, and methionine under low available forms of P (Figure 5). These observations are consistent with other studies that have also shown that increased amounts of amino acids play an important role in chelating metals, thus enhancing the mobility of P in the soil.59,60 For instance, amino acids such as glutamic acid and aspartic acids can release a total of three protons, two protons

found to depend primarily on root exudation to enhance P acquisition, whereas Zea mays, Triticum aestivum, and Brassica napus exhibited changes in root morphology under P deficiency.55 This difference suggests that P mobilization strategy could be highly species dependent. The confined rooting volume in our study might also have affected the rooting morphology of the A. virginicus. However, across the treatments, the lower 10 cm of the growth-tubes contained only citric acid > malic acid > fumaric acid > amino acids. Although the possibility of amino acids being involved in the chelation of metal ions has been discussed in previous studies,61,71 observations from the current study indicate that, compared to organic acids, amino acids have a lower effect on the release of U from uranyl-phosphate. The ligands selected for this study contain COOH and NH2 groups as electron pair donor sites that attract electron-deficient metals. The net charges of individual ligands vary depending on the degree of dissociation of these functional groups, which is a function of the pH of the media.72 Most chelating agents are less reactive at low pH,73 and pH range 5−6 generally leads to the dissociation of the carboxyl groups, and as the pH increases further, the amino groups are deprotonated at pH 9−10.72 The organic acids used in this study carry 2−3 negatively charged carboxyl groups,

Figure 5. Average concentration of identified organic acids and amino acids in the root exudates of A. virginicus exposed to P treatments (KH2PO4, FePO4, Ca3(PO4)2, and no P) in hydroponic media at 72 h. Bars represent means ± SE (n = 4).

from carboxyl groups and one proton from amine group, enabling formation of soluble complexes with metal ions that are cocomplexed with P, thereby releasing P for plant uptake.61 In addition, amino acids detected in root exudates could also affect the growth of soil microorganisms, which in turn could enhance the mobilization and uptake of P.62,63 For organic acids, the reverse was the case, where plants exposed to the KH2PO4 treatment had a higher abundance of 11 organic acids in roots. Increased exudation of organic acids under P limiting conditions has been demonstrated in several studies.62−64 Organic acid anions in root exudates lower rhizosphere pH, perhaps mobilizing the metal-bound P, thereby increasing P acquisition efficiency in plants. Furthermore, it is thought that an increase in the levels of organic acids in the rhizosphere will also chelate metal ions, which are usually associated with phosphates, thus allowing the inorganic P to be released for plant uptake.64 Thus, the ability of organic acids to solubilize P is attributed to acidification, chelation, and ligand exchange reaction.65 Organic acids can release adsorbed phosphate onto soils and Fe oxides through ligand exchange reaction. However, at the smaller adsorbed-P concentration, the predominant mechanism of organic-acid induced P release from iron oxide is ligand-enhanced dissolution of the Fe oxide rather than ligand exchange reaction.66 In this study, the lower abundance of organic acids in the root tissues of FePO4 and Ca3(PO4)2 treatments could be due to the greater release of organic acids from roots to the rhizosphere. This is supported by the analysis of the rhizosphere soils that shows that the organic acids were abundant in the rhizosphere of plants exposed to FePO4 and Ca3(PO4)2 (Supporting Information Figure S2). Similar results were obtained from hydroponic culture studies in which A. virginicus was exposed to various P treatments. Under hydroponics, organic acids were the most abundant in the root exudates of plants exposed to FePO4 and Ca3(PO4)2. Among organic acids, specifically citric acid, malic acid (P < 0.001), and oxalic acid (P = 0.023) were higher in plants treated with low available forms of P (Supporting Information Figure S3). These results suggest that the organic acids in the root exudates appear to play a crucial role in solubilizing P by this species. Organic acids such as malic acid, citric acid, oxalic acid, succinic acid, and fumaric acid have been detected in root exudates under nutrient-stress conditions.67 7658


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exudates collected from A. virginicus exposed to less available forms of P treatments (FePO4) resulted in 70% dissolution of U from Chernikovite (Figure 6b). The amount of U dissolution could be partly explained by the root exudate composition: plants exposed to no-P and KH2PO4 treatments had a lower concentration of organic acids, whereas the organic acids in the root exudates of plants exposed to FePO4 treatments were higher. The hydroponic study was not conducted in an axenic condition because the presence of microbial cells and metabolites is required to elicit natural root exudation. Thus, the possibility that part of the observed mobilization could have been from microbial exudates in the medium cannot be ruled out. The batch dissolution study using the root exudates from hydroponic experiment, though instrumental in elucidating the potential of multicompound root exudates in mobilizing U from mineral forms, would overestimate the root exudate mobilization of U from soil matrixes. This is because in soils the compounds in root exudates will be actively degraded by microbes, sorbed on to soil minerals, or reabsorbed by plants, which in turn reduce the amount of chelating compounds in the rhizosphere. The percent dissolution of U from Chernikovite by the root exudates was 25% lower than the sum total of the dissolution potential of individual organic compounds (Supporting Information Figure S5). While the concentration of individual organic compounds used for the batch dissolution study was ∼0.5 mM, total organic acid exuded under low P availability (FePO4) was 10 times greater (Supporting Information Figure S3). Under P stress, chelation appears to be the major mechanism for P solubilization from its metal-bound complexes (FePO4, Ca3(PO4)2), and thus would reduce the chelating compounds that are available to complex with U. For instance, experimental results show that presence of citric acid increases dissolution of U due to the formation of U(VI)−citrate complexes. However, the formation of U(VI)−citric acid complexes could be regulated by the dissolution of iron from FePO4 in the hydroponic solution as a consequence of the formation of iron−citric acid complexes. Citrate can also form complexes with other micronutrients in the hydroponic solution such as manganese, zinc, and copper.74 Thus, the presence of other metal elements in the system can be one of the factors limiting the mobilization and potential leaching of U in the soil. In addition, the natural exudates from plant roots are a complex mixture of different classes of biomolecules37 and will vary substantially from the individual organic ligands in function and character. Only small proportions of compounds in the exudates, mostly low molecular substances such as organic acids, sugars, and amino acids, can be identified with a greater molecular-level accuracy, whereas polymeric compounds such as proteins and carbohydrates are often difficult to characterize. This has been shown previously for real and artificial root exudates (ARE), where ARE contained substantially higher DOC than plant root exudates.37 Thus, there could be many more compounds in the root exudates of A. virginicus, some of which could synergize or antagonize the overall dissolution of U form uranyl-phosphate, which would have resulted in the observed lower mobilization of U from uranyl-phosphate by root exudates. Although heavy metal dissolution as a function of root exudation has been studied before,74 our study links the plant stress response to U mobilization via changes in the composition of root exudates. Overall, the results indicate that when exposed to moderate nutrient stress, contributed by

Figure 6. Uranium dissolution from uranyl-phosphate at pH 5 following treatment with different solution matrix for 24 h: (a) 0.5 mM organic ligands; (b) hydroponic solution from 72 h of P treatments (KH2PO4, FePO4, Ca3(PO4)2, and No P). Different letters indicate significant difference between treatments (Tukey’s HSD multiple comparison at P ≤ 0.05). Bars represent means ± SE (n = 4).

which form strong chelates with metals.72 Thus, at the pH used in this study (pH 6), the dissolution potential of organic acids was higher than that of the other ligands. In addition, types and position of functional groups in the organic acids are most important in regulating metal dissolution and subsequent leaching.73 For instance, comparing three dicarboxylic acids such as malic acid, fumaric acid, and succinic acid; malic acid was the most effective in mobilizing the U from uranylphosphate due to the presence of α-hydroxyl group. These results emphasize the importance of type of organic acids produced under P stress in dissolving/mobilizing U from uranyl-phosphate in the soil. Differential Influence of Root Exudates Produced under Varying P Bioavailability on U Dissolution. Root exudates present a complex, multicompound matrix, where individual compounds could facilitate or antagonize the chelating capacity of the companion compounds. This, in turn, will alter the chelation capacity of the overall exudate matrix. Batch dissolution studies were conducted with the root exudates of A. virginicus exposed to different P treatments to determine the differential ability of the overall exudate matrix to influence the dissolution of Chernikovite. Root exudates from plants grown under no-phosphate and KH2PO4 had a lower effect on U dissolution (20% dissolution). However, the root 7659



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low available forms of P, plants increase the amounts of chelating compounds in root exudates to mobilize the sparingly available form of P in the soil. This, in turn, could potentially facilitate the mobilization of U from uranyl-phosphate. Our results suggest that nutrient stress could be one of the key factors that regulate plant-mediated mobilization of radionuclides in soils. However, there are multiple fates for root exudates, including microbial degradation, and only a smaller fraction of the overall exuded compounds take part in nutrient foraging. Hence, the process of root exudate mediated mobilization of U would be slower under field conditions, but could contribute significantly to the soil transport of U at decadal time scale.Although plant nutrient foraging processes could inadvertently enhance the mobility of U from uranylphosphate, there are multiple fates associated with mobilized U under natural environmental conditions.75,76 Thus, future studies are still needed to gain better insight into the relationship between soil P status in terms of plant bioavailability and the associated U uptake and transport in plants and soil.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b05836. Section S1: Preparation of soil inoculum, tissue phosphorus analysis. Section S2: GC parameters. Section S3: Modified Hoagland solution. Section S4: ECF derivatization. Image S1: Root image from hydroponic culture study. Image S2: Mycorrhizal staining. Figure S1: Effect of phosphorus treatment on root to shoot weight ratio. Figure S2: Abundance of organic acids in the rhizosphere sand. Figure S3: Concentration of identified organic acids and amino acids in the root exudates in hydroponic media. Figure S4: Dissolution of U form uranyl-phosphate using different concentration of citric acid and uranyl-phosphate. Figure S5: Comparison between observed U dissolution with root exudate matrix and expected dissolution with all individual ligands together. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: (864)656-4453; E-mail: [email protected]. ORCID

Dawn Montgomery: 0000-0002-6381-818X Brian A. Powell: 0000-0003-0423-0180 Nishanth Tharayil: 0000-0001-6866-0804 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Vidya Suseela for assisting with the photosynthesis measurements and analysis of root metabolomics data. This material is based upon work supported by the U.S. Department of Energy Office of Science, Office of Basic Energy Sciences and Office of Biological and Environmental Research under Award Number DE-SC-0001253, and DE-SC-0010832. This is a technical contribution No. 6661 of the Clemson University Experiment Station. 7660


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Phosphorus Stress-Induced Changes in Plant Root Exudation Could

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