Article pubs.acs.org/est
Arsenic Induced Phytate Exudation, and Promoted FeAsO4 Dissolution and Plant Growth in As-Hyperaccumulator Pteris vittata Xue Liu,† Jing-Wei Fu,† Dong-Xing Guan,† Yue Cao,† Jun Luo,† Bala Rathinasabapathi,‡ Yanshan Chen,*,† and Lena Q. Ma*,†,§ †
State Key Lab of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210023, China Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, United States § Soil and Water Science Department, University of Florida, Gainesville, Florida 32611, United States ‡
S Supporting Information *
ABSTRACT: Arsenic hyperaccumulator Pteris vittata (PV) is efficient in taking up As and nutrients from As-contaminated soils. We evaluated the mechanisms used by PV to mobilize As and Fe by examining the impacts of As and root exudates on FeAsO4 solubilization, and As and Fe uptake in four plants: Ashyperaccumulators PV and Pteris multif ida (PM), nonhyperaccumulator Pteris ensiformis (PE), and angiosperm plant tomato (Solanum lycopersicum). Phytate and oxalate were dominant in fern plants (>93%), which were 50−83, 15−42, and 0−32 mg kg−1 phytate and 10−15, 7−26, and 4−12 mg kg−1 oxalate for PV, PM, and PE respectively, with higher As inducing greater phytate exudation and no phytate being detected in tomato exudates. PV treated with phytate+FeAsO4 had higher As and Fe contents and larger biomass than phytate or FeAsO4 treatment, which were 340 vs 20 and 130 mg kg−1 As in the fronds and 7900 vs 1600 and 4100 mg kg−1 Fe in the roots. We hypothesized that As-induced phytate exudation helped PV to take up Fe and As from insoluble FeAsO4 and promoted PV growth. Our study suggests that phytate exudation may be special to fern plants, which may play an important role in enhancing As and nutrient uptake by plants, thereby increasing their efficiency in phytoremediation of As-contaminated soils.
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INTRODUCTION Arsenic (As) is of environmental concern due to its toxicity and ubiquity in the environment. Chinese Brake fern (Pteris vittata; PV) is the first-known As-hyperaccumulator.1 In contaminated soils, it can accumulate up to 23 g kg−1 As in the fronds. Even in uncontaminated soils, it can take up 744 mg kg−1 As, which is much greater than typical plants at ≤10 mg kg−1.1,2 Arsenic in soils exists primarily in its oxidized form arsenate (AsV), which is a chemical analogue for phosphate (P). In soils, As and P often bind with metals including Ca, Al, and Fe,3 making them unavailable for plant uptake.4 As-hyperaccumulator P. vittata prefers to grow in alkaline soils, which are characterized by low available Fe and P.5 This suggests that PV may have evolved strategies to mobilize Fe and P. However, limited information is available about Fe and P nutrition and their associated rhizosphere processes. Plants release root exudates to enhance their uptake of nutrients and metals.6 Among root exudates, low molecular weight organic acids (LMWOA) are the most common and important due to their ability to mobilize nutrients like Fe and P, and heavy metals like Zn and Cd.7,8 Exudation of LMWOA is often induced under nutrient deficient conditions, especially Fe and P.9 For example, Bao et al.10 found that Fe-deficiency © XXXX American Chemical Society
induces higher exudation of malate and acetate by black nightshade, leading to more Cd accumulation. Dong et al.8 observed P-deficiency increases secretion of oxalate and malate in soybean plants. The data suggest that nutrient-deficiency induces secretion of organic acids, helping element mobilization and plant accumulation. Citrate and oxalate are typical LMWOA in plant root exudates.7,11 Unlike typical plants, phytate has been detected in the root exudates of P. vittata.12 Phytate, known as inositol hexaphosphate or phytic acid, is the principal storage form of organic P in soils and plants.13 Phytic acid chelates metals such as Ca, Mg, and Fe, forming insoluble phytate and reducing their availability.14 While it exists as Fe- and Al-phytate in soils, it is mainly present as phytin (Ca−Mg−K phytate) in cereals.15 However, its presence in root exudates has not been reported until the study of Tu et al.12 The study showed that phytate is effective in releasing As from As-contaminated soil. However, due to the complexity of a soil system, they were unable to Received: March 17, 2016 Revised: July 21, 2016 Accepted: August 2, 2016
A
DOI: 10.1021/acs.est.6b00668 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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oxalic or citric acid at organic acid:FeAsO4 molar ratios of 1:1, 2:1, 5:1, 10:1, 20:1, or 50:1. Chloramphenicol at 30 mg L−1 was added to prevent microbial decomposition,12 with 10 mM NaClO4 electrolyte (pH = 6.0) as a control. Preliminary experiment showed that 30 mg L−1 chloramphenicol had no significant effect on the dissolution of FeAsO4 compared to 10 mM NaClO4 electrolyte, with released Fe being at 1.51 and 1.37 μM, respectively (data not shown). The solutions were shaken at 200 rpm and 28 °C for 1 d and centrifuged at 10 621 g for 10 min. The supernatant was analyzed for Fe with flame atomic absorption spectrophotometry (FAAS; PinAAcle 900T, PerkinElmer) and for As with inductively coupled plasma mass spectrometry (ICP-MS; NexION300X, PerkinElmer). Effect of Phytic Acid on FeAsO4 Dissolution, Plant Fe, and As Uptake and Growth of P. vittata. Although phytic acid can solubilize FeAsO4 via complexation with Fe3+, Fephytate complex can be insoluble (log k = 17.6 at pH = 6).17 To better understand the mechanism of Fe uptake by PV, we studied the role of phytic acid in Fe- and As-mobilization from insoluble FeAsO4 and their subsquent plant uptake. P. vittata with 5−6 fronds and ∼12 cm in height were cultured in hydroponic systems in an incubator at 26 °C with following treatments (four replicates): (1) Fe- and P-free 0.2strength HS control; (2) 1 mM phytic acid; (3) 100 μM FeAsO4; and (4) 1 mM phytic acid +100 μM FeAsO4. Sterile Fe- and P-free 0.2-strength HS was used as medium in all treatments and 30 mg L−1 chloramphenicol was added to inhibit microbial activity. Phytic acid concentration at 1 mM is consistent with the reported organic acid concentrations in soil solution, which are 0.01−1 mM.18 After treatment for 2 weeks, plant fresh biomass was recorded after washing the roots with ice-cold phosphate buffer and Milli-Q water to remove surface adsorbed As and Fe. Plants were separated into the fronds and roots, lyophilized and stored at −80 °C. Freeze-dried plant materials were digested with HNO3/H2O2 using USEPA Method 3050B.12 Total As and Fe concentrations were analyzed with ICP-MS and FAAS. Plant P was analyzed using a modified molybdenum blue method to overcome arsenate interference via cysteine reduction.19 Plant As, Fe, and P concentrations were expressed on a dry weight (dw) basis. Phytate and oxalate in the plant tissues were extracted according to Carneiro et al.20 Briefly, weighing 0.2 g of ground, freeze-dried material, transferring it to 4 mL of 1.0 M HCl and shaking vigorously during 3 h at room temperature. After centrifugation at 5000 rpm for 10 min, the supernatant was filtered (0.45 μm) for organic acid analysis. Quantification and Identification of Phytic Acid by HPLC and TOF-MS. Organic acids in root exudates were analyzed by high performance liquid chromatography (HPLC; Waters, Milford, U.S.A.) equipped with a reversed-phase dC18 anion-exchange analytical column (Agilent Zorbax-Aq 4.6 × 250 mm, 5-Micron, Germany) and a multiwavelength UV detector at 210 nm. Before HPLC analysis, the lyophilized residue was redissolved in 1.5 mL of mobile phase (20 mM NaH2PO4, pH 2.7), which was filtered through a 0.22 μm filter and degassed. The sample injection volume was 10 μL with a mobile phase flow rate of 0.5 mL min−1 and column temperature at 30 °C. Representative organic acids in plant root exudates including phytic, oxalic, D-tartaric, quinic, D-malic, citric, fumaric, and succinic acids (purity ≥99%; Sigma) were used as standards.12,21 The organic acid concentrations were expressed on a fresh root weight (fw) basis.
identify the underlying mechanism. Besides, although the roles of root exudates in metal mobilization have been widely studied, little is known regarding their roles in As and Fe mobilization and uptake by P. vittata. Therefore, the present study uses a hydroponic system to investigate the mechanisms of As and Fe release by phytate and their effects on PV growth. The goal of this study was to elucidate the mechanisms used by P. vittata to mobilize As and Fe, and their subsequent uptake and translocation. The specific objectives were to (1) determine the differences in As-induced exudation of organic acids in fern and angiosperm plants; (2) quantify the ability of organic acids in FeAsO4 solubilization; and (3) examine plant As and Fe uptake, and plant growth treated with phytic acid and/or FeAsO4. The results may shed light on the role of special root exudate phytate in dissolving Fe−As mineral and enhancing As uptake by P. vittata, which has implication for efficient phytoremediation of As-contaminated soils.
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MATERIALS AND METHODS Plant Materials and Growth Conditions. To examine the effect of As on plant root exudates, As-hyperaccumulators P. vittata (PV) and P. multif ida (PM), nonhyperaccumulator P. ensiformis (PE) and angiosperm S. lycopersicum (tomato) were used. Prior to As exposure, plants of uniform height (∼20 cm) were acclimatized for 3−4 weeks in aerated 0.2-strength Hoagland nutrient solution (HS; Table S1 of the Supporting Information, SI). The solutions were replenished with Milli-Q water daily and renewed biweekly (Millipore, U.S.A.). The plants were grown in a greenhouse under a 14 h photoperiod at 26 °C/20 °C day/night temperature, 75% relative humidity, and a light intensity of 180 μmol m−2 s−1. Effect of As on Plant Root Exudates. Acclimated plants were transferred to 250 mL opaque plastic pots containing 200 mL 0.2-strength HS spiked with 0, 5, or 20 mg L−1 of As with three replicates (Na2HAsO4·7H2O; Sigma-Aldrich, St. Louis, U.S.A.). After 2 d of treatment, root exudates were collected. Briefly, plant roots were soaked in antibiotic solution (30 mg L−1 chloramphenicol; Sigma) for 2 h to minimize microbial growth and washed with sterile Milli-Q water.12 Subsequently, plants were transferred to 20 mL of sterile Milli-Q water to collect root exudates for 12 h under sterile conditions. The flasks were covered with black plastic cloth to reduce photodegradation. The collected solutions were immediately filtered (0.45 μm), lyophilized, and stored at −80 °C (FreezZone 12, LabConco, Kansas City, U.S.A.). Effect of Organic Acids on FeAsO4 Mineral Dissolution. Once it became clear that PV produced phytate and oxalate, we examined their ability in FeAsO4 solubilization with citrate as a comparison. Citric and oxalic acids were from Sinopharm Chemical Reagent Co. and phytic acid was from Aladdin Industrial Inc. (Shanghai, China). FeAsO4 was synthesized according to Hess and Blanchar,16 washed free of salts, freeze-dried, and verified with X-ray diffraction (data not shown). The ability of root exudates in solubilizing FeAsO4 was measured by determining As and Fe concentrations in the media. The dissolution experiment was conducted in 10 mM NaClO4 electrolyte with pH adjusted to ∼6 with 0.5 M MES buffer, which was maintained at ∼6 after addition of LMWOA root exudates (phytic, oxalic, or citric acid) using 0.01 M NaOH or HCl and solution redox potential was maintained at ∼67.2 mV. Then, 100 μM FeAsO4 was mixed with phytic, B
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Figure 1. Arsenic concentrations in the fronds (A) and roots (B), and phytate (C) and oxalate (D) concentrations in root exudates of four plant tissues after growing for 2 d in 0.2-strength Hoagland solution containing 0, 5, or 20 mg L−1 of As. The bars indicate the standard error of triplicates, means marked with different letters indicate significant differences (p < 0.05) and PV = P. vittata, PM = P. multif ida, and PE = P. ensiformis.
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RESULTS AND DISCUSSION Arsenic Accumulation in Plants. Four plant species (PV, PM, PE, and tomato) were exposed to 0, 5, or 20 mg L−1 As for 2 d in hydroponic systems. All fern plants under As exposure grew well; however, 20 mg L−1 As caused toxicity symptoms in tomato. Higher As in the media induced greater As uptake by plants (Figure 1AB). As expected, hyperaccumulators (PV and PM) took up and transported much more As than the nonhyperaccumulator PE. For example, after exposure to 5 and 20 mg L−1 As, As accumulation by PV and PM were 63−117 and 32−43 mg kg−1 in the fronds, 6.5−7 and 2.4−3.5 fold that of PE (Figure 1A). Compared to the ferns, tomato plant had the lowest As with shoot and root As being 3−4.5 and 2.6−6.1 mg kg−1 (Figure 1AB). The results indicated that fern plants were more resistant to As, especially hyperaccumulators, with more As being translocated from the roots to the fronds. Arsenic-Induced Phytate Secretion in Fern Plants. The HPLC chromatograms of organic acid standards are shown in Figure 2A. The retention time of phytic acid was 4.90 ± 0.2 min (Figure 2A−C; Table S2). On the basis of phytic acid standard addition method and coupling HPLC (Figure 2B) with molecular weight determination using TOF−MS (m/z = 659) (Figure S1), we confirmed that phytic acid was present in the root exudates of three fern plants, but not in tomato plant. This was consistent with the fact that phytate as a root exudate has not been reported in plants except in fern plants PV and Nephrolepis exaltata.12 In the root exudates, phytate and oxalate were the predominant LMWOA in fern plants, whereas oxalate was predominant in tomato plant (Figure 1CD). The composition of root exudates of fern plants was consistent with Tu et al.12 While phytate was detected in two As-hyperaccumulators PV and PM at 5 mg L−1 As, it was absent in nonhyperaccumulator PE where only oxalate was detected. Besides, phytate
Organic acids were identified by comparing the retention times of the samples against the standards and their concentrations were calculated based on sample peak area.22 Their detection limit was 0.5 mg L−1 except for phytic acid, which was 12.5 mg L−1. As a result, low concentrations of phytate were analyzed according to Dost and Tokul23 using HPLC/UV−vis method based on metal replacement of phytate from colored complex (Fe3+−thiocyanate), and separation and monitoring of decreased concentration of colored complex. The mobile phase was a mixture of 30% acetonitrile in water including 0.1 M HNO3 and flow rate was 0.5 mL min−1. Standards and samples were injected at 10 μL and separation was performed on an octyldecylsilane column. The peak of Fe3+−thiocyanate was detected at 460 nm, with a detection limit of 1.5 mg L−1. The phytate collected from root exudates was identified by HPLC with standard addition method and further confirmed by TOF-MS analysis. Briefly, the peak effluent separated with HPLC was determined by a Triple TOF 5600 mass spectrometer equipped with a duo-spray ion source. Sample (10 μL) was injected into the source using an Agilent 1100 G1329A-1 auto sampler, and the mobile phase consisting of water (60%) and methanol (40%) was eluted at 200 μL min−1 by an Agilent 1260 G1312B-1 pump. Mass spectra were recorded in a negative ion mode at following parameters: capillary voltage, 4.5 kV; drying gas temperature, 550 °C; nebulizing gas pressure, 55 psi; and heating gas pressure, 55 psi. The detection was performed considering a mass range of 100− 2000 m/z. Statistical Analysis. Data are presented as the mean of three or four replicates with standard error. Analysis of variance (ANOVA) and Tukey’s mean grouping were used to determine significance of the interactions among treatments. All statistical analyses were performed with SAS statistical software (version 9.1.3, NC, U.S.A.). C
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exaltata).12 In our work, we found that PV exuded 262% more phytate compared to PE under 5 mg L−1 As (Figure 1C). The data indicated that phytate exudation may be correlated with their ability in As accumulation in fern plants. Generally, organic acids are secreted by plants to enhance nutrient uptake or reduce metal uptake by forming stable complexes.6 Zhu et al.7 observed that Cd-resistant tomato plants secreted oxalate to exclude Cd from entering the roots, contributing to its Cd resistance. In buckwheat plants, oxalic acid forms Al-oxalate complexes, rendering Al less toxic to the plant.26 Phytate exudation by PV may also relate to its nutrient uptake and/or adaption to As-rich environment, although the specific mechanism is still unclear. Phytic Acid was Highly Efficient in FeAsO4 Solubilization. Phytate and oxalate were two main organic acids in the root exudates of fern plants (Figure 2B). Iron is an important nutrient for plants and exudation of LMWOA is often induced under Fe deficient conditions. Arsenic often binds Fe to form insoluble minerals in soils,27 thus Fe mobilization inevitably releases Fe-bound As.28 While the role of oxalate in mobilizing metals (e.g., Cd and Al) has been reported,29,30 the role of phytate and oxalate in As and Fe mobilization is not well understood. Therefore, we examined the role of phytate and oxalate in FeAsO4 solubilization with citrate as a comparison. As expected, solubilization of As and Fe increased with increasing organic acid concentrations (Figure 3AB). Among the three organic acids, phytic acid was the most effective at low organic acid:FeAsO4 molar ratios (1:1 and 2:1) while it was comparable with citric acid at high ratios (10:1, 20:1, and 50:1), with oxalic acid being the least effective across all ratios (Figure 3A). For example, at ratios of 1:1 and 2:1, phytic acid solubilized the highest amount of As from FeAsO4 at 39−43 μM compared to 32−41 μM for citric acid, and 10−15 μM for oxalic acid (Figure 3A). Since 100 μM FeAsO4 was used, it would have produced 100 μM As and Fe if it were all dissolved. The data indicated that at organic acid:FeAsO4 molar ratio of 1:1, phytic acid solubilized 39% of FeAsO4 while citric and oxalic acid solubilized 32 and 10%, indicating the much stronger chelation ability of phytic acid. The ability of phytic acid to solubilize FeAsO4 may attribute to its stronger complex stability with Fe, with higher concentration of organic acid leading to higher Fe dissolution from FeAsO4 (Table S4). In addition, phytic acid contains six orthophosphate moieties with 12 dissociable protons (pKa = 1.1−12; Table S3), thus it has high chelation potential for cations over a wide pH range.31 Its binding affinity increases exponentially with cation valence, making it effective in chelating Fe in soils.32 Different from the highest soluble As in solution (39−82 μM), phytic acid produced the lowest soluble Fe (1.6-31 μM) among three acids except at molar ratio of 50:1 (91 μM) where all acids produced similar Fe concentrations (Figure 3B). The soluble As:Fe molar ratio for oxalic and citric acids was ∼1 across all treatments, however, it decreased from 24:1 to 1:1 for phytic acid as organic acid/FeAsO4 ratios increased from 1:1 to 50:1. The data indicated that Fe solubilized by oxalic and citric acids was present mainly in soluble form whereas it was probably precipitated with phytic acid as Fe−phytate. On the basis of soluble As and Fe concentrations in the growth media, 96, 92, 87, 75, 58, and 1% of Fe was sequestered as insoluble Fe−phytate at phytic acid/FeAsO4 ratios of 1:1, 2:1, 5:1, 10:1, 20:1, and 50:1 (Table S4). According to Nielsen et al.,33 monoferric phytate is water-soluble, but tetraferric phytate is not, indicating the solubility of Fe-phytate increases with
Figure 2. Mixed standards of organic acids (A), and chromatogram of P. vittata (PV) root exudates with and without phytic acid standard addition based on HPLC (B) and phytate peaks in concentration range of 10−20 000 mg L−1 (C). P. vittata was grown in 0.2-strength Hoagland solution containing 20 mg L−1 As for 2 d.
concentration increased with increasing As levels in fern plants. For example, phytate levels in PV and PM exudates were 50 and 15 mg kg−1 in the control, which increased to 84 and 37 mg kg−1 under 5 mg L−1 As (Figure 1C). Although there was no phytate detected in PE at As ≤ 5 mg L−1, it was detected at 32 mg kg−1 under 20 mg L−1 As. However, for tomato plant, there was no phytate detected across all As levels (Figure 1C). Tomato plant mainly produced oxalate (Figure 1D), with low levels of malate, succinate, citrate, and fumarate (data not shown). The results indicated that phytate as root exudate was probably a special property of fern plants and its exudation was related to As. Positive responses of organic acid exudation to external stress have been observed among plants. It was reported that Alresistant wheat plants exuded 5−10 fold more malate than Alsensitive genotypes.24 Besides, enhanced citrate exudation was found in Al-resistant buckwheat plants than the Al-sensitive cultivars.25 In the study of Tu et al.,12 they observed that Ashyperaccumulator PV exuded 46−106% more phytate compared to the nonhyperaccumulator Boston fern (N. D
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Figure 3. Influence of organic ligands on As (A) and Fe (B) solubilization from FeAsO4 mineral at different organic acid/FeAsO4 ratios, and effect of phytate on dissolution of FeAsO4 mineral (C), and plant growth of P. vittata (D) in FeAsO4−phytic acid−P. vittata system after 14 d of growth in sterile Fe- and P-free 0.2-strength Hoagland media containing phytic acid and/or FeAsO4. Means marked with different letters indicate significant (p < 0.05) differences. The bars indicate the standard error of quadruplicates.
soluble Fe after 14 d of continuous Fe uptake by PV (Figure 3C). The data suggested that PV root exudates were effective in continuously releasing Fe from FeAsO4. Since phytate constituted 83% of PV root exudates (Figure 1C) and was more effective in releasing As from FeAsO4 than oxalate (Figure 3A), it was probably the main contributor in FeAsO4 dissolution. The formed Fe−phytate complex can be soluble or insoluble depending on the number of Fe3+ ions bound by phytic acid.33 In the phytic acid−FeAsO4 system (10:1 molar ratio), 75% of Fe released from FeAsO4 was probably present as insoluble Fephytate (Figure 3AB). However, in the phytic acid−FeAsO4− PV system, the soluble Fe concentration was doubled (51 vs 27 μM; Figure 3C). The data again suggested that PV root exudates were effective in FeAsO4 dissolution and Fe was probably solubilized from the insoluble Fe-phytate before being taken up by PV. It was possible that phytase released by PV roots may have played a role. Phytase is the only known enzyme capable of decomposing phytic acid. Although most plants produce phytase to release P from phytic acid, this process is normally observed inside the plant tissues.5 Unlike other plants, phytase has been detected in PV root exudates with a reported activity at 20 nmol P mg−1 protein min−1.5 Our hypothesis was supported by our data where a large amount of P was also released in the media containing phytic acid with and without FeAsO4 (135 and 120 μM) (Figure 3C). Lessl et al.5 showed that PV roots were efficient in releasing phytase to solubilize P from phytate, which was probably the case here. Attributing to the presence of phytase, it was understandable that little P was detected in P-free 0.2-strength Hoagland media control while 120−135 μmole P in control containing phytate. However, 58 μmole P was detected in FeAsO4 treatment, which was probably released from PV roots.
decreasing Fe in the complex. Our data indicated that, with similar concentrations of phytic acid and Fe at phytic acid/ FeAsO4 ratio of 1:1 (100 and 39 μM; Table S4), phytic acid probably bound 4 Fe3+ to form insoluble tetraferric phytate. However, with excess phytic acid at ratio of 50:1 (5000 and 83 μM of phytic acid and Fe; Table S4), water-soluble monoferric phytate was probably formed.34 The results implied that Fephytate complex was insoluble at low phytic acid:Fe ratios and became soluble when excess phytic acid was present. This was supported by Trela32 who observed >90% reduction in Fe concentration at phytic acid:Fe ratio of 1:1. Hurrell et al.35 suggested that Fe binds to phytic acid to form insoluble Fe− phytate. However, there is no confirmed value on its formation constant.22 The insoluble Fe−phytate was probably responsible for low Fe concentrations (1.6 and 3.3 μM) at low phytic acid/ FeAsO4 ratios (1:1 and 2:1) (Figure 3B). In soils, phytic acid is mainly present as insoluble Fe- and Alphytate15,36 and total soluble Fe concentration is estimated at 10−10.4 M, which is too low to sustain plant growth at 10−8 M.37 To better understand how PV takes up Fe-phytate, we tested the effect of phytic acid on FeAsO4 dissolution and Fe uptake in a phytic acid−FeAsO4−PV system. P. vittata Grew Better in Media Amended with FeAsO4 and Phytic Acid. Given the low solubility of Fe-phytate in soils, we investigated the growth, and As and Fe uptake of PV in Fe- and P-free 0.2-strength Hoagland media amended with 100 μM FeAsO4 and/or 1 mM phytic acid as the sole source of Fe and P. The selected concentrations of phytic acid and FeAsO4 corresponded to phytic acid/FeAsO4 ratio of 10:1 in organic acid−FeAsO4 system, which produced 27 μM soluble Fe (Figure 3B), comparable to typical 0.2-strength Hoagland media containing 37 μM EDTA-Fe (Table S1). Interestingly, without phytic acid, the media with PV and FeAsO4 had 12 μM E
DOI: 10.1021/acs.est.6b00668 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Under As stress, more phytate was exudated and being hydrolyzed by phytase, which was consistent with our data (Figures 1C and 3C). With comparable P in the media, PV biomass increase in phytic acid+FeAsO4 or FeAsO4 treatment was 6-fold of that in the phytic acid treatment (Figure 3D). The remarkable difference suggested that improved P nutrition may not play an important role in As-induced PV growth as hypothesized previously.38 FeAsO4 significantly enhanced PV growth, which was more apparent when coupled with phytic acid (0.57 and 0.67 g plant−1) whereas PV under phytic acid treatment had far less growth (0.11 g plant−1) (Figure 3D). The data suggested that PV biomass increase was more closely correlated with plant Fe and As contents than P (R2 = 0.80, 0.83, and 0.05, respectively). This further illustrated the more important role of Fe and As in PV growth promotion than P. However, it was unclear if it was the Fe or As that actually promoted PV growth. Arsenic has been reported to promote PV growth even at high concentrations,38,39 however, the associated mechanisms are still unclear and warrant further study. Phytic Acid Enhanced Fe, As, and P Uptake by P. vittata. While phytic acid is efficient in complexing Fe, its impact on Fe uptake by PV is unclear as Fe-phytate can be soluble or insoluble. In our experiment, PV accumulated 2-fold higher Fe in phytic acid+FeAsO4 treatment than FeAsO4 in both the roots and fronds (8 and 2.1 vs 4 and 0.9 mg g−1) (Figure 4A), which corresponded to the Fe concentrations in the media, i.e., 51 vs 12 μM (Figure 3C). Besides, PV accumulated 2.7- and 26-fold higher As in the roots and fronds from phytic acid+FeAsO4 than FeAsO4 treatment (160 vs 60 and 340 vs13 mg kg−1) (Figure 4B). This suggested that phytic acid enhanced both Fe and As accumulation in PV probably by complexing Fe and simultaneously releasing As. This was supported by Tu et al.12 who found 1.5 mM phytic acid released 58% of As from 43 mM FeAsO4, which was 33 times higher than that of oxalic acid. This was also consistent with Porter and Peterson40 who found significant correlation between As and Fe in As-tolerant plants; however, no correlation was found between As and other elements including P. Moreover, the large amount of P released from phytic acid (Figure 3C) resulted in greater P accumulation in PV roots and fronds than 0.2-strength Hoagland control (4.1 vs 1.5 and 7.2 vs 4.0 mg g−1) (Figure 4C). While 61−75% of P was transported to the fronds, which was consistent with Ghosh et al.41 and Xu et al.,38 75−82% of Fe was stored in the roots in this study (Figure 4A). Generally, Fe is transported to plant shoots for its role in chlorophyll synthesis.42 Apparently, PV was inefficient in translocating Fe, which was consistent with 385 and 20 mg kg−1 Fe in the roots and fronds observed by Ghosh et al.41 High root Fe concentrations in the tens of mg kg−1 range have also been found in plants by others.43,44 Gramlich et al.45 suggested that root-to-shoot metal translocation may be affected by organic ligands in plants via complex formation. We tested this hypothesis by external application of phytic acid to PV, which increased phytate and oxalate concentrations in PV (Figure S1). Contrary to enhanced oxalate in the fronds, phytate increase occurred mainly in the roots. Phytate has a high affinity with Fe and it seemed that stronger complexion between phytic acid and Fe made it more stable in the roots where they were highly correlated (R2 = 0.998) (Figure 4A and Figure S1B). Whether Fe−phytate is present in the root sap is
Figure 4. Total Fe (A), As (B), and P (C) contents in P. vittata tissues after 14 d of growth in sterile Fe- and P-free 0.2-strength Hoagland solution containing 1 mM phytic acid and/or 100 μM FeAsO4. Means marked with different letters indicate significant (p < 0.05) differences for fronds or roots. The bars indicate the standard error of quadruplicates.
currently being examined based on gel filtration chromatography and will be presented in a separate publication. Environmental Implication. Quantitative and qualitative differences were observed in the composition of root exudates of four plant species, with phytate being unique in fern plants. As-hyperaccumulators (PV and PM) with more As accumulation excreted higher concentrations of phytate and oxalate than nonhyperaccumulator PE, whereas tomato plant exudated no phytate across all As levels. In addition, phytate induced higher As-solubilization from FeAsO4 mineral than oxalate and citrate. As a result, PV grown in phytic acid+FeAsO4 had higher As and Fe contents and larger biomass than phytic acid or FeAsO4 treatment. Large amount of P was taken up by PV when phytic acid was supplied, indicating the ability of PV in releasing phytase, which probably solubilized P from phytate. Though similar amount of P was taken up by PV, the fact that larger PV biomass was obtained in phytic acid+FeAsO4 than phytic acid treatment suggested the importance of Fe and/or As for PV growth. PV was effective in taking up Fe, but little was translocated to the fronds possibly due to formation of Fephytate in the roots. The presence of FeAsO4 in the media induced P release to the growth media by PV, possibly responding to As stress. In short, phytate exudation was probably a special property in fern plants, which was related to F
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Environmental Science & Technology
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As stress and probably plays an important role in As hyperaccumulation in P. vittata. Our data identified phytate as a dominant root exudate from fern plants, which was stimulated under As stress. However, it is counterintuitive that a plant would exudate phytate to counter As stress. Our previous research has shown that under As stress,12 PV stored more P in the roots than the fronds, indicating that P was needed to reduce As stress as As interferes with P metabolism in plants. Therefore, it would be interesting to investigate the molecular mechanisms behind As-induced exudation of phytate by PV and other fern plants. In addition, our data also clearly showed the role of phytic acid in releasing Fe and As from insoluble FeAsO4 and enhancing their uptake by PV. However, the mechanisms of Fe uptake by PV and whether Fe-phytate was taken up by PV are still unclear. Though phytic acid solubilized both Fe and As from FeAsO4, part of the Fe was probably present as insoluble Fe-phytate. The fact that soluble P was present in the growth media suggested the presence of phytase in releasing P from phytic acid and/or P/Fe from Fe-phytate. As such, soluble Fe was probably taken up by PV, which was complexed with phytic acid in the roots as both Fe and phytic acid were detected and highly correlated in the roots. Better understanding of how PV solubilizes Fe and P from insoluble minerals and the associated molecular mechanisms of their uptake by PV may be useful to increase plant uptake of Fe, P, and As, which has implication for more efficient phytoremediation of As-contaminated soils.
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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.6b00668. Composition of 0.2-strength Hoagland nutrient solution (Table S1), information on organic acid standards (Table S2), pKa data for myo-inositol hexakisphosphate (Table S3), soluble As and Fe concentrations released from FeAsO4 by phytic acid at various phytic acid/FeAsO4 molar ratios (Table S4), TOF−MS spectra of phytate root exudate (Figure S1), and effect of external phytic acid on phytate and oxalate contents in the fronds and roots of P. vittata (Figure S2) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +86 025 8968 0631; e-mail:
[email protected] (Y.C.). *Phone: +86 025 8968 0631; e-mail: lqma@ufl.edu (L.Q.M.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the National Natural Science Foundation of China (No. 21277070) and Jiangsu Provincial Innovation Fund.
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REFERENCES
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DOI: 10.1021/acs.est.6b00668 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.est.6b00668 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
