Plant and Soil 258: 9–19, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Root exudates and arsenic accumulation in arsenic hyperaccumulating Pteris vittata and non-hyperaccumulating Nephrolepis exaltata Shuxin Tu1, Lena Ma1 & Thomas Luongo Soil and Water Science Department, University of Florida, Gainesville, FL 32611-0290, USA. 1 Corresponding authors∗ Received 31 January 2003. Accepted in revised form 22 July 2003
Key words: arsenic, hyperaccumulation, mobilization, organic acid, root exudates
Abstract This study compared the roles of root exudates collected from two fern species, the As hyperaccumulating Chinese Brake fern (Pteris vittata L.) and the As-sensitive Boston fern (Nephrolepis exaltata L.), on As-mobilization of two As minerals (aluminum arsenate and iron arsenate) and a CCA (chromated copper arsenate)-contaminated soil as well as plant As accumulation. Chinese Brake fern exuded 2 times more dissolved organic carbon (DOC) than Boston fern and the difference was more pronounced under As stress. The composition of organic acids in the root exudates for both ferns consisted mainly of phytic acid and oxalic acid. However, Chinese Brake fern produced 0.46 to 1.06 times more phytic acid than Boston fern under As stress, and exuded 3–5 times more oxalic acid than Boston fern in all treatments. Consequently, root exudates from Chinese Brake fern mobilized more As from aluminum arsenate (3–4 times), iron arsenate (4–6 times) and CCA-contaminated soil (6–18 times) than Boston fern. Chinese Brake fern took up more As and translocated more As to the fronds than Boston fern. The molar ratio of P/As in the roots of Chinese Brake fern was greater than in the fronds whereas the reverse was observed in Boston fern. These results suggested that As-mobilization from the soil by the root exudates (enhancing plant uptake), coupled with efficient As translocation to the fronds (keeping a high molar ratio of P/As in the roots), are both important for As hyperaccumulation by Chinese Brake fern. Abbreviations: CCA – chromated copper arsenate; DOC – dissolved organic carbon; HMW – high molecular weight; LMW – low molecular weight; TF – translocation factor Introduction Root exudates are plant metabolites that are released to the root surface or into the rhizosphere to enhance plant nutrient uptake (Curl and Truelove, 1986). They are generally classified into two types, high molecular weight (HMW) and low molecular weight (LMW) materials. The first includes mucilage (mainly polysaccharides and polyuronic acid) and ectoenzymes; the latter mainly consists of organic acids, sugars, phenols and various amino acids, including non-protein amino acids such as phytosiderophores (Marshner, 1995). The composition and quantity of root exudates vary ∗ FAX No: 353-392-3902. E-mail:
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from plant to plant with two factors being important. One is a plant’s inherent biology, such as plant species, growth and developmental period and nutrient status. The other is the external environment for plant growth, i.e., soil and its elemental content (Gregory and Atwell, 1991). Approximately 28–59% of the photosynthetic product in a plant is translocated underground, of which up to 70% is excreted into the soil (Lynch and Whipps, 1990). Such a large amount of organic material released into the soil produces significant changes to the soil chemistry, especially in the rhizosphere. Numerous studies have probed into the identification, characterization and functions of root exudates, which include the movement of root exudates in the
10 rhizosphere (Donald et al., 1993), weathering of soil minerals (Hinsinger et al., 1993), mobilization of soil nutrients (Subbarao et al., 1997), enhancement of nutrient uptake (Jones et al., 1994) and stress resistance to acid and toxic metals (Jones, 1998; Lasat, 2002). Phytoextraction, based on the efficient uptake function of the roots of living plants, has proven to be an effective technique to remediate contaminated soils and groundwater. Since soil metals/metalloids are often insoluble and unavailable for plant uptake, plants that posses mechanisms to increase metal/metalloid bioavailability in the rhizosphere have the potential for metal/metalloid uptake. Such mechanisms mainly include acidification, complexation/chelation and reduction/oxidation (Marshner, 1995). For instance, it was found that some plants, especially dicotyledons, acidify the rhizosphere via organic acid extrusion from the roots to increase metal and phosphate uptake (Marschner et al., 1986). An acidic environment usually enhances metal reduction, such as changing Fe3+ to Fe2+ , which is then easily taken up by the plants (Bienfait et al., 1982). The most common mechanism for mobilizing heavy metals by plants is complexation. One study has found that Fe deficient wheat (Triticum aestivum L. cv. Pika), which exudes phytosiderophores, a powerful chelator of metals, shows enhanced uptake of Zn, Ni and Cd (Volker and Fikry, 2000). Similar results were also reported for other metals, such as Cu, Mn, Pb, and Co (Dousset et al., 2001). The mechanisms related to metal/metalloid hyperaccumulation in plants are variable. Research on Thlaspi caerulescens, a Zn/Cd hyperaccumulator, indicates that the plant mobilizes Zn and Cd efficiently from soils. However, this ability may not be due to rhizosphere acidification by root exudates (Knight et al., 1997). Similar results were obtained for a Ni hyperaccumulator, Thlaspi goesingense (Krämer et al., 2000). Zhao et al. (2001) reported that root exudates from the hydroponically-grown T. caerulescens did not significantly enhance metal mobilization from Cu- or Zn-loaded resins, indicating that certain hyperaccumulators are not necessarily more efficient in extracting metals from the non-labile pools in soils than non-hyperaccumulating plants. Arsenic (As) is a metalloid of environmental concern due to its toxicity and ubiquity. Chinese Brake fern (Pteris vittata L.) was recently found to hyperaccumulate As (Ma et al., 2001). In As-spiked soils, it can accumulate 23 g kg−1 of As in its aboveground biomass (fronds). Even in uncontaminated soils, this
fern can take up as much as 744 mg kg−1 of As. This is much greater than most other plants, which normally take up ≤10 mg kg−1 of As (Matschullat, 2000). Research on its As uptake kinetics found that Chinese Brake fern displayed a high As uptake influx rate as compared to Boston fern (Nephrolepis exaltata L.), an As non-hyperaccumulator (Tu et al., 2003). These results clearly demonstrate that this hyperaccumulator may be equipped with both an efficient As uptake and a detoxification system. Arsenate is a chemical analogue of phosphate, whose bioavailability has been found to be enhanced by plant root exudates (Dinkelaker et al., 1989; Ohwaki and Hirata, 1992). This may suggest that root exudates could be important in the mobilization of soil As and accumulation of As by the plant. Therefore, the objectives of this research were to characterize the root exudates of two ferns: Chinese Brake fern and Boston fern and examine their effects on soil As mobilization and plant As accumulation.
Materials and methods Plant materials and plant growth condition Two ferns, Chinese Brake fern and Boston fern were used in this study. Four-month old Chinese Brake fern, propagated in the growth room of our laboratory and 4-month old Boston fern, procured from a nursery (Milestone Agriculture, Inc., FL, USA) were used in this experiment. The plants were transferred to our hydroponic systems in a growth room at 23–28 ◦ C and 70% humidity. A 14-h photoperiod with a daily photosynthetic photon flux of 350 µmol m−2 s−1 at plant canopy was supplied by an assembly of both cool-white and warm-white fluorescent lamps. Both ferns were allowed to grow for two weeks to initiate new root growth. The nutrition solution used was half-strength HoaglandArnon solution (Hoagland and Arnon, 1938), which comprised of 3 mM KNO3 , 0.5 mM NH4 H2 PO4 , 2.0 mM Ca(NO3 )2 , 1.0 mM MgSO4 .7H2 O, 4.5 µM MnCl2 .4H2 O, 23 µM H3 BO3 , 0.4 µM ZnSO4 .7H2 O, 0.15 µM CuSO4 .5H2 O, 0.05 µM H2 MoO4 .H2 O, and 22 µM EDTA-Fe. The solution pH in this study was adjusted to 6.0 with dilute HCl and NaOH. All chemicals were reagent of trace metal grade. The solution was aerated continually and replenished twice a week.
11 Collection of root exudates Before the collection of the root exudates, both fern plants which were acclimated in a hydroponic system for 2 weeks were transferred and grown for an additional 2 days in half-strength Hoagland nutrition solution spiked with 0, 67, 267 or 1068 µM of As (Na2 HAsO4 . 7H2 O) (SIGMA). Nine plants for each treatment were treated in a 1.5-L opaque plastic pot containing 1 L of the solution. The solution was aerated and replaced at the second day. The plant roots were then washed with tap water, and soaked in an antibiotic solution [30 mg L−1 chloramphenicol (SIGMA)] for 2-h to minimize microbial growth (Subbarao et al., 1997), followed by washing with tap water and sterilized, deionized water. Three plants from each treatment after As-exposure were placed into 800 mL of sterilized, deionized water to collect root exudates for 6-h. Each treatment had three replicates. The root exudates solution was immediately filtered, lyophilized (FreezZone 12, LABCONCO) to 10 mL and stored at −20 ◦ C for further analysis. Arsenic mobilization from As minerals and CCA soil by root exudates In most soils under aerobic conditions, arsenate (AsO4 3− ) is the predominant form of As which is mostly bound to clay minerals, Fe and Mn-oxides/hydroxides, and organic substances (Matschullat, 2000). AlAsO4 ·2H2 O (Al-As) and FeAsO4 ·2H2 O (Fe-As) are the most common As minerals in soils (Rochette et al., 1998). Arsenicmobilization from the two common As minerals and a CCA (chromated copper arsenate)-contaminated soil by root exudates was measured by determining As concentrations in the root exudates solution. Al-As and Fe-As were synthesized via the method of Hess and Blanchar (1976), washed free of salts, and verified with x-ray diffraction and total chemical analysis since they are not available from commercial sources. The CCA-contaminated soil was collected from an abandoned CCA wood preservation site in central Florida. Its selected physical and chemical properties were: sand 896 g kg−1 , silt 79 g kg−1, clay 25 g kg−1 (Pipette method; Day, 1965), pH (1:1 soil/water) 6.88, CEC 0.78 mmol kg−1 (Ammonium acetate method; Thomas, 1982), total organic matter 15.7 g kg−1 (Walkley Black method; Nelson and Sommers, 1982), total As 254 mg kg−1 , water soluble As 6.25 mg kg−1 , reactive Fe 313 mg kg−1 (Sodium hydroxide extrac-
tion; Yuan and Fiskell, 1959), and reactive Al 219 mg kg−1 (Acid ammonium oxalate extraction; Schwertmann, 1964). Two mL of root exudate solution and 2 mL of deionized water were added to a test tube with 20.0 mg of the As minerals or 100 mg of the CCA soil. Deionized water was used as a control. Fifty µL of chloroform was added to prevent microbial decomposition of root exudates. The solutions were mechanically shaken at 25 ◦ C for 24-h and centrifuged at 10 000 g for 15-min. The supernatant was acidified using concentrated HNO3 (50 µL) for total As determination. To compensate for the influence of root weight between treatments, the net As-mobilization from minerals or soil expressed on a dry root weight (d. wt) basis was calculated as follows: Net As − mobilization =
AsS − AsRE − AsC , AsT × DW
where, AsS is the amount of As in the supernatant of root exudate solution; AsRE is the amount of As contained in root exudate solution added; AsC is the amount of As solubilzed by control; AsT is the amount of As in the minerals or the soil weight; and DW is dry weight of the roots. Arsenic-mobilization kinetics from soil and minerals by LMW organic acids Phytic acid and oxalic acid are the two main LMW organic acids identified in the root exudates of the Chinese Brake ferns in this study. The role of oxalic acid in mobilizing mineral elements has been widely reported (Jones, 1998), however, the role of phytic acid on As-mobilization was not well understood. Arsenic dissolution kinetics from two As minerals (Al-As and Fe-As) and one CCA soil were carried out in a batch experiment: Into a 50-mL centrifuge tube 0.200 g of Al-As or Fe-As or 1.000 g of the soil was weighed. This was followed by adding 20 mL of a phytic acid (myo-inositol-1,2,3,4,5,6hexakis-dihydroge nphosphate) (SIGMA), or oxalic acid (EASTMAN) solution with concentrations ranging from 0 to 1.5 mm and 50 µL of chloroform to inhibit microbial activity. Deionized water was used as a control. The centrifuge tube was capped and shaken mechanically at room temperature for 24-h. The solution was filtered and centrifuged at 10 000 g for 15-min. The supernatant was removed and acidified with 50 µL of concentrated HNO3 for total As determination.
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Figure 1. Dissolved organic carbon (DOC) in root exudates of Chinese Brake fern and Boston fern expressed on a unit root dry weight basis. The root exudates were collected from the fern after being treated with different levels of As (0, 67, 267 and 1068 µM) for 2 days. The error bars are the standard errors of the means from three replicates. DOC at 1068 µM As in root exudates of Boston fern was not determined due to severe As toxicity.
Plant As uptake The ferns grown in the hydroponic system for 2 weeks were removed and their roots were washed carefully first with tap water followed by deionized water. Thereafter, the uptake studies were initiated by placing three plants (using Styrofoam sheet with holes on the surface as a plant supporter) in a 1-L opaque plastic pot containing 800 mL of half-strength Hoagland solution spiked with 0, 67, 267 or 1068 µM As (Na2 HAsO4 .7H2 O), resulting in 8 treatments (including both ferns). Each treatment was replicated three times. The solution pH was adjusted to 6.0 with dilute HCl and NaOH, and the solution was aerated vigorously. Since Boston fern is sensitive to As, the ferns were allowed to grow in As medium for only 2 days. Then, plant roots were washed with tap water followed by an ice-cold phosphate buffer containing 1 mM Na2 HPO4 , 10 mM MES and 0.5 mM Ca(NO3 )2 to ensure desorption of As from the root surface and the root free space (Asher and Reay, 1979). Thereafter, the plants were rinsed in tap water followed by deionized water. The fern plants were then separated into roots and fronds, and dried at 65 ◦ C for later determination of total As and P.
Figure 2. Net As-mobilization from aluminum arsenate [Al-As, (a)], iron arsenate [Fe-As, (b)] and a CCA (chromated copper arsenate) contaminated soil (c) by root exudates collected from Chinese Brake fern and Boston fern after being treated with 0, 67, 267 or 1068 µM As for 2 days. The error bars are the standard errors of the means from three replicates. The legends indicate different levels of As treatments for plants. Missing data at 1068 µM As in Boston fern was due to severe As toxicity.
13 Chemical analysis As determination Plant samples were digested using USEPA Method 3050A. Briefly, 0.1–0.5 g of ground plant material was weighed directly into a polypropropylene digestion vessel, mixed with 10 mL of 1:1 (v/v) nitric acid (trace metal grade) and covered with a clean polypropylene ribbed watch glass. The samples were placed into a temperature-controlled digestion block (Environmental Express, Mt. Pleasant, S.C.) and heated for 16 h at 45 ◦ C. The temperature was then raised to 105 ◦ C for 3 h, with more 1:1 nitric acid added as needed to maintain a minimum of 5 mL in the vessel. The samples were then removed from the block and two 0.5-mL aliquots of 30% H2 O2 were added. After having been returned to the block for another 20 min, the samples were cooled, brought to a final volume of 50 mL, filtered and capped for determination of As. Analysis was performed with a transversely heated, Zeeman background correction equipped graphite furnace atomic absorption spectrophotometer (Perkin-Elmer SIMAA 6000, Norwalk, CT). Palladium nitrate (1000 mg L−1 ) was used as the modifier. Pyrolysis temperature was 1200 ◦ C and atomization was carried out at 2100 ◦ C. The standard reference material was carried through the digestion and analyzed as part of the quality assurance–quality control protocol. Reagent blanks and internal standards were used where appropriate to ensure accuracy and precision in the analysis of As. Plant P determination Plant samples were digested using a H2 SO4 /H2 O2 method(Jones et al., 1991). Because arsenate interferes with P determination using the molybdenum blue method (Murphy and Riley, 1962), P was determined using a modified method of Carvalho et al. (1998). Briefly, the pH of the digestion solution was adjusted to around 5 with NaOH and HCl. Ten mL of the solution was pipetted into a 20 mL glass test tube, and to this 0.5 mL of L-cysteine (5% w/v in 0.6 M HCl) was added. The test tube was capped tightly to allow arsenate reduction for 5 min at 80 ◦ C. The solution was cooled to room temperature, and P was determined by the molybdenum blue method. Determination of dissolved organic carbon (DOC) DOC in the root exudate solution was analyzed using TOC-5050A total organic carbon analyzer (Shimadzu) equipped with an autosampler.
Determination of organic acids in root exudates The LMW organic acids were identified and analyzed using HPLC. The concentrated root exudates were first centrifuged at 10 000 g for 15 min. An aliquot (1–5 mL) of supernatant was removed and diluted with DI water (1/10) and poured onto an Analytichem Silica-based, anion-exchange (SAX) column (quaternary amine Bond Elut column, 500 mg/3 mL, Varian, Harbor City, CA). The column was washed with 2 mL of deionized water, and the organic acids were eluted with 2 mL of 2 M HCl. The eluate was freeze-dried (maintaining up to 24-h) (FreezZone 12, LABCONCO). The residue was dissolved in a ultrasonic bath for 6 min with 1 mL of the HPLC mobile phase solution, 5 mM H2 SO4 . The mixture was filtered through 0.45 µm filter to remove suspended material prior to injection into the HPLC. HPLC analysis was performed using a Waters 2690 Separations Module equipped with an auto sampling system, a SUPELCOGEL C610H HPLC column (30cm × 7.8 mm ID) and a Waters 2478 Dual λ absorbance detector set at 210 nm with a flow rate of 30 mL h−1 . Since the quantification of phytic acid using HPLC methods results in overestimation of the actual concentrations due to the problems associated with the quantitating peaks that coincide with solvent front (Lee and Abendroth, 1983; Lehrfeld, 1994), the quantification of phytic acid in the root exudates was carried out by the modified colorimetric method of Haug and Lantzsch (1983). Briefly, 0.5 mL of root exudate solution (3–30 µg mL−1 phytate P) was pipetted into a test tube fitted with a ground-glass stopper. One mL of ferric solution [0.2 g ammonium iron (III) sulphate.12 H2 O (ACROS ORGANICS, NJ) in 100 mL 2 M HCl and make up to 1 L] was added. The tube was covered with the stopper and fixed with a clip. The tube was then heated in a boiling water bath (ISOTEMP 210, Fisher Scientific) for 30 min. After cooling in ice water for 15 min, the tube was allowed to adjust to room temperature. The contents of the tube was mixed and centrifuged for 10 min at 10 000 g. One mL of the supernatant was transferred to another test tube and 1.5 mL of the 2,2 -Bipyridine solution [dissolve 10 g 2,2 -Bipyridine (ACROS ORGANICS, NJ) and 10 mL thioglycollic acid (ACROS ORGANICS, NJ) in deionized water and make up to 1 L] was added. A calibration curve was prepared using the same procedure with standard solutions of sodium phytate (SIGMA). The absorbance was measured at 519 nm against a control (deionized water) using a Shimadzu 160U spectrometer.
14 Statistical analysis Analysis of variance was carried out with the ANOVA procedure of SAS Software. The Tukey procedure was used for mean separation.
Results Composition of root exudates Concentrations of dissolved organic carbon (DOC) in the root exudates of Chinese Brake fern expressed on a root dry weight basis were significantly greater than Boston fern (Figure 1). In the control treatment, DOC in the root exudates of Chinese Brake fern was approximately 2 times greater than Boston fern. The difference in the amount of DOC exudated by the two ferns was even more pronounced with the addition of As, which caused DOC levels to increase in Chinese Brake fern and to decrease in Boston fern. Root exudates from the two ferns had similar compositions in terms of LMW organic acids. HPLC analysis identified that the dominating LMW organic acids were phytic acid and oxalic acid (Table 1). The presence of phytic acid in the root exudate solution was also confirmed and quantified by the colorimetric method of Haug and Lantzsch (1983). Citric acid, ascorbic acid, succinic acid and fumaric acid were found in small quantities in some samples (data not shown). Although no significant difference in phytic acid exudation was found between the two ferns in the absence of As, Chinese Brake fern exuded 46–106% more phytic acid than Boston fern under As stress. Additionally, Chinese Brake fern exuded 3–5 times more oxalic acid than Boston fern in all treatments. No difference was found in phytic and oxalic acid concentrations among As treatments, except that exudation of phytic acid by Boston fern was reduced significantly under As stress (Table 1). Net As-mobilization from Al-As, Fe-As and CCA soil by root exudates and LMW organic acids Root exudates from both ferns dissolved significant amounts of As from Al-As, Fe-As and CCA soil. The amount of As mobilized from Al-As was significantly (approximate 5–8 times) greater than that from Fe-As due to the high solubility of Al-As (Figure 2a,b). Root exudates from Chinese Brake fern mobilized about 3–4 times more As from Al-As, 4–6 times more As from Fe-As, and 6–18 times more As from CCA soil
than Boston fern, suggesting that Chinese Brake fern possesses greater ability to solubilize As than Boston fern. However, pre-treatment of the ferns with As did not have any significant effects on the mobilization capacity of root exudates on Al-As (Figure 2a) and Fe-As (Figure 2b). But pre-treatment did decrease As mobilization from CCA soil (Figure 2c). Phytic acid and oxalic acid were both effective in mobilizing As from Fe-As and Al-As minerals as well as from CCA soil (Figure 3). The amount of As-mobilization increased as the concentrations of organic acids increased. At the same concentrations, however, phytic acid was more effective in mobilizing As than oxalic acid. The increases in As-mobilization by phytic acid were 0.7–4.6 times greater for Al-As, 13.6–32.8 times for Fe-As and 4.4–5.6 times for CCA soil.
Arsenic uptake and distribution of As and P in two fern species The addition of As to the hydroponic solution promoted significant As uptake for both ferns (Table 2). However, Chinese Brake fern took up much more As than Boston fern under the same conditions. After 2 days of exposure to 67–1068 µM As, As accumulation by Chinese Brake fern increased by 103–392 mg kg−1 in the fronds, a 2.5–18 fold increase over Boston fern, and 18–155 mg kg−1 (1.1–2.0 fold increase over Boston fern) in the roots. Translocation factor (TF), defined as the ratio of As concentration in fronds to that in roots, was calculated to compare As distribution among fern species (Table 2). The results showed that Chinese Brake fern transported relatively more As to the fronds (TF=2.2– 5.1) than Boston fern (TF=0.67–0.87). For Chinese Brake fern, P was evenly distributed between its fronds and roots with P concentration ranging from 2600 to 3300 mg kg−1 . Unlike Chinese Brake fern, P in Boston fern was more concentrated in the fronds than the roots, with frond P concentration being as high as ∼8500 mg kg−1 , which was 7 times greater than its root P concentration (∼1500 mg kg−1 ). The concentration and distribution patterns of As and P were further confirmed by the molar ratios of P/As in the two ferns (Figure 4). Clearly, the molar ratios of P/As in the roots of Chinese Brake fern were greater than those in the fronds whereas the opposite was observed in Boston fern.
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Figure 3. Influence of different concentrations of LMW organic acids, phytic acid and oxalic acid, on As-mobilization from aluminum arsenate (Al-As), iron arsenate (Fe-As) and a CCA (chromated copper arsenate) contaminated soil.
Figure 4. Molar ratio of P/As in fronds and roots of Chinese Brake fern and Boston fern grown for 2 days in half-strength Hoagland nutrition solution spiked with different levels of As. Symbols As67, As367 and As1068 represent As concentrations of 67, 267, and 1068 µM, respectively. The error bars indicate the standard errors of the means from three replicates.
Discussion In comparison with Boston fern, Chinese Brake fern excreted large amounts of DOC when expressed on the basis of unit root dry weight (Figure 1). DOC in the root exudates includes different organic compounds, which can trigger a range of chemical reactions and biological transformation in the rhizosphere (Pinton et al., 2001). In this sense, the roots of Chinese Brake fern exhibited, directly or indirectly via exudation, a greater capability in mobilizing As. In fact, we found that the root exudates from Chinese Brake fern mobil-
ized significantly greater amounts of As from both As minerals and CCA soil than did Boston fern (Figure 2). Therefore, the increased As-mobilization due to high root exudation could play an important role in As hyperaccumulation by Chinese Brake fern. In addition, root exudation might be involved with As detoxification in Chinese Brake fern, as it has been found that root exudates sometimes help to reduce metal toxicity, such as Al toxicity in Maize (Pellet et al., 1995) and wheat (Pellet et al., 1996) and Ni toxicity in Maize and ryegrass (Yang et al., 1997). However, since As is a metalloid, the mechanisms for As detoxification by root exudates may be different from other metals. Therefore, further research is needed to understand the relationship between As detoxification and root exudation. Chinese Brake fern exuded 40–106% more phytic acid under As stress and 300–500% times more oxalic acid across all treatments than Boston fern (Table 1). Some of the samples contained small amounts of citric acid, ascorbic acid, succinic acid and fumaric acid as well (data not shown). Oxalic acid is a typical LMW organic acid in plant root exudates (Pinton et al., 2001). Since it possesses the capability of both proton donation and ion complexation, oxalate/oxalic acid has been widely used as an extractant for plant available nutrients, including phosphate, from soil (Fransson, 2001; Sagoe et al., 1998). It was interesting to note that large amounts of phytic acid existed in the root exudates of both ferns. Phytic acid is a common constituent of cereal grains and cereal products. In germinating legume seeds, it constitutes >85% of
16 Table 1. Concentrations of phytic acid and oxalic acid in root exudates of Chinese Brake fern and Boston fern expressed on a root dry weight (d. wt) basis. Before the 6-h collection of root exudates, both ferns were pre-cultured in half-strength Hoagland nutrition solution for 2 weeks to grow new roots and then were exposed in half-strength Hoagland nutrition solution spiked by different levels of As (0, 67, 267 and 1068 µM) for 2 days Ferns
As levels (µM)
Phytic acid (µg g−1 D. wt)
Oxalic acid (µg g−1 D. wt)
Chinese Brake fern
0 67 267 1068
396.4 (15.9)a 383.1 (29.5) 386.7 (13.5) 373.0 (22.4)
A A A A
55.3 (6.0) 48.2 (3.7) 43.3 (15.8) 53.1 (20.3)
A A A A
Boston fern
0 67 267 1068b
380.4 (36.7) 262.2 (22.1) 187.6 (23.7) /
A B C
13.7 (1.8) 11.5 (1.5) 7.2 (0.7) /
B B B
a Data in parenthesis are the standard errors of three duplications. The means
with the same letter in a column are not significantly different at P < 0.05 based on Tukey’s test. b Samples were not collected due to severe As toxicity in Boston fern after 2 days exposure at 1068 µM As.
the total P (Reddy et al., 1989). Thus, phytic acid is considered a major form for P storage. Some fern plants, such as Lemna gibba, L. minor and Wolffiella floridana, are known to have high capacity for phytic acid synthesis (Roberts and Loewus, 1968; Van Steveninck et al., 1990). Since phytic acid contains 12 acid groups, it is able to complex with metals, such as Fe, Ca, mg and Zn, to form insoluble minerals like Mn-phytate and Zn-phytate, which have been recently proposed to account for Zn/Mn detoxification in plant cells (Otegui et al., 2002; Van Steveninck et al., 1990). Little is known about the role that organic acids play in plant As hyperaccumulation. In order to better understand the mechanism of As uptake and hyperaccumulation in Chinese Brake fern, we studied the role of phytic acid and oxalic acid in As-mobilization from two common As minerals and a CCA-contaminated soil. The concentrations of the organic acids used in the batch experiment were designed by referring literature, which reported that the concentrations of organic acids in soil solution were generally about 0.01–4 mM with most in the range of 0.01–1 mM (Jones, 1998). As expected, both organic acids were able to mobilize significant amounts of As from both the As minerals and the contaminated soil. In addition, the amounts of As solubilized increased with organic acid concentration (Figure 3) and extraction time (data not shown). Being chemical analogues, the mechan-
ism of arsenate-mobilization by organic acids may be similar to that of phosphate, as is found in many plants, like the chickpea (Cicer arietinum) and the white lupin (Lupinus albus). These plants produce relatively large quantities of organic acids in P deficient soils to enhance the bioavailability of soil P (Dinkelaker et al., 1989; Ohwaki and Hirata, 1992). Under the same concentrations, phytic acid mobilized more As from Al-As (0.7–4.6 times), Fe-As (13.6–32.8 times) and CCA soil (4.4–5.6 times) than oxalic acid. This may be due to both the greater acidity and stronger complexation capability of phytic acid than oxalic acid. Detailed principles concerning their functions in soil As-mobilization and plant As hyperaccumulation deserve further attention. The As uptake study revealed that the hydroponically-grown Chinese Brake fern took up large amounts of As in 2 days (Table 2). Compared with the Boston fern, it accumulated up to 18 times more in the fronds and up to 2 times more in the roots. In addition, Chinese Brake fern transported more As to the fronds than Boston fern. These characteristics of As hyperaccumulation by Chinese Brake fern were similar to those of Wang et al. (2002), and corroborate the results by other researchers (Komar, 1999; Ma et al., 2001) who characterized the As hyperaccumulation of the fern grown in soils.
17 Table 2. Arsenic concentrations and translocation factors (TF) for Chinese Brake fern and Boston fern. Both ferns were grown in half-strength Hoagland nutrition solution for 2 weeks to initiate new roots and then were exposed in half-strength Hoagland nutrition solution spiked by different levels of As (0, 67, 267 and 1068 µM) for 2 days Concentration of As (mg kg−1 d. wt) Fronds Roots
TFa
Ferns (µM)
As levels
Chinese Brake fern
0 67 267 1068
6.2 (1.2)b 109 (23.4) 159 (36.3) 398 (24.9)
D C B A
3.9 (0.9) 21.8 (2.0) 71.9(7.2) 159 (14.1)
D C B A
2.2 5.1 2.2 2.5
Boston fern
0 67 267 1068c
1.5 (0.4) 6.9 (1.2) 17.7 (0.6)
D D D
1.9 (0.4) 10.5 (0.9) 20.8 (2.3) /
D CD C
0.87 0.67 0.86
/
a TF was calculated as the ratio of As concentrations in fronds to that in roots. b Data in
parenthesis are the standard errors of three duplications. The means with the same letter in a column are not significantly different at P < 0.05 based on Tukey’s test. c Arsenic determination was not carried out due to severe As toxicity in Boston fern after 2 days exposure at 1068 µM As.
The mechanisms of As hyperaccumulation in Chinese Brake fern, investigated in previous experiments, showed that As uptake was significantly influenced by P nutrition (Tu et al., 2003; Tu and Ma, 2003; Wang et al., 2002). In this experiment, we determined and compared the concentrations of P and As in different parts of the two ferns under As influence and found that the molar ratios of P/As in the roots of Chinese Brake fern was greater than those in the fronds. Boston fern showed the opposite results (Figure 4). Since phosphate and arsenate are analogues and the addition of P decreases As uptake and consequently reduces As toxicity (Meharg and Macnair, 1992), greater molar ratios of P/As in the roots may help reduce As toxicity in the roots. We suggest that Chinese Brake fern is equipped with an As detoxification mechanism including both P accumulation in the roots and As translocation from the roots to the fronds, resulting in a high P/As ratios in the roots. Boston fern, however, accumulated less P in the roots and transported little As from its roots to the fronds, yielding a low ratio of P/As in the roots. Such a distribution pattern deprived Boston fern of any As detoxification mechanism and eventually led to plant death (when treated at 1068 µM As for 2 days). Therefore, the distribution pattern of As and P may be a clue to As hyperaccumulation mechanism in Chinese Brake fern. In summary, as compared to Boston fern, Chinese Brake fern was able to exude high amounts of DOC including phytic and oxalic acids, and consequently was
effective in mobilizing As from both As-minerals and As-contaminated soils. Chinese Brake fern displayed a unique capability to take up As and translocate it to aboveground biomass. The molar ratios of P/As in the roots of Chinese Brake fern were greater than those in the fronds, whereas in Boston fern the opposite was observed. These results suggested that the mechanism for As hyperaccumulation in Chinese Brake fern may include both high root exudation to enhance As-bioavailability in the growth medium and rapid translocation of As to the fronds coupled with retaining more P in the roots yielding a high molar ratio of P/As in roots.
Acknowledgements This research was supported in part by the National Science Foundation (Grant BES-0086768 and BES0132114). The authors gratefully acknowledge the assistance provided by Dr Xinde Cao for providing the CCA soil and Gina Kertulis for proof-reading the manuscript.
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