Role of Ligands in Accumulation and Fractionation of Rare Earth ...

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Role of Ligands in Accumulation and Fractionation of Rare Earth Elements in Plants Examples of Phosphate and Citrate SHIMING DING,1 TAO LIANG,*,1 CHAOSHENG ZHANG,2 JUNCAI YAN,3 ZILI ZHANG,3 AND QIN SUN4 1

Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China; 2 Department of Geography and Environmental Change Institute, National University of Ireland, Galway, Ireland; 3Department of Soil & Agrichemistry, Anhui Agricultural University, Hefei 230036, China; and 4State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing 210093, China Received August 10, 2004; Revised November 15, 2004; Accepted December 15, 2004

ABSTRACT Few studies have been carried out on the effects of ligands on rare earth element (REE) bioaccumulation processes. In this study, the effects of phosphate (Pi, an inorganic ligand) and citrate (an organic ligand) on accumulation and fractionation of REEs in wheat were investigated using aqueous culture with extraneous mixed REEs (MRE). The results show that initial Pi solution culture at various levels followed by exposure to a fixedMRE solution did not significantly change the total concentrations of REEs (ΣREE) in roots, whereas the ΣREE in leaves dramatically decreased with increasing levels of Pi applied. Simultaneous culture of wheat with mixture of MRE and citrate solutions caused obvious decreases of the ΣREE in both roots and leaves. Compared with MRE, significant fractionations of REEs were found in wheat organs when no ligand was applied. Notable middle REE (MREE) enrichment and M-type tetrad effect were observed in the roots, and heavy REE (HREE) enrichment and W-type tetrad effect existed in the leaves. Pi treatments did not significantly affect the fractionations of

*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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Ding et al. REEs in the roots, but enrichment of HREEs in the leaves slightly increased at the highest level of Pi applied. Fractionations of REEs in both roots and leaves decreased with increasing levels of citrate applied; at higher levels of citrate (≥150 µM), no above fractionation features were observed in wheat, but light REE (LREE) enrichment existed in the roots and leaves. The results indicate that ligands might play important roles in accumulation and fractionation of REEs during bioaccumulation processes. Index Entries: Accumulation; citrate; fractionation; ligand; phosphate; rare earth elements (REEs); wheat.

INTRODUCTION The biogeochemical behavior of rare earth elements (REEs) has been poorly understood because of a lack of reliable and sufficient field data. With the utilization of inductively coupled plasma–mass spectrometry (ICP-MS) (1) for high-quality and all-REE determinations in natural samples and with continual improvements in estimation of REE stability constants (2–6), it is now possible to make much more quantitative studies on the roles of REEs in bioaccumulation processes. Under natural conditions, concentrations of REEs in plants are extremely variable. For example, about 700 ng/g La was reported in a species of fern (Matteuccia) (7), but it can be less than 10 ng/g in the needles of Norwegian spruce (Picea abies) (8). Possible reasons for the difference include the difference of REE abundances in soils and the species-specific uptake of REEs by plants (9–12). Resulting from identical trivalent charge and systematic decrease in ionic radius with the increase of atomic number, REEs have very similar chemical behaviors and tend to be present in nature as a group rather than alone (13). This feature enables REEs to be useful tracers in geochemistry and can record subtle geochemical processes in natural systems (12,14,15). However, this might be complicated by fractionation among individual REEs, and an understanding of REE fractionation in natural systems is needed. Various fractionation features of REEs have been observed in bioaccumulation processes. These features range from radius or redox dependent, resulting in fractionation between light REEs (LREEs, La–Eu) and heavy REEs (HREEs, Gd–Lu) (11,16) and redox-sensitive REE anomalies (i.e., Ce and Eu anomaly) (11,12,16,17) and redox-independent, resulting in nonredox-sensitive REE anomalies (e.g., Gd, Yb, and Lu) (11,18). Moreover, a fine structure in a REE chondrite-normalized distribution pattern called the “tetrad effect” was found in plants (7,12,19). This effect can cause a split of chondrite-normalized REE patterns into four segments called tetrads (first tetrad, La–Ce–Pr–Nd; second tetrad, [Pm]–Sm–Eu–Gd; third tetrad, Gd–Tb–Dy–Ho; and fourth tetrad, Er–Tm–Yb–Lu). The overall shapes are either convex or concave and form an M-shaped distribution in solid and a W-shaped distribution in solution, respectively (20). The tetrad-effect disBiological Trace Element Research

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tribution of REEs in samples can provide useful information such as how REEs transport in environment. For example, the appearance of a W-type tetrad effect in ferns shows that REEs must have once been in a dissolved state, as the W-type tetrad effect is exclusively found in solution (7). Previous studies on REE bioaccumulation have focused on rate of accumulation and fractionation, but the processes remain unclear. This might be the result of the shortcomings in field investigation, such as extremely low concentration ratios of plant/soil, contamination of plant samples with soil and dust, and the uncertainty of the normalization procedure (8,21). In this study, cultural experiments were used to find out the effects of an inorganic ligand, phosphate (Pi), and an organic ligand, citrate (CA), on accumulation and fractionation of REEs in wheat, and their mechanisms were discussed.

MATERIALS AND METHODS Stock Solution of MRE The stock solution of 2.1 mM mixed REEs (MRE) used in this study was composed of 14 lanthanides except Pm, with identical concentration of each element (i.e. [Ln3+] = 0.15 mM, Ln represents any lanthanide). Each REE stock solution of 10 mM was prepared by dissolving REE oxides with 1 M HCl, except that Ce was made from CeCl3. The solution of each element was calibrated by ICP-MS and prepared for the MRE solution. All solutions used in the present study were made using deionized water of 18 MΩ quality or better.

Plant Materials Seeds of wheat (Triticum aestivum L. cv Jin-Dong 8) were sterilized for 15 min in a solution of 1% sodium hypochlorite, rinsed with tap water and soaked in deionized water for 24 h. Then the seeds were placed on a nylon net, which was fixed on an aerated solution containing 0.2 mM CaCl2 in a 7-L plastic container. After being kept in the dark for 5 d at 25°C, the seedlings were used for following experiments.

Phosphate-Dose Experiment The seedlings precultured in 0.2 mM CaCl2 were transplanted to 3.0L plastic pots with 20 seedlings per pot and grown in nutrient solution containing different levels of Pi. The nutrient solution was composed of the macronutrients (mM) KNO3 (1.0), Ca(NO3)2 (1.0), and MgSO4 (0.4) and the micronutrients (µM) FeCl3 (4), H3BO3 (3.0), MnCl2 (0.5), CuSO4 (0.2), ZnSO4 (0.4), and (NH4)6Mo7O24 (1.0). Pi was added in the form of KH2PO4, and the concentrations were 0, 0.04, 0.2, 1, and 5 mM, respectively. Plants were grown in controlled-environment conditions with a 14-h/25°C day Biological Trace Element Research

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and 10-h/20°C night regime and light intensity of 40 W/m2, with solution renewed every other day. After a week of growth, the roots of the seedlings were immerged in 1 mM CaCl2 solution (pH = 5.5) overnight to remove PO43– adhering on root surfaces; then the seedlings were exposed to 1 mM CaCl2 solution containing 5 µM MRE (pH = 5.5) for another 4 d. At harvest, the roots of the plants were rinsed in 1 mM CaCl2 solution (pH = 5.5) for 30 min to remove adhered REEs from root surfaces; then the roots and leaves were sampled. Only the seedlings without Pi culture showed nutrient-deficient symptoms during the treatments, reflected by the necrotic and yellow appearance in tips of the leaves.

Citrate-Dose Experiment The seedlings precultured in 0.2 mM CaCl2 were transplanted to 3.0L plastic pots with 20 seedlings per pot and grown in the aforementioned nutrient solution containing 0.2 mM Pi. Plants were grown under the aforementioned conditions, with solution renewed every other day. After a week of growth, the seedlings were placed in 0.5-L plastic cups containing 1 mM CaCl2 solution (pH = 5.5) overnight, then exposed to 1 mM CaCl2 solution (pH = 5.5) containing 5 µM MRE combined with a range of citrate. The levels of citrate (abbreviated as CA, added as pure acid) were 0, 6, 30, 150, and 750 µM, respectively. After a 4-d exposure, the wheat was cultured in 1 mM CaCl2 solution (pH = 5.5) for 30 min; then the roots and leaves were harvested.

REEs Determination and Quality Control The wheat samples were thoroughly washed with tap water and deionized water, then oven-dried at 75°C until the dry weight reached a constant value. The dry samples were ground to fine power. Each sample with 0.05 g of root, or 0.1 g of leaf, was digested with the mixture of 5 mL HNO3 and 0.5 mL HClO4 in a 50-mL beaker. The digested solution was evaporated to near dryness. The residue was dissolved by 2% HNO3 and transferred into a 10mL flask, then diluted to volume with deionized water. Prior to determination, 0.2 mL of 1 µg/mL. In solution was added. REE concentrations in the plant samples were determined by ICP-MS (PQ II Turbo, VG). The quality control was achieved with certified reference samples GBW07603 of shrub leaves from the National Research Center for Certified Reference Materials (Beijing, China). Seven duplicates were made for each sample. The results for the reference samples were in line with the reference values, with the deviations less than 5%, except for Sm (6.7%), and Lu (5.4%).

Quantification of the Tetrad Effect A proposed method was adopted to quantify the degree of the tetrad effect according to Irber (22) and Monecke et al. (23). The method was designed for REE patterns that exhibit a split into four rounded segments. Biological Trace Element Research

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Table 1 Effect of Pi Levels on Accumulation of the Total REE in Levels and Roots of Wheat

Note: Values are mean ± SE (n =3).

To describe the four segments of the REE pattern showing the tetrad effect, the tetrads are consecutively numbered as i = 1, 2, 3, and 4. The size of the tetrad effect in each segment can be given as ti =

xBi xCi xAi xDi

(1)

where XAi, XBi, XCi, and XDi are the concentrations of the first to the fourth elements in the tetrad, respectively. The second tetrad (Pm to Gd) cannot be calculated because of the missing Pm in nature. The values of ti determined from the remaining three segments can be averaged to produce an overall value of T: Size of the tetrad effect = T = (t1 × t3 × t4)1/3

(2)

The tetrad effect can be characterized with an M-shape (T>1) or a Wshape (T leaves > seeds (11). In this study, less than 1% of the absorbed REEs were transported to the leaves. Because of extremely low concentrations, this part of REEs should be highly sensitive to phosphate variation along the transport path. This might explain the decrease of Σ REE in leaves with increasing levels of Pi applied and the development of the tetrad effect at the highest level of Pi applied (Table 1, Fig. 3A), as phosphate concentration in the transport path should be successively increased with increasing addition of Pi in root medium. A radius-dependent fractionation of REEs was observed in leaves, characteristic of notable HREE enrichment (Fig. 2A). This fractionation, which has been observed in our previous experiments, is suspected to result from the combination of solution complexation and other processes such as cell wall absorption. For many organic and inorganic ligands, the REE–ligand stability constants increase with the increase of atomic number across lanthanides (2–6). In this study, the wheat was precultured in nutrient solution containing various inorganic ions. Among these ions, Pi had stronger ability to complex REEs and the stability constants increase with increasing atomic number of REEs (Table 3). Increasing application of Pi in root medium, therefore, should enhance the complexation of Pi with Biological Trace Element Research

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Ding et al. Table 3 First Stability Constants of REEs with PO43– and CA (Ionic Strength = 0 for PO43– and 0.1 for CA, 25°C)

Source: Data from refs. 5 and 6.

HREEs and cause HREEs to become more dissolved in solution before the phosphate precipitation occurred, and, correspondingly, the LREEs should be easier to be absorbed by plant cell walls. As a result, the HREEs became easier to be transported to leaves compared to the LREEs, as indicated by the significant increase (p < 0.05) of HREE/LREE at the highest level of Pi applied (5 mM) when compared with the control (Fig. 4A). It clearly demonstrates that phosphate complexation might partly contribute to the enrichment of HREEs in leaves. However, this complexation effect cannot be considered to be a dominant process because the enrichment of HREEs in wheat leaves only increased to a small degree at the highest level of Pi (Fig. 4A). The mechanisms responsible for the enrichment of HREEs in wheat leaves deserve further studies.

Effects of CA on Accumulation and Fractionation of REEs in Wheat The increasing addition of CA in MRE-fixed solution had significant influences on accumulation and fractionation of REEs in wheat, characterized by the overall reduction of Σ REE and the decrease of REE fractionations in both roots and leaves (Table 2, Figs. 1B, 2B, 3B, and 4B). Qualitatively, kinetically labile species such as the hydrated metal ions or inorganic complexes are bioavailable to plants, and organic ligand complexation of metals normally leads to decreases in their bioavailability (25). One possible explanation for the above phenomena is that CA might form stable citrate–REE complexes and decrease the bioavailability of REEs to the wheat. On the other hand, the added CA might strongly compete with Pi for REEs and reduce phosphate precipitation of REEs in the roots. It is important to note that the effects caused by CA were much stronger for HREEs than for LREEs because of higher affinity of HREEs with CA (Table 3). As a consequence, the accumulation of HREEs in the wheat decreased more than that of the LREEs with the increasing application of CA, which finally led to the occurrence of the LREE-enrichment feature in roots and leaves at higher levels of CA applied (≥150 µM) (Figs. 1B and 2B). In accordance with results, the addition of citric acid inhibited the Pb uptake by the radish (26) and rice and wheat (27). By contrast, Mailer et al. (28) observed greater Cd accumulation in roots and old leaves of lettuce exposed to higher Cd–EDTA concentrations. Moreover, in the research on Biological Trace Element Research

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lead accumulation in Brassica juncea, it has been demonstrated that application of synthetic chelators, such as EDTA, both in soil (29) and hydroponic culture conditions (30) increased its uptake to plants by forming Pb–EDTA complexes. These discrepant results might be caused by ligand, metal-, and species-specific differences.

Roles of Ligands in Accumulation and Fractionation of REEs in Soil–Plant Systems Inorganic ligands, such as NO3–, Cl–, SO42–, and PO43–, are involved in many soil–plant processes. However, because of the higher affinity between phosphate and REEs and the considerable increase of their complexation constants with the increase of atomic number across lanthanides, it seems that only PO43– plays important roles in REE bioaccumulation processes. Generally, PO43– concentration in soil solution is extremely low [0.6–5 µM (31)] and it is close to that of the Σ REE [with an average value of 9 µM in 33 types of soil solutions in China (32)]. However, inorganic phosphorus in plants and xylem solution can be raised several hundred times via active absorption by plants (33,34), which largely stimulates the interaction between PO43– and REEs. Hence, this study might reveal a possible process responsible for the acropetal decrease of REE accumulation and the development of the tetrad effect observed in plants (7,19), although the process might be less important than others, such as cell wall absorption. Under field conditions, the contaminants, such as soil particles and aerosols adhering on plant surfaces, however, cannot be sufficiently removed and drastically complicated the data. This might partly explain why the Pi-triggered fractionations of REEs (e.g., MRE enrichment in roots and the tetrad effect in roots and shoots) are not widely observed in plants, especially in roots. It has been hypothesized that organic acids with low molecular weight might play key roles in the mobilization and uptake of poorly soluble nutrients by plants, such as Mn, Cu, Zn, Fe and Pi (35,36). Similar effects of organic acids were also found in REE accumulation and fractionation processes, as indicated in this work. The concentrations of organic acids in a soil solution across a broad range of ecosystems have been found to range from 1 to 50 µM (36–39). At the root–soil interface, the concentrations can be increased to between 5 and 150 µM (40). Both are within the concentration range used in this study. It was shown with wheat that the presence of CA even at the lowest of level (6 µM) had obvious effects on REE fractionation (Fig. 2B). Other low-molecular-weight organic acids, such as malate and oxalate, which are important root exudates in addition to CA, should also have similar effects because of their high complexing abilities with metals and the considerable increase of complexation constants with the increase of atomic number across lanthanides (6). This has also been partly verified by us in other experiments (data not shown). The complexing effects produced by organic acids, Biological Trace Element Research

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therefore, should widely exist in REE bioaccumulation processes and might be strengthened within the plants because of high concentrations of organic acids (35). Results in this study have shown that organic acid complexation could produce the LREE-enrichment feature and simultaneously reduce the Pi-triggered fractionations in plant organs (Figs. 1B and 2B). Thus, these observations might provide available evidences for why LREE-enrichment feature was widely observed in plant organs compared to the soils and why the Pi-triggered fractionations of REEs were insignificant in plants in most studies (7,11,16,17,19). In addition, it should be noted that the desorption process needs to be considered to evaluate the roles of organic acids in soil–plant system. Complexation of these ligands in soil can release REEs from the solid phase and increase concentrations of REEs in soil solution, which would contribute to the uptake of REEs by plants (41). The combination of the aforementioned processes finally determines how low-molecular-weight organic acids influence the accumulation and fractionation of REEs during bioaccumulation processes.

ACKNOWLEDGMENT This work was sponsored by the Key Project of National Natural Science Foundation of China (grant no. 40232022).

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