Subcellular Distribution and Chemical Forms of Pb in Corn: Strategies

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Subcellular Distribution and Chemical Forms of Pb in Corn: Strategies Underlying Tolerance in Pb Stress Jianling Sun† and Liqiang Luo*,‡ †

Beijing Municipal Research Institute of Environmental Protection, Beijing 100037, People’s Republic of China National Research Center for Geoanalysis, Beijing 100037, People’s Republic of China

‡

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S Supporting Information *

ABSTRACT: Studying the accumulation position and forms of heavy metals (HMs) in organisms and cells is helpful to understand the transport process and detoxification mechanism. As typical HMs, lead (Pb) subcellular content, localization, and speciation of corn subcellular fractions were studied by a series of technologies, including transmission electron microscopy, inductively coupled plasma mass spectrometry, and X-ray absorption near edge structure. The results revealed that the electrodense granules of Pb were localized in the cell wall, intercellular space, and plasma membranes. About 71% Pb was localized at the cell wall and soluble fraction. In cell walls, the total amount of pyromorphite and Pb carbonate was about 80% and the remaining was Pb stearate. In the nuclear and chloroplast fraction, which demonstrated significant changes, major speciations were Pb sulfide (72%), basic Pb carbonate (16%), and Pb stearate (12%). Pb is blocked by cell walls as pyromorphite and Pb carbonate sediments and compartmentalized by vacuoles, which both play an inportant role in cell detoxification. Besides, sulfur-containing compounds form inside the cells. KEYWORDS: corn, subcellular separation, Pb, chemical speciation, synchrotron radiation, X-ray absorption near edge structure technology

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radiation X-ray fluorescence spectroscopy (SRXRF) microprobe were used to study the distribution of the elements in tissue cross sections of pakchoi (Brassica chinensis L.) under stress of elevated Pb and Cr. The results also demonstrated that the majority of the two elements was in vacuoles and cell walls.10 AAS is a common technology for trace element analysis, which has the advantages of a simple pretreatment process and suitable for analysis of solid and high-viscosity liquid samples, whereas AAS has some obvious disadvantages, such as matrix interference, and is also not suitable for multielemental analysis. Speciation identification of elements in plants can provide important information for the study of the metabolic mechanism of toxic HMs in the organism. An important point in elemental speciation analysis is to avoid chemical transformation of the samples; therefore, it is of great advantage if it only requires minimal sample preparation. Various publications describe the method of differential centrifugation analysis for the speciation of elements in samples, but the process is usually very long and also requires the use of special reagents.11−13 In comparison to chemical approaches,9,14,15 X-ray absorption spectroscopy near edge structure (XANES) technology is a new technology that can provide the local structure information around the selected elements and is considered to be a powerful approach for the identification of element forms with a low content. Pb LIII edge analysis with XANES was performed on root and leaf samples

INTRODUCTION Heavy metal (HM) pollution is a global environmental problem. Lead (Pb) is one of the most serious and ubiquitously distributed toxic metals. Mining, electronicwaste disposal, and sewage treatment can contribute to Pb occurrence in the environment.1 Pb enters the human body through the food chain and causes damage to the immune system, nervous system, etc.2 In plants, it can also inhibit cell division, photosynthesis, and nitrogen fixation.3 The capability of plants to enrich and tolerate HMs is closely related to the absorption and transportation of HMs as well as the cellular and subcellular localization of HMs,4 which can provide the necessary information for the HM detoxification approach and the tolerance mechanism.5 Moreover, the different forms of HMs always show different toxicities for cells and organisms. Therefore, studying the accumulation area and forms of Pb is helpful to understand the mechanism of action and then discuss the tolerance mechanism under Pb stress in organisms. Differential centrifugation as the more popular method is used to separate subcellular fractions from samples, and atomic absorption spectrometry (AAS) is used to determine the content of HMs.6−8 Related research mainly focuses on the tolerance and enrichment mechanism of hyperaccumulators, especially the content of HMs and forms in crops, which relate to human health. For Athyrium wardii, the mining ecotype (ME) was more tolerant in the environment with a high Pb content than the non-mining ecotype (NME). The Pb concentrations in shoots and roots of ME were 3.2−8.6 and 3.0−24.6 times higher than those of the NME, respectively. At the subcellular level, 77.4−88.8% of total Pb was stored in the cell walls of ME and 9.0−18.9% of total Pb was stored in soluble fractions.9 Differential centrifugation and a synchrotron © XXXX American Chemical Society

Received: March 22, 2018 Revised: June 6, 2018 Accepted: June 10, 2018

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DOI: 10.1021/acs.jafc.7b03605 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

process is shown in Figure 1. In brief, plant tissues were homogenized in extraction buffer [50 mM 4-(2-hydroxyethyl)-1-piperazineethane-

from four different plants and a lichen that were collected from an abandoned mining site of the Eifel Mountain in Germany. It was found that one Pb2+ could either coordinate with nine oxygen atoms in the first coordination shell similar to outersphere complexation or just three oxygen atoms similar to inner-sphere complexation.16 Using transmission electron microscopy (TEM) and synchrotron X-ray microdiffraction (μ-XRD), the author found Pb present as pyromorphite, Pb5[PO4]3(Cl,OH), in both Pinus sylvestris roots and needles and the extracellular embedding of pyromorphite within plant cell walls was interpreted as a defense mechanism of the plant against lead pollution.17 Tian et al. using synchrotron techniques found that Pb was mainly associated with sulfur in Sedum alfredii.18 Previous studies have focused on Pb speciation in plant organs; however, subcellular Pb forms were not involved. In this study, the location of the Pb sediment in corn roots and leaves was determined using TEM. The subcellular distribution and chemical forms of Pb were determined by inductively coupled plasma mass spectrometry (ICP−MS) and XANES, respectively. Finally, the transport process and detoxification mechanism were discussed.

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MATERIALS AND METHODS

Figure 1. Flowchart of subcell separation of plant samples.

Corn Seed Cultivation. Corn seeds (JINXIANGNUO 1), as the locally grown broader species, were purchased from the Yunnan seed bank. First, plump seeds were chosen and sterilized with 3% (w/v) hydrogen peroxide for 30 min, followed by rinsing 3 times with MilliQ water and then soaking for 6 h. Seeds with integrated episperm were selected and placed in each glass culture dish with double filter papers. Subsequently, the seeds were wetted with Milli-Q water and germinated in a constant temperature incubator for 6 days at 27 °C under a light/dark regime of 12/12 h. The germinated seeds were moved to a conical flask and fixed on foam with sprouts upward and the roots immersed in the culture solution. The control group was treated with Hoagland solution, and Pb(NO3)2 was added with final concentrations of 5, 10, and 20 μg/g. Thereafter, the nutrient solution was changed every 3 days, and after 18 days, the plants were harvested. Table 1 shows the experimental parameters of the artificial climate chamber.

sulfonic acid (HEPES), 1.0 mM dithiothreitol (DTT), 500 mM sucrose, 5.0 mM ascorbic acid, and 1.0% (w/v) Polyclar AT polyvinylpolypyrrolidone (PVPP), adjusted to pH 7.5 with NaOH]. The homogenate was filtered through a nylon cloth (φ 120 μm), and the residue was washed twice with the extraction medium. The washing solution, together with the first filtrate, were centrifuged at 300g for 1 min. The precipitate combined with the residue of the nylon cloth filtration was mainly cell walls and wall debris, and this fraction was denoted as F1. The supernatant was centrifuged at 2000g for 15 min, and the precipitate was the nuclear and chloroplast fraction, denoted as F2. Then, the third centrifugation step was performed at 10000g for 30 min, and the mitochondria fraction was obtained, denoted as F3. The supernatant was finally centrifuged at 100000g for 30 min, and the precipate was the nucleoprotein (membrane) fraction, denoted as F4, while the supernatant containing mainly vacuole solution was referred to as the soluble fraction, denoted as F5. All steps were performed at 4 °C. The fractions were dried and digested with a mixture of HNO3 and HClO4 (5:3, v/v). HM concentrations in the fractions were determined by ICP−MS (Agilent 7500 A).12 At the same time, 2.00 g of fresh sample was frozen, ground into powder under liquid nitrogen, and then digested for the determination of the total Pb content. Standard substances (GSB-3, GSB-11, and GSB-1) were used as quality control samples. Pb Speciation in Plant Samples. Pb speciation in different plant tissues was determined by XANES. Samples were collected from two kinds of cultivation modes. One was collected from a water culture described above with the Pb concentration of 20 μg/g, and the other was planted in pots with field soil collected from a Lanping (Yunnan, China) Pb−Zn mine. The field soil was spiked with Pb(NO3)2 by 3 cycles of saturation with DI water and air drying for 10 days, before being remixed and vegetated. During harvest, plants were gently removed from soil and washed until no soil. Both samples were washed with DI H2O several times. Roots and leaves were further separated with scissors and fixed on the sample holder by 3M tape. Other samples were freeze-dried, ground to powder under liquid N2, pressed into slices, and fixed on the sample holder by 3M tape. The separated fractions (F1−F5) of corn plant grown in 20 μg/g of Pb2+ nutrient solution were treated with the same way above. The XANES experiments were carried out at beamline 14W1 at the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) and beamline 1W1B at the Beijing Synchrotron Radiation Facility (BSRF, Beijing, China). For calibration, the energy of the Pb LIII

Table 1. Artificial Climate Chamber Parameters for Hydroponic Experiments time interval

illumination intensity (Lx)

temperature (°C)

humidity (% RH)

20:00−4:00 4:00−12:00 12:00−16:00 16:00−20:00

0 8000 20000 8000

27 27 30 27

50 50 50 50

Microscopy. The roots and leaves were washed with tap water and rinsed with deionized (DI) H2O 3 times. The middle of each leaf and the root tips were cut into 1 × 2 mm2 and 2 mm long samples, respectively. The samples were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.2) for 4 h and then thoroughly washed with the same buffer 3 times, followed by post-fixation with 1% osmium tetroxide in the same buffer for 2 h. The samples were subsequently dehydrated in a graded series of acetone (30, 50, 70, 80, 90, 95, and 100%) and embedded in Spurr overnight. Ultrathin sections (ca. 60− 90 nm) were cut on a Leica EM UC 6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and then photographed by TEM (HITACHI-750).19 Tissue Fractionation. Frozen materials were pretreated according to the method described by Lozano-Rodriguez et al.20 The method of separating tissues into five fractions was suggested by Fu et al.21 The B


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Figure 2. Golgi apparatus in corn root epidermal cells. Corn grown in different Pb solution (left, 0 μg/g; right, 20 μg/g) for 18 days and the root tips were collected to make into ultrathin sections, photographed by TEM (HITACHI-750).

Figure 3. Gradual ultrastructure damages of the corn mesophyll cell. Corn grown in different Pb solution (A, 0 μg/g; B, 5 μg/g; C, 10 μg/g; and D, 20 μg/g) for 18 days and the middle of the leaf were collected to make into ultrathin sections, photographed by TEM (HITACHI-750). edge was set to 13 035 eV using the first maximum of the first derivative of the metal foil spectrum. The powder reference compounds were measured in transmission mode. Otherwise, all plant samples were measured in fluorescence mode using a seven element Si (Li) solid-state detector for the low content with a germanium (Ge) filter. All measurements were performed in an air environment at room temperature. The XANES spectra were measured with 0.5 eV equidistant energy steps in the edge region from 12 985 to 13 155 eV. The obtained data were normalized using a

first-order function for the pre-edge region and quadratic polynomial for the post-edge region and analyzed by Athena software.

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RESULTS AND DISCUSSION

Ultrastructure Damage of Corn Root and Leaf. Pb toxicity can cause ultrastructure damage of corn roots and leaf cells, leading to cellular metabolism disturbance. C


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Figure 4. Gradual ultrastructure damages of the corn bundle sheath cell. Corn grown in different Pb solution (A, 0 μg/g; B, 5 μg/g; C, 10 μg/g; and D, 20 μg/g) for 18 days and the middle of the leaf were collected to make into ultrathin sections, photographed by TEM (HITACHI-750).

Root Ultrastructure Damage. There were no significant differences in root ultrastructure between three experimental groups and the control group. The cells, nuclei, and nuclear membrane were all intact. The number of organelles was a little more than in normal cells. The vacuole disappeared in some cells under 20 μg/g of Pb stress, and similar results were obtained when the nutrient solution contained 10 μg/g of Pb. In comparison to the control group, the degree of intracellular vacuolization in the treatment group increased with the increase of the Pb concentration, and the number of transfer vesicles was also increased (the pictures show in Figure S1 of the Supporting Information). More interestingly, epidermal cells from corn roots contained a large number of golgi apparatus under 20 μg/g of Pb stress, and the volume was larger than the one in the control group, shown in Figure 2. Moreover, the lamellar structure was relatively complete. The golgi apparatus is a central organelle for secretory pathways in cells. Secretory vesicles can gradually move to the cell surface and then combine with the plasma membrane to excrete the inclusions. Therefore, the speculation is that corn root cells produce many golgi apparatus, which pack Pb and Pb compounds inside to reduce the toxicity or clear them from cells when they are exposed to Pb stress. As speculated by Jarvis and Leung, there is a probability that dictyosome-mediated exocytosis occurred in a cytosis event.22 Leaf Ultrastructure Damage. The phenomenon of the plasmolysis generally appeared in the treatment group, but no cell wall was ruptured completely, even though they were exposed to 20 μg/g of Pb. Membrane structure damage from Pb exposure was more serious than that of the cell walls. Figures 3 and 4 show the gradual ultrastructure damages of the

corn mesophyll cell and corn bundle sheath cell, respectively. Under moderate and severe Pb stress, there were no complete golgi apparatus and endoplasmic reticulum. With an increased Pb concentration, the chloroplast, cell nucleus, and mitochondria were injured in different degrees, such as membrane structure injury, internal structure deformation, or organelle vacuolization. Chloroplasts, mitochondria, and ribosomes are the key organelles in the plant cell for major cell-life activities.23,24 Their ultrastructure changes and defects would have a strong impact on the normal cell physiological reactions. Studies have shown that HMs have a strong impact on antioxidant enzyme systems in plants, such as significantly altered superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) activities. and glutathione reductase (GR),25 glutathione (GSH), oxidized glutathione (GSSG),26 and soluble protein production,27 as well as significant growth inhibition and content of chlorophyll a and b proteins.28 In this study, corn leaf cell mitochondria vacuolated29 or mitochondrial cristae and matrix density decreased under Pb stress,30 which resulted in the reduction of the attached surface area of enzymes, which is required for the tricarboxylic acid cycle. Cellular respiration intensity decreased accordingly, resulting in blocked aerobic metabolism of sugar.31 The integrity and order of the chloroplast ultrastructure are the basis of normal photosynthesis. The destruction of the thylakoid membrane structure directly affects the electron transfer of photosynthesis.32 HMs can cause irreversible damage to the nucleus, such as condensation of chromatin, dispersion of nucleoli, and disruption of the nuclear membrane. As a result, the nucleic acid metabolism of the nucleus was disordered.30 The damage of the cell membrane D


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Figure 5. Deposition of Pb in corn root cells. Corn grown in 20 μg/g of Pb solution for 18 days and the root tips were collected to make into ultrathin sections, photographed by TEM (HITACHI-750). A, plasma membranes; B, middle lamella; C, transport vesicles; D, fragmentary endoplasmic reticulum; E, dictyosome; and F, lamella structure of mitochondria.

structure caused by Pb exposure was more serious than that of the cell wall. Endoplasmic reticulum and golgi apparatus reduction or absence may also affect the roles, including manufacture, transport, and connection in cells.33 Subcellular Localization of Pb in the Root Cells. Using TEM, the electrodense granules of Pb were studied in corn root cell under 20 μg/g of Pb. Figure 5 illustrates deposition of Pb granules on the root wall and along their plasma membranes (A). The intercellular space between three vacuolated cells and regions of the middle lamella (B) contained dense granules. Evidence of smaller Pb grains can be seen in the Pb-laden, transport vesicles (C), fragmentary endoplasmic reticulum (D), dictyosome (E), and lamella structure of mitochondria (F) of the cells. Possible detoxification mechanisms include metal compartmentalization and sequestration in cell walls and intercellular space34 or chelation to form complexes by plants.35 These results were consistent with previous studies of Pb distribution in and around the Sesbania drummondii cells.36 HM Content and Subcellular Proportions. Samples were collected from farmland around the Lanping mining area in Yunnan province. Three corn leaves with higher Pb content were chosen for the subcellular fraction, and three replicates were performed for each sample. The proportions of interesting elements in leaf subcellular fractions (w %) was calculated by this formula: w % = Cfi/∑Cfi. Cfi refers to the element concentration in subcellular fractions, and i values were 1−5. The average value (ω %) was then calculated for the three replicates. ω % of toxic elements are listed in Table 2 in different corn subcellular fractions. In this study, about 71% Pb is localized at the cell wall and soluble fractions, followed by the nuclear and chloroplast fraction, which contained

Table 2. Subcellular Proportions of HMs in Corn Leaves (ω %)a fraction F1 F2 F3 F4 F5

52

Cr

77 11 4 4 3

65

Cu

37 6 4 3 51

66

Zn

54 12 9 2 23

75

As

42 7 4 2 44

114

Cd

52 11 8 3 27

208

Pb

35 25 2 2 36

a F1, F2, F3, F4, and F5 stand for the cell wall fraction, the nuclear and chloroplast fraction, the mitochondria fraction, the nucleoprotein (membrane) fraction, and the soluble fraction, respectively.

approximately 25% Pb. The mitochondria and nucleoprotein fractions have the lowest Pb content, only about 2%. The distribution of arsenic (As) was similar to Pb. Cadmium (Cd) and zinc (Zn) have similar chemical properties because they have the same electron configuration. The concentration followed the orders F1 > F5 > F2 > F3 > F4. The highest copper (Cu) concentration in the soluble fraction was 51%, and the second was in the cell wall fraction, approximately 37%, whereas the Cu content in the remaining three fractions was about 5%. The chromium (Cr) content in different subcellular fractions had the following order: F1 > F2 > F3 = F4 > F5. The distribution of Cr in cell walls was up to 77%, and the concentration in the nucleus fraction declined sharply to 11%. The most interesting elements in corn were predominantly accumulated in the cell wall and vacuoles, which may suggest the following process of corn cell absorption and accumulation of HMs: First, the metal elements (e.g., Pb, As, Cu, Cd, and Zn) are blocked by cell walls as the result of ligand chelation. Second, excessive toxic metal cations will enter plant cells E


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which were measured by XANES directly. Because of low Pb content, unsmooth and low-quality XANES spectra were obtained for some samples, especially corn leaf. The plot shows that all of the samples have the same edge position and were almost overlapping, which suggests that the same chemical speciation was obtained from the two cultured models and two pretreatment methods. Only the root cell wall fraction (corn root F1) and the nuclear and chloroplast fraction (corn root F2) absorption spectra can be measured; the remaining subcellular fractions cannot be used to obtain absorption spectra because of their low Pb contents. White peak energy of the Pb nitrate cultured solution was 13 048.45 eV, while corn Pb absorption spectral energy was 13 052.90 eV, increased by 4.45 eV. In comparison to Pb nitrate, Pb absorption edge energies of corn samples were displaced, suggesting that corn could transform Pb nitrate in the growth medium into other Pb chemical species and store it in cells and tissues. Pb is always present in different species in unknown samples. Thus, all XANES spectra have been fitted with LCF of Pb standard spectra using Athena software. A total of 11 references of Pb(C2H5)3Cl, PbS, Pb(C17H35COO)2, Pb(NO 3 ) 2 , PbSO 4 , PbO, Pb 3 O 4 , Pb(CH 3 COO) 2 ·3H 2 O, 2PbCO3·Pb(OH)2, Pb5(PO4)3Cl, and Pb3(PO4)2 were used in LCF, and at most, three components were combined for LCF with a normalization condition, with the fitting range set from −20 to +50 eV relative to the edge. Table 3 shows the

through the plasma membrane and be sequestered mostly by vacuoles within the cells to protect organelles from their toxicity.37 The cell wall of the root is the first barrier against HM stress, which can restrict HM uptake by the root cells.15,38 The plant cell wall contains protein and polyoses, including cellulose, hemicellulose, lignin, and mucilage glue, which provide many potential ligands, such as hydroxyl, carboxyl, amino, and thiol groups,39 that can form complexes with metal cations to restrict their transmembrane transportation.40 Recent studies have shown that Pb accumulates mainly in the cell walls and vacuoles of Brassica chinensis L., and the concentration of Cr in root cell walls was about 75.0%.10 Lozano-Rodriguez et al. found that most Cd accumulated in the maize cell walls and vacuoles.20 The results of this study are consistent with previous reports. Therefore, the cell walls and vacuoles act as two key storage compartments of metals in plants. 41 Instead of AAS, ICP−MS can analyze the concentration of multi-elements in about 2 min, with high sensitivity and low detection limits. The method of HClO4− HNO3 can overcome the digestion problem caused by the large amount of sugar existing in subcellular separated fractions. Therefore, in further studies, it is the fast and effective analysis method for the synergistic effect and antagonistic effect between HMs at the subcellular level. Pb XANES Speciation. The cultured samples and subcellular fractions were measured by XANES with a 3 × 3 μm2 spot. Figure 6 shows Pb LIII-edge XANES spectra of corn

Table 3. Best Fitting Results of Standard Pb for Corn Root and Separated Subcellular Samples sample corn root corn root F1

corn root F2

compound

weight

R factor

Pb3(PO4)2 Pb(C17H28COO)2 PbCO3·Pb(OH)2 Pb5(PO4)3Cl Pb(C17H28COO)2 PbS PbCO3·Pb(OH)2 Pb(C17H28COO)2

0.889 0.111 0.269 0.527 0.204 0.715 0.164 0.121

0.00021 0.000077

0.00095

results of the processed fits and the quality-of-fit parameter, R factor. The smaller the R factor, the better the results could be obtained.42,43 Figure 7 shows the best fitting spectra for corn root and subcellular samples. The corn root is only listed here because of corn samples having the same spectra. The best fitting of Pb in corn root is pyromorphite combined with Pb stearate. The result is coincident with other works44,45 and our previous research46 very well. The cell wall sample primarily consisted of pyromorphite-type Pb, and approximately 53% of the XANES spectra were defined by the pyromorphite structure. Remaining Pb bound to the cell wall was comprised mainly of basic Pb carbonate and Pb stearate (27 and 20%, respectively). This indicates that the corn cell wall physiologically transforms the starting material, Pb nitrate, into insoluble pyromorphite and Pb carbonate, which play a leading role in reducing the toxicity of Pb. Pb composition of the nuclear and chloroplast fraction changed significantly, mainly composed of Pb sulfide (72%), basic Pb carbonate (16%), and Pb stearate (12%). These results may reveal that there are two different approaches to reducing the toxicity of Pb in cell walls and inside cells.

Figure 6. Pb-LIII edge XANES spectra of corn root, leaf, and subcellular fractions. The XANES experiments were carried out at beamline 14W1 of SSRF and beamline 1W1B of BSRF. Samples were measured with a 3 × 3 μm2 spot in fluorescence mode using a 7 element Si (Li) solid-state detector for the low content with a germanium (Ge) filter. The energy steps were 0.5 eV, from 12 985 to 13 155 eV. All spectra were fitted with LCF of Pb standard spectra use Athena software.

root, leaf, and subcellular fractions compared to the model compound Pb(NO3)2. The spectra are displaced vertically for clarity. The vertical solid line marks the white line (strongest absorption peak) of the Pb spectrum of corn samples to visualize the edge shifts. The corn leaf was labeled “Cornleaf Water culture” grown in nutrient solution containing Pb. “Cornroot”, “Cornleaf”, and “Cornrootfresh” refer to samples collected from the soil culture described above, but they were divided into freeze-dried root and leaf as well as fresh root, F



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jianling Sun: 0000-0002-7911-4179 Funding

This research was commissioned and financed by the National Natural Science Foundation of China (Grants 20775018 and 41201527) and the National High Tech R&D Program (Grant 2007AA06Z124). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Professors Yuying Huang and Aiguo Li of the Shanghai Synchrotron Radiation Facility beamlines 15U and 14W1 and Professor Dongliang Chen of the Beijing Synchrotron Radiation Facility beamlines 4W1B and 1W1B. In addition, the authors thank Binbin Chu, Yuan Zeng, Jing Yuan, Jie Liu, and Yanhong Ma for their help during the experiments.

Figure 7. Best fitting spectra for corn root and subcellular samples. Sample spectra fitted with LCF of 11 Pb standard spectra use Athena software. Two to three components were used for fitting and normalization (Table 3 shows the results). The fitting range was set from −20 to +50 eV relative to the edge.

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The cell wall and cell membrane contain specialized protein ion channels and receptors that permit the influx and efflux of small molecules and inorganic ions. There is no proprietary channel for Pb entering the plant cell because it is a nonessential element; therefore, Pb must enter the plants through the channels of essential elements. It has been documented that low-soluble Pb can deposit around the plant roots in phosphate and sulfate forms47 or be bound to the cell wall and form a lot of pyromorphite and Pb carbonate precipitation.48,49 Unbonded Pb enters into plant cells through calcium ion channels.50 The strategy for plants to tolerate Pb is the complexation with strong ligands, such as thiol groups provided by phytochelatins and glutathione or combined with C/O/N ligands, likely provided by the cell wall and cortex.51,52 In this study, Pb existed in different chemical forms among different subcellular fractions, even though XANES spectra were only obtained for two fractions. The Pb−S content increased drastically in the nucleus and chloroplast fraction, indicating that both of them contained some S ligands, which can combine with Pb to form a chelate. Although the Pb concentration in the cell wall fraction is almost equal to the soluble fraction, which contains a large amount of extraction buffer, it cannot produce XANES spectra as a result of serious scattering. This work is still at a preliminary stage, and further analysis is required to examine the distribution of Pb in other subcellular fractions, especially in liquid samples, and to improve the understanding of Pb effects on the cell ultrastructure within the plant. Meanwhile, the interaction mechanisms between the interesting elements in crops can be investigated.

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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.jafc.7b03605. Ultrastructure damages of corn root cells (Figure S1) (PDF) G


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Subcellular Distribution and Chemical Forms of Pb in Corn: Strategies

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