Article pubs.acs.org/est
Charge, Size, and Cellular Selectivity for Multiwall Carbon Nanotubes by Maize and Soybean Guangshu Zhai,*,† Sarah M. Gutowski,† Katherine S. Walters,‡ Bing Yan,§ and Jerald L. Schnoor*,† †
Department of Civil and Environmental Engineering and IIHR Hydroscience and Engineering, The University of Iowa, Iowa City, Iowa 52242, United States ‡ Central Microscopy Research Facility, The University of Iowa, Iowa City, Iowa 52242, United States § School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China S Supporting Information *
ABSTRACT: Maize (Zea mays) and soybean (Glycine max) were used as model food-chain plants to explore vegetative uptake of differently charged multiwall carbon nanotubes (MWCNTs). Three types of MWCNTs, including neutral pristine MWCNT (p-MWCNT), positively charged MWCNTNH2, and negatively charged MWCNT-COOH, were directly taken-up and translocated from hydroponic solution to roots, stems, and leaves of maize and soybean plants at the MWCNT concentrations ranging from 10.0 to 50.0 mg/L during 18-day exposures. MWCNTs accumulated in the xylem and phloem cells and within specific intracellular sites like the cytoplasm, cell wall, cell membrane, chloroplast, and mitochondria, which was observed by transmission electron microscopy. MWCNTs stimulated the growth of maize and inhibited the growth of soybean at the exposed doses. The cumulative transpiration of water in maize exposed to 50 mg/L of MWCNT-COOHs was almost twice as much as that in the maize control. Dry biomass of maize exposed to MWCNTs was greater than that of maize control. In addition, the uptake and translocation of these MWCNTs clearly exhibited cellular, charge, and size selectivity in maize and soybean, which could be important properties for nanotransporters. This is the first report of cellular, charge, and size selectivity on the uptake by whole food plants for three differently charged MWCNTs.
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INTRODUCTION Carbon nanotubes (CNTs) are a carbon allotrope, including single-wall carbon nanotubes (SWCNTs) and multiwall carbon nanotubes (MWCNTs). CNTs are widely used in nanomedicine, pigments, electronics, optics, cosmetics, and energy and environmental technologies due to their unique chemical, physical, optical, and magnetic properties.1−3 In 2010, CNTs were ranked as one of the top 10 most produced engineered nanomaterials (ENMs) by mass in the world, reaching 3200 t/ year. Most of CNTs end up in landfills (∼80%) and soils (∼15%).1 Importantly, more and more CNTs are being increasingly utilized in bioapplications for the transport and delivery of therapeutic agents.4−9 Their manufacturing and release will undoubtedly increase the exposure to humans and the environment and potentially cause biological effects on living organisms. Plants might accumulate CNTs from soils and water to threaten human and ecological health via food chains. On the other hand, uptake of carbon nanotubes with different properties and potential to penetrate individual cells may prove important in medical and agrochemical technologies. Therefore, it is of practical and fundamental importance to understand the interaction of CNTs and plants. MWCNTs have created a concern regarding toxicity to humans and animals.10−13 However, reports on MWCNT toxicity and interaction between MWCNTs and plants in vitro and in vivo are extremely limited. MWCNTs exhibited a detrimental effect on the growth of rice cells (Oryza sativa L.) © XXXX American Chemical Society
by the increase of reactive oxygen species (ROS), and caused a decrease of cell viability and cell density.14,15 Moreover, agglomerates of MWCNTs were toxic to Arabidopsis T87 suspension plant cells because they decreased cell dry weights, viabilities, chlorophyll contents, and superoxide dismutase (SOD) activity.16 Contrary to plant cells in vitro, MWCNTs have not demonstrated much apparent toxicity to whole plants in vivo. For example, MWCNTs did not exhibit any influence on wheat growth in a hydroponic study,17 and MWCNT suspensions (even at a high concentration of 2000 mg/L) did not show any toxicity to root growth of radish, rape, ryegrass, lettuce, maize, or cucumber.18 The penetration of MWCNTs into plant protoplasts, cells, and whole plants have produced mixed results in the experimental literature. In rice cells exposed at 20−80 mg/L, the MWCNTs remained associated with the cell wall and were blocked from entering the cells.14 But in Catharanthus roseus plant protoplasts (plant cell walls removed via enzymatic treatment), MWCNTs moved right through the cell membrane into the cytoplasm.19 For whole plants like wheat, MWCNTs were observed to pierce the epidermal and wheat root hair cell walls and enter the root hair and root cap of the wheat root. Received: March 5, 2015 Revised: May 14, 2015 Accepted: May 26, 2015
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DOI: 10.1021/acs.est.5b01145 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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selected and grown in the vial with preholed caps. The vial was filled with 20 mL of 0.1 strength Hoagland solution and sealed with Parafilm. The germinated maize and soybean seeds were grown in Percival Plant Growth Chamber with 16/8 day/night cycle at 25 °C, 59% relative humidity (RH), and light intensity of 120−180 μmol m−2 s−1 for 7 days. The healthy vigorously growing maize and soybean seedlings were selected for the carbon nanotube exposure experiments after 7 days of growth. Different concentrations of MWCNTs were added into 20 mL of 0.1 strength Hoagland solution in the vials. Blank maize and soybean controls (one seedling per vial) included four seedlings (three for mass measurement and one for TEM) without the addition of MWCNTs; four healthy maize and soybean seedlings were exposed to different concentrations of MWCNTs (one seedling per vial) and were used to test the transport and behavior of MWCNTs in these plants. Due to generally poor solubility of MWCNTs used, we selected 50 mg/L as the highest concentration because our experiments showed that at this concentration, all MWCNTs were reliably suspended and stable for 2−3 weeks (Figure S2, Supporting Information). Therefore, the final exposure concentrations of pMWCNT, MWCNT-NH2s, and MWCNT-COOHs selected were 10.0, 20.0, and 50.0 mg/L for maize, except for the blank control without any MWCNTs. The concentration of pMWCNT, MWCNT-NH2s, and MWCNT-COOHs utilized for exposure to soybean was 20.0 mg/L except for the blank control without any MWCNTs. These concentrations and the method of exposure are representative of the literature, although our concentrations of MWCNTs were lower than most studies.14,18 All exposure vials were wrapped with aluminum foil. A Percival Plant Growth Chamber was used for plant growth at 25 °C, 59% RH. The photoperiod was set 16 h per day under fluorescent lighting with a light intensity between 120 and 180 μmol m−2 s−1, and 0.1 strength Hoagland solution was injected into the vials every day to compensate for the evapotranspiration loss, which was determined by monitoring weight loss of the vials. Cumulative evapotranspiration for each plant was the sum of the evapotranspiration during the 18-day exposure. Method for TEM. Maize and soybean plants were separated into the root, stem, and leaf parts to elucidate the transport of MWCNTs (Figure S3, Supporting Information). Maize exposed to 50 mg/L of MWCNTs and soybean exposed to 20 mg/L of MWCNTs, and their relevant controls were used for TEM determination. Samples of root, stem, and leaf were removed from the plants and quickly transferred to 1/2 strength Karnovsky’s fixative.24 These samples were cut to a size of less than 1 mm3 and allowed to remain in the fixative at 4 °C until further processing could occur (>1 h). After the primary fixation, several buffer washes of 0.1 M Cacodylate solution removed the excess fixative. The samples were then postfixed for 90 min in 1% osmium tetroxide in 0.1 M Cacodylate buffer containing 1.5% potassium ferrocyanide.25 Several changes of wash buffer and one of distilled water followed. Samples incubated for 1 h with an en-bloc stain of 2.5% uranyl acetate26 at this point followed by a very slow dehydration in increasing concentrations of acetone starting at 15% acetone. When the samples had subjected to at least 3 changes of 100% acetone, a 1:1 solution of acetone and propylene oxide was applied for 30 min. Two incubations of 1 h in 100% propylene oxide prepared the tissues for a mixture of propylene oxide and Spurr’s epoxy resin. A gradual infiltration of the resin occurred by small incremental increases of Spurr’s resin27,28 until a 100% Spurr’s
MWCNTs traversed the cell walls and entered the cellular cytoplasm up to 4 μm distance.17 However, in whole rice plants, the uptake of MWCNTs was found to be insignificant in the rice plant root tissues.20 The charge of nanomaterials influences their toxicity, bioavailability, environmental behavior and fate. SWCNTs induced root elongation in six crop species (cabbage, carrot, cucumber, lettuce, onion, and tomato) and exhibited both charge- and plant species-dependence. Negatively charged SWCNTs (functionalized with poly-3-aminobenzenesulfonic acid) inhibited root elongation in lettuce, but no influence was seen on cabbage and carrots. Pristine SWCNTs enhanced root elongation in onion and cucumber and inhibited root elongation in tomato but did not affect the root elongation of cabbage and carrots.21 Zhu et al. reported that positively charged gold nanoparticles (AuNPs) were most readily taken up by plant roots, while negatively charged AuNPs were most efficiently transported to plant shoots (including stems and leaves) from the roots.22 However, up to now, there have been no reports regarding the uptake and translocation mechanisms of differently charged MWCNTs in whole plants. In this hydroponic study, we investigated the uptake and translocation mechanisms of three differently charged MWCNTs, including neutral pristine MWCNT (p-MWCNT), positively charged amine (NH2)functionalized MWCNT (MWCNT-NH2) and negatively charged carboxylate (COOH)-functionalized MWCNT (MWCNT-COOH) in two whole plants and food species: maize and soybean.
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MATERIALS AND METHODS Reagents. Pristine multiwall carbon nanotubes (pMWCNTs), amine (NH 2 )-functionalized MWCNTs (MWCNT-NH2s) and carboxylate (COOH)-functionalized MWCNTs (MWCNT-COOHs) were characterized as described previously.23 The detailed characteristics of three types of MWCNTs are showed in the Supporting Information. The sizes of these nanotubes were approximately 20−30 nm in diameter and 0.05−2.0 μm in length (Figure S1, Supporting Information). The p-MWCNT, MWCNT-NH 2 s and MWCNT-COOHs were neutral, positively charged, and negatively charged at pH 7.0. Deionized water (18.3 MΩ) was from an ultrapure water system (Barnstead International, Dubuque, IA). Deionized water, glass Petri dishes, vials, and filter paper were sterilized in a high-pressure sterilizer (TOMY ES-315). The solid MWCNT lumps were pressed into fine powder and then soaked in the deionized water for 2 days. The soaked MWCNTs were sonicated for 10 min or more in an ultrasonic bath to get a uniform suspension of MWCNTs for further use. All other chemicals and reagents used in this experiment were of analytical reagent grade or better. Maize (Zea mays) seeds were purchased from Lake Valley Seed Company, Inc. (Colorado), and Soybean (Glycine max) seeds were from Botanical Interests, Inc. (Colorado). Plant Germination, Growth, and Treatment. Maize and soybean seeds were surface-sterilized by a 5 min soak in autoclaved deionized water, two rinses in autoclaved deionized water, and a 1 min soak in hydrogen peroxide (30%), followed by three rinses in autoclaved deionized water. The maize and soybean seeds were separately transferred into the dampened filter paper in glass Petri dishes to germinate in the dark at 25 °C for 4 days. Deionized water was added every day to keep the filter paper dampened. Germinated seeds of similar sizes were B
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Figure 1. (A) Cumulative transpiration of maize exposed to different concentrations of MWCNTs; (B) cumulative transpiration of soybean exposed to 20 mg/L of MWCNTs; (C) total dry mass of maize exposed to different concentrations of MWCNTs at day 18; and (D) total dry mass of soybean exposed to 20 mg/L of MWCNTs at day 18. Asterisk (*) indicates a significant difference of exposed samples compared to control.
transpiration of water (by volume) of maize exposed to 50 mg/L of MWCNT-COOHs was 41.7 ± 1.9 mL, which was almost twice as much as that of the maize control (23.9 ± 1.6 mL). Dry biomass of maize exposed to 20 mg/L of MWCNTCOOHs was also greater than that of maize control, corroborating the statistically significant biostimulation observed based on cumulative transpiration. In addition, a dose− response relationship was evident. The degree of stimulation of MWCNTs to maize increased from a concentration of 10−50 mg/L, as measured by the increase in the cumulative volume of transpiration. However, p-MWCNTs showed only a slight stimulation of maize growth compared to controls, and the effect decreased slightly from 10 to 50 mg/L. Contrary to maize, all three types of MWCNTs at 20 mg/L produced inhibition to soybean transpiraton and growth of dry weight biomass. MWCNT-COOHs demonstrated the least inhibitory effect (Figure 1B,D), and the inhibition was not statistically significant change compared to the controls. These data show that the influence of MWCNTs on plant growth was charge- and plant species-dependent. In the literature, MWCNTs were also reported to stimulate or inhibit the growth of other plant species. For example, pMWCNTs have been found to enhance the growth of tobacco cell cultures via the upregulation of genes involved in cell division/cell wall formation and water transport,30 and to penetrate the seed coats of maize, barley, and soybean producing positive effects on seed germination, growth, and development of corn, barley and soybean.31 However, MWCNTs did not significantly inhibit or enhance wheat root
solution was reached. To ensure complete infiltration, samples were placed in three successive changes of Spurr’s resin over a 6 h period. Finally, the plant samples were placed into flat silicon embedding molds and cured at 70 °C for 48 h. The resulting blocks were cut at 80 nm using a Leica EM UC6 Ultramicrotome (Solms, Germany) and placed on grids coated with Formvar (Electron Microscopy Sciences, Hatfield, Pennsylvania #15820). The samples were imaged using a JEOL JEM-1230 transmission electron microscope (JEOL USA, Inc.) Statistical Analyses. The data of statistical analysis of MWCNTs are presented for significant differences by one way ANOVA with Tukey test at p < 0.05.
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RESULTS AND DISCUSSION Stimulation and Inhibition of Plant Growth by MWCNTs. The effect of MWCNTs on maize and soybean was inferred by the rate and extent of transpiration and biomass growth, which is a reliable indicator of the relative health of plants grown under hydroponic conditions.29 The concentration and charge of MWCNTs greatly influenced cumulative water transpiration and biomass growth of maize in the 18-day exposure (Figure 1A,C). Specifically, MWCNTs stimulated maize growth. Maize seedlings also showed charge- and concentration-dependence of the MWCNTs based on their cumulative water transpiration volume and dry biomass weight. MWCNT-COOHs (negatively charged) were the strongest growth stimulator for maize compared to MWCNT-NH2s (positively charged) and p-MWCNTs (neutral) at the same exposure concentration, in that order. The cumulative C
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Figure 2. (A) Image of pristine MWCNTs in the root; (B) image of pristine MWCNTs in the root; (C and D) magnification of the highlighted rectangle area in panels A and B, respectively; (E and F) magnification of cell No.7 and cell Nos. 3−6, respectively, in panel A; (G and H) magnification of the highlighted areas in panel F. (Nos. 1−10) Cell numbers for easy explanations of the cell relationship; (CC) companion cell; (CW) cell wall; (PC) parenchyma cell; (ST) sieve tube member; and (X) xylem.
or shoot growth.17 Furthermore, the influence of SWCNTs on plant growth showed similar charge- and plant speciesdependence as did MWCNTs. Negatively charged SWCNTs (functionalized with poly-3-aminobenzenesulfonic acid) inhibited root elongation in lettuce, but did not influence cabbage and carrots; pristine SWCNTs enhanced root elongation in onion and cucumber and inhibited root elongation in tomato, but did not affect the root elongation of cabbage and carrots.21 Uptake and Translocation of MWCNTs. Transmission electron microscopy (TEM) was utilized to identify MWCNTs in plants according to their special tubular (spiky) shape. TEM imaging confirmed that the original size range of the MWCNT standard was from 0.05 to 2 μm (Figure S1, Supporting Information). As expected, MWCNTs were not observed by TEM in the maize and soybean control samples where none were added (Figure S3, Supporting Information). However, TEM observations did demonstrate the presence and distribution of three types of MWCNT in various treatments, providing micrographic evidence that all three of these differently charged MWCNTs were taken up and translocated in maize and soybean (Figures 2−8 and Figures S5 and S6, Supporting Information). In our experiments, MWCNTs were found to be sorbed to the external surface of primary roots and secondary roots, which was visible to the naked eye via a black coloration of
white roots, likely due to the high affinity of MWCNTs for the epidermis and the waxy casparian strips of the roots. MWCNTs were more abundant in the roots than leaves because the roots were in direct contact with the solution containing the MWCNTs. In addition, p-MWCNTs, MWCNT-NH2s and MWCNT-COOHs were found in the specific cells of roots, stems, and leaves of maize and soybean (Figures 2−8 and Figures S5 and S6. Supporting Information), such as the root phloem complex (including companion cells and sieve tube members) and the xylem (Figures 2−6). The specific intracellular sites where p-MWCNTs, MWCNT-NH2s, and MWCNT-COOHs accumulated in the maize root, stem, and leaf cells were the cytoplasm, cell wall, cell membrane, chloroplast, and on the surface of unidentified organelles. In particular, p-MWCNTs appeared to freely enter the whole xylem cells and also partially destroyed the maize root cell wall (Figure 2G). Furthermore, they created a hole which accumulated p-WMCNTs in the maize root cell wall (Figure 2H). For soybean, p-MWCNTs, MWCNT-NH2s, and MWCNTCOOHs accumulated in the cytoplasm, cell wall, cell membrane, chloroplast, mitochondria, around the plasmodesmata, and on the surface of unidentified organelles and substances. However, fewer p-MWCNTs, MWCNT-NH2s, and MWCNT-COOHs were detected in both maize and soybean stems compared to the roots and the leaves, which D
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Figure 3. (A) Image of MWCNT-NH2s in the maize root; (B1) image of MWCNT-NH2s in the maize root cell No.1 and 2; (B2−B5) magnification of the highlighted rectangle area in panels B1, B2, and B4, respectively; (C1) image of MWCNT-NH2s in the root cell No.1 and 3; (C2 and C3) magnification of the highlighted rectangle areas in panel C1; (Nos.1−3) cell numbers for easy explanations of the cell relationship; (CW) cell wall; (CP) cytoplasm; (UO) unidentified organelle; and (X) xylem.
not the others. Xylem cells and phloem cells (including sieve tube members and companion cells) showed a special propensity for the uptake of MWCNTs. Figure 2 clearly shows that maize xylem cells preferentially took up a large number of p-MWCNTs (cells No. 1, 2, 8, 9 in Figure 2A−D). In addition, p-MWCNTs were sorbed and accumulated on maize cell membranes of sieve tube members and on the maize companion cell nucleus. However, p-MWCNTs were not observed to be accumulated in the parenchyma cells, though they are adjacent to and between both xylem and phloem. The cellular difference between maize xylem cells, phloem cells, and parenchyma cells might be due to their different functions during cellular translocation. In the xylem cells, pMWCNTs were taken up and translocated with water and nutrients from the hydroponic solution to roots, stems, and leaves (unidirectional), such that they filled the whole cells. However, the phloem (including sieve tube members and companion cells) mainly translocates food and nutrients from leaves to storage organs and the fastest-growing parts of the plant. Although transportation of material is bidirectional in the phloem, the main flow direction is from top to bottom, so that p-MWCNTs was transported gradually from bottom to top by xylem and from top to bottom by phloem. During this transport of water and nutrients, MWCNTs accumulated
suggests that p-MWCNTs, MWCNT-NH2s, and MWCNTCOOHs rapidly passed through the stems from the roots to the leaves. Therefore, it follows that p-MWCNTs, MWCNT-NH2s, and MWCNT-COOHs were taken-up from the hydroponic solution by maize and soybean roots, and then they were translocated to the leaves via the stems. Once p-MWCNTs, MWCNT-NH2s, and MWCNT-COOHs entered the plant cells, they stimulated maize growth and inhibited soybean growth (Figure 1). This is the first report that MWCNTs can be taken up and translocated all the way from roots to leaves in whole plants, as other studies have reported that the uptake of MWCNTs into whole plants was blocked by the cell wall.17,20 Our method of exposing the plants hydroponically to MWCNTs was very similar to the referenced studies. It is of importance because soybean and maize are food plants. Cellular Selectivity for MWCNTs in Maize and Soybean. Cellular selectivity for nanomaterials is an important characteristic of distinguishing nanotransporters in medical and agricultural fields. Therefore, the cellular selectivity for pMWCNTs, MWCNT-NH2s, and MWCNT-COOHs was investigated in maize and soybean. The uptake and translocation of p-MWCNTs, MWCNT-NH2s, and MWCNTCOOHs in maize and soybean revealed a clear cellular selectivity. First, we only found MWCNTs in some cells and E
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Figure 4. (A) Image of MWCNT-COOHs in the maize root; (B) magnification of the highlighted rectangle area in panel A.
Figure 5. (A) Image of p-MWCNTs in maize leaf; (A1 and A2) the magnification of the highlighted rectangle areas in panel A; (B) image of MWCNT-NH2s in maize leaf; (B1 and B2) magnification of the highlighted rectangle areas in panel B; (C) image of MWCNT-COOHs in maize leaf; (C1−C5) magnification of the highlighted rectangle areas in panel C.
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Figure 6. (A) Image of p-MWCNTs in soybean root; (A1 and A2) magnification of the highlighted rectangle areas in panel A; (B) image of MWCNT-NH2s in soybean root; (B1 and B2) magnification of the highlighted rectangle areas in panel B.
throughout the course of the experiment, albeit it at gradually decreasing concentration due to uptake. Therefore, the different charge characteristics of p-MWCNTs, MWCNTNH2s, and MWCNT-COOHs led to different behaviors in maize and soybean (Figures 2−4). Positively charged MWCNT-NH2s were observed to be sorbed and clustered on the surface of negatively charged membranes. It can be seen from Figure 3 that a large number of nanotubes (MWCNT-NH2) accumulated around the maize root cell membrane and other unidentified organelles (UO), but there were almost no MWCNT-NH2s detected in the cytoplasm. MWCNT-NH2s were arranged in a pattern of layers, like iron filings attracted to a magnet. Contrary to positively charged MWCNT-NH 2 s, negatively charged MWCNT-COOHs were spatially distributed (dispersed) in the xylem presumably because of the repulsion with the negatively charged cell membrane (Figure 4). The p-MWCNTs are neutral, and they exhibited an “in-between” behavior. They were partially accumulated and partially dispersed in the maize root cells (Figure 2). In contrast, after the “long-distance” translocation of positively and negatively charged MWCNTs, some were dispersed in the cell wall and the cytoplasm and around the cell membrane. Less charge selectivity was observed in maize stems and leaves for neutral p-MWCNTs (Figure 5 and Figure S5, Supporting Information). The charge selectivity of plant cells for MWCNTs was plantdependent. Accumulation of MWCNTs in various cells and parts of the plant showed a different pattern in soybean compared to maize. In soybean, a small number of p-MWCNTs and MWCNT-NH2s preferentially aggregated in the roots (Figure 6). However, there were very few MWCNT-COOHs detected by TEM in roots, presumably due to repulsion with the negative surface charge of roots (images not shown). In addition, few nantotubes of any of the three types, p-MWCNT, MWCNT-NH2s, and MWCNT-COOHs, were found in the stems of soybean, suggesting they rapidly were translocated through the soybean stem to the leaves, and not apparently retained by the cellular tissues (images not shown). However, all the nanotubes, p-MWCNT, MWCNT-NH 2 s, and MWCNT-COOHs, showed a clear charge selectivity in
around the cell membranes and were subsequently sorbed onto the cell membranes. Parenchyma cells, specialized for food storage including starch, protein, and fats, are influenced by transport from the leaves to the roots. The necessity of MWCNTs first being translocated up in the xylem and then gradually down by the phloem resulted in the observation that there were no pMWCNTs in the root parenchyma cells (none detected by TEM imaging). These results also suggest that there was no direct lateral transport between xylem, parenchyma cells and phloem. Maize xylem cells showed a clear selectivity for uptake and translocation of MWCNT-NH2s and MWCNT-COOHs (Figures 3 and 4). MWCNT-NH2s were only found around the cell wall in cell No.1 among all the cells observed in the maize root (Figure 3), while MWCNT-COOHs were detected in specific maize root xylem cells (Figure 4). However, pMWCNTs, MWCNT-NH2s and MWCNT-COOHs exhibited less cellular selectivity in the maize stems and leaves than for roots, and they seemed to be dispersed randomly in the cytoplasm, cell membrane, and cell wall of some root cells (Figure 5 and Figure S5, Supporting Information). The uptake and translocation of p-MWCNTs, MWCNTNH2s and MWCNT-COOHs in soybean also showed a cellular selectivity on the part of the plant in which only special (minor) cells contained the nanotubes, and we did not detect any carbon nanotubes in the vast majority of cells (Figures 6−8 and Figure S6, Supporting Information). Therefore, these images suggest that certain maize and soybean cells demonstrate the selection of MWCNTs during their uptake and translocation. Charge Selectivity of MWCNTs in Maize and Soybean. The uptake and translocation of MWCNTs were greatly influenced by their charges because differently charged MWCNTs possess different surface properties and capacity for aggregation. On the other hand, cells also have a certain preference for charged particles due to their negatively charged cell membrane. For example, neutral p-MWCNTs could not be well dispersed in water in our experiments. However, positively charged MWCNT-NH2s and negatively charged MWCNTCOOHs were well dispersed in water and remained that way G
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Figure 7. (A) Image of MWCNT-NH2s in the soybean leaf; (B1−H1) magnification of the highlighted areas in panel A; (B2, C2, D2−5, E2−3, F2− 3, G2−4, and H2−4) magnification of panels B1−H1; (CW) cell wall; (MC) mesophyll cell; (PL) plasmodesma; and (SC) sclerenchyma cell.
soybean leaves (Figures 7 and 8 and Figure S6, Supporting Information). Positively charged MWCNT-NH2 was found to be deposited in a group of sclerenchyma cells (Figure 7). Sclerenchyma cells are supporting tissue with different shapes in the two plants. These cells have thick cell walls composed of cellulose, lignin, and hemicellulose. Two types of sclerenchyma cells exist: fibers (Figure 7D1, E1, G1, and H1) and sclereids (Figure 7B1 and F1). In fibers, MWCNT-NH2s tended to be attached to the fibrous stuff inside the soybean cell; and in sclereids, MWCNTNH2s formed a cluster attached to the soybean cell membrane, presumably because of the positive surface charge properties of MWCNT-NH2s. The plasmodesmata are likely channels for MWCNT-NH2s to pass through the thick soybean sclerenchyma cell wall (Figure 7D2 and F3). In addition, MWCNT-NH2s
were also found in the mesophyll cells of soybean leaves (Figure 7C1). On the other hand, neutral p-MWCNTs and negatively charged MWCNT-COOHs were observed in sporadic distributions in soybean leaves (Figure 8 and Figure S6, Supporting Information), suggesting that neutral p-MWCNTs and negatively charged MWCNT-COOHs were less mobile than positively charged MWCNT-NH2s from soybean roots to leaves. To our knowledge, there have been no previous reports regarding the surface-charge selectivity of plants for MWCNTs. However, charge selectivity of other nanomaterials has been previously reported in Caco-2 cells32 and whole plants.22,33 Bannunah et al. found that both positively and negatively charged blue aminated latex nanoparticles (50 nm) and orange H
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Figure 8. (A) Image of MWCNT-COOHs in soybean leaf; (B and C) magnification of the highlighted rectangle areas in panel A; (D) image of MWCNT-COOHs in soybean leaf; (E and F) magnification of the highlighted rectangle area in panel D.
demonstrate that maize and soybean roots took up a wide range of sizes of p-MWCNT, MWCNT-NH2s, and MWCNTCOOHs in roots, but only a narrow range (50−100 nm) of MWCNTs could be translocated to the leaves via the stems. The size selectivity of MWCNTs by whole plants has not been reported so far, but the size-based selection of other nanomaterials has been frequently reported in plants,19,35 and our results are consistent with previous studies. Serag et al. observed that short p-MWCNTs (
Charge, Size, and Cellular Selectivity for Multiwall Carbon Nanotubes by Maize and Soybean Guangshu Zhai,*,† Sarah M. Gutowski,† Katherine S. Walters,‡ Bing Yan,§ and Jerald L. Schnoor*,† †
Department of Civil and Environmental Engineering and IIHR Hydroscience and Engineering, The University of Iowa, Iowa City, Iowa 52242, United States ‡ Central Microscopy Research Facility, The University of Iowa, Iowa City, Iowa 52242, United States § School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China S Supporting Information *
ABSTRACT: Maize (Zea mays) and soybean (Glycine max) were used as model food-chain plants to explore vegetative uptake of differently charged multiwall carbon nanotubes (MWCNTs). Three types of MWCNTs, including neutral pristine MWCNT (p-MWCNT), positively charged MWCNTNH2, and negatively charged MWCNT-COOH, were directly taken-up and translocated from hydroponic solution to roots, stems, and leaves of maize and soybean plants at the MWCNT concentrations ranging from 10.0 to 50.0 mg/L during 18-day exposures. MWCNTs accumulated in the xylem and phloem cells and within specific intracellular sites like the cytoplasm, cell wall, cell membrane, chloroplast, and mitochondria, which was observed by transmission electron microscopy. MWCNTs stimulated the growth of maize and inhibited the growth of soybean at the exposed doses. The cumulative transpiration of water in maize exposed to 50 mg/L of MWCNT-COOHs was almost twice as much as that in the maize control. Dry biomass of maize exposed to MWCNTs was greater than that of maize control. In addition, the uptake and translocation of these MWCNTs clearly exhibited cellular, charge, and size selectivity in maize and soybean, which could be important properties for nanotransporters. This is the first report of cellular, charge, and size selectivity on the uptake by whole food plants for three differently charged MWCNTs.
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INTRODUCTION Carbon nanotubes (CNTs) are a carbon allotrope, including single-wall carbon nanotubes (SWCNTs) and multiwall carbon nanotubes (MWCNTs). CNTs are widely used in nanomedicine, pigments, electronics, optics, cosmetics, and energy and environmental technologies due to their unique chemical, physical, optical, and magnetic properties.1−3 In 2010, CNTs were ranked as one of the top 10 most produced engineered nanomaterials (ENMs) by mass in the world, reaching 3200 t/ year. Most of CNTs end up in landfills (∼80%) and soils (∼15%).1 Importantly, more and more CNTs are being increasingly utilized in bioapplications for the transport and delivery of therapeutic agents.4−9 Their manufacturing and release will undoubtedly increase the exposure to humans and the environment and potentially cause biological effects on living organisms. Plants might accumulate CNTs from soils and water to threaten human and ecological health via food chains. On the other hand, uptake of carbon nanotubes with different properties and potential to penetrate individual cells may prove important in medical and agrochemical technologies. Therefore, it is of practical and fundamental importance to understand the interaction of CNTs and plants. MWCNTs have created a concern regarding toxicity to humans and animals.10−13 However, reports on MWCNT toxicity and interaction between MWCNTs and plants in vitro and in vivo are extremely limited. MWCNTs exhibited a detrimental effect on the growth of rice cells (Oryza sativa L.) © XXXX American Chemical Society
by the increase of reactive oxygen species (ROS), and caused a decrease of cell viability and cell density.14,15 Moreover, agglomerates of MWCNTs were toxic to Arabidopsis T87 suspension plant cells because they decreased cell dry weights, viabilities, chlorophyll contents, and superoxide dismutase (SOD) activity.16 Contrary to plant cells in vitro, MWCNTs have not demonstrated much apparent toxicity to whole plants in vivo. For example, MWCNTs did not exhibit any influence on wheat growth in a hydroponic study,17 and MWCNT suspensions (even at a high concentration of 2000 mg/L) did not show any toxicity to root growth of radish, rape, ryegrass, lettuce, maize, or cucumber.18 The penetration of MWCNTs into plant protoplasts, cells, and whole plants have produced mixed results in the experimental literature. In rice cells exposed at 20−80 mg/L, the MWCNTs remained associated with the cell wall and were blocked from entering the cells.14 But in Catharanthus roseus plant protoplasts (plant cell walls removed via enzymatic treatment), MWCNTs moved right through the cell membrane into the cytoplasm.19 For whole plants like wheat, MWCNTs were observed to pierce the epidermal and wheat root hair cell walls and enter the root hair and root cap of the wheat root. Received: March 5, 2015 Revised: May 14, 2015 Accepted: May 26, 2015
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DOI: 10.1021/acs.est.5b01145 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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selected and grown in the vial with preholed caps. The vial was filled with 20 mL of 0.1 strength Hoagland solution and sealed with Parafilm. The germinated maize and soybean seeds were grown in Percival Plant Growth Chamber with 16/8 day/night cycle at 25 °C, 59% relative humidity (RH), and light intensity of 120−180 μmol m−2 s−1 for 7 days. The healthy vigorously growing maize and soybean seedlings were selected for the carbon nanotube exposure experiments after 7 days of growth. Different concentrations of MWCNTs were added into 20 mL of 0.1 strength Hoagland solution in the vials. Blank maize and soybean controls (one seedling per vial) included four seedlings (three for mass measurement and one for TEM) without the addition of MWCNTs; four healthy maize and soybean seedlings were exposed to different concentrations of MWCNTs (one seedling per vial) and were used to test the transport and behavior of MWCNTs in these plants. Due to generally poor solubility of MWCNTs used, we selected 50 mg/L as the highest concentration because our experiments showed that at this concentration, all MWCNTs were reliably suspended and stable for 2−3 weeks (Figure S2, Supporting Information). Therefore, the final exposure concentrations of pMWCNT, MWCNT-NH2s, and MWCNT-COOHs selected were 10.0, 20.0, and 50.0 mg/L for maize, except for the blank control without any MWCNTs. The concentration of pMWCNT, MWCNT-NH2s, and MWCNT-COOHs utilized for exposure to soybean was 20.0 mg/L except for the blank control without any MWCNTs. These concentrations and the method of exposure are representative of the literature, although our concentrations of MWCNTs were lower than most studies.14,18 All exposure vials were wrapped with aluminum foil. A Percival Plant Growth Chamber was used for plant growth at 25 °C, 59% RH. The photoperiod was set 16 h per day under fluorescent lighting with a light intensity between 120 and 180 μmol m−2 s−1, and 0.1 strength Hoagland solution was injected into the vials every day to compensate for the evapotranspiration loss, which was determined by monitoring weight loss of the vials. Cumulative evapotranspiration for each plant was the sum of the evapotranspiration during the 18-day exposure. Method for TEM. Maize and soybean plants were separated into the root, stem, and leaf parts to elucidate the transport of MWCNTs (Figure S3, Supporting Information). Maize exposed to 50 mg/L of MWCNTs and soybean exposed to 20 mg/L of MWCNTs, and their relevant controls were used for TEM determination. Samples of root, stem, and leaf were removed from the plants and quickly transferred to 1/2 strength Karnovsky’s fixative.24 These samples were cut to a size of less than 1 mm3 and allowed to remain in the fixative at 4 °C until further processing could occur (>1 h). After the primary fixation, several buffer washes of 0.1 M Cacodylate solution removed the excess fixative. The samples were then postfixed for 90 min in 1% osmium tetroxide in 0.1 M Cacodylate buffer containing 1.5% potassium ferrocyanide.25 Several changes of wash buffer and one of distilled water followed. Samples incubated for 1 h with an en-bloc stain of 2.5% uranyl acetate26 at this point followed by a very slow dehydration in increasing concentrations of acetone starting at 15% acetone. When the samples had subjected to at least 3 changes of 100% acetone, a 1:1 solution of acetone and propylene oxide was applied for 30 min. Two incubations of 1 h in 100% propylene oxide prepared the tissues for a mixture of propylene oxide and Spurr’s epoxy resin. A gradual infiltration of the resin occurred by small incremental increases of Spurr’s resin27,28 until a 100% Spurr’s
MWCNTs traversed the cell walls and entered the cellular cytoplasm up to 4 μm distance.17 However, in whole rice plants, the uptake of MWCNTs was found to be insignificant in the rice plant root tissues.20 The charge of nanomaterials influences their toxicity, bioavailability, environmental behavior and fate. SWCNTs induced root elongation in six crop species (cabbage, carrot, cucumber, lettuce, onion, and tomato) and exhibited both charge- and plant species-dependence. Negatively charged SWCNTs (functionalized with poly-3-aminobenzenesulfonic acid) inhibited root elongation in lettuce, but no influence was seen on cabbage and carrots. Pristine SWCNTs enhanced root elongation in onion and cucumber and inhibited root elongation in tomato but did not affect the root elongation of cabbage and carrots.21 Zhu et al. reported that positively charged gold nanoparticles (AuNPs) were most readily taken up by plant roots, while negatively charged AuNPs were most efficiently transported to plant shoots (including stems and leaves) from the roots.22 However, up to now, there have been no reports regarding the uptake and translocation mechanisms of differently charged MWCNTs in whole plants. In this hydroponic study, we investigated the uptake and translocation mechanisms of three differently charged MWCNTs, including neutral pristine MWCNT (p-MWCNT), positively charged amine (NH2)functionalized MWCNT (MWCNT-NH2) and negatively charged carboxylate (COOH)-functionalized MWCNT (MWCNT-COOH) in two whole plants and food species: maize and soybean.
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MATERIALS AND METHODS Reagents. Pristine multiwall carbon nanotubes (pMWCNTs), amine (NH 2 )-functionalized MWCNTs (MWCNT-NH2s) and carboxylate (COOH)-functionalized MWCNTs (MWCNT-COOHs) were characterized as described previously.23 The detailed characteristics of three types of MWCNTs are showed in the Supporting Information. The sizes of these nanotubes were approximately 20−30 nm in diameter and 0.05−2.0 μm in length (Figure S1, Supporting Information). The p-MWCNT, MWCNT-NH 2 s and MWCNT-COOHs were neutral, positively charged, and negatively charged at pH 7.0. Deionized water (18.3 MΩ) was from an ultrapure water system (Barnstead International, Dubuque, IA). Deionized water, glass Petri dishes, vials, and filter paper were sterilized in a high-pressure sterilizer (TOMY ES-315). The solid MWCNT lumps were pressed into fine powder and then soaked in the deionized water for 2 days. The soaked MWCNTs were sonicated for 10 min or more in an ultrasonic bath to get a uniform suspension of MWCNTs for further use. All other chemicals and reagents used in this experiment were of analytical reagent grade or better. Maize (Zea mays) seeds were purchased from Lake Valley Seed Company, Inc. (Colorado), and Soybean (Glycine max) seeds were from Botanical Interests, Inc. (Colorado). Plant Germination, Growth, and Treatment. Maize and soybean seeds were surface-sterilized by a 5 min soak in autoclaved deionized water, two rinses in autoclaved deionized water, and a 1 min soak in hydrogen peroxide (30%), followed by three rinses in autoclaved deionized water. The maize and soybean seeds were separately transferred into the dampened filter paper in glass Petri dishes to germinate in the dark at 25 °C for 4 days. Deionized water was added every day to keep the filter paper dampened. Germinated seeds of similar sizes were B
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Figure 1. (A) Cumulative transpiration of maize exposed to different concentrations of MWCNTs; (B) cumulative transpiration of soybean exposed to 20 mg/L of MWCNTs; (C) total dry mass of maize exposed to different concentrations of MWCNTs at day 18; and (D) total dry mass of soybean exposed to 20 mg/L of MWCNTs at day 18. Asterisk (*) indicates a significant difference of exposed samples compared to control.
transpiration of water (by volume) of maize exposed to 50 mg/L of MWCNT-COOHs was 41.7 ± 1.9 mL, which was almost twice as much as that of the maize control (23.9 ± 1.6 mL). Dry biomass of maize exposed to 20 mg/L of MWCNTCOOHs was also greater than that of maize control, corroborating the statistically significant biostimulation observed based on cumulative transpiration. In addition, a dose− response relationship was evident. The degree of stimulation of MWCNTs to maize increased from a concentration of 10−50 mg/L, as measured by the increase in the cumulative volume of transpiration. However, p-MWCNTs showed only a slight stimulation of maize growth compared to controls, and the effect decreased slightly from 10 to 50 mg/L. Contrary to maize, all three types of MWCNTs at 20 mg/L produced inhibition to soybean transpiraton and growth of dry weight biomass. MWCNT-COOHs demonstrated the least inhibitory effect (Figure 1B,D), and the inhibition was not statistically significant change compared to the controls. These data show that the influence of MWCNTs on plant growth was charge- and plant species-dependent. In the literature, MWCNTs were also reported to stimulate or inhibit the growth of other plant species. For example, pMWCNTs have been found to enhance the growth of tobacco cell cultures via the upregulation of genes involved in cell division/cell wall formation and water transport,30 and to penetrate the seed coats of maize, barley, and soybean producing positive effects on seed germination, growth, and development of corn, barley and soybean.31 However, MWCNTs did not significantly inhibit or enhance wheat root
solution was reached. To ensure complete infiltration, samples were placed in three successive changes of Spurr’s resin over a 6 h period. Finally, the plant samples were placed into flat silicon embedding molds and cured at 70 °C for 48 h. The resulting blocks were cut at 80 nm using a Leica EM UC6 Ultramicrotome (Solms, Germany) and placed on grids coated with Formvar (Electron Microscopy Sciences, Hatfield, Pennsylvania #15820). The samples were imaged using a JEOL JEM-1230 transmission electron microscope (JEOL USA, Inc.) Statistical Analyses. The data of statistical analysis of MWCNTs are presented for significant differences by one way ANOVA with Tukey test at p < 0.05.
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RESULTS AND DISCUSSION Stimulation and Inhibition of Plant Growth by MWCNTs. The effect of MWCNTs on maize and soybean was inferred by the rate and extent of transpiration and biomass growth, which is a reliable indicator of the relative health of plants grown under hydroponic conditions.29 The concentration and charge of MWCNTs greatly influenced cumulative water transpiration and biomass growth of maize in the 18-day exposure (Figure 1A,C). Specifically, MWCNTs stimulated maize growth. Maize seedlings also showed charge- and concentration-dependence of the MWCNTs based on their cumulative water transpiration volume and dry biomass weight. MWCNT-COOHs (negatively charged) were the strongest growth stimulator for maize compared to MWCNT-NH2s (positively charged) and p-MWCNTs (neutral) at the same exposure concentration, in that order. The cumulative C
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Figure 2. (A) Image of pristine MWCNTs in the root; (B) image of pristine MWCNTs in the root; (C and D) magnification of the highlighted rectangle area in panels A and B, respectively; (E and F) magnification of cell No.7 and cell Nos. 3−6, respectively, in panel A; (G and H) magnification of the highlighted areas in panel F. (Nos. 1−10) Cell numbers for easy explanations of the cell relationship; (CC) companion cell; (CW) cell wall; (PC) parenchyma cell; (ST) sieve tube member; and (X) xylem.
or shoot growth.17 Furthermore, the influence of SWCNTs on plant growth showed similar charge- and plant speciesdependence as did MWCNTs. Negatively charged SWCNTs (functionalized with poly-3-aminobenzenesulfonic acid) inhibited root elongation in lettuce, but did not influence cabbage and carrots; pristine SWCNTs enhanced root elongation in onion and cucumber and inhibited root elongation in tomato, but did not affect the root elongation of cabbage and carrots.21 Uptake and Translocation of MWCNTs. Transmission electron microscopy (TEM) was utilized to identify MWCNTs in plants according to their special tubular (spiky) shape. TEM imaging confirmed that the original size range of the MWCNT standard was from 0.05 to 2 μm (Figure S1, Supporting Information). As expected, MWCNTs were not observed by TEM in the maize and soybean control samples where none were added (Figure S3, Supporting Information). However, TEM observations did demonstrate the presence and distribution of three types of MWCNT in various treatments, providing micrographic evidence that all three of these differently charged MWCNTs were taken up and translocated in maize and soybean (Figures 2−8 and Figures S5 and S6, Supporting Information). In our experiments, MWCNTs were found to be sorbed to the external surface of primary roots and secondary roots, which was visible to the naked eye via a black coloration of
white roots, likely due to the high affinity of MWCNTs for the epidermis and the waxy casparian strips of the roots. MWCNTs were more abundant in the roots than leaves because the roots were in direct contact with the solution containing the MWCNTs. In addition, p-MWCNTs, MWCNT-NH2s and MWCNT-COOHs were found in the specific cells of roots, stems, and leaves of maize and soybean (Figures 2−8 and Figures S5 and S6. Supporting Information), such as the root phloem complex (including companion cells and sieve tube members) and the xylem (Figures 2−6). The specific intracellular sites where p-MWCNTs, MWCNT-NH2s, and MWCNT-COOHs accumulated in the maize root, stem, and leaf cells were the cytoplasm, cell wall, cell membrane, chloroplast, and on the surface of unidentified organelles. In particular, p-MWCNTs appeared to freely enter the whole xylem cells and also partially destroyed the maize root cell wall (Figure 2G). Furthermore, they created a hole which accumulated p-WMCNTs in the maize root cell wall (Figure 2H). For soybean, p-MWCNTs, MWCNT-NH2s, and MWCNTCOOHs accumulated in the cytoplasm, cell wall, cell membrane, chloroplast, mitochondria, around the plasmodesmata, and on the surface of unidentified organelles and substances. However, fewer p-MWCNTs, MWCNT-NH2s, and MWCNT-COOHs were detected in both maize and soybean stems compared to the roots and the leaves, which D
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Figure 3. (A) Image of MWCNT-NH2s in the maize root; (B1) image of MWCNT-NH2s in the maize root cell No.1 and 2; (B2−B5) magnification of the highlighted rectangle area in panels B1, B2, and B4, respectively; (C1) image of MWCNT-NH2s in the root cell No.1 and 3; (C2 and C3) magnification of the highlighted rectangle areas in panel C1; (Nos.1−3) cell numbers for easy explanations of the cell relationship; (CW) cell wall; (CP) cytoplasm; (UO) unidentified organelle; and (X) xylem.
not the others. Xylem cells and phloem cells (including sieve tube members and companion cells) showed a special propensity for the uptake of MWCNTs. Figure 2 clearly shows that maize xylem cells preferentially took up a large number of p-MWCNTs (cells No. 1, 2, 8, 9 in Figure 2A−D). In addition, p-MWCNTs were sorbed and accumulated on maize cell membranes of sieve tube members and on the maize companion cell nucleus. However, p-MWCNTs were not observed to be accumulated in the parenchyma cells, though they are adjacent to and between both xylem and phloem. The cellular difference between maize xylem cells, phloem cells, and parenchyma cells might be due to their different functions during cellular translocation. In the xylem cells, pMWCNTs were taken up and translocated with water and nutrients from the hydroponic solution to roots, stems, and leaves (unidirectional), such that they filled the whole cells. However, the phloem (including sieve tube members and companion cells) mainly translocates food and nutrients from leaves to storage organs and the fastest-growing parts of the plant. Although transportation of material is bidirectional in the phloem, the main flow direction is from top to bottom, so that p-MWCNTs was transported gradually from bottom to top by xylem and from top to bottom by phloem. During this transport of water and nutrients, MWCNTs accumulated
suggests that p-MWCNTs, MWCNT-NH2s, and MWCNTCOOHs rapidly passed through the stems from the roots to the leaves. Therefore, it follows that p-MWCNTs, MWCNT-NH2s, and MWCNT-COOHs were taken-up from the hydroponic solution by maize and soybean roots, and then they were translocated to the leaves via the stems. Once p-MWCNTs, MWCNT-NH2s, and MWCNT-COOHs entered the plant cells, they stimulated maize growth and inhibited soybean growth (Figure 1). This is the first report that MWCNTs can be taken up and translocated all the way from roots to leaves in whole plants, as other studies have reported that the uptake of MWCNTs into whole plants was blocked by the cell wall.17,20 Our method of exposing the plants hydroponically to MWCNTs was very similar to the referenced studies. It is of importance because soybean and maize are food plants. Cellular Selectivity for MWCNTs in Maize and Soybean. Cellular selectivity for nanomaterials is an important characteristic of distinguishing nanotransporters in medical and agricultural fields. Therefore, the cellular selectivity for pMWCNTs, MWCNT-NH2s, and MWCNT-COOHs was investigated in maize and soybean. The uptake and translocation of p-MWCNTs, MWCNT-NH2s, and MWCNTCOOHs in maize and soybean revealed a clear cellular selectivity. First, we only found MWCNTs in some cells and E
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Figure 4. (A) Image of MWCNT-COOHs in the maize root; (B) magnification of the highlighted rectangle area in panel A.
Figure 5. (A) Image of p-MWCNTs in maize leaf; (A1 and A2) the magnification of the highlighted rectangle areas in panel A; (B) image of MWCNT-NH2s in maize leaf; (B1 and B2) magnification of the highlighted rectangle areas in panel B; (C) image of MWCNT-COOHs in maize leaf; (C1−C5) magnification of the highlighted rectangle areas in panel C.
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Figure 6. (A) Image of p-MWCNTs in soybean root; (A1 and A2) magnification of the highlighted rectangle areas in panel A; (B) image of MWCNT-NH2s in soybean root; (B1 and B2) magnification of the highlighted rectangle areas in panel B.
throughout the course of the experiment, albeit it at gradually decreasing concentration due to uptake. Therefore, the different charge characteristics of p-MWCNTs, MWCNTNH2s, and MWCNT-COOHs led to different behaviors in maize and soybean (Figures 2−4). Positively charged MWCNT-NH2s were observed to be sorbed and clustered on the surface of negatively charged membranes. It can be seen from Figure 3 that a large number of nanotubes (MWCNT-NH2) accumulated around the maize root cell membrane and other unidentified organelles (UO), but there were almost no MWCNT-NH2s detected in the cytoplasm. MWCNT-NH2s were arranged in a pattern of layers, like iron filings attracted to a magnet. Contrary to positively charged MWCNT-NH 2 s, negatively charged MWCNT-COOHs were spatially distributed (dispersed) in the xylem presumably because of the repulsion with the negatively charged cell membrane (Figure 4). The p-MWCNTs are neutral, and they exhibited an “in-between” behavior. They were partially accumulated and partially dispersed in the maize root cells (Figure 2). In contrast, after the “long-distance” translocation of positively and negatively charged MWCNTs, some were dispersed in the cell wall and the cytoplasm and around the cell membrane. Less charge selectivity was observed in maize stems and leaves for neutral p-MWCNTs (Figure 5 and Figure S5, Supporting Information). The charge selectivity of plant cells for MWCNTs was plantdependent. Accumulation of MWCNTs in various cells and parts of the plant showed a different pattern in soybean compared to maize. In soybean, a small number of p-MWCNTs and MWCNT-NH2s preferentially aggregated in the roots (Figure 6). However, there were very few MWCNT-COOHs detected by TEM in roots, presumably due to repulsion with the negative surface charge of roots (images not shown). In addition, few nantotubes of any of the three types, p-MWCNT, MWCNT-NH2s, and MWCNT-COOHs, were found in the stems of soybean, suggesting they rapidly were translocated through the soybean stem to the leaves, and not apparently retained by the cellular tissues (images not shown). However, all the nanotubes, p-MWCNT, MWCNT-NH 2 s, and MWCNT-COOHs, showed a clear charge selectivity in
around the cell membranes and were subsequently sorbed onto the cell membranes. Parenchyma cells, specialized for food storage including starch, protein, and fats, are influenced by transport from the leaves to the roots. The necessity of MWCNTs first being translocated up in the xylem and then gradually down by the phloem resulted in the observation that there were no pMWCNTs in the root parenchyma cells (none detected by TEM imaging). These results also suggest that there was no direct lateral transport between xylem, parenchyma cells and phloem. Maize xylem cells showed a clear selectivity for uptake and translocation of MWCNT-NH2s and MWCNT-COOHs (Figures 3 and 4). MWCNT-NH2s were only found around the cell wall in cell No.1 among all the cells observed in the maize root (Figure 3), while MWCNT-COOHs were detected in specific maize root xylem cells (Figure 4). However, pMWCNTs, MWCNT-NH2s and MWCNT-COOHs exhibited less cellular selectivity in the maize stems and leaves than for roots, and they seemed to be dispersed randomly in the cytoplasm, cell membrane, and cell wall of some root cells (Figure 5 and Figure S5, Supporting Information). The uptake and translocation of p-MWCNTs, MWCNTNH2s and MWCNT-COOHs in soybean also showed a cellular selectivity on the part of the plant in which only special (minor) cells contained the nanotubes, and we did not detect any carbon nanotubes in the vast majority of cells (Figures 6−8 and Figure S6, Supporting Information). Therefore, these images suggest that certain maize and soybean cells demonstrate the selection of MWCNTs during their uptake and translocation. Charge Selectivity of MWCNTs in Maize and Soybean. The uptake and translocation of MWCNTs were greatly influenced by their charges because differently charged MWCNTs possess different surface properties and capacity for aggregation. On the other hand, cells also have a certain preference for charged particles due to their negatively charged cell membrane. For example, neutral p-MWCNTs could not be well dispersed in water in our experiments. However, positively charged MWCNT-NH2s and negatively charged MWCNTCOOHs were well dispersed in water and remained that way G
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Figure 7. (A) Image of MWCNT-NH2s in the soybean leaf; (B1−H1) magnification of the highlighted areas in panel A; (B2, C2, D2−5, E2−3, F2− 3, G2−4, and H2−4) magnification of panels B1−H1; (CW) cell wall; (MC) mesophyll cell; (PL) plasmodesma; and (SC) sclerenchyma cell.
soybean leaves (Figures 7 and 8 and Figure S6, Supporting Information). Positively charged MWCNT-NH2 was found to be deposited in a group of sclerenchyma cells (Figure 7). Sclerenchyma cells are supporting tissue with different shapes in the two plants. These cells have thick cell walls composed of cellulose, lignin, and hemicellulose. Two types of sclerenchyma cells exist: fibers (Figure 7D1, E1, G1, and H1) and sclereids (Figure 7B1 and F1). In fibers, MWCNT-NH2s tended to be attached to the fibrous stuff inside the soybean cell; and in sclereids, MWCNTNH2s formed a cluster attached to the soybean cell membrane, presumably because of the positive surface charge properties of MWCNT-NH2s. The plasmodesmata are likely channels for MWCNT-NH2s to pass through the thick soybean sclerenchyma cell wall (Figure 7D2 and F3). In addition, MWCNT-NH2s
were also found in the mesophyll cells of soybean leaves (Figure 7C1). On the other hand, neutral p-MWCNTs and negatively charged MWCNT-COOHs were observed in sporadic distributions in soybean leaves (Figure 8 and Figure S6, Supporting Information), suggesting that neutral p-MWCNTs and negatively charged MWCNT-COOHs were less mobile than positively charged MWCNT-NH2s from soybean roots to leaves. To our knowledge, there have been no previous reports regarding the surface-charge selectivity of plants for MWCNTs. However, charge selectivity of other nanomaterials has been previously reported in Caco-2 cells32 and whole plants.22,33 Bannunah et al. found that both positively and negatively charged blue aminated latex nanoparticles (50 nm) and orange H
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Figure 8. (A) Image of MWCNT-COOHs in soybean leaf; (B and C) magnification of the highlighted rectangle areas in panel A; (D) image of MWCNT-COOHs in soybean leaf; (E and F) magnification of the highlighted rectangle area in panel D.
demonstrate that maize and soybean roots took up a wide range of sizes of p-MWCNT, MWCNT-NH2s, and MWCNTCOOHs in roots, but only a narrow range (50−100 nm) of MWCNTs could be translocated to the leaves via the stems. The size selectivity of MWCNTs by whole plants has not been reported so far, but the size-based selection of other nanomaterials has been frequently reported in plants,19,35 and our results are consistent with previous studies. Serag et al. observed that short p-MWCNTs (
