Organic Pollutant Clustered in the Plant Cuticular Membranes

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Organic Pollutant Clustered in the Plant Cuticular Membranes: Visualizing the Distribution of Phenanthrene in Leaf Cuticle Using Two-Photon Confocal Scanning Laser Microscopy Qingqing Li†,‡ and Baoliang Chen†,‡,* †

Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Zhejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Plants play a key role in the transport and fate of organic pollutants. Cuticles on plant surfaces represent the first resistance for the uptake of airborne toxicants. In this study, a confocal scanning microscope enhanced with a twophoton laser was applied as a direct and noninvasive probe to explore the in situ uptake of a model pollutant, phenanthrene (PHE), into the cuticular membrane of a hypostomatic plant, Photinia serrulata. On the leaf cuticle surfaces, PHE forms clusters instead of being evenly distributed. The PHE distribution was quantified by the PHE fluorescence intensity. When PHE concentrations in water varying over 5 orders of magnitude were applied to the isolated cuticle, the accumulated PHE level by the cuticle was not vastly different, whether PHE was applied to the outer or inner side of the cuticle. Notably, PHE was found to diffuse via a channel-like pathway into the middle layer of the cuticle matrix, where it was identified to be composed of polymeric lipids. The strong affinity of PHE for polymeric lipids is a major contributor of the fugacity gradient driving the diffusive uptake of PHE in the cuticular membrane. Membrane lipids constitute important domains for hydrophobic interaction with pollutants, determining significant differentials of fugacities within the membrane microsystem. These, under unsteady conditions, contribute to enhance net transport and clustering along the z dimension. Moreover, the liquid-like state of polymeric lipids may promote mobility by enhancing the diffusion rate. The proposed “diffusive uptake and storage” function of polymeric lipids within the membrane characterizes the modality of accumulation of the hydrophobic contaminant at the interface between the plant and the environment. Assessing the capacity of fugacity of these constituents in detail will bring about knowledge of contaminant fate in superior plants with a higher level of accuracy.

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INTRODUCTION The uptake of organic compounds by plant is of importance for their regional/global cycling and in their ultimate environmental fate.1−3 Organic pollutants emitted into the air are eventually transferred to and transformed in the terrestrial ecosystem.4−7 The plant cuticle is considered to be the main interface for the exchange of organic compounds between air and vegetation and the first barrier to prevent chemical uptake by plant tissues.1,2,8−10 Aerial parts of vascular plants are covered by this continuous extra-cellular membrane, which is a mixture of waxes, polymeric lipids, and polysaccharides.10−12 Due to its elaborate micro- and nanostructures, hydrophobic composition and interconnection with the inner part of the plant, the cuticle performs multiple functions essential to plant life and serves as a plant−air interface crucial for biochemical processes.13,14 Among the various substances that reach the cuticle, airborne organic pollutants are of concerns because of their strong affinity to the cuticle and potential toxicity.15,16 © 2014 American Chemical Society

Numerous studies have been conducted in order to explore the interaction between organic pollutants and the plant cuticle.1,17−20 Through batch sorption experiments, the plant cuticle shows an overwhelming capacity in organic compound accumulation, although the lipophilicities of its components vary.21−23 Chen et al.21 found that depolymerized lipids (cutin) are the major reservoir of organic pollutants, while the extractable lipids (wax) act as an antiplasticizer by suppressing the uptake by cutin, and the polysaccharides serve as a plasticizer to regulate the sorption properties of the polymeric lipids (cutin and cutan). Cuticle components have been isolated with enzymes and chemical solvents to investigate the sorption capacities of Received: Revised: Accepted: Published: 4774

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method.21 Elemental (C, H, N) analysis was conducted via an EA 112 CHN elemental analyzer (Thermo Finnigan). The oxygen contents were determined based on mass balance. Detailed contents of the cuticle elements are available in the Supporting Information (SI). The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of the outer and inner surfaces of the adaxial and abaxial cuticular membranes were recorded in the 4000−650 cm−1 on a Nicolet 6700 FTIR spectrometer (Thermo Scientific, U.S.A.) equipped with an attenuated total reflectance (ATR) accessory, iS10 Smart iTR, which allows samples to be examined directly in the solid or liquid state without further preparation. Resolution was set 1.0 cm−1. Phase transitions of adaxial and abaxial cuticles were measured by a modulated differential scanning calorimeter (MDSC Q100, TA Instruments) under an ultrahigh-purity nitrogen atmosphere. Surface morphology was examined with a field emission scanning electron microscope (FE-SEM, S-4800; Hitachi, Tokyo, Japan) operated at an accelerating voltage of 3 kV. The specimens for SEM examination were prepared following a reported method after modification,37 available in the SI. Staining and Contamination Procedures for Leaf Cuticle. Auramine O is a commonly used lipophilic fluorescent dye, which has distinct affinities to different cuticle components. Cuticles were stained with auramine O (Sigma; 0.01% w/v in 0.05 M Tris/HCl, pH 7.2) for 15 min and rinsed with distilled water. Slides were mounted with the outer side upward in distilled water with a coverslip and viewed quickly (sample preparing time less than 1 h). Phenanthrene contamination was conducted by adding 3 mL of PHE solution to the Petri dishes (6 cm in diameter) to fully cover the dish surfaces using the following concentrations: 80, 8, 0.8, 0.08, and 0.0008 μg/L. Round cuticle slices 6 mm in diameter were quickly tiled on the PHE solution surfaces with the outer side in contact with the solution. A dish containing 0.8 μg/L PHE solution was used to conduct the reversal sorption with the inner surface contacting the PHE solution instead of the outer surface. The Petri dishes were all sealed with plastic films. The contamination lasted 12 h at 25 °C, and then the cuticles were removed and rinsed with distilled water to remove unabsorbed PHE. Slides were mounted using the same method used for the stained cuticles and viewed quickly. Two-Photon Laser Confocal Scanning Microscopy (TPLCSM). The cuticle slides were analyzed under a Zeiss LSM 710 NLO confocal laser scanning microscope with a LD LCI Plan-Apochromat 25 × /0.8 DIC water immersion objective and a Plan-Apochromat 63 × /1.4 DIC oil immersion objective (Zeiss) for auramine O and PHE imaging, respectively. Auramine O was excited at 430 nm and detected at 500 nm to obtain laser confocal scanning microscopy (LCSM) micrographs. PHE was imaged by excitation of two photons at 700 nm and emitted fluorescence was detected at 435 ± 15 nm to obtain TPLCSM micrographs. Threedimensional images of reconstructed cuticles and PHE distribution were acquired by z-stack projections with 0.35 and 1 μm z-resolution, respectively. The mean gray value (pixel per unit area) of each scanned layer was recorded by an image processing software ImageJ (version 1.46) without extra processing and represented the fluorescence intensity. Integration of the fluorescence intensity was used to quantify the content of PHE on each scanning layer. Because the maximum

the components in vitro. However, these methods are unable to detect the uptake processes involved with micro- and nanostructures of cuticles when the components function integrally. Moreover, there is no evidence that the isolated cuticle components display the same properties as those before isolation. Thus, the distribution and transport of organic chemicals on intact plant cuticular membranes should be investigated. Furthermore, the abundant microstructures of leaf cuticle could result in adaxial and abaxial cuticular topographies that are distinct from each other.24,25 This variability could result in different organic pollutant uptake behaviors by adaxial and abaxial cuticles. Currently, little attention has been given to the differences in the adaxial and abaxial cuticles in the literature. In recent years, the ability to detect target chemicals in cells, tissues, and organisms via their excited fluorescence or that of their combined biomarkers has greatly increased knowledge on the interaction between organisms and xenobiotic chemicals. This approach makes it possible to accurately track chemicals such as gene therapy drugs26−28 and environmental pollutants29−34 in biological organisms. Furthermore, it may be able to shed some light on the complex fate of various contaminants within biological tissues. Two-photon excitation microscopy was first utilized to accomplish such a goal by Wild et al.,29 who tracked trace organic contaminants in living plant tissues to make real-time observations.33−35 The targets were extended to nanomaterials30 and, more recently, mycelia.32 In previous studies, a highly focused streaming phenomenon was observed, resulting in a distribution of organic pollutants, forming clusters and stripes both in the root and leaf cuticles. The interpretation between the uneven distribution of organic pollutants and the structural deposition of polymeric lipids,23 however, requires more direct evidence and further confirmation. The main objective of the current study is to explore the mechanisms of organic pollutant uptake into plant cuticles and to probe differences in organic pollutant uptake among cuticle components in situ. Two-photon laser confocal scanning microscopy (TPLCSM) was employed to observe the penetration and distribution of an excitable fluorescent pollutant, phenanthrene (PHE), in the adaxial and abaxial cuticles of a hypostomatic plant, Photinia serrulata.

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EXPERIMENTAL SECTION Plant Cuticle Preparation and Characterization. Mature leaves of Photinia serrulata sampled from the Zijingang campus, Zhejiang University, China, were collected and washed with distilled water to remove the dust on the surface. Cuticles were detached using the method described in an earlier report,36 although several modifications were made, including lowering the incubation temperature and cutting the reaction time appropriately to maintain the integrity of the cuticle. Airdried leaves were weighed and put into a 1:1 mixture of 30% H2O2 and CH3COOH under a 60 °C water bath until the color of the leaves faded and became transparent (8−12 h). After being washed with distilled water, using thin-tipped tweezers, leaves were torn along the margins and mesophylls were smoothly removed using a soft brush. The remaining tissues were washed away, and the surface water was carefully blotted. Then the air-dried adaxial and abaxial cuticles were preserved in pairs in clean envelopes. The contents of the cuticle components (waxes, polymeric lipids, polysaccharides, and cutan) were determined by isolating the cuticular fractions via a modification of an earlier reported 4775

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and inner sides. The bands at 2917, 2850, 1460, and 1370 cm−1 are assigned mainly to CH2 units of aliphatic components (wax, cutin, and cutan).21,38,39 The peaks at 1730 and 1169 cm−1 are assigned to CO and C−O stretching vibrations of ester bonds of cutin, respectively. The bands at 1018 and 1047 cm−1 are assigned to aliphatic C−O−C, which represents the oxygenated functional groups of polysaccharides.40 For both adaxial and abaxial cuticular membranes (Figure 1C), the band intensities for aliphatic CH2 and ester CO were stronger for the outer surfaces (ad-o and ab-o) than the respective inner surfaces (ad-i and ab-i), while the band intensities of the polysaccharide components were identical for the outer side and the inner side of a given cuticular membrane (adaxial cuticle or abaxial cuticle). Detailed analysis of IR spectra is available in the SI. To further investigate the relative distribution of lipidcomponents and polysaccharides, the ATR-FTIR results were semiquantified by calculating the relative vibration intensities of aliphatic functional groups/polysaccharides. The ratios were applied to characterize the relative contents of the aliphatic components and polysaccharides on the cuticle surfaces and are presented in SI Table S-2. The ratios for the outer side of the cuticles are observably higher than those of the inner side ones. Take νas(−CH2)/ν(polysaccharides) for example, the ratio for the ad-o and ab-o surfaces are 1.00 and 0.83, respectively, which are considerably higher than the ratio of the ad-i (0.20) and the ab-i surfaces (0.38). This confirms that the aliphatic components are generally piled on the outer side of the leaf cuticles, while polysaccharides stick to the epidermal cell wall on the inner side of the cuticles. Stratification of Plant Cuticle. Imaging of leaf cuticles resulted in two- and three-dimensional micrographs. SEM was applied to observe the surface morphology of the plant cuticles (Figure 2). The adaxial cuticle surface was slightly wrinkled (Figure 2A,C), and the higher magnification revealed tiny wax flakes on the rough cuticle surface (Figure 2E). Protruding stomata are irregularly spread on the abaxial side, and the surface is significantly undulate (Figure 2B,D). Scattered

pixel value was never larger than 255, the saturated fluorescence was not detected in any of the experiments.

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RESULTS AND DISCUSSION Characterization of the Detached Cuticle. The distinct mass fractions of the cuticle components (i.e., wax, cutin, cutan, and polysaccharide) on the adaxial and abaxial surfaces are displayed in Table 1. The amount of wax in the adaxial cuticle Table 1. Percentages of Cuticular Components of Leaf Adaxial and Abaxial Surfaces of Photinia serrulata. cuticle

waxes%a

cutin%a

cutan%b

polysaccharides%a

adaxial surface abaxial surface

12.46 19.50

32.64 21.43

19.70 20.50

35.21 38.94

a

Cuticular fractions were isolated from the leaf cuticle by an earlier reported method.21 Contents of waxes, polysaccharide, and cutin were calculated based on mass balance. bContent of cutan was calculated by the mass difference.

(12.46%) was less than that in the abaxial cuticle (19.50%), and the polysaccharide content in the adaxial cuticle (35.21%) was also less than that in the abaxial cuticle (38.94%). Meanwhile, the amount of cutin was observably larger in the adaxial cuticle (32.64%) than in the abaxial cuticle (21.43%). The content of cutan in the adaxial and abaxial cuticles was almost the same. Correspondingly, the ratio of polymeric lipids (cutin and cutan) to polysaccharides was relatively high for the adaxial cuticle (1.49) in comparison with that for the abaxial cuticle (1.08). The distinct chemical composition leads to different elemental characteristics of the adaxial and abaxial cuticles (see SI Table S-1). The polarity index (O + N)/C indicates that more polar components are present in the abaxial cuticle. A pair of relatively intact cuticle membranes (Figure 1B) separated from the original leaflet (Figure 1A) was prepared. The selected ATR-FTIR spectra of the four surfaces of the leaf cuticle were initially presented (Figure 1C) to aid in understanding the differences in the functional group distribution of the adaxial and abaxial cuticles on the outer

Figure 1. Photographs of the adaxial and abaxial surfaces of a Photinia serrulata leaf (A) showing that the cuticle slide was detached from them (B), and selected ATR-FTIR spectra of the adaxial and abaxial cuticles on the outer and the inner side (C). The symbols of ad-o, ad-i, ab-o, and ab-i are abbreviations of adaxial-outer, adaxial-inner, abaxial-outer, and abaxial-inner, respectively. 4776

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Figure 2. SEM micrographs of plant surface morphology of Photinia serrulata on the adaxial (A,C,E) and abaxial cuticle (B, D, F) at different magnifications of 200× (A, B), 500× (C, D), and 3000× (E, F).

abaxial cuticle, regions around the stomata strongly fluoresce, which may be due to the protruding and pore-like topography of the stomata that could be easily stained. Visualizing PHE Distribution in Leaf Cuticle by TPLCSM. Three-dimensional distributions of PHE using serial concentrations were demonstrated in SI Figure S-1. The fluorescence intensity as an indication of PHE concentrations at various depths of leaf cuticles by TPLCSM is shown in Figure 4. Different colored dots represent the different concentrations of PHE applied to the cuticles. Distribution of 0.8 μg/L PHE in Photinia serrulata on their x−y, x−z, and y−z planes by MIP were presented as insets and magnification of projections along the z-depth were also displayed in Figure 4 (C−F). Unexpectedly, there is no regular pattern detected related to the concentrations, even though the concentration range covered 5 orders of magnitude. It seems that the difference in pollutant concentration does not drive accumulation, as neither the highest fluorescence intensities in the adaxial cuticles nor in the abaxial cuticles correlate to the highest concentration. The lack of a relationship between the level of PHE in the membrane and the exposure levels suggest that the uptake experiment was suspended long before the membrane constituents reached an equilibrium. Therefore, the observed distribution of PHE along the z-depth is not simply the result of the contaminant diffusing out of the membrane after exposure was interrupted to allow microscopy analysis. As illustrated, there is an obvious accumulation of PHE at the

minuscule wax fragments were also clearly observed at higher magnification (Figure 2F). In order to reconstruct the three-dimensional matrix of the cuticles and to differentiate the associated components, auramine O, a lipophilic fluorescent dye, having different affinities for the various cuticle components41,42 was added. The 3D structures of the cuticles are presented in Figure 3. With the topography of the cuticle surfaces clearly illustrated, a contrast in fluorescence intensity was observed in both the adaxial and abaxial cuticles. The middle layer of the cuticle architecture strongly fluoresces, while the side regions look relatively dim. To amplify this difference, the color “green_sat” was subjected to the maximum intensity projection (MIP, Figure 3), resulting in sharply contrasting fluorescence intensity to show a sandwich-like vertical distribution, presented in Figure 3(E−H). Considering both the characteristics of the cuticle components measured by ATR-FTIR and well-defined stratification of cuticle components by auramine O, the two dimer layers could be reasonably defined as waxes and polysaccharides and the strong fluorescent region as polymeric lipids. The distribution agrees with the hypothetical stratification of the plant cuticle, which is widely accepted.43,44 Some variations between the adaxial and abaxial cuticles are also detected. In the adaxial cuticle, auramine O favored the polygonal boundaries, which are actually cuticular pegs that fill in the intercellular spaces during biosynthesis of cuticles. In the 4777

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Figure 4. PHE uptake in the adaxial (A) and abaxial (B) cuticle of Photinia serrulata at various depths, where the mean gray value is an indicator for PHE fluorescence intensity. Cuticles contaminated with 80 μg/L PHE solution (a, b), with 8 μg/L PHE solution (c, d), with 0.8 μg/L PHE solution (e, f), with 0.08 μg/L PHE solution (g, h), and with 0.0008 μg/L PHE solution (i, j). Distribution of 0.8 μg/L PHE in the plant cuticles of Photinia serrulata on their x−y, x−z (up), and y−z (right) planes by maximum intensity projection were presented as the insets of parts A and B. The magnifications of x−z and y−z planes of MIP images were presented in parts C and D for the adaxial cuticles and in E and F for the abaxial cuticles.

plants, as the pore structures in the abaxial cuticle surface would offer a rapid access of PHE to the deeper cuticle parts.45 Nevertheless, according to the current data, organic pollutants were able to penetrate and accumulate in the adaxial cuticle to result in a higher concentration even without such topography. Hence, it is apparent that accessible topography is not decisive for the pollutant accumulation; instead, the chemical affinity is the real control factor. This is supported by the decreased (O + N)/C ratio in the adaxial cuticle compared to that in the abaxial one, as the more lipophilic adaxial cuticle has the higher affinity to the organic pollutants. Moreover, it was reported earlier that cutin is the main reservoir of organic pollutants,21 and consistently, the mass fractions of the cuticle components in the present study showed that the adaxial cuticle had a higher cutin content (32.64%), and the accumulation of PHE increased accordingly. Three-dimensional distributions using one concentration of the series, 0.8 μg/L, is shown in Figure 5. Different distributions in three-dimensional spaces are displayed. On the adaxial cuticle, the irregular polygonal penetration pathway is displayed. PHE distributed unevenly and penetrated deeper along the anticlinal pegs. On the abaxial cuticle, PHE accumulation was highlighted in the stomata and around the guard cells that are considered to be active spots for substance exchange, attributed to their very approachable topography. The phenomenon was also consistent with an earlier investigation.45 The channel-like penetration pathway of PHE

Figure 3. LCSM micrographs of reconstructed adaxial (A) and abaxial (B) cuticles of Photinia serrulata stained by auramine O and their x−y, x−z (up), and y−z (right) planes by maximum intensity projection (C and D). Parts E, F, G, and H were magnifications of MIP images. Scale bars of A and B were not labeled, but the scale sizes can be informed by ones given in C and D, as they were projections of A and B.

depth between 8 to 13 μm in the adaxial cuticle and 9 to 16 μm in the abaxial cuticle. According to the projected images in Figure 4(C−F), the strong accumulation of PHE in the middle layer of the cuticles was compatible with the distribution of lipophilic matrix (cutin) along z-depth as presented in Figure 3(E−H). The maximal fluorescence intensities in the adaxial cuticles at each PHE concentration are higher than those in abaxial cuticles (Figure 4). On the basis of the results in Figure 4, the highest intensity value in the adaxial cuticle (126.01) is nearly twice as much as that in the abaxial cuticle (69.19). It was considered that stomata contribute significantly in the uptake of chemicals by 4778

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Figure 5. TPLCSM-3D distribution and fluorescence intensities of phenanthrene in adaxial (A, A′) and abaxial (B, B′) cuticles of Photinia serrulata by forward (A, B) and reverse (A′, B′) penetration, and the fluorescence intensities on the cuticle layers.

Figure 6. MDSC heating curves of adaxial (A) and abaxial (B) cuticles of Photinia serrulata at a heating rate of 20 °C min−1.

little ability to impede the transport of PHE into the middle compartment. It is clear that the driving force controlling the transport of PHE into and through the membrane is the gradient of PHE fugacity, and transport of the hydrophobic contaminant is only due to molecular diffusion through the cuticular membrane. In this context, lipids in the membrane simply have an important contribution in the build up capacity of fugacity and speeding molecular diffusion. The uptake of organic chemicals by cuticle components is a multistage dynamic process. Not only does the chemical nature of the cuticle components matter, but also their physical state, which can be very different across the various constituents. To get insights into the uptake process, the MDSC measurements of the adaxial and abaxial cuticles were conducted; the results are shown in Figure 6. Two distinct transitions are illustrated, i.e., the glass transition of cutin polyester at −21.82 °C for the adaxial cuticle and −18.17 °C for the abaxial cuticle, and the melting of waxes at 47.05 °C for the adaxial cuticle and 48.05 °C for the abaxial cuticle. The MDSC results for the abaxial cuticle were similar to the adaxial ones. According to the MDSC results, waxes were in an amorphous solid state and polymerized lipids were in a liquidlike viscous state at room temperature when the contamination experiment was conducted. It is reported that polysaccharides contribute to the stiffness of the cuticle47 and would change phase above room temperature. This suggests that polysaccharides were in the solid state at room temperature. Hence in this microinterface process, organic pollutants may not only encounter a solid resistance (waxes) when they diffuse through

in the cuticular membrane was consistent with the reports in several earlier studies where it was observed that the organic pollutant was highly concentrated and formed streams longitudinally toward the shoot and root.33 Another similar phenomenon detected in leaf cuticles was that PHE accumulated in clusters and stripes and the visible transport pathway formed irregular polygons.35 The laterally uneven distribution was attributed to the varied topography and microstructures on the cuticle surfaces, and the longitudinal uneven distribution could be ascribed to the distinct chemical composition. Similarly, Wild et al. initially observed that polycyclic aromatic hydrocarbons, which were uniformly distributed in pure oil and waxes at the beginning of the experiment, formed clusters over time.46 Organic pollutants exchange dynamically on the interface between cuticle and the ambient environment, and the behavior seasonally affects the regional air pollution level.7,17 To further investigate the penetration reversibility of PHE, reverse contamination from the inner to outer cuticle side at the concentration of 0.8 μg/L was presented alongside the forward contamination results in Figure 5. The maximum fluorescence intensities of the forward penetrations (80.34 and 66.41 for adaxial and abaxial cuticle, respectively) were found to be similar to those of the reverse penetrations (89.93 vs 69.19 for adaxial vs abaxial cuticle). Combined with the above-mentioned stratification of cuticle components and their different affinities to organic chemicals, the results suggest that PHE was diffused in and was retained by polymeric lipids in the middle layer of the cuticle, while the soluble lipids and polysaccharides had 4779

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the cuticle and into the plant, but also a diffusion enhancing viscous matrix (polymeric lipids) layered or imbedded in the cuticles enhances transmittance and storage capacity. When a material is at a temperature above its glass transition temperature, various properties are changed, and one of the most important changes is the exponential increase of molecular mobility.48 On the basis of this reasoning, the diffusion of organic pollutants from waxes to polymeric lipids is supposed to be more efficient as the liquid-like polymeric lipids was in a semisolid phase, whereas the diffusion from polymeric lipids to polysaccharides was likely dynamically limited. At environmentally relevant temperatures, diffusivity of a hydrophobic pollutant is expected to be significantly higher in the viscous quasi-liquid matrix of polymeric lipids than in the solid waxes. The role of the heterogeneous transport media and their affinity with the hydrophobic chemicals should be considered when modeling diffusion transport through plant cuticles. Present results provide insights that may be useful for detailing the uptake of contaminants in plants49,50 with a higher level of accuracy. In summary, the clustering of PHE inside plant cuticle was investigated in relation to the distribution of cuticle components using an in situ method. Polymeric lipids served as preferential diffusion pathways for uptake and transfer of organic pollutants. The detailed information presented here may be useful to improve current understanding and model of uptake of hydrophobic contaminants by plants. To extend the insights gained into the uptake and within-leaf behavior of semivolatile organics, other volatile or semivolatile organics with differing physio-chemical properties should be considered in future investigations.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

Sample preparation procedure for SEM measurement and ATR-FTIR analysis; elemental analysis and atomic ratios of cuticles (Table S-1); relative contents of selected aliphatic functional groups/polysaccharides; (Table S-2); TPLCSM-3D of the distribution of PHE of serial concentrations in cuticles (Figure S-1); and the sorption kinetic curves of sieved adaxial and abaxial cuticles (Figure S-2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0086-571-88982587; fax: 0086-571-88982587; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Basic Research Program of China (2014CB441106), the National Natural Science Foundation of China (Grant Nos. 21277120, 41071210, and 20977081), and the Doctoral Fund of Ministry of Education China (Grant No. J20130039). We would like to express our sincere gratitude to the reviewers of the paper for their valuable comments and constructive suggestions, which greatly contributed toward improving the quality of the paper. 4780

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dx.doi.org/10.1021/es404976c | Environ. Sci. Technol. 2014, 48, 4774−4781