Article

pubs.acs.org/est

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 two-
photon 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.

■ INTRODUCTION
The uptake of organic compounds by plant is of importance for
their regional/global cycling and in their ultimate environ-
mental 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
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: November 8, 2013
Revised: March 23, 2014
Accepted: March 30, 2014
Published: March 31, 2014

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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 pollu-
tants29−34 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.
■
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. Air-
dried 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
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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. Three-
dimensional 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. Integra-
tion of the fluorescence intensity was used to quantify the
content of PHE on each scanning layer. Because the maximum
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pixel value was never larger than 255, the saturated fluorescence
was not detected in any of the experiments.
■ 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 12.46 32.64 19.70 35.21
abaxial surface 19.50 21.43 20.50 38.94
a available in the SI.
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.
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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
To further investigate the relative distribution of lipid-
components 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
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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).
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 strat-
ification 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
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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
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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
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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
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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.
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
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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 liquid-
like 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 poly-
saccharides 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
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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 hydro-
phobic 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.
■
ASSOCIATED CONTENT
* Supporting Information
S biophysical design of plant cuticles: An overview. New Phytol. 2011,
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.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 0086-571-88982587; fax: 0086-571-88982587; e-mail:
blchen@zju.edu.cn.
Notes
The authors declare no competing financial interest.
■
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.
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