Journal of Colloid and Interface Science 271 (2004) 507510

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Letter to the Editor
Cutin and suberin monomers are membrane perturbants
Jean-Paul Douliez
Unit de Recherche sur les Protines Vgtales et leurs Interactions, INRA, rue de la Graudire, 44316 Nantes, France
Received 11 August 2003; accepted 10 December 2003
Abstract
The interaction between cutin and suberin monomers, i.e., -hydroxylpalmitic acid, , -hexadecanedioic acid, , -hexadecanediol, 12-
hydroxylstearic acid, and phospholipid vesicles biomimicking the lipid structure of plant cell membranes has been studied by optical and
transmission electron microscopy, quasielastic light scattering, differential scanning calorimetry, and
31
P solid-state NMR. Monomers were
shown to penetrate model membranes until a molar ratio of 30%, modulating their gel to uid-phase transition, after which monomer crystals
also formed in solution. These monomers induced a decrease of the phospholipid vesicle size from several micrometers to about 300 nm.
The biological implications of these ndings are discussed.
2004 Elsevier Inc. All rights reserved.
Keywords: DMPC membranes; Bola amphiphiles; Vesicles; Fatty acids; Cutin; Suberin
1. Introduction
Cutin and suberin are plant polymers consisting of ester-
ied hydroxyl and dicarboxylic fatty acids with 16 and 18
atoms of carbon [13]. They are localized at the surface of
organs and their surface properties are of particular impor-
tance [4]. Whereas the biosynthesis of the monomers has
been the focus of numerous studies [5], their routing path-
way in the plant from the cell where they are synthesized
to the external layers where they are polymerized is still
unclear. The participation of lipid colloids, i.e., monomer
corpuscles, has been formulated [1]. However, the cutin and
suberin monomers are poorly water soluble, suggesting the
participation of proteins or other lipids as cosolubilizing
agents.
Another important question is related to the potential ac-
tivity of cutin monomers as messengers in plantpathogen
interactions [1]. Upon pathogen attack, cutin is hydrolyzed,
releasing monomers which can be perceived by plant cells
for the induction of resistance [1,6]. How this process occurs
is still an enigma but monomers could induce strong pertur-
bations in the membrane of the polymer juxtaposing cells,
potentially modulating its functions and that of membrane
proteins.
E-mail address: douliez@nantes.inra.fr.
Several cutin and suberin monomers as -hydroxylhexa-
decanoic acid (-OHplm) , -hexadecanedioic acid (di-
COplm), , -hexadecanediol (diOHplm), and 12-hydroxy-
stearic acid (12OHSt) are commercial and were used as
models of cutin and suberin monomers in this study. They
were mixed with dimyristoyl phosphatidylcholine (DMPC),
a phospholipid forming membrane vesicles as a synthetic
simplied membrane model of bilayers, i.e., a biomimetic
system of cell membranes. Optical microscopy was used to
observe the texture of the lipid mixtures, light scattering and
transmission electron microscopy for determining the vesi-
cle size, and DSC and
31
P solid-state NMR to follow the
thermotropism and phase behavior of the mixtures.
2. Materials and methods
2.1. Lipids
All lipids used in this study were from Sigma. Because
cutin and suberin monomers are not water soluble, their
interaction with DMPC cannot be probed on preformed li-
posomes of this phospholipid and previous cosolubiliza-
tion in a solvent is required as in the case of mixtures of
phospholipids and cholesterol [7]. As a consequence, the
vesicle-size distribution is polydisperse and is driven by
the monomer/phospholipid interactions. A stock solution
of DMPC was then prepared in methanol, vortexed under
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doi:10.1016/j.jcis.2003.12.020
508 J.-P. Douliez / Journal of Colloid and Interface Science 271 (2004) 507510
low-power ultrasonic conditions, and further aliquoted at
10 mg/ml in glass tubes. Monomers were previously sol-
ubilized in methanol and added with the phospholipid. All
samples were dried with a stream of nitrogen, hydrated with
pure water, freeze-thawed three times, and then freeze-dried.
This allowed removal of any trace of methanol. The lipid dis-
persions were further realized with 1 ml (full hydration) of
a 30 mM TrisHCl, pH 7.5, buffer. Samples were vortexed,
freeze-thawed three times, and conserved at 20

C. Prior
to use, each sample was heated to 50

C for 30 min.
2.2. Solid-state NMR
31
P solid-state NMR experiments were performed at sev-
eral temperatures on a 400 MHz Bruker spectrometer using
a static probe, the sample coil of which was adapted to load
a 7-mm rotor such as those used with magic-angle-spinning
probes. Typically, 350 l of lipid dispersion was poured in
the rotor. A Hahn echo sequence was used with an inter-
pulse delay of 30 s, and 1k points in 12k accumulations
were done with a 90

pulse and a spectral width of 7 s

and 100 kHz, respectively. Free induction decay signals were
zero-lled to 4k points prior to Fourier transform after a
broad line exponential multiplication of 100 Hz.
2.3. Differential scanning calorimetry
Experiments were performed on a microDSCIII fromSe-
taram (Caluire, France). The amount of 0.85 ml of the lipid
solution (10 mg/ml) was accurately weighed in a Hastelloy
C276 vessel. The sample was scanned between 1 and 60

C
upon two successive heating and cooling cycles at 1

C/min.
The DSC traces from the second heating steps were used.
2.4. Light microscopy
Observations were made at 20 magnication using
an optical microscope in the phase-contrast mode (Nikon
Eclipse E-400, Tokyo, Japan) equipped with a 3-CCD JVC
camera allowing digital images (768 512 pixels) to be
collected. A drop of the lipid dispersion (about 50 l) was
deposited on the glass-slide surface (76 26 1.1 mm,
RS France) and covered with a cover slide (22 22 mm,
Menzel-Glaser, Germany). With such a lipid volume, the
dispersion was not conned between glass plates, prevent-
ing the formation of tubules or giant vesicles [8]. The cover
slides were cleaned with ethanol and acetone.
2.5. Transmission electron microscopy (TEM)
Samples were deposited on copper grids and further
stained with either uranyl acetate or phosphotungstic acid.
This procedure is widely used in the literature and is not
expected to affect the results, i.e., the vesicle size. Prepa-
rations were examined with a transmission electron micro-
scope JEOL 100S operating at 80 keV. Observations were
made at various magnications.
2.6. Light scattering
Quasielastic light-scattering experiments were performed
with an HPPS instrument from Malvern (UK) allowing
particle-size determination from 0.6 nm to 6 m in turbid
medium. The scattered beam at 173

was analyzed and the

correlation function treated by CONTIN software. Scatter-
ing intensities are reported as a function of particle size.
Samples were diluted 10 times.
3. Results and discussion
The pure cutin and suberin monomers used in this study
are not water soluble at neutral pH and remained dispersed
as crystals. Phase-contrast microscopy and visual inspec-
tion (not shown) were used to investigate their solubility
in DMPC membranes at four different molar ratios, fatty
acids/DMPC of R
i
= 0.01, 0.1, 0.5, and 1. As commented
above, the interaction between monomers with DMPC can-
not be probed on well-dened preformed phospholipid vesi-
cles. As a consequence, polydisperse vesicles were observed
in the case of pure DMPC [8] as expected from the prepa-
ration method since no sizing down as extrusion or soni-
cation was used. At a molar ratio of 0.1, solutions became
less turbid and vesicles exhibited a lower diameter than for
R
i
= 0.01. At R
i
= 0.5 small vesicles were still present as
small dots in addition to crystals of about 10 m size. Then,
it can be estimated that DMPC incorporates about 30% mo-
lar cutin or suberin monomers prior to yield a two-phase sys-
tem in which crystals coexist. This behavior is similar to that
encountered in the case of cholesterol which also forms crys-
tals at ratios higher than 50%molar in model membranes [7].
Interestingly, monocarboxylic fatty acids have been shown
to incorporate in phospholipid membranes at a molar ratio
of 2 [9]. This reveals that the specic chemical structure of
the cutin and suberin monomers, that is, the presence of a
second carboxylic moiety or hydroxyl group, hampers the
cosolubilization in model membranes.
DSC was then used to monitor the effect of monomers
on the thermotropism of DMPC. This phospholipid when
pure exhibits a weak pretransition at 15

C from a gel phase

to a ripple phase and a main transition at 24

C to a uid
phase [10]. Increasing the amount of monomers induced a
shift toward higher temperatures, a broadening or a split-
ting into two overlapping peaks of the main transition, and
the disappearance of the pretransition (Fig. 1). First, this
suggests a possible phase separation, which is further sug-
gested from
31
P NMR results. Next, this clearly shows
that monomers are incorporated in the DMPC membranes,
modulating the interaction between the phospholipids and
their phase transition. This behavior is very similar to that
observed in the case of mixtures of DPPC and palmitic
acid [9] or cholesterol for which the main transition is
markedly broadened [7] or upon incorporation of linoleic
acid in DMPC membranes [11]. Moreover, a similar thermo-
J.-P. Douliez / Journal of Colloid and Interface Science 271 (2004) 507510 509
Fig. 1. DSC thermograms obtained upon incorporation of various amounts
of -OHplm in DMPC. The molar ratio is indicated on the left.
gram was observed in the case of sonicated vesicles, a pro-
cedure which is known to induce small unilamellar vesi-
cles [12], in agreement with the present observations made
by phase-contrast microscopy. Moreover, vesicle-shape tran-
sitions have been widely studied in lipid mixtures [13,14]
and may originate from lipid-phase segregation in the mem-
brane as in the case of raft formation [15]. Then, the present
results suggest that cutin and suberin monomers, especially
because of their molecular shape, induce phase segregation
in DMPC membranes, resulting in a marked modication of
the phase transitions and the spontaneous vesicle curvature.
Further investigations were done by
31
P solid-state NMR,
which is a powerful technique for monitoring the coexis-
tence of various phases in phospholipid mixtures. At a molar
ratio of 0.01,
31
P NMR produced a bilayer powder pattern in
coexistence with an isotropic line, the intensity of which in-
creased with the temperature and molar ratio as shown Fig. 2
in the case of -OHplm. The presence of superimposed
two components on the NMR spectra indicates the pres-
ence of a two-phase system in slow exchange at the NMR
time scale. The powder component can be undoubtedly as-
cribed to the large vesicles as observed by phase-contrast
microscopy whereas the isotropic line may occur for sev-
eral reasons: particular orientation of the phospholipid polar
head, local motions in the membrane, phospholipids embed-
ded in a cubic phase, and more probably, small size colloids
as previously suggested.
These mixtures were then investigated by quasielastic
light-scattering experiments to determine the particle size
(not shown). In the case of pure DMPC and at a molar ra-
tio of 0.01, a scattering broad peak corresponding to vesicles
Fig. 2. Selected
31
P solid-state NMR spectra of the mixture of DMPC and
-OHplm at various molar ratios and temperatures. (A) 0.01 at 308 K; (B)
0.01 at 323 K, and (C) 0.5 at 323 K.
of diameter higher than 2 m was obtained. At a molar ra-
tio of 0.1, the large vesicles were still observed together
with colloids having a diameter of about 300 nm with a
broad polydispersity. The presence of these small size col-
loids is consistent with DSC data and with the isotropic line
as observed by solid-state NMR. At higher molar ratios, the
small colloids were still observed together with large ob-
jects, the latter corresponding to the presence of fatty acid
crystals.
Fig. 3 shows the lipid dispersion as viewed by TEM at a
molar ratio of 1 in the case of -OHplm after sedimentation
of the fatty acid crystals. Vesicles having a diameter of about
300 nm were observed. This is in very good agreement with
510 J.-P. Douliez / Journal of Colloid and Interface Science 271 (2004) 507510
Fig. 3. Transmission electron microscopy of a mixture of DMPC and
-OHplm at a molar ratio of 1, stained with uranyl acetate after sedimenta-
tion of the fatty acid crystals. Scale bar corresponds to 1 m.
the light-scattering data and prove that small-size colloids
are formed upon addition of cutin or suberin monomers to
membranes.
Altogether, these results are evidence that cutin and
suberin monomers are membrane perturbants. Interestingly,
most of these monomers are known as bola-amphiphiles [16]
and the perturbing effect may arise from the hydroxyl or
carboxyl moieties localized at both extremities of the alkyl
chain. Note that the membrane hydrophobic thickness in the
case of DMPC is about 25 [17] while the cutin and suberin
hydrophobic string length is about 14 . In other words, the
perturbation of the DMPC membranes by such monomers
can be associated with the hydrophobic mismatch. However,
the positioning and localization of the monomers within the
phospholipid membrane in the small colloids remain to be
determined.
Beyond these fundamental aspects, the present ndings
are of particular interest to bio-mimic what happens in
plants: rst, for the elucidation of the path followed by
monomers when transported out of the cell to the polymer-
ization site (it is shown here that these insoluble monomers
can be cosolubilized with phospholipids as small-size col-
loids which can serve as vehicles through the plant compart-
ments); and second, to predict the effect of free monomers
that are released upon pathogen attack on the cell mem-
branes localized in proximity of cutin or suberin polymers.
This work shows that even at low concentrations, cutin and
suberin monomers induce a change in the membrane cur-
vature. Although cells are certainly not transformed into
small colloids, monomers can more probably induce strong
perturbations in the cell membrane, potentially modulat-
ing its functions and that of associated membrane pro-
teins.
4. Conclusions
It is shown here that cutin and suberin monomers pene-
trate model membranes and exhibit a limit of solubility in the
phospholipid matrix. These monomers are membrane per-
turbants and induce a marked decrease of the vesicle size,
forming small colloids. The perturbation is supposed to orig-
inate from phase segregation in the phospholipid bilayer be-
cause of the hydrophobic mismatch between the membrane
thickness and the monomer string length. Finally, the present
ndings bring valuable information on the routing as small
colloids of these monomers in plants and on their potential
effect on cell membranes.
Acknowledgments
I thank J. Davy (LPCM, INRA, Nantes) for having per-
formed the DSC experiments, L. Lachmanski from Malvern
for giving me the opportunity to perform the QLS experi-
ments, C. Mangavel and B. Bouchet for the realization and
help with the TEM experiments, and D. Marion for sugges-
tions and helpful discussions.
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