New

Review Phytologist

Research review
The biophysical design of plant
cuticles: an overview

Author for correspondence: Eva Domı́nguez1, José Alejandro Heredia-Guerrero2 and Antonio
Antonio Heredia
Tel: +34 952131940
Heredia1
1
Email: heredia@uma.es Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM) ‘La Mayora’, Centro Mixto
CSIC-Universidad de Málaga, E-29760 Algarrobo-Costa, Málaga, Spain; 2Instituto de Ciencias de
Received: 31 August 2010
Accepted: 9 October 2010 Materiales de Sevilla, Centro Mixto CSIC-Universidad de Sevilla, E-41092 Sevilla, Spain

Summary

New Phytologist (2011) 189: 938–949
doi: 10.1111/j.1469-8137.2010.03553.x
Key words: biomechanics, cell wall, cuticle,
cutin, cutin structure, hydration.
The outer surfaces of epidermal cell walls are impregnated with an extracellular
matrix called the cuticle. This composite matrix provides several functions at the
interface level that enable plants to thrive in different habitats and withstand
adverse environmental conditions. The lipid polymer cutin, which is the main con-
stituent of the plant cuticle, has some unique biophysical properties resulting from
its composition and structure. This review summarizes the progress made towards
understanding the biophysical significance of this biopolymer with special focus on
its structural, thermal, biomechanical, and hydric properties and relationships. The
physiological relevance of such biophysical properties is discussed in light of exist-
ing knowledge on the plant cuticle.

‘Omnem plantae superficiem cingit lamina tenuis et
pellucida, quae cuticula dicitur, haec densa est et in
maceratione plantarum non dissoluitur’
C. G. Ludwig (1757)
The cuticle, a ubiquitous and composite
biopolymer
A group of green algae initiated the invasion of dry land
450 million yr ago, and in order to do so, they protected
themselves from desiccation by covering their aerial parts
with an extracellular membrane called the cuticle (Graham,
1993). Thus, the main function ascribed to the cuticle is
protection against water loss, together with regulation of gas
exchange. However, the cuticle has evolved other functions,
such as protection against mechanical injury from the envi-
ronment or in association with an attack of microorganisms
or pests, attenuation of UV light sorption, and generation
of a microenvironment suitable for certain organisms
(phyllosphere) (Kerstiens, 1996a; Riederer, 2006).
The presence of a distinctive layer covering the outer
surface of leaves and fruits was mentioned as early as the
fourth century BC by the Greek botanist Theophrastus.
This layer was regarded as the ‘skin of plant tissues’, analogous
to animal skin. Later on, this layer was called the epidermis
or ‘cuticle’ and was believed to be composed of several cell
layers. In 1757, C. G. Ludwig suggested the existence of a
delicate membrane covering the cuticle. This membrane or
pellicle was first isolated by Brongniart (1830, 1834) and
Henslow (1831) after long maceration of plant tissues. This
discovery led to the (sometimes confusing) definition of
a delicate membrane covering the thin cuticle membrane. It
took some time for the term ‘cuticle’ to evolve into its
modern usage, meaning ‘an extracellular membrane cover-
ing aerial organs of the plants’ (Riederer, 2006). Nowadays,
this definition should be modified to clearly reflect the
intimate relationship between the cuticle and the under-
lying cell wall.
The cuticle can be considered as a cutinized cell wall,
stressing the composite nature of the cuticle and the physio-
logically crucial interaction between the cuticle itself and

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the cell wall underneath. This cutinization process can be
seen in Fig. 1. This transmission electron microscopy
(TEM) image is of the epidermis of a young tomato fruit,
and the cuticle can be seen as an electron-dense layer that is
being constructed using the outermost electron-translucent
cell wall region as a framework. An extensive study on the
cuticle ultrastructure can be found in Jeffree (2006, and ref-
erences therein). Light microscopy images of tomato fruit
epidermis cross-sections are shown in Fig. 2. The different
combination of dyes allows the visualization of cellulose
(Fig. 2a), cuticle (Fig. 2b), and pectin (Fig. 2c), together
with the above-mentioned interconnection between the
cuticle and epidermal cell wall material (Fig. 2c,d).
The composition of the cuticle has been recently
reviewed (Jeffree, 2006; Pollard et al., 2008) and will only
be mentioned briefly. Fig. 3(a) shows a schematic drawing
of a cuticle cross-section with its different components. The
first characteristic that should be pointed out is the hetero-
geneous chemical composition and structure of such a layer.
Epicuticular waxes are deposited on the outer surface as a
more or less uniform and amorphous layer or in the form of
discontinuous crystals. The cuticle matrix underneath is
chiefly composed (40–80% weight) of cutin (Frémy, 1859),
a polymer constituted by a network of oxygenated C16
and ⁄ or C18 fatty acids cross-linked by ester bonds.
Depending on the species, the amount of cutin may vary
from a few to over 1000 lg cm–2 and its thickness can range
from submicrons to 10 lm or more (Walton, 1990;
Heredia, 2003).
Fig. 1 Transmission electron micrograph of the tomato fruit
epidermis at an early stage of development. The cuticle is present as
a continuous electron-dense outer layer surrounding the epidermal
cell walls and impregnating the radial walls as well. cut, cuticle; cw,
cell wall; ep, epidermal cell. Bar, 5 lm. (The picture is from the
authors’ laboratory.)
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Intracuticular waxes and phenolics intrude into the
cutin matrix. While intracuticular waxes are known to be
embedded and can be extracted with organic solvents, it is
still open to debate whether or not phenolic compounds
are chemically bound or trapped in the cutin matrix. The
principal constituents of the phenolic fraction are cinnam-
ic acids and flavonoids (Hunt & Baker, 1980), although
in some gymnosperm cuticles a lignin-like fraction has
been detected (Reina et al., 2001). Waxes, either epi- or
intracuticular, are mainly mixtures of C20-C40 n-alcohols,
n-aldehydes, very long-chain fatty acids and n-alkanes. On
the inner side of the cuticle, cutin is mixed with polys-
accharide material from the epidermal cell wall. This
polysaccharide fraction is chiefly composed of cellulose,
hemicellulose and pectin in ratios similar to those found
in the primary cell walls of tomato fruit pericarp (López-
Casado et al., 2007). The degree of interconnection and
the chemical bonds between cutin and cuticle polysaccha-
rides, if present, should be studied in detail since they
may have a major impact on biophysical properties of the
plant cuticle.
Monomer composition determines the
macromolecular structure of plant cutin
The first attempts to analyze cutin composition date back
to Frémy & Urbain (1882), but it was in the 1970s that
cutin monomers were identified after polymer degradation
by alkaline hydrolysis, transesterification and other methods
(Walton, 1990; Kolattukudy, 2001). Cutin can be com-
posed of C16, C18, or a mixture of C16 and C18 fatty acids.
Thus, the main components of C16 cutin are 9(10),16-
dihydroxyhexadecanoic acid and 16-hydroxyhexadecanoic
acid, and only in some cases are 16-hydroxy-10-oxo-C16
acid and 16-oxo-9 or 10-hydroxy-C16 acid present. C18
cutin is composed of 18-hydroxy-9,10-epoxyoctadecanoic
acid and 9,10,18-trihydroxyoctadecanoic acid together with
their monounsaturated homologs (Fig. 3b). In some
species, glycerol has also been found in the cutin matrix
(Graça et al., 2002). Arabidopsis thaliana (L) Heynh has
been widely used as a model in cutin biosynthesis studies,
although its composition is atypical and not representative
of the majority of plant cutins (Franke et al., 2005; Pollard
et al., 2008).
Cutan is another lipid polymer sometimes present in
plant cuticles, either as an alternative to or in combination
with cutin (Kolattukudy, 1996). It is composed of polyun-
saturated fatty acid derivatives, mostly linked to each other
by ether bonds (Villena et al., 1999). These types of bond
render a polymer matrix resistant to chemical degradation.
Unfortunately, little is known about distribution of cutan
among plant species and the potential advantages for plant
growth and survival associated with a more chemically resis-
tant cuticle matrix based on cutan.
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(a) (b)

(c) (d)

Fig. 2 Light microscopy photographs of tomato fruit epidermis at mature green (a–c) and red ripe (d) stages. (a) Calcofluor white stain is used
to visualize polysaccharides. (b) The same section stained with Sudan IV to see the cuticle. (c) Combination of (a) and (b) to provide an image
of the intimate association between the cuticle and epidermal cell wall material. (d) Ruthenium red stain used to visualize pectin material. The
cuticle can be observed because of the color contrast given by the flavonoids accumulated at the red ripe stage. Bar, 20 lm. (Pictures are from
the authors’ laboratory.)

(a) (c)

(b)

Fig. 3 (a) Scheme of a transverse section of the cuticle representing the different components and main structural features. (b) Major
monomers present in C16 and C18 cutins. (c) Schematic representation of a cutin network showing the free carboxyl and hydroxyl groups and
the ester bonds that link the monomers.

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Monomer composition and linkage are the first steps
towards understanding cutin architecture. While only half
of midchain hydroxyl groups of monomers take part in
side-chain ester cross-linking, most primary hydroxyl
groups are involved in ester bonds and, consequently, there
is a very low number of unesterified carboxyl functional
groups present in the polymer (Kolattukudy, 1996)
(Fig. 3c). This implies that there is a low number of reactive
centers available in the cutin polymer, since most hydroxyl
and carboxyl groups are chemically bound.
Although monomer composition and partial cutin
degradation analyses have been carried out in various inves-
tigations (Walton, 1990; Kolattukudy, 1996 and references
therein), some aspects related to the characteristics of cutin
monomers have not been studied in detail. Learning about
the chemical properties of cutin monomers is key since they
may influence or determine the resulting macromolecular
structure and have an impact on certain properties of the
polymer. Cutin monomers have bifunctional chemical
groups with the potential to bind, a property which, accord-
ing to polymer science, indicates that they are able to gener-
ate a nonlinear, amorphous and cross-linked polymer. In
the resulting polymer, relatively polar groups such as ester
bonds would not be statistically significant when compared
with abundant aliphatic methylene chains (-CH2-). The
rotational freedom of methylene chains may allow a
multitude of conformations to the polymer, although the
significant amount of in-chain hydroxyl groups will con-
strain chain linearity and weak chain–chain interactions.
Moreover, the types of functional groups and their location
in cutin monomers, especially primary and secondary
hydroxyl groups, confer the monomers’ self-assembly prop-
erties. Cutin monomers, under the right orientation and at
a given molecular density, generate structures based on
short-range interactions such as hydrogen bonding and
other weak interactions between monomers followed by
chemical polymerization. The self-assembly and self-poly-
merization properties of cutin monomers under specific
chemical conditions have been recently described (Benı́tez
et al., 2007, 2008; Heredia-Guerrero et al., 2008, 2009)
and open a new and promising field of study to comple-
ment current knowledge on cutin architecture. In this sense,
involvement of self-assembled polyhydroxy fatty acid parti-
cles (i.e. cutinsomes) in tomato cutin formation during the
early stages of development was recently confirmed with
antibodies raised against these supramolecular particles
(Domı́nguez et al., 2010).
Molecular dynamic (MD) calculations can provide theo-
retical information to interpret the molecular structure of
macromolecules. Such MD calculations have been applied
to study the three-dimensional structure of a cutin oligomer
based on the molecular characteristics of the monomers
described earlier. The model suggests that cutin constitutes
a moderately flexible network with motional constraints
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mainly located at the cross-link sites of functional ester
groups (Matas & Heredia, 1999), which ties in with the
amorphous and hydrophobic characteristics of cutin.
Additionally, a molecular basal spacing of 0.4–0.5 nm
between the methylene groups of an oligomeric chain and
the formation of holes or cavities within the structure has
been estimated (Matas & Heredia, 1999). These cavities are
void-free volumes resulting from the final three-dimen-
sional structure. The presence of such cavities is an intrinsic
characteristic of amorphous and cross-linked polymers and
may play a pivotal role in explaining the interactions
between cutin and low-molecular-weight compounds,
which can be either exogenous, such as adjuvants and pesti-
cides, or endogenous, such as phenolics and flavonoids.
Mobility and transport across the cutin matrix of these low-
molecular-weight compounds could also be reinterpreted
based on the presence of these cavities.
On the other hand, a polymer network can have, and
indeed has, emergent properties that cannot be extrapolated
from the monomers themselves. Furthermore, soluble olig-
omers obtained after depolymerization do not always repre-
sent the intact polymer, which justifies the need to use
nondestructive analytical techniques. Fourier transform
infrared (FT-IR) spectroscopy is a noninvasive tool that has
been extensively applied in our laboratory to check the
quality of the cuticle isolation procedure (Ramı́rez et al.,
1992; Luque et al., 1995a). This structural method allows
the identification of the functional groups present in the
sample and can be used to analyze the depolymerization of
specific cuticle components (Villena et al., 2000). Solid-
state nuclear magnetic resonance (NMR) analyses have been
extensively performed on cuticle and cutin (Batteas &
Stark, 2005; Stark & Tian, 2006). This technique has
confirmed the amorphous and flexible nature of the cutin
network with motional constraints at particular cross-linked
sites. Approximately 36% of the cutin methylene chains are
located in a flexible molecular domain, whereas the rest are
in a more rigid domain (Batteas & Stark, 2005). NMR
relaxation measurements have also provided interesting
information on the molecular dynamics of cutin chains
(Stark & Tian, 2006). Two-dimensional NMR spectro-
scopy has been used to investigate the changes induced by
the partial depolymerization of cutin, using enzymatic or
chemical reagents (Fang et al., 2001). Such methodology
has confirmed structural features derived from indirect and
classical chemical studies, that is, the cutin polyester is held
together mostly by primary hydroxyl ester linkages, with
about half of the secondary hydroxyl groups involved in
ester cross-links. Recent works on cutin NMR (Deshmukh
et al., 2003, 2005; Sachleben et al., 2004) have provided
new information on the molecular arrangement of tomato
fruit and Agave americana leaf cutin. Consequently,
alfa-branched hydroxyl fatty acids and their corresponding
esters have been identified in cutin. Further studies will be
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necessary to elucidate the role of this monomer in cutin
structure. Cutan structure and composition should also be
mentioned. Contrary to the early model described by
Villena et al. (1999), which postulated the presence of ether
bonds as the key points of an aliphatic cutan structure and
stability, Sachleben et al. (2004) and Deshmukh et al.
(2005) recently proposed a structure based on large aro-
matic domains linked by methylene chains of variable
length and esters of a small number of carbon atoms.
X-ray diffraction has been used to study the cutin matrix
and the associated waxes. Although this powerful tool has
provided interesting information on the structure of epicu-
ticular plant waxes (Casado & Heredia, 1999), its use has
been limited in the case of isolated cutin. X-ray diffraction
of tomato fruit cuticle corroborates the amorphous nature
of cutin suggested by polymer science and MD analysis.
Two broad and major hydrophobic interplanar spaces, c.
1.0 and 0.45 nm, were observed in tomato cuticles by X-
ray diffraction (Luque et al., 1995a), the latter attributed to
spacing between methylene chains predicted by MD analy-
sis. The 1 nm spacing between polymer chains was sensitive
to incorporation of ions to the cutin network and could be
assigned to regions rich in aromatic hydroxyl groups capa-
ble of ionic exchange (Luque et al., 1995a). This ionic
modulation of the 1 nm basal distance may modify water
mobility across the cuticle, either favoring or hindering it
(Luque et al., 1995a). The ionic exchange capacity of cuti-
cles was first studied by Schönherr & Bukovac (1973).
They reported that cuticles can be visualized as sieves with a
moderate ionic exchange capacity mainly ascribed to the
polysaccharide fraction, although the cutin matrix may also
play a minor role.
The ionic exchange capacity of the cuticle can also be
analyzed from an electrodynamic point of view. The plant
cuticle can be regarded as an electrically asymmetric mem-
brane (Heredia & Benavente, 1991; Tyree et al., 1991). In
an aqueous electrolyte solution, a clear electrokinetic gradi-
ent is established across isolated cuticles. Whereas the outer
surface appears mainly uncharged, the inner surface sup-
ports a net negative charge described by a Donnan-like
membrane potential associated with polysaccharides present
in the inner part of the cuticle (Heredia & Benavente,
1991). This fixed charge is an important physiological char-
acteristic that influences sorption, uptake and transport of
ions and charged molecules and should be taken into
account when considering cuticular transpiration, agri-
cultural spray applications and related ecotoxicological
problems (Kerstiens, 1996b).
Impedance spectroscopy (dielectric spectroscopy) deter-
mines the dielectric properties of a system as a function of
electric frequency. It is based on the interaction of an exter-
nal field with the overall electric dipole moment of the sam-
ple. As mentioned earlier, the plant cuticle is a complex
asymmetric composite that can be polarized. Measurement
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of impedance provides information about the energy stor-
age (capacitance, C) and dissipation (resistance, R) capacity
of a sample. Dewaxed cuticles and cutin display both energy
storage and dissipation properties, like many biological tis-
sues (Ramos-Barrado et al., 1993; Benavente et al., 1998b).
Nevertheless, remarkable differences in the impedance plots
and in their associated equivalent circuits were obtained for
dewaxed cuticles and cutin, indicating a different electroki-
netic macromolecular behavior. A simple RC equivalent cir-
cuit was associated with cutin, which implies that the
polymer itself has homogeneous dielectric properties
(Benavente et al., 1998b). The dewaxed cuticle (cutin and
polysaccharides) showed a different electrical response
(Benavente et al., 1998b), in this case a modified equivalent
circuit, where one of the components of the system is
hydrated and allows proton diffusion and charge move-
ment. This behavior can be assigned to the polysaccharides
present in the cuticle. Again, the polysaccharide fraction of
the cuticle seems to be not only charged, but also in control
of the movement of charged molecules.
Nanotechnological, noninvasive tools have rarely been
used to investigate plant cuticles and cutin structure. The
three-dimensional arrangement and surface architecture of
outer plant surfaces at the nanoscopic level have been
analyzed by atomic force microscopy (AFM) (Canet et al.,
1996; Round et al., 2000; Wisniewska et al., 2003; Batteas
& Stark, 2005). A comprehensive AFM study in combina-
tion with infrared and NMR spectroscopy was applied to
investigate the influence of the cross-linking degree in the
macromolecular arrangement of cutin (Benı́tez et al.,
2004). Molecular topography analyses of the outer surface
of cutin samples from immature and ripe tomato fruits
showed a correlation between the cross-linking degree and
the texture of the outer surfaces. Cross-linked ripe tomato
cutin presented a flatter and more globular texture together
with elongated and orientated superstructures (Benı́tez
et al., 2004). Fig. 4 shows an example of this AFM topo-
graphy. Emergent nanoscopic approaches combine AFM
with ultrasound or Kelvin probes. Thus, atomic force
acoustic microscopy (AFAM) allows identification of voids,
inclusions, or cracks based on different elastic properties of
the sample surface, while the Kelvin probe detects micro-
and nanoscopic changes in the polarity of surfaces. These
techniques could provide useful additional information and
should be explored in future cuticular investigations.
The interesting and physiologically relevant information
derived from these structural techniques has regrettably
been limited to a few investigations in comparison with the
existing number of analytical studies on cuticle composi-
tion. The plant cuticle is a composite where each fraction
has a complex composition. This complexity masks
assignment of measured properties to a specific fraction or
the major contribution of certain compounds within a cuti-
cle fraction. In this sense, thorough studies on how these
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18 nm
9.15 nm
0 nm
2203 nm
2203 nm
1102 nm
1102 nm
0 nm
0 nm
Fig. 4 Atomic force microscopy (AFM) three-dimensional
topographic image (contact mode) showing the morphology of the
outer surface of ripe tomato fruit cutin at 2.2 · 2.2 lm2 resolution.
A globular and flattened relief with a maximum high of 18 nm can
be observed. (AFM topography courtesy of Dr José J. Benı́tez.)
properties change during cuticle development and in species
with known cuticle differences are required.
Hydration and temperature: major players in
biophysical cuticle behavior
Land plants survive in an environment characterized by
changes in temperature, hydration and light intensity.
Despite this, most plants live within a relatively narrow
range of these parameters; however, some plants can also
survive under extreme conditions. As a subtle polymer bar-
rier, the cuticle responds to changes in these parameters by
modulating its characteristics. The behavior of a polymer
under variable thermal and hydration conditions is crucial
to understanding the polymer itself.
Once synthesized and formed, a cutinized cell wall is
sensu stricto an extracellular nonliving membrane, and the
driving forces and mechanisms involved in the sorption and
active transport can be expected to differ from those found
within the cell. Since the 1970s, several studies have assessed
water and solute permeability and diffusion through cuticles
and cutin from a wide variety of plants, as revised in detail
by Fernández & Eichert (2009). In general, astomatous
cuticles have a low water permeability, which is a thousand
times lower than the plant cell wall and remains in a range
comparable to that of some hydrophobic synthetic polymers
(Nobel, 1991; Schreiber & Schönherr, 2009). The inter-
ested reader should refer to Kerstiens (2006), Fernández &
Eichert (2009) and the recent book by Schreiber &
Schönherr (2009), which covers this topic extensively, as
well as the very recent review by Schreiber (2010) extended
to the transport barrier properties of cutinized and suber-
ized cell walls. Briefly, these studies consider the cuticle and
cutin as solution-diffusion membranes for water; that is,
individual water molecules follow a random pathway in a
mostly lipophilic environment. It has also been suggested
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that water transport occurs via aqueous pores or channels,
whose presence seems to be highly dependent on relative
humidity (RH) and temperature (Kerstiens, 2006;
Schreiber & Schönherr, 2009). The next challenge in this
area will be to characterize the diffusion mechanisms that
take place in the cuticle and also to explain the thousand-
fold differences in permeability between species observed in
some investigations (Kerstiens, 2006).
By contrast, few studies have been focused on the sorp-
tion capacity of the cuticle and its major compounds, that
is, cutin and polysaccharides. The significant water sorption
capacity of plant cuticles as shown in some investigations
has important consequences for the foliar uptake of pesti-
cides, which constitutes a complex and empirical research
area. The first studies on this subject were carried out by
Chamel et al. (1991) with fruit and leaf cuticles and showed
a water sorption between 1 and 8% of the initial cuticle
DW. Wax extraction did not affect the water sorption
capacity, but cutin showed a drastic reduction (c. 63%) in
comparison to the intact cuticle (Chamel et al., 1991).
Luque et al. (1995b) reported a similar sorption behavior
for the tomato fruit cuticle and predicted water clustering
(accumulation of water in a liquid state) above 60% RH
and the existence of water-binding sites of variable strength.
These sites suggest the existence of polymer regions of dif-
ferent accessibility and the creation of new binding sites as
the cuticle is hydrated. Cluster formation may explain a
good number of transport phenomena in polymers, since it
reduces the effective mobility of water by increasing the size
of the diffusing molecular group and the tortuosity of the
diffusion path (Luque et al., 1995b). There is only one
investigation dealing with the sorption capacity of a gymno-
sperm plant cuticle. Water sorption and desorption were
studied in Araucaria bidwillii Hook cuticles and also with
regard to its components (Reina et al., 2001). Such a
cuticle, as observed in many gymnosperms, has significant
polysaccharide (40% DW) and lignin-like domains (c. 27%
DW). Water sorption-desorption of isolated cuticles and
their components exhibited hysteresis and, noticeably, the
lignin fraction showed the highest sorption and water reten-
tion capacity of all the cuticular components (Reina et al.,
2001). However, more research is needed to understand the
role of this lignin-like fraction in gymnosperm cuticles. The
substantial reduction in water sorption after the removal of
polysaccharides suggests that this fraction is the one mainly
responsible for cuticle water uptake. Domı́nguez & Heredia
(1999) studied the water sorption of the polysaccharide
fraction of A. americana and Clivia miniata cuticles and
confirmed that they can sorb up to 50% of the initial DW,
a value similar to the one recorded for isolated cell walls
(Mercado et al., 2004). Water clustering, in this case, was
predicted to occur at 48% RH (Domı́nguez & Heredia,
1999). The noteworthy difference in water sorption
observed between an intact cuticle (1–8%) and its
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polysaccharide fraction (50%) is an example of the synergis-
tic effect of cutin and polysaccharides.
Temperature plays a central role in the biophysical
properties of a polymer, since it can produce changes in its
structure and modify the hydration profile. These structural
changes can be of first order (i.e. crystallization and fusion)
or second order (i.e. glass transitions). Several methodolo-
gies, such as latent heats (Eckl & Gruler, 1980) and volume
expansion coefficients (Schreiber & Schönherr, 1990), have
been used to determine the influence of temperature in
plant cuticles. However, the most accurate method for
monitoring thermal events is the evaluation of the heat
capacity or specific heat (Cp), a thermodynamic parameter
very sensitive to structural changes. Unfortunately, the
literature available on this topic is limited, even though
temperature-dependent changes in the cuticle, cutin and
wax structure of several species have been reported.
Plant cuticle and cutin have a significantly higher specific
heat (Casado & Heredia, 2001) than other polymers. For
example, the specific heat of cutin ranges between 2 and
2.5 J K)1 g)1, whereas cellulose, one of the main compo-
nents of the plant cell wall, has a specific heat of
1.5 J K)1 g)1 (Boraston, 2005). A high value means that a
greater amount of heat is required to raise the polymer’s
temperature by 1ºC. Although the cuticle makes a minor
contribution to the overall mass of leaves and fruits, it could
play an important role as a thermoregulator between the
plant and the environment, especially when plants are
exposed to extreme temperatures.
Temperature transitions of tomato fruit cuticle and cutin
during growth have been investigated using differential
scanning calorimetry (Luque & Heredia, 1997; Matas
et al., 2004b). Both samples presented a broad glass
transition temperature, Tg (c. 23C), that did not change
significantly during growth. The Tg of a polymer is a
second-order thermal transition characterized by solid-like
changes in the physical properties, for example, changes in
viscosity, rigidity, and heat capacity. This temperature, usu-
ally present in amorphous polymers, defines two physical
stages: a stiff stage (like a glass) and a liquid-like very viscous
stage with less restricted translational movements and rota-
tional and vibrational degrees of freedom. A glass transition
temperature is the temperature at which macromolecular
chains increase their flexibility and fluidity. The fact that
cutin shows this transition at environmental temperature
has obvious physiological implications, since it involves
conformational changes in its amorphous structure. Below
the Tg, the cuticle is rigid and restricts the mobility of exog-
enous compounds across it. Above this temperature the
cuticular matrix appears more viscous, facilitating the
mobility of the compounds.
The changes observed in cuticle permeability to water,
low-molecular-weight molecules and ions as a function
of temperature could be explained by a second-order
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transition. The break point above 30C observed in the
plots of permeability against temperature involves a change
in the polymer structure and a decrease in the activation
energy of the process (Schönherr et al., 1979; Benavente
et al., 1998a). Moreover, water sorption at high RH lowers
this glass transition temperature, since it decreases the
energy required to obtain a more relaxed and flexible poly-
mer network, which implies that water plasticizes the cutin
matrix (Matas et al., 2004b). These data are in good agree-
ment with those previously reported by Round et al. (2000)
on tomato fruit cutin using AFM and NMR. NMR has also
been applied to study the changes in the cuticle and cutin as
a function of water content and temperature. Hydration
and temperature enhance the segmental motions of the
methylene chains, (CH2)n, and other minor functional
groups indicating a decrease in the potential resistance of
the polymer to deformation (Stark & Tian, 2006; Stark
et al., 2008).
Water content and temperature appear as key elements
that modulate the macromolecular structure and the energy
and mass transfer between the environment and the plant
cell. These two properties have an important effect on the
physiological response of the cuticle to the application of
adjuvants (Knoche & Bukovac, 2004), the deleterious
effects of frost and heat, and even in some physiological dis-
orders such as fruit cracking (Beyer & Knoche, 2002; Beyer
et al., 2005).
Little work has been done on the role of light radiation at
the plant surface level (see Pfündel et al., 2006 for a review).
The phenolic compounds present in most cuticles (i.e. cin-
namic acids, flavonoids and flavonols) can absorb UV light,
providing the cuticle with a screening function against UV
radiation (Kolb & Pfündel, 2005). This can result in pat-
terns of UV reflection, depending on the spatial distribution
of phenolics that might increase pollinating insect attrac-
tion. No such optical properties have been observed for
cutin monomers or cell wall polysaccharides (Pfündel et al.,
2006). Surface architecture can also modify the optical
effects, and hence epicuticular wax composition and
deposition could be of importance in the study of the light
properties of the cuticle. The effect of light intensity and
wavelength on cuticle monomer synthesis and accumulation
is another topic that deserves further attention.
Cutin biomechanics: the polymer flows
‘The tension of the outer surface is mechanical, whilst
that of the inner surface is vital’
C. R. Darwin (1875)
Plants living in aquatic or aerial environments are subjected
to mechanical stresses often in the form of water or air
currents. Therefore, the cuticle protecting the surface may
modify but should not restrain the mechanical properties of
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Phytologist
the tissue. Early scientists noticed the relationship between
cell wall extensibility and plant growth and much work has
been done since then on the biomechanics of plant cell walls
(see Cosgrove, 1993 for a review on the different techniques
applied) and methods of analysis and interpretation of data
(Thompson, 2001). From the early work of Kraus in 1867
and others (for a review, see Kutschera & Niklas, 2007), it
was postulated that the inner tissues provide the driving
force for elongation, whereas the outer cells (i.e. the epider-
mis) restrict and control the rate of growth by imposing a
mechanical constraint (Kutschera, 1989; Kutschera &
Niklas, 2007; Savaldi-Goldstein et al., 2007). Hence, the
cuticle surrounding the epidermis must play a role concern-
ing the mechanical behavior of plant organs. For example,
in staminal filament thigmonasticity, it has been suggested
that the cuticle provides the force for early contraction of
filaments in vivo, while, as the load increases, the cuticle can
no longer sustain all deformation and cell walls are strained
(Hasenstein et al., 1993).
The biomechanical behavior of the skin and its isolated
cuticle has usually been studied in tomato fruits, and the
rheological behavior of the skin mirrors that of the cuticle,
both being isotropic, viscoelastic and strain-hardening
materials (Matas et al., 2004a; Bargel & Neinhuis, 2005).
Matas et al. (2004a) showed that the elastic modulus (E) of
the cuticle is higher than that of the peel tissue, whereas the
work of fracture, the amount of energy required to propa-
gate a crack, exhibited an opposite trend. From these
results, it can be concluded that the cuticle stiffens the cell
walls, while the epidermal cell walls strengthen the cuticle,
better sustaining superficial cracks. By contrast, Bargel &
Neinhuis (2005) obtained a higher E, breaking stress and
breaking strain for the skin. These differences could be
attributed to the types of tomato fruits (cherry vs normal-
sized varieties) or to the liquid employed to keep the
samples hydrated: tomato juice (Matas et al., 2004a) and
distilled water (Bargel & Neinhuis, 2005).
The biomechanical and rheological behavior of the plant
cuticle, like most plant materials, can be described as visco-
elastic; that is, the relationship between stress and strain is
time-dependent (Niklas, 1992), with an elastic component
at small deformations. In some species, the stress–strain
curves show two phases with different slopes, the first corre-
sponding to the linear elastic phase and the second to the
nonlinear viscoelastic phase (Matas et al., 2005; López-
Casado et al., 2007). Elasticity implies that the deformation
strain increases linearly with increasing stress applied and
that the instant deformation is not time-dependent.
Rheology studies the flow of matter and hence is applied to
materials that cannot sustain a stress in static equilibrium,
without changing their viscosity. It is concerned with the
relationship between the flow or deformation (mechanical
behavior) of a material and its internal structure; for exam-
ple, the orientation and elongation of polymer molecules. A
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Research review Review 945
thorough compilation of the principles and theories of plant
biomechanics can be found in the superb book written by
Karl Niklas (1992).
The biomechanical nature of the isolated plant cuticle
was first studied by Petracek & Bukovac (1995), who
reported the viscoelastic nature of the tomato fruit cuticle,
later confirmed by other authors and for different species
(Wiedemann & Neinhuis, 1998; Edelmann et al., 2005;
see Bargel et al., 2006 for a review). Most of the biome-
chanical analyses have been performed with tomato fruit
cuticles and, unless indicated differently, all of the investiga-
tions mentioned in the following have been carried out on
tomato. The complex composition of the cuticle is accom-
panied by a complex biomechanical performance. Thus, it
is of special interest to understand the contribution of each
cuticle fraction to the overall biomechanics. Petracek &
Bukovac (1995) reported a decrease in the breaking stress
and an increase in plastic behavior after wax removal,
suggesting a role of waxes as fillers. As such, waxes would
reduce cutin matrix mobility, acting as compounds that
increase rigidity. López-Casado et al. (2007) showed that
the polysaccharide fraction of the cuticle is mainly responsi-
ble for the high elastic modulus (i.e. the stiffness) and the
linear elastic behavior of the cuticle, whereas the cutin
matrix has a low elastic modulus and high strain values. A
role of flavonoids concerning the mechanical properties of
the cuticle was first suggested by Bargel et al. (2006) and
observed in the y tomato mutant (Adato et al., 2009).
Flavonoids contribute to the elastic phase, reinforcing the
elastic contribution of the polysaccharide fraction and
increasing cuticle rigidity, that is, they play a role similar to
that already suggested for waxes (Domı́nguez et al., 2009).
Few preliminary works focusing on fruit growth and
ripening reported an increase in E and a decrease in strain as
the fruit matured (Bargel & Neinhuis, 2005; López-Casado
et al., 2007; Domı́nguez et al., 2009). From a physiological
point of view and considering the potential agronomic
implications for pest and disease attack and control, fruit
quality or irrigation and fertilization strategies, to cite some
examples, it will be key to further improve our understand-
ing of the evolution of cuticular biomechanics during
growth. Such information could help to explain how the
mechanical properties are modified by the occurrence of
internal pressure changes and in relation to the assembly of
the different cuticle components during plant organ
development.
Water and temperature are two factors that largely influ-
ence every aspect of the biomechanical properties, as well as
interacting with each other. The role of water and tempera-
ture as modifiers of the mechanical properties of synthetic
and natural polymers is widely recognized. The effect of
water as a plasticizer of the plant cuticle was first observed
by Petracek & Bukovac (1995) and later confirmed by sev-
eral authors in isolated cuticles (Edelmann et al., 2005;
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946 Review Research review
Matas et al., 2005) and also in the cutin matrix (López-
Casado et al., 2007). The effect is strongly dependent on
the degree of hydration of the material. Water molecules
probably interact with cutin and polysaccharide fractions,
decreasing the viscosity of these polymers and hence favor-
ing the displacement of the polymer macromolecular
segments. The cuticle responds to increases in temperature
by decreasing its strength and rigidity (Edelmann et al., 2005;
Matas et al., 2005). The observed effect of temperature
on cuticular rheology consisted of two temperature-
independent phases separated by a transition temperature
between 23 and 30C. The cutin matrix exhibits a glass
transition at this temperature. The combined effect of
water and temperature on the elastic modulus is presented
in Fig. 5. The effect of transition temperature on E
diminishes with increasing RH. It can be concluded that
the biomechanical properties of the cuticle change within a
physiological range of temperature and RH.
The physiological implications of the mechanical proper-
ties of the cuticle cannot be underestimated, since it may
explain some modifications of plant growth and develop-
ment, or even tissue failures. An interesting approach for
studying how modifications of the cuticle may affect the
rheological properties would be the use of mutants and
transformants. In this sense, two papers have reported the
mechanical study of tomato mutants (Bargel & Neinhuis,
2004; Adato et al., 2009), although more basic research is
still needed. Thus, Bargel & Neinhuis (2004) studied the
mechanical differences during tomato fruit growth between
the cuticle of wild-type (wt) and the nonripening (nor)
mutant. An increase in stiffness and breaking strength was
observed during fruit growth and ripening in the cuticle of
wt fruits, whereas in nor fruits the cuticle was significantly
less stiff and weaker. Hydration generally decreased the
elastic modulus and strength, while the breaking strain was
Fig. 5 Variation of elastic modulus with temperature at two values
relative humidities (RH): 40% RH (solid line), and wet (dashed line)
(taken from Matas et al., 2005). To visualize the second-order
transition, the change of heat capacity, Cp, with temperature is also
shown (red dots). (Taken from Matas et al., 2004b.)
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only significantly affected in nor fruits. On the other hand,
the tomato y mutation (colorless fruit epidermis) is character-
ized by a cuticle that lacks the yellow flavonoid naringenin
chalcone and showed a significant shortening of the
elastic phase of the corresponding strength–strain curve in
comparison with the corresponding wt cuticle samples
(Adato et al., 2009).
Nanotechnology has been employed in the study of cuti-
cle surface mechanics using AFM nanoindentation (Round
et al., 2000; Isaacson et al., 2009). Although the mechani-
cal parameters are not comparable to those obtained with
an extensiometer, this interesting tool could generate a
mechanical topography of the cuticle and help to identify
regions prone to surface cracks. Recently, this technique has
been used to study contact mechanics at the plant–insect
interface and has shown that the ability of insects to attach
to plant surfaces depends in part on the mechanical stability
of the wax surface (Voigt et al., 2008).
Conclusions
Hydric, thermal, and biomechamical properties constitute
the basis for understanding the biophysical behavior of iso-
lated plant cuticles. This behavior could vary, within limits,
between plant species or even between genotypes of the
same species. The influence of some abiotic and hormone
stresses on cuticle synthesis has been reported in the last
decade. During growth and development, the plant cuticle
responds to these stresses by changes in cuticle thickness
and deposition, and also by modifying the ratio of some
cuticular components. Such alterations have been noticed,
for example, as a result of the influence of plant hormone
application (Knoche & Peschel, 2007), water deficiency
(Kosma et al., 2009), in association with iron deficiency
chlorosis (Fernández et al., 2008) and as a result of the
direct effect of UV-B light (Paoletti, 2005). A biophysical
analysis of such modified cuticles would be of interest for
understanding how these properties may vary in response to
these cuticle modifications and the role of different compo-
nents or morphological traits such as thickness in cuticle
biophysics. In this sense, several cuticle mutants have been
identified, especially in Arabidopsis and tomato, and their
cuticles have been chemically analyzed (see Pollard et al.,
2008 for a review and, more recently, Isaacson et al., 2009;
Lü et al., 2009). The use of cuticle mutants to assess the
contribution of the different cuticular fractions and selected
components to the properties addressed in this review will
help to improve our understanding of the biochemical and
biophysical scenarios that determine the performance of the
plant cuticle.
Current knowledge of the biophysical properties of the
cuticle has been obtained from analyses performed on iso-
lated cuticles. How this information correlates with the
physiological behavior of the epidermis and, ultimately, of
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Fig. 6 Flowchart representing the interactions observed in the
cuticle between the thermal, hydric, and mechanical properties
discussed in this update. Tg, glass transition temperature; RH,
relative humidity.
the whole plant is a subject to consider. Several questions
remain to be answered. What is the actual hydration degree
of a cuticle attached to the epidermal cell wall? Is the degree
of hydration constant along a given cuticle thickness or is
there a RH gradient? How does the attachment to the
epidermis modify the properties of the cuticle? And, finally,
how can this all be measured? Another topic that should be
stressed is the need to corroborate the information gained
from studies on different plant species. It would also be
advisable to normalize the methodologies and instrumenta-
tion used to investigate cuticle properties such as
permeability and biomechanics to enable data comparison.
Most of the physiological functions of the cuticle are a
consequence of the physical properties of the polyester
cutin, its interaction with the cell wall and the subtle
regulation of other minor compounds. These key physical
properties, belonging to three basic areas of physics – thermo-
dynamics, hydrodynamics and mechanics – are not isolated
but they largely influence each other’s performance. The
design requirements of each property can be incompatible
with the properties needed for the others to maximize their
functions, leading to a necessary compromise. Fig. 6 shows
graphically the major features of the complex interactions
between thermal, hydric, and mechanical properties of the
cuticle, allowing us to summarize the main consequences of
their sometimes conflicting requirements. Therefore, the
resulting functions of the cuticle and cutin would be a
design compromise between the interactions of these intrin-
sic physical properties. In the words of the biochemist
François Jacob, they could be interpreted as the result of
‘the game of the possible’, that is, chemical and biological
evolution.
In the revolutionary era of ‘omics’, which is still in its
early stages, the biophysical approach should be taken into
consideration and applied to obtain more detailed knowl-
edge of the plant cuticle. Only under such a generative and
holistic view will we be able to comprehend and not only
apprehend the physiology of the plant cuticle.
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Research review Review 947
Acknowledgements
The authors dedicate this work to Prof. Dr M. J. Bukovac
(Michigan State University). The authors would like to
thank Dr Victoria Fernández for helpful discussions and
Plan Nacional I+D (MEC, Spain) for several grants that
have supported their research over the last 20 yr.
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