The biophysical design of plant cuticles: an overview

Summary

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 constituent 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 existing 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 environment 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 covering aerial organs of the plants’ (Riederer, 2006). Nowadays, this definition should be modified to clearly reflect the intimate relationship between the cuticle and the underlying cell wall.

The cuticle can be considered as a cutinized cell wall, stressing the composite nature of the cuticle and the physiologically crucial interaction between the cuticle itself and 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 references 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).

Figure 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 μm. (The picture is from the authors’ laboratory.)

Figure 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 μm. (Pictures are from the authors’ laboratory.)

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 heterogeneous 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 μg cm^–2 and its thickness can range from submicrons to 10 μm or more (Walton, 1990; Heredia, 2003).

Figure 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.

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 cinnamic 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-C₄₀n-alcohols, n-aldehydes, very long-chain fatty acids and n-alkanes. On the inner side of the cuticle, cutin is mixed with polysaccharide 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 polysaccharides, 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 composed 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 polyunsaturated 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 resistant cuticle matrix based on cutan.

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 investigations (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, according to polymer science, indicates that they are able to generate 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 constrain 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 properties. 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-polymerization properties of cutin monomers under specific chemical conditions have been recently described (Benítez et al., 2007, 2008; Heredia-Guerrero et al., 2008, 2009) and open a new and promising field of study to complement current knowledge on cutin architecture. In this sense, involvement of self-assembled polyhydroxy fatty acid particles (i.e. cutinsomes) in tomato cutin formation during the early stages of development was recently confirmed with antibodies raised against these supramolecular particles (Domínguez et al., 2010).

Molecular dynamic (MD) calculations can provide theoretical 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 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-dimensional 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 pesticides, 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 oligomers obtained after depolymerization do not always represent 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 (Ramírez 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 spectroscopy 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 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 aromatic 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 epicuticular 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 analysis. 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 capable 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 cuticles 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 membrane (Heredia & Benavente, 1991; Tyree et al., 1991). In an aqueous electrolyte solution, a clear electrokinetic gradient is established across isolated cuticles. Whereas the outer surface appears mainly uncharged, the inner surface supports 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 characteristic that influences sorption, uptake and transport of ions and charged molecules and should be taken into account when considering cuticular transpiration, agricultural spray applications and related ecotoxicological problems (Kerstiens, 1996b).

Impedance spectroscopy (dielectric spectroscopy) determines the dielectric properties of a system as a function of electric frequency. It is based on the interaction of an external field with the overall electric dipole moment of the sample. As mentioned earlier, the plant cuticle is a complex asymmetric composite that can be polarized. Measurement of impedance provides information about the energy storage (capacitance, C) and dissipation (resistance, R) capacity of a sample. Dewaxed cuticles and cutin display both energy storage and dissipation properties, like many biological tissues (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 electrokinetic macromolecular behavior. A simple RC equivalent circuit 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 movement. 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 combination with infrared and NMR spectroscopy was applied to investigate the influence of the cross-linking degree in the macromolecular arrangement of cutin (Benítez 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 (Benítez et al., 2004). Fig. 4 shows an example of this AFM topography. 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.

Figure 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 μm² resolution. A globular and flattened relief with a maximum high of 18 nm can be observed. (AFM topography courtesy of Dr José J. Benítez.)

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 composition. 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 cuticle fraction. In this sense, thorough studies on how these 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 barrier, 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 interested 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 suberized 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 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 sorption 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 pesticides, 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 different 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 gymnosperm 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 retention 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. Domínguez & 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 (Domínguez & Heredia, 1999). The noteworthy difference in water sorption observed between an intact cuticle (1–8%) and its polysaccharide fraction (50%) is an example of the synergistic 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 methodologies, 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 (C_p), 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 components 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, T_g (c. 23°C), that did not change significantly during growth. The T_g 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, usually 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 rotational 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 T_g, the cuticle is rigid and restricts the mobility of exogenous 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 transition. The break point above 30°C 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 polymer network, which implies that water plasticizes the cutin matrix (Matas et al., 2004b). These data are in good agreement 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 disorders 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. cinnamic 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 patterns of UV reflection, depending on the spatial distribution of phenolics that might increase pollinating insect attraction. 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 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 epidermis) 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 concerning 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 propagate 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 viscoelastic; 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 corresponding 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 example, the orientation and elongation of polymer molecules. A 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 biomechanical analyses have been performed with tomato fruit cuticles and, unless indicated differently, all of the investigations mentioned in the following have been carried out on tomato. The complex composition of the cuticle is accompanied 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 responsible 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 (Domínguez 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; Domínguez 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 understanding 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 influence every aspect of the biomechanical properties, as well as interacting with each other. The role of water and temperature 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 several authors in isolated cuticles (Edelmann et al., 2005; 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 favoring 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 30°C. 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.

Figure 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, C_p, with temperature is also shown (red dots). (Taken from Matas et al., 2004b.)

The physiological implications of the mechanical properties of the cuticle cannot be underestimated, since it may explain some modifications of plant growth and development, 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 only significantly affected in nor fruits. On the other hand, the tomato y mutation (colorless fruit epidermis) is characterized 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 cuticle surface mechanics using AFM nanoindentation (Round et al., 2000; Isaacson et al., 2009). Although the mechanical 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 isolated 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 components 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; Lüet 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 isolated cuticles. How this information correlates with the physiological behavior of the epidermis and, ultimately, of 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 instrumentation 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 – thermodynamics, 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 intrinsic 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.

Figure 6.  Flowchart representing the interactions observed in the cuticle between the thermal, hydric, and mechanical properties discussed in this update. T_g, glass transition temperature; RH, relative humidity.

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 knowledge 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.

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.