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Biomechanics of isolated tomato (Solanum lycopersicum L.) fruit cuticles: the role of the cutin matrix and polysaccharides

Biomechanics of isolated tomato (Solanum lycopersicum L.) fruit cuticles: the role of the cutin... Abstract The mechanical characteristics of the cuticular membrane (CM), a complex composite biopolymer basically composed of a cutin matrix, waxes, and hydrolysable polysaccharides, have been described previously. The biomechanical behaviour and quantitative contribution of cutin and polysaccharides have been investigated here using as experimental material mature green and red ripe tomato fruits. Treatment of isolated CM with anhydrous hydrogen fluoride in pyridine allowed the selective elimination of polysaccharides attached to or incrusted into the cutin matrix. Cutin samples showed a drastic decrease in elastic modulus and stiffness (up to 92%) compared with CM, which clearly indicates that polysaccharides incorporated into the cutin matrix are responsible for the elastic modulus, stiffness, and the linear elastic behaviour of the whole cuticle. Reciprocally, the viscoelastic behaviour of CM (low elastic modulus and high strain values) can be assigned to the cutin. These results applied both to mature green and red ripe CM. Cutin elastic modulus, independently of the degree of temperature and hydration, was always significantly higher for the ripe than for the green samples while strain was lower; the amount of phenolics in the cutin network are the main candidates to explain the increased rigidity from mature green to red ripe cutin. The polysaccharide families isolated from CM were pectin, hemicellulose, and cellulose, the main polymers associated with the plant cell wall. The three types of polysaccharides were present in similar amounts in CM from mature green and red ripe tomatoes. Physical techniques such as X-ray diffraction and Raman spectroscopy indicated that the polysaccharide fibres were mainly randomly oriented. A tomato fruit CM scenario at the supramolecular level that could explain the observed CM biomechanical properties is presented and discussed. Cuticle, cutin matrix, plant biomechanics, polysaccharides, Solanum lycopersicum Introduction Epidermal cells of the aerial parts of higher plants are covered by a continuous extracellular membrane of soluble and polymerized lipids called the cuticle, or cuticular membrane (CM). The cuticle is basically composed of waxes that are embedded within (intracuticular), or deposited on the surface of (epicuticular), a matrix of cutin; an insoluble and amorphous biopolymer that consists largely of hydroxylated and epoxy-hydroxylated C16 and C18 esterified fatty acids (Heredia, 2003). Cuticular waxes are mixtures of homologous series of long-chain aliphatics, such as alkanes, alcohols, aldehydes, fatty acids, and esters, together with varying amounts of cyclic compounds such as triterpenoids and hydroxycinnamic acid derivatives (Bianchi, 1995). In addition, it has been observed that when wax and cutin components are removed from isolated cuticle preparations, there is usually some residual material that, for many species, is predominantly polysaccharidic and represents the portion of the epidermal cell walls to which the cuticular membrane was attached (Jeffree, 2006). The main function ascribed to the CM is to minimize water loss (Riederer and Schreiber, 2001), but the CM also limits the loss of substances from plant internal tissues, protects the plant against physical, chemical, and biological attack and provides mechanical support to maintain plant organ integrity (Niklas, 1992).The mechanical and rheological properties of plant cuticles are thus of great interest to plant biologists (Bargel et al., 2006) and in some cases have considerable economic importance. For example, the cuticles of fruit such as tomato not only influences post-harvest shelf life but are also associated with quality traits disorders such as fruit cracking (Matas et al., 2004a; Edelman et al., 2005). Despite these well-established and diverse functional roles, only a few reports to date have addressed the structure–function relationship of the cuticle and many aspects of cuticle composition, organization, and biomechanical properties are poorly understood. Existing studies have typically focused on specific characteristics of cuticles from leaves or from fruits, such as tomato. For example, the biomechanical properties of CM isolated from leaves of several plant species and from tomato fruit were studied in some detail by Wiedemann and Neinhuis (1998), revealing large variation between species and that, importantly for tomato fruit, the CM provides structural support for those fruits without hard internal tissue. In addition, the viscoelastic nature of plant cuticles and their physiological importance has been characterized (Marga et al., 1991; Petracek and Bukovac, 1995; Round et al., 2000; Matas et al., 2005), as have the effects of hydration and temperature on the mechanical properties of the CM in isolated tomato fruit cuticles (Round et al., 2000; Edelmann et al., 2005; Matas et al., 2005). It has also been suggested that some biomechanical characteristics of the tomato fruit exocarp can be explained by the different degree of cuticle invagination to subepidermal cell walls present in various cultivars (Matas et al., 2004a). However, the relative contributions of specific components of plant cuticles, such as cutin and the polysaccharides incorporated in the cuticular matrix, are unknown. The CM is a complex composite biomaterial and a clearer picture of the molecular arrangement of the constituent components, as well as their physical and molecular properties, is needed. The present work focuses on the biomechanical behaviour and quantitative contribution of the two main components present in the isolated cuticles of tomato fruit; the bipolyester cutin, and the polysaccharide material that is intimately associated with the cuticle. The main objective was to evaluate the mechanical parameters of isolated tomato fruit cutin matrix and how they change under controlled conditions of humidity and temperature. The contribution of the polysaccharide fraction to the mechanical properties of the cuticle is inferred as the difference between cuticle and cutin mechanical parameters. A preliminary study of the polysaccharide chemical and molecular characteristics is also presented. Analyses of cuticles from two stages of tomato fruit development, mature green and red ripe, were performed, based on previous results indicating cuticle changes in these stages of development, as well as the fact that most tomato fruit cracking takes place during the ripening period in cherry tomatoes (Thiagu et al., 1993). In addition, since the accumulation of flavonoids in the cuticle is ripening-related, a comparison of these stages provided insight into the role of flavonoids on cuticle biology during fruit ripening. Materials and methods Culture and sampling Solanum lycopersicum L. plants (cv. Cascada) were grown in a commercial polyethylene greenhouse in Málaga, Spain (36°40’ N, 4°29’ W) from mid-February to mid-June 2004, without supplemental heating or lighting. Seeds were sown in vermiculite on 12 January and, when seedlings had developed five true leaves (14 February), 40 plants were transplanted one each into 20 l pots filled with sand (3 mm diameter). Pots were arranged in rows with 1 m between rows and 2 pots m−1 within rows. Standard nutrient solution (10 mM N, 7 mM K, 0.9 mM P, 5 mM Ca, 2 mM Mg, and microelements) was supplied to the plants from transplanting to maturity by an automatic trickle irrigation system that dispensed 2.0 l h−1 per plant. Plants were grown as single stems by removing side shoots at weekly intervals. Flowers were shaken daily to facilitate pollination, labelled at the time of blooming, and monitored subsequently to evaluate fruit development and ripening. Fifty fruits were selected at the mature green and red ripe stages for CM isolation and further quality studies. Cuticles were isolated from each fruit within 5 h from harvesting. Cuticle isolation The CM samples were enzymatically isolated from mature green and red ripe tomato fruit following the protocol of Orgell (1955), as modified by Yamada et al. (1965; Petracek and Bukovac, 1995) using an aqueous solution of a mixture of fungal cellulase (0.2% w/v, Sigma, St Louis, Missouri, USA) and pectinase (2.0% w/v, Sigma), and 1 mM NaN3 to prevent microbial growth in sodium citrate buffer (50 mM, pH 4.0). A vacuum was applied to facilitate enzyme penetration, and suspensions were incubated with continuous agitation at 30 °C for 7–10 d. The CM was then separated from the epidermis, rinsed in distilled water, and stored under dry conditions. Selective elimination of the hydrolysable compounds Polysaccharide material was selectively removed by immersion of the enzymatically isolated cuticles in anhydrous hydrogen fluoride (HF) in pyridine (Aldrich, Milwaukee, Wis. USA) for 4 h at 55 °C (Villena et al., 1999). Isolated cuticle samples were weighed before and after the treatment in order to evaluate the weight lost. Histological study Enzymatically isolated cuticles were fixed in a formaldehyde, acetic acid and ethanol solution (1:1:18 by vol.); dehydrated in a series of ethanol dilutions (70–95%), and embedded in commercial resin (Leica Historesin Embedding Kit, Heidelberg, Germany). Cross-sections were cut into 4 μm slices, stained with Calcofluor White and monitored using a fluorescence microscope (Nikon, Eclipse E800). Mechanical tests The CM mechanical properties were measured following the previous work of Matas et al. (2005), using an extensiometer equipped with a linear displacement transducer (Mitutoyo, Kawasaki, Japan) that was customized to work with the CM samples (resolution of ±1 μm). The equipment is very similar to that designed and reported by Kutschera and Schopfer (1986). Rectangular uniform segments (3 mm×9 mm) of isolated CM were removed using a metal block and inspected microscopically to confirm the absence of small cracks, before mechanical testing. The dry CM segments were fixed between the ends of two hollow stainless-steel needles, with a small amount of fast-drying super glue, such that the CM formed a plane surface (see Fig. 1 in Matas et al., 2005). A container was attached to the extensiometer so the samples could be equilibrated in a buffer solution of 20 mM sodium citrate (pH 3.2) with 1 mM NaN3 in order to inhibit bacterial and fungal growth (Kutschera and Schopfer, 1986; Petracek and Bukovac, 1995). The system was enclosed in an environment controlled chamber that allowed control of temperature and relative humidity (RH). Each CM sample was held inside the extensiometer chamber for at least 30 min to equilibrate the temperature and humidity with the medium before beginning the extension test. Fig. 1. Open in new tabDownload slide Sections of isolated cuticle (A) and cutin matrix obtained after treatment with anhydrous hydrogen fluoride (B). The samples were stained with Calcofluor White and visualized under UV light with a fluorescence microscope. Magnification ×40. Fig. 1. Open in new tabDownload slide Sections of isolated cuticle (A) and cutin matrix obtained after treatment with anhydrous hydrogen fluoride (B). The samples were stained with Calcofluor White and visualized under UV light with a fluorescence microscope. Magnification ×40. The cross-sectional area of the samples was measured by optical microscopy and image analysis software (Visilog v. 6.2, Noesis, France) and the length of the exposed surface of the sample between the two supports was measured before mechanical extension tests. The mechanical tests were performed as a transient creep test to determine the changes in length of a CM segment by maintaining samples in uniaxial tension, under a constant load, for 1200 s, during which time the longitudinal extension of each sample was recorded by a computer system every 3 s. Each sample was tested repeatedly using an ascending sequence of sustained tensile forces (from 0.098 N to breaking-point by 0.098 N load increments) without recovery time (Matas et al., 2005). To determine stresses, the tensile force exerted along the sample was divided by the representative cross-sectional area of the sample. To obtain the corresponding stress–strain curve and elastic modulus (E), the applied stress was plotted against the total change in length after 20 min. Breaking stress (σmax) and maximum strain (εmax) at the breaking stress were also determined for each sample. Strain-time and the corresponding stress–strain curves were calculated for a set of 5–7 samples of CM equilibrated at each combination of temperature (23 °C and 35 °C) and hydration (40% RH and wet). Polysaccharide isolation, characterization, and fractionation Isolated tomato fruit cuticles were dewaxed by refluxing the samples in chloroform:methanol (2:1, v/v) for 5 h at 50 °C. Further, the samples were thoroughly washed in methanol. Dewaxed isolated tomato fruit cuticles were submerged in a KOH (1%, w/w) methanolic solution at 35–40 °C for 48 h in order to hydrolyse the cutin matrix. After this time, residual material appeared as continuous and fragile films. The polysaccharide nature of this material was analysed by infrared techniques: Fourier-transform infrared spectroscopy (Perkin–Elmer 1760 Fourier-transform infrared spectrometer) and micro Raman spectroscopy (Renishaw Raman) using a 785 nm wavelength as excitation line. Intact samples of the residue were analysed by X-ray diffraction (Siemens D-501 diffractometer, Germany) using graphite-monochromatic CuKα radiation. The fractionation of the polysaccharides associated with tomato fruit isolated cuticles was based on previously described protocols to fractionate fruit cell wall polysaccharides (Redgwell et al., 1988; Huysamer et al., 1997). Between 60 mg and 70 mg of the above-mentioned polysaccharide fraction obtained from isolated tomato fruit cuticles was first continuously stirred in 30 ml of water for 2 d at room temperature. After centrifuging the sample at 6000 g for 15 min (twice), the supernatant was filtered through glass fibre paper and the pellet was stored for next step. The filtered extracts were dialysed extensively (7 kDa molecular mass cut-off) against distilled water at 4 °C for a week and the dialysed samples then frozen and lyophilized. The pellet obtained after the second centrifugation was then washed twice (by centrifugation) with a 0.05 M trans-1,2-diaminocyclohexane-N,N,N′N′-tetra-acetic acid solution, allowing the separation of the ionically bound pectin from the cuticle. Pectin that was covalently bound to the cuticle and the hemicellulose were then sequentially extracted, using a 0.05 M Na2CO3 solution, and a 4 M KOH solution, respectively. The final residue corresponded to a cellulose rich fraction. Each fraction was weighed and normalized with respect to the starting amount of polysaccharides associated with the isolated cuticle. Statistics Simple and multiple regression analyses and all pairwise comparisons of the E, breaking stress, and maximum stress means were used to determine whether the measured characteristics of the CM samples varied significantly, and predictably as a function of stage of fruit ripening, humidity, and/or temperature. All analyses were performed using the JMP software package (SAS Institute, Inc.). Data are presented as means and SE with a level of significance of 5% (P=0.05). Results Cutin matrix isolation and characterization Enzymatically isolated CM samples were treated with anhydrous hydrogen fluoride in pyridine selectively to eliminate any polysaccharide material attached or embedded in the cutin matrix, as was similarly used with isolated cuticles of Agave americana and Clivia miniata leaves (Villena et al., 1999). There are two main methods to remove polysaccharide material from isolated plant cuticles. The standard method commonly used in the literature is based in the hydrolysis of polysaccharides using HCl or H2SO4 aqueous solutions under chemically harsh conditions: high acid concentration, long time reflux, stirring, and high temperature. Under these conditions, the isolates obtained are fragmented and usually have microscopic holes. Moreover, reflux in H2SO4 aqueous solution at high temperatures chemically modified the phenolic material present in the cutin matrix of many plant cuticles. On the other hand, the removal of polysaccharides using HF is effective (Fig. 1), fast and, at the microscopical level, the isolates appear intact and their original size and shape remain without affecting the lipid remaining material. This point is important for further biomechanical tests. On the other hand, spectroscopical analyses of HF-treated samples always indicated that no modifications in the chemical nature of the cutin had occurred (data not shown). At the nanoscopical level, topographic studies using Atomic Force Microscopy (AFM) on cutin samples of different plant species subjected to HF treatment never revealed any noticeable structural changes or modifications (data not shown). The HF treatment resulted in a net weight loss of approximately 28% of the initial weight for both mature green and red ripe fruit cuticles. The reaction was monitored by Fourier-transform infrared spectroscopy (data not shown) and the infrared spectra of the treated cuticles confirmed the absence of absorbances that indicate polysaccharides. The procedure, due to the use of an organic solvent, also removed the epicuticular waxes of the corresponding cuticle samples (Villena et al., 1999); thus, in the case of the tomato fruit cuticles, a standard cutin matrix was obtained after treatment. The effectiveness of the procedure was also checked by microscopy analysis. Figure 1 shows control and treated CM after staining with Calcofluor White, a fluorescence probe to detect cellulose and hemicellulose. In cross-sections of control CM samples (Fig. 1A) the cellulose fibrils were mainly located bordering the internal zone of the cuticle where the cuticular material penetrated into the epidermis. By contrast, the white colour associated with Calcofluor fluorescence was absent in cross-sections of treated CM samples, this zone did not show Calcofluor fluorescence, indicating that polysaccharide materials were removed by the treatment (Fig. 1B). Mechanical behaviour of isolated tomato fruit cuticles and their cutin matrices Figure 2A shows representative examples of strain-time curves of red ripe (RR) tomato fruit cuticles and their corresponding cutin matrix at 23 °C and 40% RH. The time-course of creep of isolated cuticle (black line) showed two clear phases when loaded in tension by load increases of 0.098 N at intervals of 1200 s and finished with the instantaneous breaking of the sample. There was a first phase that lasted from 0–0.49 N of load (5 load increases) in which CM responded to each load by instantaneous extension (about 0.5% strain in every load) but with no further extension recorded until the next load was added; the strain in this phase was purely elastic. There was a second phase, at loads greater than 0.49 N, in which CM responded by instantaneous extension (elastic strain) and by some additional extension (viscoelastic strain) during the time that the load was maintained. The transition from elastic to viscoelastic was gradual with elastic strain predominating over viscoelastic strain from 0.49–0.784 N (5–8 loads) and viscoelastic being predominant at loads greater than 0.784 N. When strain-time curves for the corresponding RR cutin sample (dark grey line) at the same temperature and RH conditions were studied, the cutin showed only one phase similar to the second phase observed in the cuticle, with instantaneous extension (elastic strain) followed by a time-dependent additional extension (viscoelastic strain). Fig. 2. Open in new tabDownload slide Mechanical response of red ripe tomato fruit cuticle and cutin under tension at 23 °C. (A) Representative example of strain-time curve of isolated cuticle (black line) and cutin (grey line) samples at 40% RH. (B) Stress–strain diagrams of the isolated cuticle (black line) and cutin matrix (grey line) at 40% RH (black circle) and wet conditions (white circle). Fig. 2. Open in new tabDownload slide Mechanical response of red ripe tomato fruit cuticle and cutin under tension at 23 °C. (A) Representative example of strain-time curve of isolated cuticle (black line) and cutin (grey line) samples at 40% RH. (B) Stress–strain diagrams of the isolated cuticle (black line) and cutin matrix (grey line) at 40% RH (black circle) and wet conditions (white circle). Figure 2B shows representative examples of stress–strain curves of red ripe (RR) tomato fruit cuticles and their corresponding cutin matrix at 23 °C under two experimental hydration conditions: 40% RH and wet. A biphasic behaviour for the cuticle sample was observed in the stress–strain curve at 40% RH, where the relationship can be defined by two phases with different slopes (Fig. 2B). The stress–strain curves allowed the calculation of elastic modulus, E, from the slope of the linear elastic phase of the curve from 0% to about 5% strain. When the cuticle was submerged in aqueous solution, the stress–strain curves were monophasic, corresponding to viscoelastic strain (Matas et al., 2005). When polysaccharides were removed from the cuticle samples, the resulting curve was no longer biphasic. Cutin matrix samples consistently showed the same behaviour: their stress–strain curves were linear with noticeable differences in the slope for the two hydration conditions. At 35 °C, cuticles and their corresponding cutin showed similar curves to those at 23 °C, although the slopes were somehow lower, especially in the case of cuticle at 40% RH (data not shown). A comparison of isolated cuticles and cutin matrix from RR fruit revealed that the E value of the cutin matrix was substantially lower (92%) than that of the cuticle at 23 °C and 40% RH (Fig. 3A) and a similar differences was observed under wet conditions and at 35 °C. High humidity and temperature reduced cuticle E and, to a lesser extent, cutin E; the effect of RH was higher than the effect of temperature because, in wet conditions, the E of cuticle and cutin were clearly lower than at 40% RH. However, the effect of temperature on the cuticle E was relatively small under wet conditions and disappeared on the cutin E. Conversely, cuticle breaking stress showed a similar pattern, with far higher values for the cuticle than cutin under all the environmental conditions tested and lower values under high RH and temperature (Fig. 3B). Cutin strain was equal or higher than cuticle strain, the differences being significant at 40% RH but not under wet conditions (Fig. 3C). High relative humidity tended to result in high cuticle strain values and no temperature effect was observed. Fig. 3. Open in new tabDownload slide Elastic modulus (A), breaking stress (B), and strain (C) of isolated cuticle (grey bars) and cutin matrix (black bars) of red ripe fruit (RR). Tests were developed at two temperatures (23 °C and 35 °C) and two relative humidities (40% RH and wet). Data are presented as means ±standard error (SE); n=5–7. Fig. 3. Open in new tabDownload slide Elastic modulus (A), breaking stress (B), and strain (C) of isolated cuticle (grey bars) and cutin matrix (black bars) of red ripe fruit (RR). Tests were developed at two temperatures (23 °C and 35 °C) and two relative humidities (40% RH and wet). Data are presented as means ±standard error (SE); n=5–7. As with the RR samples, the four environmental conditions tested had less of an effect on the elastic modulus and strength of mature green (MG) cutin when compared to MG cuticle (Fig. 4A, B). High humidity reduced cuticle E and cuticle breaking stress of MG fruit, as was observed for RR cuticles. However, temperature had no effect on E of MG fruit cuticles. MG cutin strain values were higher than those of cuticles (Fig. 4C) and under wet conditions were higher than at 40% RH. Temperature had no influence on cuticle strain of MG fruit. Fig. 4. Open in new tabDownload slide Elastic modulus (A), breaking stress (B), and strain (C) of isolated cuticle (grey bars) and cutin matrix (black bars) of mature green fruit (MG). Tests were developed at two temperatures (23 °C and 35 °C) and two relative humidities (40% RH and wet). Data are presented as means ±standard error (SE); n=5–7. Fig. 4. Open in new tabDownload slide Elastic modulus (A), breaking stress (B), and strain (C) of isolated cuticle (grey bars) and cutin matrix (black bars) of mature green fruit (MG). Tests were developed at two temperatures (23 °C and 35 °C) and two relative humidities (40% RH and wet). Data are presented as means ±standard error (SE); n=5–7. The mechanical parameters of the isolated cutin matrix from MG and RR fruit are shown in Table 1. The significantly lower E of MG cutin compared to RR cutin samples was particularly notable, while the strain followed the opposite trend. The cutin strength of MG and RR fruit samples was similar except for 23 °C and 40% RH. High humidity (wet conditions) reduced E and strength at 23 °C, but had no significant effect at 35 °C. Similarly, high temperature (35 °C) reduced E and strength at 40% RH, but had no significant effect in wet conditions. Table 1. Elastic modulus, breaking stress, and strain of isolated cutin matrix from mature green (MG) and red ripe (RR) tomato fruit Elastic modulus (E, MPa) Breaking stress (σmax, MPa) Strain (εb, %) MG RR MG RR MG RR 23 °C–40% RH 15.0±1.6 45.0±5.4 6.0±0.6 12.3±1.4 37.7±5.3 27.0±5.3 23 °C–Wet 8.2±0.6 20.4±2.6 3.4±0.4 4.3±0.3 34.0±4.5 20.9±0.6 35 °C–40% RH 9.9±1.1 32.4±3.9 4.3±0.7 6.3±0.5 39.5±5.3 23.3±2.8 35 °C–Wet 8.0±0.3 22.3±7.3 4.3±0.3 4.5±0.9 49.1±3.9 15.9±1.4 Elastic modulus (E, MPa) Breaking stress (σmax, MPa) Strain (εb, %) MG RR MG RR MG RR 23 °C–40% RH 15.0±1.6 45.0±5.4 6.0±0.6 12.3±1.4 37.7±5.3 27.0±5.3 23 °C–Wet 8.2±0.6 20.4±2.6 3.4±0.4 4.3±0.3 34.0±4.5 20.9±0.6 35 °C–40% RH 9.9±1.1 32.4±3.9 4.3±0.7 6.3±0.5 39.5±5.3 23.3±2.8 35 °C–Wet 8.0±0.3 22.3±7.3 4.3±0.3 4.5±0.9 49.1±3.9 15.9±1.4 Tests were developed at two temperature (23 °C and 35 °C) and two relative humidity (40% RH and wet) conditions. Data are means ±SE; n=5–7. Open in new tab Table 1. Elastic modulus, breaking stress, and strain of isolated cutin matrix from mature green (MG) and red ripe (RR) tomato fruit Elastic modulus (E, MPa) Breaking stress (σmax, MPa) Strain (εb, %) MG RR MG RR MG RR 23 °C–40% RH 15.0±1.6 45.0±5.4 6.0±0.6 12.3±1.4 37.7±5.3 27.0±5.3 23 °C–Wet 8.2±0.6 20.4±2.6 3.4±0.4 4.3±0.3 34.0±4.5 20.9±0.6 35 °C–40% RH 9.9±1.1 32.4±3.9 4.3±0.7 6.3±0.5 39.5±5.3 23.3±2.8 35 °C–Wet 8.0±0.3 22.3±7.3 4.3±0.3 4.5±0.9 49.1±3.9 15.9±1.4 Elastic modulus (E, MPa) Breaking stress (σmax, MPa) Strain (εb, %) MG RR MG RR MG RR 23 °C–40% RH 15.0±1.6 45.0±5.4 6.0±0.6 12.3±1.4 37.7±5.3 27.0±5.3 23 °C–Wet 8.2±0.6 20.4±2.6 3.4±0.4 4.3±0.3 34.0±4.5 20.9±0.6 35 °C–40% RH 9.9±1.1 32.4±3.9 4.3±0.7 6.3±0.5 39.5±5.3 23.3±2.8 35 °C–Wet 8.0±0.3 22.3±7.3 4.3±0.3 4.5±0.9 49.1±3.9 15.9±1.4 Tests were developed at two temperature (23 °C and 35 °C) and two relative humidity (40% RH and wet) conditions. Data are means ±SE; n=5–7. Open in new tab Cuticle polysaccharide characterization and fractioning Continuous and fragile films of non-lipid compounds attached or incorporated into the cuticle were obtained after slow hydrolysis of dewaxed isolated cuticles from tomato fruits under mild experimental conditions (see Materials and methods). The morphological characteristics of the isolated cuticles were preserved in the films, with positive staining for cellulose and pectin (results not shown) and their Fourier-transform infrared and micro Raman spectra indicated a clear polysaccharidic nature (Fig. 5). The absence of an absorption band at 1730 cm−1 indicated the elimination of the ester links that characterize the polyester cutin and the presence of the clear ‘fingerprint’ polysaccharide infrared region around 1000 cm−1 validated the isolation procedure (Villena et al., 2000). The X-ray diffraction pattern of the samples showed a very low degree of crystallinity and basal orientation (data not shown). Fig. 5. Open in new tabDownload slide Fourier-transform infrared (A) and Raman (B) spectra of the polysaccharide fraction isolated from red ripe tomato fruit cuticles. See text for further explanations. Fig. 5. Open in new tabDownload slide Fourier-transform infrared (A) and Raman (B) spectra of the polysaccharide fraction isolated from red ripe tomato fruit cuticles. See text for further explanations. Chemical fractionation of the samples allowed a quantitative assessment of the relative amounts of pectin, hemicellulose and cellulose (Fig. 6). Figure 6 shows that polysaccharide ascribed to isolated cuticles from MG and RR tomatoes fruit had similar relative amounts of pectin, hemicellulose, and cellulose, the main polymers associated with plant cell walls. As far as we are aware this is the first report describing the polysaccharide composition present in cuticles. Fig. 6. Open in new tabDownload slide Percentages of pectin (white bar), hemicellulose (grey bar) and cellulose (black bar) of the polysaccharides associated with the mature green (MG) and red ripe (RR) tomato fruit cuticles. Fig. 6. Open in new tabDownload slide Percentages of pectin (white bar), hemicellulose (grey bar) and cellulose (black bar) of the polysaccharides associated with the mature green (MG) and red ripe (RR) tomato fruit cuticles. Discussion As it was presented in the Introduction, plant cuticles can be regarded as composite biopolymers mainly comprising an amorphous polyester (cutin) and small amounts of waxes, together with hydrolysable polysaccharides. The macromolecular arrangement of these biopolymers is of fundamental importance for both the elastic and viscoelastic behaviour of the CM; however, this information has been difficult to obtain (Heredia, 2003) and, in this sense, this study has been mainly focused on investigating the correlation between the CM components, their molecular structure, and biomechanical properties. In this complex scenario, one of the main objectives of this work has been to clarify the role of the cutin matrix, the major component of plant cuticles. As far as we are aware, this current study presents the first analysis of the mechanical properties of a polysaccharide-free cutin matrix. Previous results indicated that the mechanical properties of the isolated tomato fruit CM depend on the relative humidity and temperature (Matas et al., 2005; Edelman et al., 2005). The tensile modulus and maximum stress of the CM samples tested here varied with RH and temperature: the higher the RH and temperature the lower the stiffness and strength, confirming previous results (Edelman et al., 2005; Matas et al., 2005). Interestingly, the elastic modulus and strength of cutin show the same pattern with changes in environmental conditions. Moreover, all cutin samples investigated here showed monophasic stress–strain curves, a clear indication of a viscoelastic nature, with low elastic modulus and strength but high strain. The differences in elastic modulus and stress of the cutin matrix samples to temperature can be related, as was previously suggested for isolated cuticles (Matas et al., 2005), to the presence of a temperature transition in the cutin matrix. The existence of a second order, or glass, transition temperature around 30 °C in the tomato fruit cutin matrix of isolated tomato CM was previously reported by our laboratory by differential scanning calorimetry (Matas et al., 2004b). This glass transition could explain the mechanical behaviour of the tomato cuticles, namely a higher E modulus, associated with a glass state, below the transition temperature and a plastic behaviour, related to a more viscous state, above the transition temperature. Data obtained in the present study also gave insights into the ripening-related mechanical properties of the isolated cutin matrix. The elastic modulus was always significantly higher for the ripe cutin samples, regardless of the temperature and degree of hydration. Previous reports have indicated that the degree of polymerization and amounts of lipid cuticle constituents such as cuticular waxes, cutin monomers, and degree of cutin cross-linking are similar at the two developmental stages (Baker et al., 1982; Luque and Heredia, 1994), with the exception of the abundance and increase of phenolics compounds during ripening, which comprise mainly flavonoid precursors such as naringenin and chalconaringenin (Laguna et al., 1999). Some data indicate that these phenolics form molecular clusters, included or trapped between the amorphous cross-linking of the cutin network (Luque and Heredia, 1994; Laguna et al., 1999). Taken together with the results of this current study, it is proposed that the amount of phenolics in the cutin network is correlated with a more rigid cutin matrix, restricting segmental mobility of the polyester chains and possibly reducing the free volume within the network, thereby increasing the overall matrix rigidity. This increase in rigidity would make the cutin matrix less elastic during ripening, as is reflected by the low strain values of cutin samples from the red ripe samples. These results support the hypothesis made recently by Bargel et al. (2006) that the amount of phenolic compounds is correlated with a rigid cutin matrix at full maturity. The dramatic decrease in stiffness of the cuticle isolates after selective hydrolysis of the polysaccharide compounds strongly suggests that polysaccharides that are intimately associated with, and incorporated into, the cutin matrix contribute to the linear elastic behaviour of the whole cuticle and to the high modulus of stiff cuticles. This is particularly evident in dry conditions, although in wet conditions, in spite of the modulus value being significantly lower, the same conclusions apply. In contrast, the viscoelastic behaviour of the isolated cuticle, defined by low E modulus and high strain values, can be attributed to the cutin matrix fraction. The fact that the polysaccharide material ascribed to fruit cuticles is the major factor responsible for the elastic modulus and stiffness of these samples, gives to these compounds an important and critical role in the mechanical behaviour of cuticles and, subsequently, the epidermis of fruits and leaves. Polysaccharides are integral constituents of virtually all cuticles studied to date (Jeffree, 2006), although essentially nothing has previously been reported regarding their composition, molecular characteristics or their physical and chemical behaviour. Tomato fruit cuticles isolated from MG and RR fruit contain significant amounts of the three major polysaccharides classes, hemicellulose, cellulose, and pectin. The relative amounts of each are similar to those reported for sequentially extracted cell wall from the total pericarp of tomato fruit (O'Neill and York, 2003; Reinders and Thier, 1999). These data indicate that cuticle formation during fruit growth involves progressive cutinization of the epidermal cell wall. Cellulose microfibils are inherently stiff, especially under dry conditions, and a cutinized cell wall containing cellulose would certainly stiffen the cuticular membrane, as is indicated in models of fibre-reinforced composite materials (Courtney, 1990; Bargel et al., 2006). The mechanical parameters presented here agree well with this argument. In this sense, and in clear agreement with Matas et al. (2004a), differences in cell wall components associated with the cuticle provide different cultivar-specific mechanical properties of the tomato fruit cuticle. The results section also contains structural and molecular data on the polysaccharide fraction associated with tomato fruit cuticles that open new perspectives of research for the future. The X-ray diffraction pattern of the polysaccharide fraction showed a low degree of crystallinity, mainly assigned to cellulose, whereas detailed analysis of the micro Raman spectrum of this material confirmed its low crystallinity and orientation of the cellulose fibres revealed by the intensity ratio at 1462 cm−1 and 1481 cm−1 (Fischer et al., 2005) and at 1090 cm−1 and 1120 cm−1, according Edwards et al. (1997) and Jahn et al. (2002), respectively. The orientation of the polysaccharide fibres with respect to the force expanding the fruit is crucial to avoid cracking. The maximum contribution of the polysaccharide fibres to counteract the fruit expanding force would operate if the fibre axes were perpendicular to the force. To summarize the results presented in this paper, the structure of the tomato fruit cuticle at the supramolecular level can be envisaged as a lipid cutin matrix deposited and/or embedded in an epidermal cell wall constituted by long fibres that are mainly orientated at random. This complex mixture possesses both elastic and viscoelastic characteristics that can be attributed to the polysaccharide fraction and cutin matrix, respectively, and while the stiffness of the cuticle is primarily provided by the polysaccharides, the cutin matrix imparts plasticity. Certain processes such as fruit ripening can result in the introduction of new molecules (e.g. phenolics) into the cuticle that may increase the rigidity and reduce the stiffness. Nevertheless the category of composite material assigned to the cuticle does not exclude the possibility of the emergence of mechanical characteristics stronger than those of either individual component, although this aspect could not be investigated because it was not possible to measure the mechanical properties of the isolated polysaccharides. Abbreviations Abbreviations CM cuticular membrane E modulus of elasticity FT-IR Fourier-transform infrared spectroscopy MG mature green RR red ripe The authors would like to thank JKC Rose and HC Spatz whose comments helped to improve this paper. This work has been partially supported by grant AGL2006-12494 (Plan Nacional de I+D, Ministerio de Educación y Ciencia, Spain) and Fundación Cajamar (Almería, Spain). References Baker EA , Bukovac MJ , Hunt GM . Cutler DF , Alvin DF , Price CE . 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Biomechanics and anatomy of Lycopersicon esculentum fruit peels and enzyme-treated samples , American Journal of Botany , 2004 , vol. 91 (pg. 352 - 360 ) Google Scholar Crossref Search ADS PubMed WorldCat Matas AJ , Cuartero J , Heredia A . Phase transitions in the biopolyester cutin isolated from tomato fruit cuticles , Thermochimica Acta , 2004 , vol. 409 (pg. 165 - 168 ) Google Scholar Crossref Search ADS WorldCat Matas AJ , Lopez-Casado G , Cuartero J , Heredia A . Relative humidity and temperature modify the mechanical properties of isolated tomato fruit cuticles , American Journal of Botany , 2005 , vol. 92 (pg. 462 - 468 ) Google Scholar Crossref Search ADS PubMed WorldCat Niklas KJ . , Plant biomechanics. an engineering approach to plant form and function , 1992 Chicago, London The University of Chicago Press Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC O'Neill MA , York WS . Rose JKC . The composition and structure of plant cell walls , The plant cell wall , 2003 , vol. Vol. 8 Oxford Blackwell Publishing CRC Press (pg. 4 - 7 ) Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Orgell WH . The isolation of plant cuticle with pectic enzymes , Plant Physiology , 1955 , vol. 30 (pg. 78 - 80 ) Google Scholar Crossref Search ADS PubMed WorldCat Petracek PD , Bukovac MJ . Rheological properties of enzymatically iIsolated tomato fruit cuticle , Plant Physiology , 1995 , vol. 109 (pg. 675 - 679 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Redgwell RJ , Melton LD , Brasch DJ . Cell-wall polysaccharides of kiwifruit (Actinidia deliciosa): chemical features in different tissue zones of the fruit at harvest , Carbohydrate Research , 1988 , vol. 182 (pg. 241 - 258 ) Google Scholar Crossref Search ADS WorldCat Reinders G , Thier HP . Non-starch polysaccharides of tomatoes. I. Characterizing pectins and hemicelluloses , European Food Research and Technology , 1999 , vol. 209 (pg. 43 - 46 ) Google Scholar Crossref Search ADS WorldCat Riederer M , Schreiber L . Protecting against water loss: analysis of the barrier properties of plant cuticles , Journal of Experimental Botany , 2001 , vol. 52 (pg. 2023 - 2032 ) Google Scholar Crossref Search ADS PubMed WorldCat Round AN , Yan B , Dang S , Estephan R , Stark RE , Batteas JD . The influence of water on the nanomechanical behavior of the plant biopolyester cutin as studied by AFM and solid-state NMR , Biophysical Journal , 2000 , vol. 79 (pg. 2761 - 2767 ) Google Scholar Crossref Search ADS PubMed WorldCat Thiagu R , Chand N , Ramana KVR . Evolution of mechanical characteristics of tomatoes of 2 varieties during ripening , Journal of the Science of Food and Agriculture , 1993 , vol. 62 (pg. 175 - 183 ) Google Scholar Crossref Search ADS WorldCat Villena JF , Dominguez E , Stewart D , Heredia A . Characterization and biosynthesis of non-degradable polymers in plant cuticles , Planta , 1999 , vol. 208 (pg. 181 - 187 ) Google Scholar Crossref Search ADS PubMed WorldCat Villena JF , Dominguez E , Heredia A . Monitoring biopolymers present in plant cuticles by FT-IR spectroscopy , Journal of Plant Physiology , 2000 , vol. 156 (pg. 419 - 422 ) Google Scholar Crossref Search ADS WorldCat Wiedemann P , Neinhuis C . Biomechanics of isolated plant cuticles , Botanica Acta , 1998 , vol. 111 (pg. 28 - 34 ) Google Scholar Crossref Search ADS WorldCat Yamada Y , Wittwer SH , Bukovac MJ . Penetration of organic compounds through isolated cuticles with special reference to C14 urea , Plant Physiology , 1965 , vol. 40 (pg. 170 - 175 ) Google Scholar Crossref Search ADS PubMed WorldCat Author notes * Present address: Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA. © 2007 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) © 2007 The Author(s). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Biomechanics of isolated tomato (Solanum lycopersicum L.) fruit cuticles: the role of the cutin matrix and polysaccharides

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Oxford University Press
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Copyright © 2022 Society for Experimental Biology
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0022-0957
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10.1093/jxb/erm233
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17975209
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Abstract

Abstract The mechanical characteristics of the cuticular membrane (CM), a complex composite biopolymer basically composed of a cutin matrix, waxes, and hydrolysable polysaccharides, have been described previously. The biomechanical behaviour and quantitative contribution of cutin and polysaccharides have been investigated here using as experimental material mature green and red ripe tomato fruits. Treatment of isolated CM with anhydrous hydrogen fluoride in pyridine allowed the selective elimination of polysaccharides attached to or incrusted into the cutin matrix. Cutin samples showed a drastic decrease in elastic modulus and stiffness (up to 92%) compared with CM, which clearly indicates that polysaccharides incorporated into the cutin matrix are responsible for the elastic modulus, stiffness, and the linear elastic behaviour of the whole cuticle. Reciprocally, the viscoelastic behaviour of CM (low elastic modulus and high strain values) can be assigned to the cutin. These results applied both to mature green and red ripe CM. Cutin elastic modulus, independently of the degree of temperature and hydration, was always significantly higher for the ripe than for the green samples while strain was lower; the amount of phenolics in the cutin network are the main candidates to explain the increased rigidity from mature green to red ripe cutin. The polysaccharide families isolated from CM were pectin, hemicellulose, and cellulose, the main polymers associated with the plant cell wall. The three types of polysaccharides were present in similar amounts in CM from mature green and red ripe tomatoes. Physical techniques such as X-ray diffraction and Raman spectroscopy indicated that the polysaccharide fibres were mainly randomly oriented. A tomato fruit CM scenario at the supramolecular level that could explain the observed CM biomechanical properties is presented and discussed. Cuticle, cutin matrix, plant biomechanics, polysaccharides, Solanum lycopersicum Introduction Epidermal cells of the aerial parts of higher plants are covered by a continuous extracellular membrane of soluble and polymerized lipids called the cuticle, or cuticular membrane (CM). The cuticle is basically composed of waxes that are embedded within (intracuticular), or deposited on the surface of (epicuticular), a matrix of cutin; an insoluble and amorphous biopolymer that consists largely of hydroxylated and epoxy-hydroxylated C16 and C18 esterified fatty acids (Heredia, 2003). Cuticular waxes are mixtures of homologous series of long-chain aliphatics, such as alkanes, alcohols, aldehydes, fatty acids, and esters, together with varying amounts of cyclic compounds such as triterpenoids and hydroxycinnamic acid derivatives (Bianchi, 1995). In addition, it has been observed that when wax and cutin components are removed from isolated cuticle preparations, there is usually some residual material that, for many species, is predominantly polysaccharidic and represents the portion of the epidermal cell walls to which the cuticular membrane was attached (Jeffree, 2006). The main function ascribed to the CM is to minimize water loss (Riederer and Schreiber, 2001), but the CM also limits the loss of substances from plant internal tissues, protects the plant against physical, chemical, and biological attack and provides mechanical support to maintain plant organ integrity (Niklas, 1992).The mechanical and rheological properties of plant cuticles are thus of great interest to plant biologists (Bargel et al., 2006) and in some cases have considerable economic importance. For example, the cuticles of fruit such as tomato not only influences post-harvest shelf life but are also associated with quality traits disorders such as fruit cracking (Matas et al., 2004a; Edelman et al., 2005). Despite these well-established and diverse functional roles, only a few reports to date have addressed the structure–function relationship of the cuticle and many aspects of cuticle composition, organization, and biomechanical properties are poorly understood. Existing studies have typically focused on specific characteristics of cuticles from leaves or from fruits, such as tomato. For example, the biomechanical properties of CM isolated from leaves of several plant species and from tomato fruit were studied in some detail by Wiedemann and Neinhuis (1998), revealing large variation between species and that, importantly for tomato fruit, the CM provides structural support for those fruits without hard internal tissue. In addition, the viscoelastic nature of plant cuticles and their physiological importance has been characterized (Marga et al., 1991; Petracek and Bukovac, 1995; Round et al., 2000; Matas et al., 2005), as have the effects of hydration and temperature on the mechanical properties of the CM in isolated tomato fruit cuticles (Round et al., 2000; Edelmann et al., 2005; Matas et al., 2005). It has also been suggested that some biomechanical characteristics of the tomato fruit exocarp can be explained by the different degree of cuticle invagination to subepidermal cell walls present in various cultivars (Matas et al., 2004a). However, the relative contributions of specific components of plant cuticles, such as cutin and the polysaccharides incorporated in the cuticular matrix, are unknown. The CM is a complex composite biomaterial and a clearer picture of the molecular arrangement of the constituent components, as well as their physical and molecular properties, is needed. The present work focuses on the biomechanical behaviour and quantitative contribution of the two main components present in the isolated cuticles of tomato fruit; the bipolyester cutin, and the polysaccharide material that is intimately associated with the cuticle. The main objective was to evaluate the mechanical parameters of isolated tomato fruit cutin matrix and how they change under controlled conditions of humidity and temperature. The contribution of the polysaccharide fraction to the mechanical properties of the cuticle is inferred as the difference between cuticle and cutin mechanical parameters. A preliminary study of the polysaccharide chemical and molecular characteristics is also presented. Analyses of cuticles from two stages of tomato fruit development, mature green and red ripe, were performed, based on previous results indicating cuticle changes in these stages of development, as well as the fact that most tomato fruit cracking takes place during the ripening period in cherry tomatoes (Thiagu et al., 1993). In addition, since the accumulation of flavonoids in the cuticle is ripening-related, a comparison of these stages provided insight into the role of flavonoids on cuticle biology during fruit ripening. Materials and methods Culture and sampling Solanum lycopersicum L. plants (cv. Cascada) were grown in a commercial polyethylene greenhouse in Málaga, Spain (36°40’ N, 4°29’ W) from mid-February to mid-June 2004, without supplemental heating or lighting. Seeds were sown in vermiculite on 12 January and, when seedlings had developed five true leaves (14 February), 40 plants were transplanted one each into 20 l pots filled with sand (3 mm diameter). Pots were arranged in rows with 1 m between rows and 2 pots m−1 within rows. Standard nutrient solution (10 mM N, 7 mM K, 0.9 mM P, 5 mM Ca, 2 mM Mg, and microelements) was supplied to the plants from transplanting to maturity by an automatic trickle irrigation system that dispensed 2.0 l h−1 per plant. Plants were grown as single stems by removing side shoots at weekly intervals. Flowers were shaken daily to facilitate pollination, labelled at the time of blooming, and monitored subsequently to evaluate fruit development and ripening. Fifty fruits were selected at the mature green and red ripe stages for CM isolation and further quality studies. Cuticles were isolated from each fruit within 5 h from harvesting. Cuticle isolation The CM samples were enzymatically isolated from mature green and red ripe tomato fruit following the protocol of Orgell (1955), as modified by Yamada et al. (1965; Petracek and Bukovac, 1995) using an aqueous solution of a mixture of fungal cellulase (0.2% w/v, Sigma, St Louis, Missouri, USA) and pectinase (2.0% w/v, Sigma), and 1 mM NaN3 to prevent microbial growth in sodium citrate buffer (50 mM, pH 4.0). A vacuum was applied to facilitate enzyme penetration, and suspensions were incubated with continuous agitation at 30 °C for 7–10 d. The CM was then separated from the epidermis, rinsed in distilled water, and stored under dry conditions. Selective elimination of the hydrolysable compounds Polysaccharide material was selectively removed by immersion of the enzymatically isolated cuticles in anhydrous hydrogen fluoride (HF) in pyridine (Aldrich, Milwaukee, Wis. USA) for 4 h at 55 °C (Villena et al., 1999). Isolated cuticle samples were weighed before and after the treatment in order to evaluate the weight lost. Histological study Enzymatically isolated cuticles were fixed in a formaldehyde, acetic acid and ethanol solution (1:1:18 by vol.); dehydrated in a series of ethanol dilutions (70–95%), and embedded in commercial resin (Leica Historesin Embedding Kit, Heidelberg, Germany). Cross-sections were cut into 4 μm slices, stained with Calcofluor White and monitored using a fluorescence microscope (Nikon, Eclipse E800). Mechanical tests The CM mechanical properties were measured following the previous work of Matas et al. (2005), using an extensiometer equipped with a linear displacement transducer (Mitutoyo, Kawasaki, Japan) that was customized to work with the CM samples (resolution of ±1 μm). The equipment is very similar to that designed and reported by Kutschera and Schopfer (1986). Rectangular uniform segments (3 mm×9 mm) of isolated CM were removed using a metal block and inspected microscopically to confirm the absence of small cracks, before mechanical testing. The dry CM segments were fixed between the ends of two hollow stainless-steel needles, with a small amount of fast-drying super glue, such that the CM formed a plane surface (see Fig. 1 in Matas et al., 2005). A container was attached to the extensiometer so the samples could be equilibrated in a buffer solution of 20 mM sodium citrate (pH 3.2) with 1 mM NaN3 in order to inhibit bacterial and fungal growth (Kutschera and Schopfer, 1986; Petracek and Bukovac, 1995). The system was enclosed in an environment controlled chamber that allowed control of temperature and relative humidity (RH). Each CM sample was held inside the extensiometer chamber for at least 30 min to equilibrate the temperature and humidity with the medium before beginning the extension test. Fig. 1. Open in new tabDownload slide Sections of isolated cuticle (A) and cutin matrix obtained after treatment with anhydrous hydrogen fluoride (B). The samples were stained with Calcofluor White and visualized under UV light with a fluorescence microscope. Magnification ×40. Fig. 1. Open in new tabDownload slide Sections of isolated cuticle (A) and cutin matrix obtained after treatment with anhydrous hydrogen fluoride (B). The samples were stained with Calcofluor White and visualized under UV light with a fluorescence microscope. Magnification ×40. The cross-sectional area of the samples was measured by optical microscopy and image analysis software (Visilog v. 6.2, Noesis, France) and the length of the exposed surface of the sample between the two supports was measured before mechanical extension tests. The mechanical tests were performed as a transient creep test to determine the changes in length of a CM segment by maintaining samples in uniaxial tension, under a constant load, for 1200 s, during which time the longitudinal extension of each sample was recorded by a computer system every 3 s. Each sample was tested repeatedly using an ascending sequence of sustained tensile forces (from 0.098 N to breaking-point by 0.098 N load increments) without recovery time (Matas et al., 2005). To determine stresses, the tensile force exerted along the sample was divided by the representative cross-sectional area of the sample. To obtain the corresponding stress–strain curve and elastic modulus (E), the applied stress was plotted against the total change in length after 20 min. Breaking stress (σmax) and maximum strain (εmax) at the breaking stress were also determined for each sample. Strain-time and the corresponding stress–strain curves were calculated for a set of 5–7 samples of CM equilibrated at each combination of temperature (23 °C and 35 °C) and hydration (40% RH and wet). Polysaccharide isolation, characterization, and fractionation Isolated tomato fruit cuticles were dewaxed by refluxing the samples in chloroform:methanol (2:1, v/v) for 5 h at 50 °C. Further, the samples were thoroughly washed in methanol. Dewaxed isolated tomato fruit cuticles were submerged in a KOH (1%, w/w) methanolic solution at 35–40 °C for 48 h in order to hydrolyse the cutin matrix. After this time, residual material appeared as continuous and fragile films. The polysaccharide nature of this material was analysed by infrared techniques: Fourier-transform infrared spectroscopy (Perkin–Elmer 1760 Fourier-transform infrared spectrometer) and micro Raman spectroscopy (Renishaw Raman) using a 785 nm wavelength as excitation line. Intact samples of the residue were analysed by X-ray diffraction (Siemens D-501 diffractometer, Germany) using graphite-monochromatic CuKα radiation. The fractionation of the polysaccharides associated with tomato fruit isolated cuticles was based on previously described protocols to fractionate fruit cell wall polysaccharides (Redgwell et al., 1988; Huysamer et al., 1997). Between 60 mg and 70 mg of the above-mentioned polysaccharide fraction obtained from isolated tomato fruit cuticles was first continuously stirred in 30 ml of water for 2 d at room temperature. After centrifuging the sample at 6000 g for 15 min (twice), the supernatant was filtered through glass fibre paper and the pellet was stored for next step. The filtered extracts were dialysed extensively (7 kDa molecular mass cut-off) against distilled water at 4 °C for a week and the dialysed samples then frozen and lyophilized. The pellet obtained after the second centrifugation was then washed twice (by centrifugation) with a 0.05 M trans-1,2-diaminocyclohexane-N,N,N′N′-tetra-acetic acid solution, allowing the separation of the ionically bound pectin from the cuticle. Pectin that was covalently bound to the cuticle and the hemicellulose were then sequentially extracted, using a 0.05 M Na2CO3 solution, and a 4 M KOH solution, respectively. The final residue corresponded to a cellulose rich fraction. Each fraction was weighed and normalized with respect to the starting amount of polysaccharides associated with the isolated cuticle. Statistics Simple and multiple regression analyses and all pairwise comparisons of the E, breaking stress, and maximum stress means were used to determine whether the measured characteristics of the CM samples varied significantly, and predictably as a function of stage of fruit ripening, humidity, and/or temperature. All analyses were performed using the JMP software package (SAS Institute, Inc.). Data are presented as means and SE with a level of significance of 5% (P=0.05). Results Cutin matrix isolation and characterization Enzymatically isolated CM samples were treated with anhydrous hydrogen fluoride in pyridine selectively to eliminate any polysaccharide material attached or embedded in the cutin matrix, as was similarly used with isolated cuticles of Agave americana and Clivia miniata leaves (Villena et al., 1999). There are two main methods to remove polysaccharide material from isolated plant cuticles. The standard method commonly used in the literature is based in the hydrolysis of polysaccharides using HCl or H2SO4 aqueous solutions under chemically harsh conditions: high acid concentration, long time reflux, stirring, and high temperature. Under these conditions, the isolates obtained are fragmented and usually have microscopic holes. Moreover, reflux in H2SO4 aqueous solution at high temperatures chemically modified the phenolic material present in the cutin matrix of many plant cuticles. On the other hand, the removal of polysaccharides using HF is effective (Fig. 1), fast and, at the microscopical level, the isolates appear intact and their original size and shape remain without affecting the lipid remaining material. This point is important for further biomechanical tests. On the other hand, spectroscopical analyses of HF-treated samples always indicated that no modifications in the chemical nature of the cutin had occurred (data not shown). At the nanoscopical level, topographic studies using Atomic Force Microscopy (AFM) on cutin samples of different plant species subjected to HF treatment never revealed any noticeable structural changes or modifications (data not shown). The HF treatment resulted in a net weight loss of approximately 28% of the initial weight for both mature green and red ripe fruit cuticles. The reaction was monitored by Fourier-transform infrared spectroscopy (data not shown) and the infrared spectra of the treated cuticles confirmed the absence of absorbances that indicate polysaccharides. The procedure, due to the use of an organic solvent, also removed the epicuticular waxes of the corresponding cuticle samples (Villena et al., 1999); thus, in the case of the tomato fruit cuticles, a standard cutin matrix was obtained after treatment. The effectiveness of the procedure was also checked by microscopy analysis. Figure 1 shows control and treated CM after staining with Calcofluor White, a fluorescence probe to detect cellulose and hemicellulose. In cross-sections of control CM samples (Fig. 1A) the cellulose fibrils were mainly located bordering the internal zone of the cuticle where the cuticular material penetrated into the epidermis. By contrast, the white colour associated with Calcofluor fluorescence was absent in cross-sections of treated CM samples, this zone did not show Calcofluor fluorescence, indicating that polysaccharide materials were removed by the treatment (Fig. 1B). Mechanical behaviour of isolated tomato fruit cuticles and their cutin matrices Figure 2A shows representative examples of strain-time curves of red ripe (RR) tomato fruit cuticles and their corresponding cutin matrix at 23 °C and 40% RH. The time-course of creep of isolated cuticle (black line) showed two clear phases when loaded in tension by load increases of 0.098 N at intervals of 1200 s and finished with the instantaneous breaking of the sample. There was a first phase that lasted from 0–0.49 N of load (5 load increases) in which CM responded to each load by instantaneous extension (about 0.5% strain in every load) but with no further extension recorded until the next load was added; the strain in this phase was purely elastic. There was a second phase, at loads greater than 0.49 N, in which CM responded by instantaneous extension (elastic strain) and by some additional extension (viscoelastic strain) during the time that the load was maintained. The transition from elastic to viscoelastic was gradual with elastic strain predominating over viscoelastic strain from 0.49–0.784 N (5–8 loads) and viscoelastic being predominant at loads greater than 0.784 N. When strain-time curves for the corresponding RR cutin sample (dark grey line) at the same temperature and RH conditions were studied, the cutin showed only one phase similar to the second phase observed in the cuticle, with instantaneous extension (elastic strain) followed by a time-dependent additional extension (viscoelastic strain). Fig. 2. Open in new tabDownload slide Mechanical response of red ripe tomato fruit cuticle and cutin under tension at 23 °C. (A) Representative example of strain-time curve of isolated cuticle (black line) and cutin (grey line) samples at 40% RH. (B) Stress–strain diagrams of the isolated cuticle (black line) and cutin matrix (grey line) at 40% RH (black circle) and wet conditions (white circle). Fig. 2. Open in new tabDownload slide Mechanical response of red ripe tomato fruit cuticle and cutin under tension at 23 °C. (A) Representative example of strain-time curve of isolated cuticle (black line) and cutin (grey line) samples at 40% RH. (B) Stress–strain diagrams of the isolated cuticle (black line) and cutin matrix (grey line) at 40% RH (black circle) and wet conditions (white circle). Figure 2B shows representative examples of stress–strain curves of red ripe (RR) tomato fruit cuticles and their corresponding cutin matrix at 23 °C under two experimental hydration conditions: 40% RH and wet. A biphasic behaviour for the cuticle sample was observed in the stress–strain curve at 40% RH, where the relationship can be defined by two phases with different slopes (Fig. 2B). The stress–strain curves allowed the calculation of elastic modulus, E, from the slope of the linear elastic phase of the curve from 0% to about 5% strain. When the cuticle was submerged in aqueous solution, the stress–strain curves were monophasic, corresponding to viscoelastic strain (Matas et al., 2005). When polysaccharides were removed from the cuticle samples, the resulting curve was no longer biphasic. Cutin matrix samples consistently showed the same behaviour: their stress–strain curves were linear with noticeable differences in the slope for the two hydration conditions. At 35 °C, cuticles and their corresponding cutin showed similar curves to those at 23 °C, although the slopes were somehow lower, especially in the case of cuticle at 40% RH (data not shown). A comparison of isolated cuticles and cutin matrix from RR fruit revealed that the E value of the cutin matrix was substantially lower (92%) than that of the cuticle at 23 °C and 40% RH (Fig. 3A) and a similar differences was observed under wet conditions and at 35 °C. High humidity and temperature reduced cuticle E and, to a lesser extent, cutin E; the effect of RH was higher than the effect of temperature because, in wet conditions, the E of cuticle and cutin were clearly lower than at 40% RH. However, the effect of temperature on the cuticle E was relatively small under wet conditions and disappeared on the cutin E. Conversely, cuticle breaking stress showed a similar pattern, with far higher values for the cuticle than cutin under all the environmental conditions tested and lower values under high RH and temperature (Fig. 3B). Cutin strain was equal or higher than cuticle strain, the differences being significant at 40% RH but not under wet conditions (Fig. 3C). High relative humidity tended to result in high cuticle strain values and no temperature effect was observed. Fig. 3. Open in new tabDownload slide Elastic modulus (A), breaking stress (B), and strain (C) of isolated cuticle (grey bars) and cutin matrix (black bars) of red ripe fruit (RR). Tests were developed at two temperatures (23 °C and 35 °C) and two relative humidities (40% RH and wet). Data are presented as means ±standard error (SE); n=5–7. Fig. 3. Open in new tabDownload slide Elastic modulus (A), breaking stress (B), and strain (C) of isolated cuticle (grey bars) and cutin matrix (black bars) of red ripe fruit (RR). Tests were developed at two temperatures (23 °C and 35 °C) and two relative humidities (40% RH and wet). Data are presented as means ±standard error (SE); n=5–7. As with the RR samples, the four environmental conditions tested had less of an effect on the elastic modulus and strength of mature green (MG) cutin when compared to MG cuticle (Fig. 4A, B). High humidity reduced cuticle E and cuticle breaking stress of MG fruit, as was observed for RR cuticles. However, temperature had no effect on E of MG fruit cuticles. MG cutin strain values were higher than those of cuticles (Fig. 4C) and under wet conditions were higher than at 40% RH. Temperature had no influence on cuticle strain of MG fruit. Fig. 4. Open in new tabDownload slide Elastic modulus (A), breaking stress (B), and strain (C) of isolated cuticle (grey bars) and cutin matrix (black bars) of mature green fruit (MG). Tests were developed at two temperatures (23 °C and 35 °C) and two relative humidities (40% RH and wet). Data are presented as means ±standard error (SE); n=5–7. Fig. 4. Open in new tabDownload slide Elastic modulus (A), breaking stress (B), and strain (C) of isolated cuticle (grey bars) and cutin matrix (black bars) of mature green fruit (MG). Tests were developed at two temperatures (23 °C and 35 °C) and two relative humidities (40% RH and wet). Data are presented as means ±standard error (SE); n=5–7. The mechanical parameters of the isolated cutin matrix from MG and RR fruit are shown in Table 1. The significantly lower E of MG cutin compared to RR cutin samples was particularly notable, while the strain followed the opposite trend. The cutin strength of MG and RR fruit samples was similar except for 23 °C and 40% RH. High humidity (wet conditions) reduced E and strength at 23 °C, but had no significant effect at 35 °C. Similarly, high temperature (35 °C) reduced E and strength at 40% RH, but had no significant effect in wet conditions. Table 1. Elastic modulus, breaking stress, and strain of isolated cutin matrix from mature green (MG) and red ripe (RR) tomato fruit Elastic modulus (E, MPa) Breaking stress (σmax, MPa) Strain (εb, %) MG RR MG RR MG RR 23 °C–40% RH 15.0±1.6 45.0±5.4 6.0±0.6 12.3±1.4 37.7±5.3 27.0±5.3 23 °C–Wet 8.2±0.6 20.4±2.6 3.4±0.4 4.3±0.3 34.0±4.5 20.9±0.6 35 °C–40% RH 9.9±1.1 32.4±3.9 4.3±0.7 6.3±0.5 39.5±5.3 23.3±2.8 35 °C–Wet 8.0±0.3 22.3±7.3 4.3±0.3 4.5±0.9 49.1±3.9 15.9±1.4 Elastic modulus (E, MPa) Breaking stress (σmax, MPa) Strain (εb, %) MG RR MG RR MG RR 23 °C–40% RH 15.0±1.6 45.0±5.4 6.0±0.6 12.3±1.4 37.7±5.3 27.0±5.3 23 °C–Wet 8.2±0.6 20.4±2.6 3.4±0.4 4.3±0.3 34.0±4.5 20.9±0.6 35 °C–40% RH 9.9±1.1 32.4±3.9 4.3±0.7 6.3±0.5 39.5±5.3 23.3±2.8 35 °C–Wet 8.0±0.3 22.3±7.3 4.3±0.3 4.5±0.9 49.1±3.9 15.9±1.4 Tests were developed at two temperature (23 °C and 35 °C) and two relative humidity (40% RH and wet) conditions. Data are means ±SE; n=5–7. Open in new tab Table 1. Elastic modulus, breaking stress, and strain of isolated cutin matrix from mature green (MG) and red ripe (RR) tomato fruit Elastic modulus (E, MPa) Breaking stress (σmax, MPa) Strain (εb, %) MG RR MG RR MG RR 23 °C–40% RH 15.0±1.6 45.0±5.4 6.0±0.6 12.3±1.4 37.7±5.3 27.0±5.3 23 °C–Wet 8.2±0.6 20.4±2.6 3.4±0.4 4.3±0.3 34.0±4.5 20.9±0.6 35 °C–40% RH 9.9±1.1 32.4±3.9 4.3±0.7 6.3±0.5 39.5±5.3 23.3±2.8 35 °C–Wet 8.0±0.3 22.3±7.3 4.3±0.3 4.5±0.9 49.1±3.9 15.9±1.4 Elastic modulus (E, MPa) Breaking stress (σmax, MPa) Strain (εb, %) MG RR MG RR MG RR 23 °C–40% RH 15.0±1.6 45.0±5.4 6.0±0.6 12.3±1.4 37.7±5.3 27.0±5.3 23 °C–Wet 8.2±0.6 20.4±2.6 3.4±0.4 4.3±0.3 34.0±4.5 20.9±0.6 35 °C–40% RH 9.9±1.1 32.4±3.9 4.3±0.7 6.3±0.5 39.5±5.3 23.3±2.8 35 °C–Wet 8.0±0.3 22.3±7.3 4.3±0.3 4.5±0.9 49.1±3.9 15.9±1.4 Tests were developed at two temperature (23 °C and 35 °C) and two relative humidity (40% RH and wet) conditions. Data are means ±SE; n=5–7. Open in new tab Cuticle polysaccharide characterization and fractioning Continuous and fragile films of non-lipid compounds attached or incorporated into the cuticle were obtained after slow hydrolysis of dewaxed isolated cuticles from tomato fruits under mild experimental conditions (see Materials and methods). The morphological characteristics of the isolated cuticles were preserved in the films, with positive staining for cellulose and pectin (results not shown) and their Fourier-transform infrared and micro Raman spectra indicated a clear polysaccharidic nature (Fig. 5). The absence of an absorption band at 1730 cm−1 indicated the elimination of the ester links that characterize the polyester cutin and the presence of the clear ‘fingerprint’ polysaccharide infrared region around 1000 cm−1 validated the isolation procedure (Villena et al., 2000). The X-ray diffraction pattern of the samples showed a very low degree of crystallinity and basal orientation (data not shown). Fig. 5. Open in new tabDownload slide Fourier-transform infrared (A) and Raman (B) spectra of the polysaccharide fraction isolated from red ripe tomato fruit cuticles. See text for further explanations. Fig. 5. Open in new tabDownload slide Fourier-transform infrared (A) and Raman (B) spectra of the polysaccharide fraction isolated from red ripe tomato fruit cuticles. See text for further explanations. Chemical fractionation of the samples allowed a quantitative assessment of the relative amounts of pectin, hemicellulose and cellulose (Fig. 6). Figure 6 shows that polysaccharide ascribed to isolated cuticles from MG and RR tomatoes fruit had similar relative amounts of pectin, hemicellulose, and cellulose, the main polymers associated with plant cell walls. As far as we are aware this is the first report describing the polysaccharide composition present in cuticles. Fig. 6. Open in new tabDownload slide Percentages of pectin (white bar), hemicellulose (grey bar) and cellulose (black bar) of the polysaccharides associated with the mature green (MG) and red ripe (RR) tomato fruit cuticles. Fig. 6. Open in new tabDownload slide Percentages of pectin (white bar), hemicellulose (grey bar) and cellulose (black bar) of the polysaccharides associated with the mature green (MG) and red ripe (RR) tomato fruit cuticles. Discussion As it was presented in the Introduction, plant cuticles can be regarded as composite biopolymers mainly comprising an amorphous polyester (cutin) and small amounts of waxes, together with hydrolysable polysaccharides. The macromolecular arrangement of these biopolymers is of fundamental importance for both the elastic and viscoelastic behaviour of the CM; however, this information has been difficult to obtain (Heredia, 2003) and, in this sense, this study has been mainly focused on investigating the correlation between the CM components, their molecular structure, and biomechanical properties. In this complex scenario, one of the main objectives of this work has been to clarify the role of the cutin matrix, the major component of plant cuticles. As far as we are aware, this current study presents the first analysis of the mechanical properties of a polysaccharide-free cutin matrix. Previous results indicated that the mechanical properties of the isolated tomato fruit CM depend on the relative humidity and temperature (Matas et al., 2005; Edelman et al., 2005). The tensile modulus and maximum stress of the CM samples tested here varied with RH and temperature: the higher the RH and temperature the lower the stiffness and strength, confirming previous results (Edelman et al., 2005; Matas et al., 2005). Interestingly, the elastic modulus and strength of cutin show the same pattern with changes in environmental conditions. Moreover, all cutin samples investigated here showed monophasic stress–strain curves, a clear indication of a viscoelastic nature, with low elastic modulus and strength but high strain. The differences in elastic modulus and stress of the cutin matrix samples to temperature can be related, as was previously suggested for isolated cuticles (Matas et al., 2005), to the presence of a temperature transition in the cutin matrix. The existence of a second order, or glass, transition temperature around 30 °C in the tomato fruit cutin matrix of isolated tomato CM was previously reported by our laboratory by differential scanning calorimetry (Matas et al., 2004b). This glass transition could explain the mechanical behaviour of the tomato cuticles, namely a higher E modulus, associated with a glass state, below the transition temperature and a plastic behaviour, related to a more viscous state, above the transition temperature. Data obtained in the present study also gave insights into the ripening-related mechanical properties of the isolated cutin matrix. The elastic modulus was always significantly higher for the ripe cutin samples, regardless of the temperature and degree of hydration. Previous reports have indicated that the degree of polymerization and amounts of lipid cuticle constituents such as cuticular waxes, cutin monomers, and degree of cutin cross-linking are similar at the two developmental stages (Baker et al., 1982; Luque and Heredia, 1994), with the exception of the abundance and increase of phenolics compounds during ripening, which comprise mainly flavonoid precursors such as naringenin and chalconaringenin (Laguna et al., 1999). Some data indicate that these phenolics form molecular clusters, included or trapped between the amorphous cross-linking of the cutin network (Luque and Heredia, 1994; Laguna et al., 1999). Taken together with the results of this current study, it is proposed that the amount of phenolics in the cutin network is correlated with a more rigid cutin matrix, restricting segmental mobility of the polyester chains and possibly reducing the free volume within the network, thereby increasing the overall matrix rigidity. This increase in rigidity would make the cutin matrix less elastic during ripening, as is reflected by the low strain values of cutin samples from the red ripe samples. These results support the hypothesis made recently by Bargel et al. (2006) that the amount of phenolic compounds is correlated with a rigid cutin matrix at full maturity. The dramatic decrease in stiffness of the cuticle isolates after selective hydrolysis of the polysaccharide compounds strongly suggests that polysaccharides that are intimately associated with, and incorporated into, the cutin matrix contribute to the linear elastic behaviour of the whole cuticle and to the high modulus of stiff cuticles. This is particularly evident in dry conditions, although in wet conditions, in spite of the modulus value being significantly lower, the same conclusions apply. In contrast, the viscoelastic behaviour of the isolated cuticle, defined by low E modulus and high strain values, can be attributed to the cutin matrix fraction. The fact that the polysaccharide material ascribed to fruit cuticles is the major factor responsible for the elastic modulus and stiffness of these samples, gives to these compounds an important and critical role in the mechanical behaviour of cuticles and, subsequently, the epidermis of fruits and leaves. Polysaccharides are integral constituents of virtually all cuticles studied to date (Jeffree, 2006), although essentially nothing has previously been reported regarding their composition, molecular characteristics or their physical and chemical behaviour. Tomato fruit cuticles isolated from MG and RR fruit contain significant amounts of the three major polysaccharides classes, hemicellulose, cellulose, and pectin. The relative amounts of each are similar to those reported for sequentially extracted cell wall from the total pericarp of tomato fruit (O'Neill and York, 2003; Reinders and Thier, 1999). These data indicate that cuticle formation during fruit growth involves progressive cutinization of the epidermal cell wall. Cellulose microfibils are inherently stiff, especially under dry conditions, and a cutinized cell wall containing cellulose would certainly stiffen the cuticular membrane, as is indicated in models of fibre-reinforced composite materials (Courtney, 1990; Bargel et al., 2006). The mechanical parameters presented here agree well with this argument. In this sense, and in clear agreement with Matas et al. (2004a), differences in cell wall components associated with the cuticle provide different cultivar-specific mechanical properties of the tomato fruit cuticle. The results section also contains structural and molecular data on the polysaccharide fraction associated with tomato fruit cuticles that open new perspectives of research for the future. The X-ray diffraction pattern of the polysaccharide fraction showed a low degree of crystallinity, mainly assigned to cellulose, whereas detailed analysis of the micro Raman spectrum of this material confirmed its low crystallinity and orientation of the cellulose fibres revealed by the intensity ratio at 1462 cm−1 and 1481 cm−1 (Fischer et al., 2005) and at 1090 cm−1 and 1120 cm−1, according Edwards et al. (1997) and Jahn et al. (2002), respectively. The orientation of the polysaccharide fibres with respect to the force expanding the fruit is crucial to avoid cracking. The maximum contribution of the polysaccharide fibres to counteract the fruit expanding force would operate if the fibre axes were perpendicular to the force. To summarize the results presented in this paper, the structure of the tomato fruit cuticle at the supramolecular level can be envisaged as a lipid cutin matrix deposited and/or embedded in an epidermal cell wall constituted by long fibres that are mainly orientated at random. This complex mixture possesses both elastic and viscoelastic characteristics that can be attributed to the polysaccharide fraction and cutin matrix, respectively, and while the stiffness of the cuticle is primarily provided by the polysaccharides, the cutin matrix imparts plasticity. Certain processes such as fruit ripening can result in the introduction of new molecules (e.g. phenolics) into the cuticle that may increase the rigidity and reduce the stiffness. 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This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) © 2007 The Author(s).

Journal

Journal of Experimental Botany – Oxford University Press

Published: Nov 1, 2007

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