Cuticle biomechanics during fruit growth: the importance of the cutin:polysaccharide ratio
Cuticle deposition during growth has been shown to follow different patterns in different species. For example, cuticle deposition in cherry (Prunus avium) occurred at early stages of development and the amount of cuticle decreased during fruit expansion (Peschel et al., 2007). In tomato, two different patterns have been identified, one for cherry tomatoes and another for medium-sized tomatoes. In cherry tomatoes, such as ‘Cascada’, cuticle is accumulated in the early stages of development, and a constant amount of cuticle is then maintained throughout development (Domínguez et al., 2008). In contrast, in medium tomatoes such as ‘Moneymaker’ the amount of cuticle increases during the whole growing and ripening period (Baker et al., 1982; Luque & Heredia, 1994). Table S2 shows the amount of ‘Moneymaker’ cuticle and its components during fruit growth and ripening. In accordance with the findings of previous studies, the amount (in micrograms) of cuticle, cutin, polysaccharides and waxes increased during the whole period until they reached their maxima at the red ripe stage. The cutin:polysaccharide ratio increased during development, indicating a preferential accumulation of lipid material over polysaccharides. At 15 daa, an unusual 1 : 1 ratio between cutin and polysaccharides was observed, but it was modified in the following stages mainly as a result of the accumulation of cutin and waxes. Although there is scarce literature on this topic, our results agree with those of Baker et al. (1982) and Luque & Heredia (1994), where the ratio between cutin and polysaccharides was close to 1: 1 at early stages of tomato fruit development but dramatically changed in later stages. In cherry tomato, as expected from the different pattern of cuticle accumulation, no significant changes in the cutin:polysaccharide ratio were observed at the early stages of development (Domínguez et al., 2008).
These differences in cuticle deposition during development between medium-sized and cherry tomatoes can lead to differences in their biomechanical behaviour. Several parameters are known to influence extensibility, such as density, thickness, the chemical nature and fraction of each component, and the bonds linking the different molecules (Geitmann & Ortega, 2009). Organ growth is the result of two processes: cell division that, in the case of tomato fruit, is mostly confined to the earliest stages of development, and cell expansion (Ho & Hewitt, 1986; Bertin et al., 2007). The latter is believed to be responsible for most of the organ growth, and is driven by the yielding of the cell wall that generates an irreversible increase in cell volume (Schopfer, 2006). The cuticle exhibited only elastic, reversible deformation at the earliest stage of development studied in both genotypes. This is in agreement with a period of cell division with no cell enlargement (Bertin et al., 2007; Domínguez et al., 2008). In this period, the cuticle was very thin with low amounts of material accumulated (Domínguez et al., 2008; Table S2). A viscoelastic, irreversible behaviour appeared at the next stage studied (15 daa in ‘Cascada’, and 25 daa in ‘Moneymaker’) and corresponded with the beginning of cell enlargement. Although Bertin et al. (2007) reported that cell division ceased in medium-sized tomatoes c. 30 daa, in ‘Moneymaker’, epidermal cells had already significantly decreased their number per unit surface area and increased in size at 25 daa (data not shown). In contrast, Knoche et al. (2004) found only plastic deformation in the cuticle at early stages of cherry growth while elastic deformation appeared later in development. The mechanism by which the cell wall switches between elastic and viscoelastic deformation and its genetic control are still unclear (Burgert & Keplinger, 2013). The presence of viscoelastic deformation was related to a significant accumulation of cuticle material, with twofold increases being found in both ‘Cascada’ and ‘Moneymaker’. This accumulation was accompanied by an increase in cuticle thickness, density, stiffness and deformability, suggesting a more compacted and stiff yet deformable material. This increase in cuticle density was transient, as the cuticle density decreased later, and seemed to mark a period of massive accumulation of material that was rearranged in the following stages.
Once the stage of biphasic behaviour (elastic at low stresses and viscoelastic at higher stresses) had been reached, the cuticle exhibited little biomechanical change during the remainder of the fruit expansion period: its stiffness, breaking stress and maximum strain remained constant. These results are in agreement with those of Bargel & Neinhuis (2005), who found no biomechanical differences between the cuticles of immature and mature green tomato fruits. Takahashi et al. (2012), in contrast, observed a continued increase in cuticle stiffness and breaking stress together with a decrease in maximum strain during S. alba leaf expansion. However, some differences in the yield stress and strain were observed during fruit expansion (see Fig. 1). It is known that the yield threshold is not a static mechanical property but is spatially and temporally modulated by the cell (Dumais et al., 2006). These changes corresponded to a decrease in the elastic phase at 25 and 35 daa in ‘Cascada’ and 35 daa in ‘Moneymaker’ which did not affect the final breaking stress or maximum strain; hence the duration of the viscoelastic phase was increased. It should be pointed out that Young's modulus was not modified, as both the stress and the strain at the yield point were decreased. Interestingly, at these stages the cutin : polysaccharide ratio was temporarily increased and the cuticle had more cutin than polysaccharides (see data in Domínguez et al. (2008), and Table S2). Cutin has been shown to be mostly viscoelastic, whereas polysaccharides confer stiffness to the cuticle (López-Casado et al., 2007). Thus, our data indicate that the elastic phase is susceptible not only to the amount of polysaccharides (López-Casado et al., 2007; Tsubaki et al., 2012) but also to subtle changes in the ratio between the main cuticle components. This implies a role of the cutin:polysaccharide ratio in the modulation of cuticle viscoelastic deformation, thus contributing to cell enlargement. This is an important topic with clear biological implications that deserves further research.
As was mentioned in the Discussion, density is important in determining polymer extensibility. Nevertheless, there is very little literature on this topic. Schreiber & Schonherr (1990) used a pycnometer to calculate the density of the tomato fruit cuticle. This method has the inconvenience of the substantial amount of cuticle needed per replicate. Takahashi et al. (2012) estimated cuticle density in S. alba leaves using the average cuticle thickness. Yet, in tomato fruit, cuticle pegs and cell invaginations are of significant size and vary between stages and genotypes; hence the use of thickness would lead to an overestimation of cuticle density. The methodology employed in this study takes both invaginations and pegs into account and needs only small amounts of cuticle, allowing a large number of replicates. Our results showed that tomato fruit cuticle density varied between 0.7 and 1.3 g cm−3 depending on the genotype and stage of development. These results are in accordance with those obtained by Schreiber & Schonherr (1990), who calculated a density of 1.0–1.3 g cm−3 for tomato fruit.
The ‘Cascada’ cuticle showed a significant but transient increase in cuticle stiffness and breaking stress at the first stages of cell expansion (15 and 20 dda) that was not observed in ‘Moneymaker’. This increase was related to a higher density at 15 and 20 daa compared with the following stages, which suggests that a more compacted material could be responsible for this change in stiffness and breaking stress. The gradual pattern of cuticle accumulation present in ‘Moneymaker’ probably prevented a transient stage of high density and stiffness, despite the small increase in density observed in ‘Moneymaker’ at 25 daa. Waxes, mostly intracuticular ones, have been shown to confer stiffness to plant cuticles (Petracek & Bukovac, 1995; Takahashi et al., 2012; Khanal et al., 2013) and could also participate in this temporal increase. Wax accumulation in ‘Cascada’ reached its maximum at 20 daa (Domínguez et al., 2008), but removal of waxes reduced Young's modulus similarly at 20 and 25 daa (c. 30%; data not shown), thus maintaining the differences in stiffness observed with waxes.
Cuticle biomechanics during fruit ripening: flavonoid accumulation and cutin depolymerization
Changes in tomato cuticle biomechanics during ripening have already been reported, such as an increase in stiffness and a decrease in maximum strain (Bargel & Neinhuis, 2004; López-Casado et al., 2007), and were attributed to the incorporation of flavonoids in the tomato fruit cuticle (Domínguez et al., 2009), supporting the hypothesis suggested by Bargel et al. (2006) that phenolic compounds could be related to a rigid cutin matrix at full maturity. Our results support this increase in strength and decrease in deformability. The correlation in both genotypes of cuticle stiffness with the amount of phenolic compounds present in the cuticle clearly shows their involvement in providing rigidity to the cuticle once cell walls start their degradation during ripening. Moreover, the higher stiffness during ripening can be explained as an increment in the stress needed to achieve the same elastic deformation. This increase in the length of the elastic phase is responsible for the higher breaking stress, as the length of the viscoelastic phase did not change during ripening. The decrease in the maximum strain is then attributable to a reduced viscoelastic deformation. Thus, flavonoids play two roles: one, providing strength during the elastic phase and two, reducing irreversible viscoelastic deformation. In other words, flavonoids allow the cuticle to withstand transient changes in fruit volume with little expansion. In fact, Domínguez et al. (2012) observed a significant decrease in tomato fruit growth rate during ripening.
Some differences in the mechanical responses of ‘Moneymaker’ and ‘Cascada’ cuticles were observed in this period. Flavonoid accumulation in ‘Moneymaker’ was not associated with an increase in breaking stress. In this case, there was an increase in the yield stress but the viscoelastic phase was clearly shorter. Similarly, the decrease in the viscoelastic deformation was more pronounced than in ‘Cascada’. These differences could be related to differences in the changes in cuticle density observed in the two genotypes. Whereas in ‘Cascada’ density increased during ripening and could contribute to the strengthening of the cuticle, in ‘Moneymaker’ the significant decrease in density would promote the opposite effect: lowering the breaking stress despite flavonoid accumulation.
The flavonoids naringenin and chalconaringenin are the main phenolic constituents present in the tomato fruit cuticle at red ripe, albeit some phenolic acids such as p-coumaric and ferulic acids have also been reported in low quantities (Hunt & Baker, 1980; Laguna et al., 1999). In the ‘Cascada’ cuticle, p-coumaric and p-hydroxybenzoic acids were present throughout fruit development and flavonoids only started their accumulation at 45 daa. p-hydroxybenzoic acid has been reported to be a minor component of both angiosperm and gymnosperm primary cell walls (Hartley & Harris, 1981; Carnachan & Harris, 2000; El Modafar & El Boustani, 2001). Hence, the gradual increase in stiffness between 35 and 45 daa could be attributable to the subsequent increase in p-hydroxybenzoic acid that occurred during this period in the cutin matrix. According to the literature, phenolic acids esterified to cell walls contribute to their mechanical properties, conferring stiffness and resistance (Peyron et al., 2001; Antoine et al., 2003). However, the maximum increase in cuticle stiffness and breaking stress occurred between breaker and red ripe, where p-hydroxybenzoic acid decreased and flavonoids accumulated. This suggests that the flavonoids naringenin flavanone and chalcone are the major contributors to the almost 2.5-fold increase in phenolics observed during this period.
Significant changes in the cutin matrix were observed during ripening. They corresponded to a decrease in the overall number of ester bonds and an increase in hydroxyl and carboxylic acid functional groups, which implies a rearrangement of the polymer chains into a different conformation with fewer ester or strong H bonds. These results indicate a depolymerization of the cutin matrix that did not reduce the mechanical strength of the cuticle. On the contrary, these changes were associated with an increase in cuticle stiffness, probably as a result of the role of phenolics as fillers restraining the mobility of cutin chains. A similar role as fillers has recently been reported for triterpenoids in the persimmon fruit (Diospyros kaki) cuticle (Tsubaki et al., 2013). Moreover, the ATR-FTIR spectroscopic profile showed a heterogeneous distribution of phenolic compounds within the cuticle. Thus, the highest amount of phenolics was present in the cutin, followed by the cuticle outer surface, and the lowest amount was detected in the cuticle inner surface. This heterogeneous allocation indicates that phenolics mainly interact and modify the cutin matrix and very little the polysaccharides derived from the cell wall. The significant correlation observed between the γ(C-H) aromatic band, the amount of phenolics (mostly flavonoids), and the degree of cutin esterification suggests that this band can be used to monitor these changes in the cutin matrix.
Ripening-related changes in the cuticle showed a co-regulation of flavonoid incorporation with depolymerization of the cutin matrix and modification of cuticle density. It would be of great interest to ascertain if these changes are regulated by ripening genes or are a direct/indirect consequence of flavonoid incorporation. The study of transgenic plants that do not accumulate flavonoids would be a first step towards identifying the regulatory pathway and eventually the different genes involved and their contribution to cuticle mechanics. In conclusion, cuticles from fruits at the initial stages of development are mechanically weak and only exhibit elastic deformation. Once a significant amount of cutin has been accumulated, viscoelastic behaviour appears and the mechanical behaviour of the cuticle remains mostly unaltered during the fruit expansion period. Ripening is accompanied by several changes in the cuticle, such as chemical cleavage of cutin ester bonds and incorporation of phenolic compounds. These changes modify both the elastic and viscoelastic behaviour of the cuticle which becomes much stiffer and less deformable.