Tomato fruit continues growing while ripening,affecting cuticle properties and cracking

Fruit cuticle composition and their mechanical performance have a special role during ripening because internal pressure is no longer sustained by the degraded cell walls of the pericarp but is directly transmitted to epidermis and cuticle which could eventually crack. We have studied fruit growth, cuticle modifications and its biomechanics, and fruit cracking in tomato; tomato has been considered a model system for studying fleshy fruit growth and ripening. Tomato fruit cracking is a major disorder that causes severe economic losses and, in cherry tomato, crack appearance is limited to the ripening process. As environmental conditions play a crucial role in fruit growing, ripening and cracking, we grow two cherry tomato cultivars in four conditions of radiation and relative humidity (RH). High RH and low radiation decreased the amount of cuticle and cuticle components accumulated. No effect of RH in cuticle biomechanics was detected. However, cracked fruits had a significantly less deformable (lower maximum strain) cuticle than non‐cracked fruits. A significant and continuous fruit growth from mature green to overripe has been detected with special displacement sensors. This growth rate varied among genotypes, with cracking‐sensitive genotypes showing higher growth rates than cracking‐resistant ones. Environmental conditions modified this growth rate during ripening, with higher growing rates under high RH and radiation. These conditions corresponded to those that favored fruit cracking. Fruit growth rate during ripening, probably sustained by an internal turgor pressure, is a key parameter in fruit cracking, because fruits that ripened detached from the vine did not crack.     

Altered Tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.) Fruit Cuticle Biomechanics of a Pleiotropic Non Ripening Mutant

Tomato (Lycopersicon esculentum Mill.) fruit ripening involves multiple metabolic changes resulting in softening and pigmentation. We investigated the mechanics and morphology of the enzymatically isolated cuticular membrane (CM) of cv. Ailsa Craig wild-type (wt) and nonripening mutant (nor) at three developmental stages. Cuticle thickness and degree of cutinization increased significantly from immature to fully ripe fruits for both wt and nor without differences between them. Mechanical characterization was carried out on dry and fully hydrated samples in uni-axial tension to determine their modulus of elasticity, stress, and strain at failure. Corresponding stress-strain diagrams were biphasic and showed yield for virtually all dry CM samples, while that of hydrated CM displayed considerable differences between wt and nor fruits. Concerning the mechanical properties, the CM of wt fruits was characterized by increasing stiffness and strength during fruit growth and maturation in both dry and hydrated states, whereas the CM of nor fruits was significantly less stiff and weaker at full maturity. Hydration generally caused lower moduli of elasticity and strength, while breaking strain was significantly affected only for the CM of ripe nor fruits. This plasticizing effect of water increased towards full maturity for both wt and nor, and may be related to fiber content in the CM matrix and hydration state of the cuticle. Comparative analysis of two additional wild-type tomato cultivars supported the ripening-related stiffening of the CM of Ailsa Craig wt and the altered mechanical properties of the nor mutant, as well as the plasticizing effect of water.     

Viscoelastic nature of isolated tomato (Solanum lycopersicum) fruit cuticles: a mathematical model

Viscoelastic behaviour of isolated tomato fruit cuticle (CM) is well known and extensively described. Temperature and hydration conditions modify the mechanical properties of CM. Mechanical data from previous transient‐creep analysis developed in tomato fruit cuticle under different temperature and hydration conditions have been used to propose a rheological model that describes the viscoelastic nature of CM. As a composite material, the biomechanical behaviour of the plant cuticle will depend not only on the mechanical characteristics of the individual components by themselves but also on the sum of them. Based on this previous information, we proposed a two‐element model to describe the experimental behaviour: an elastic hookean element connected in parallel to a viscous element or Voigt element that will describe the mechanical behaviour of the isolated CM and cutin under the studied conditions. The main parameters of the model, E₁ and E₂ will reflect the elastic and viscoelastic behaviour of the cuticle. Relationship between these physical parameters and the change in CM properties were discussed in order to elucidate the rheological processes taking place in CM. This model describes both the influence of temperature and hydration and the behaviour of the isolated cutin and the inferred contribution of the cuticle fraction of polysaccharides when the whole cuticle is tested.     

Regulation of tomato fruit growth by epidermal cell wall enzymes

Water relations of tomato fruit and the epidermal and pericarp activities of the putative cell wall loosening and tightening enzymes Xyloglucan endotransglycosylase (XET) and peroxidase were investigated, to determine whether tomato fruit growth is principally regulated in the epidermis or pericarp. Analysis of the fruit water relations and observation of the pattern of expansion of tomato fruit slices in vitro , has shown that the pericarp exerts tissue pressure on the epidermis in tomato fruit, suggesting that the rate of growth of tomato fruit is determined by the physical properties of the epidermal cell walls. The epidermal activities of XET and peroxidase were assayed throughout fruit development. Temporal changes in these enzyme activities were found to correspond well with putative cell wall loosening and stiffening during fruit development. XET activity was found to be proportional to the relative expansion rate of the fruit until growth ceased, and a peroxidase activity weakly bound to the epidermal cell wall appeared shortly before cessation of fruit expansion. No equivalent peroxidase activity was detected in pericarp tissue of any age. It is therefore plausible that the expansion of tomato fruit is regulated by the combined action of these enzyme activities in the fruit epidermis.     

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

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.     

A reevaluation of the key factors that influence tomato fruit softening and integrity

The softening of fleshy fruits, such as tomato (Solanum lycopersicum), during ripening is generally reported to result principally from disassembly of the primary cell wall and middle lamella. However, unsuccessful attempts to prolong fruit firmness by suppressing the expression of a range of wall-modifying proteins in transgenic tomato fruits do not support such a simple model. 'Delayed Fruit Deterioration' (DFD) is a previously unreported tomato cultivar that provides a unique opportunity to assess the contribution of wall metabolism to fruit firmness, since DFD fruits exhibit minimal softening but undergo otherwise normal ripening, unlike all known nonsoftening tomato mutants reported to date. Wall disassembly, reduced intercellular adhesion, and the expression of genes associated with wall degradation were similar in DFD fruit and those of the normally softening 'Ailsa Craig'. However, ripening DFD fruit showed minimal transpirational water loss and substantially elevated cellular turgor. This allowed an evaluation of the relative contribution and timing of wall disassembly and water loss to fruit softening, which suggested that both processes have a critical influence. Biochemical and biomechanical analyses identified several unusual features of DFD cuticles and the data indicate that, as with wall metabolism, changes in cuticle composition and architecture are an integral and regulated part of the ripening program. A model is proposed in which the cuticle affects the softening of intact tomato fruit both directly, by providing a physical support, and indirectly, by regulating water status.     

Ongoing Growth Challenges Fruit Skin Integrity

As a result of continuing volume and hence surface-area growth, the skins of most fruit species suffer ongoing strain throughout development. Maintenance of surface integrity is essential to protect the underlying tissues from desiccation and pathogen attack. Fruit skins are commonly “primary” in structure. They comprise a polymeric cuticle overlying an epidermis and a hypodermis. The cuticle is responsible for the skin's barrier function and the cellular layers for the skin's load-bearing functions. Skin failure can be just of the cuticle layer (microcracking) resulting in barrier impairment or it can involve cuticle and cellular layers (macrocracking) resulting in both barrier and structural impairment. Fruit skin failure is associated with a number of disorders including shriveling, cracking, russeting, and skin spots. All result in reduced market value. Our objective is to review the literature on the strategies adopted by fruit to cope with the challenge of continuing skin expansion. We uncover a multistep strategy to prevent or minimize the risk of fruit skin failure. This comprises: (1) area expansion of the load-bearing skin-cell layer(s) by ongoing cell division and (2) the avoidance of skin stress or strain concentrations by matching patterns of skin-cell division to those of area expansion. Also involved, (3) are the partitioning of cuticle strain into plastic and viscoelastic components at the expense of the elastic one. For this, wax and cutin are deposited in the cuticle during growth. Wax and cutin deposition “fix” the strain in the cuticle. Cutin is preferentially deposited on the inner surface of the cuticle, which fixes the strain, but it leaves the outer cuticle surface more strained. Last, (4) if the primary skin is damaged, the barrier functions are restored by the formation of a “secondary” fruit surface (periderm). Lignin can also be used to strengthen the underlying cells following structural failure.     

Biomechanics and anatomy of Lycopersicon esculentum fruit peels and enzyme-treated samples

We report the biomechanics and anatomy of fruit wall peels (before and after cellulase/pectinase treatment) from two Lycopersicon esculentum cultivars (i.e., Inbred 10 and Sweet 100 cherry tomatoes). Samples were tested before and after enzyme treatment in uniaxial tension to determine their rate of creep, plastic and instantaneous elastic strains, breaking stress (strength), and work of fracture. The fruit peels of both cultivars exhibited pronounced viscoelastic and strain-hardening behavior, but differed significantly in their rheological behavior and magnitudes of material properties, e.g., Inbred 10 peels crept less rapidly and accumulated more plastic strains (but less rapidly), were stiffer and stronger, and had a larger work of fracture than Sweet 100 peels. The cuticular membrane (CM) also differed; e.g., Sweet 100 CM strain-softened at forces that caused Inbred 10 to strain-harden. The mechanical behavior of peels and their CM correlated with anatomical differences. The Inbred 10 CM develops in subepidermal cell layers, whereas the Sweet 100 CM is poorly developed below the epidermis. Based on these and other observations, we posit that strain-hardening involves the realignment of CM fibrillar elements and that this phenomenon is less pronounced for Sweet 100 because fewer cell walls contribute to its CM compared to Inbred 10.     

NMR characterization of hydration and thermal stress in tomato fruit cuticles

In its natural environment, the plant cuticle, which is composed of the biopolymer cutin and a mixture of surface and embedded cuticular waxes, experiences a wide variety of temperatures and hydration states. Consequently, a complete understanding of cuticular function requires study of its thermal and mechanical properties as a function of hydration. Herein, we report the results of a comprehensive 13C nuclear magnetic resonance (NMR) relaxation study of hydrated tomato fruit cuticle. Cross-polarization and direct-polarization experiments serve to measure the solid-like and liquid-like components, respectively, of hydrated cuticle. Localized, high-frequency motions are probed by T1(C) spin relaxation measurements, whereas T1rho(H) and T1rho(C) experiments reflect low-frequency, lower amplitude polymer-chain motions. In addition, variable-temperature measurements of T1(C) and T1rho(C) for dry tomato cuticles are used to evaluate the impact of temperature stress. Results of these experiments are interpreted in terms of changes occurring in individual polymer motions of the cutin/wax components of tomato cuticle and in the interaction of these components within intact cuticle, both of which are expected to influence the functional integrity of this protective plant covering.     

Xyloglucan endotransglucosylase/hydrolases (XTHs) during tomato fruit growth and ripening

Depolymerization of cell wall xyloglucan has been proposed to be involved in tomato fruit softening, along with the xyloglucan modifying enzymes. Xyloglucan endotransglucosylase/hydrolases (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151) have been proposed to have a dual role integrating newly secreted xyloglucan chains into an existing wall-bound xyloglucan, or restructuring the existing cell wall material by catalyzing transglucosylation between previously wall-bound xyloglucan molecules. Here, 10 tomato (Solanum lycopersicum) SlXTHs were studied and grouped into three phylogenetic groups to determine which members of each family were expressed during fruit growth and fruit ripening, and the ways in which the expression of different SlXTHs contributed to the total XET and XEH activities. Our results showed that all of the SlXTHs studied were expressed during fruit growth and ripening, and that the expression of all the SlXTHs in Group 1 was clearly related to fruit growth, as were SlXTH12 in Group 2 and SlXTH6 in Group 3-B. Only the expression of SlXTH5 and SlXTH8 from Group 3-A was clearly associated with fruit ripening, although all 10 of the different SlXTHs were expressed at the red ripe stage. Both total XET and XEH activities were higher during fruit growth, and decreased during fruit ripening. Ethylene production during tomato fruit growth was low and experienced a significant increase during fruit ripening, which was not correlated either with SlXTH expression or with XET and XEH activities. We suggest that the role of XTH during fruit development could be related to the maintenance of the structural integrity of the cell wall, and the decrease in XTHs expression, and the subsequent decrease in activity during ripening may contribute to fruit softening, with this process being regulated through different XTH genes.     

Fruit cuticle lipid composition during development in tomato ripening mutants

Recent studies suggest that fruit cuticle is an important contributing factor to tomato (Solanum lycopersicum) fruit shelf life and storability. Moreover, it has been hypothesized that variation in fruit cuticle composition may underlie differences in traits such as fruit resistance to desiccation and microbial infection. To gain a better understanding of cuticle lipid composition diversity during fruit ontogeny and to assess if there are common features that correlate with ripening, we examined developmental changes in fruit cuticle wax and cutin monomer composition of delayed‐ripening tomato fruit mutants, ripening inhibitor (rin) and non‐ripening (nor) and delayed‐ripening landrace Alcobaça. Previous reports show that fruit ripening processes such as climacteric ethylene production, cell wall degradation and color change are significantly delayed, or do not occur, in these lines. In the study presented here, however, we show that fruits from rin, nor and Alcobaça have cuticle lipid compositions that differ significantly from normal fruits of Ailsa Craig (AC) even at very early stages in fruit development, with continuing impacts throughout ripening. Moreover, rin, nor and the Alcobaça lines show quite different wax profiles from AC and each other throughout fruit development. Although cutin monomer composition differed much less than wax composition among the genotypes, all delayed‐ripening lines possessed higher relative amounts of C₁₈ monomers than AC. Together, these results reveal new genetic associations between cuticle and fruit development processes and define valuable genetic resources to further explore the importance of cuticle in fruit shelf life.     

The Influences of Epicuticular Wax Disruption and Cutinase Resistance on Penetration of Tomatoes by Colletotrichum gloeosporioides

Conidia of Colletotrichum gloeosporioides germinated on green and ripe tomato fruit with intact epicuticular wax, and formed penetration pegs below melanized appressoria. If the delicate layer of epicuticular wax was distupted by abrasion or removed by solvents before inoculation, apparent increased diffusion of fruit substances into the inoculum stimulated fungal growth, hyphal anastomosis and the production of penetration pegs from hyaline appressoria. This was followed by cutlcle erosion centred on the penetration pegs in green fruit allowing sec-ondary growth of infection hyphae. Due to the development of cutinase resistance when the cuticle became yellow at ripening, no cuticle erosion occurred at penetrations on ripe fruit Since cuticle erosion followed penetration of the cutinase-susceptible cuticle and since penetration peg formation was not hindered by the cutinase cuticle, the process of primary penetration is regarded as mechanical.     

Inhibitor-resistant early ethylene production during tomato fruit development

In addition to the ethylene formed at the onset of tomato fruit ripening, three peaks of ethylene are produced during earlier periods of in vitro development of tomato flower to fruit. This is the first report characterizing ethylene production during early development of tomato fruit. Previous reports from this laboratory showed that VFNT Cherry tomato calyces are transformed into fruit tissue when cultured in vitro at lower temperatures (16–23 °C). Early ethylene production was also measured in these ripening calyces, as well as in fruit and calyces of other tomato cultivars cultured in vitro. Calyces from Ailsa Craig and rin tomato flowers, which are not transformed into fruit tissue at these lower temperatures, also form ethylene during early periods of in vitro culture, but to a much smaller extent. Unlike ethylene formed at the onset of fruit ripening, the earlier peaks are resistant to the inhibitors, aminovinylglycine (AVG) and CoCl₂. The data suggest that ethylene produced during earlier periods of tomato fruit development is formed by an alternative biosynthetic pathway.     

Partial purification of tomato fruit peroxidase and its effect on the mechanical properties of tomato fruit skin

Peroxidase (EC 1.11.1.7)-mediated stiffening of cell walls within the fruit skin of tomato is hypothesized to regulate fruit growth. However, to date, there is no experimental evidence demonstrating that peroxidase affects the mechanical properties of skin tissue. Here, the mechanical properties of skin strips excised from a range of fruits at different ages were determined using an 'Instron' universal material testing instrument. The stiffness of tomato fruit skin strips increases 3-fold with increasing fruit age. Application of partially-purified peroxidase from the cell walls of mature tomato fruit skin significantly increased the stiffness of fruit skin irrespective of the age of fruit. Furthermore, the application of hydrogen peroxide significantly increased the stiffness of skin strips excised from fruit of an age when endogenous peroxidase isozymes associated with the termination of growth are first detected. The results support the hypothesis that the tomato fruit skin plays an integral role in the regulation of tomato fruit growth, and that changes in its mechanical properties may be mediated by peroxidase. As far as is known, this is the first demonstration that peroxidases alter the mechanical properties of the plant cell wall.     

Relative humidity and temperature modify the mechanical properties of isolated tomato fruit cuticles

The mechanical properties of enzymatically isolated cuticular membrane (CM) from ripe tomato fruits were investigated at 10 to 45°C and relative humidity (RH) of 40 to wet. CM samples were stressed by uniaxial tension loads to determine their tensile modulus, E, breaking stress (strength), σ(max), and maximum elongation, ε(max). The CM stress-strain curves revealed a biphasic behavior when tested at RH values below wet conditions. In the first phase, CM responded to the loads by instantaneous extension with no further extension recorded until a further load was added: defined as pure elastic strain (E(e)). In the second phase, CM responded by instantaneous extension and by some additional time-dependent extension, defined as viscoelastic strain (E(v)). When CMs were submerged in aqueous solution (wet), the stress-strain curves were monophasic, with both elastic and viscoelastic strain. E(e) depended on RH and was higher than E(v), which was independent of RH. Temperature decreased E(e) and σ(max) of tomato fruit CM. Temperature response was not linear but consisted of two temperature-independent phases separated by a transition temperature. This transition zone has been related previously to the presence of a secondary phase transition in the cutin matrix of the tomato fruit CM.     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

CHS silencing suggests a negative cross-talk between wax and flavonoid pathways in tomato fruit cuticle

Tomato fruits (Solanum lycopersicum L.) accumulate flavonoids in their cuticle and epidermal cells during ripening. These flavonoids come from de novo biosynthesis due to a significant increase in chalcone synthase (CHS) activity during ripening. Virus-induced gene silencing (VIGS) of tomato fruits have been used to down-regulate SlCHS expression during ripening and analyze the effects at the epidermal and cuticle level. Besides the expected change in fruit color due to a lack of flavonoids incorporated to the cuticle, several other modifications such as a decrease in the amount of cutin and polysaccharides were observed. These indicate a role for either flavonoids or CHS in the alteration of the expression levels of some genes involved in cuticle biosynthesis. Moreover, a negative interaction between the 2 cuticle components, flavonoids and waxes, suggests a relationship between these 2 metabolic pathways.     

Rheological Properties of Enzymatically Isolated Tomato Fruit Cuticle

Rheological properties were determined for cuticular membranes (CMs) enzymatically isolated from mature tomato (Lycopersicon esculentum Mill. cv Pik Red) fruit. The cuticle responded as a viscoelastic polymer in stress-strain studies. Both CM and dewaxed CM expanded and became more elastic and susceptible to fracture when hydrated, suggesting that water plasticized the cuticle. Dewaxing of the CM caused similar changes in elasticity and fracturing, indicating that wax may serve as a supporting filler in the cutin matrix. Exposure of the cuticle to the surfactant Triton X-100 did not significantly affect its rheological properties.